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Metabolomic-guided discovery
of cyclic nonribosomal peptides
from Xylaria ellisii sp. nov., a leaf
and stem endophyte of Vaccinium
angustifolium
Ashraf Ibrahim1,7, Joey B. Tanney2,3,4, Fan Fei1, Keith A. Seifert4, G. Christopher Cutler5,
Alfredo Capretta1, J. David Miller2 & Mark W. Sumarah2,6*
Fungal endophytes are sources of novel bioactive compounds but relatively few agriculturally
important fruiting plants harboring endophytes have been carefully studied. Previously, we identied a
griseofulvin-producing Xylaria species isolated from Vaccinium angustifolium, V. corymbosum, and Pinus
strobus. Morphological and genomic analysis determined that it was a new species, described here as
Xylaria ellisii. Untargeted high-resolution LC-MS metabolomic analysis of the extracted ltrates and
mycelium from 15 blueberry isolates of this endophyte revealed dierences in their metabolite proles.
Toxicity screening of the extracts showed that bioactivity was not linked to production of griseofulvin,
indicating this species was making additional bioactive compounds. Multivariate statistical analysis
of LC-MS data was used to identify key outlier features in the spectra. This allowed potentially new
compounds to be targeted for isolation and characterization. This approach resulted in the discovery
of eight new proline-containing cyclic nonribosomal peptides, which we have given the trivial names
ellisiiamides A-H. Three of these peptides were puried and their structures elucidated by one and two-
dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and high-resolution tandem
mass spectrometry (HRMS/MS) analysis. The remaining ve new compounds were identied and
annotated by high-resolution mass spectrometry. Ellisiiamide A demonstrated Gram-negative activity
against Escherichia coli BW25113, which is the rst reported for this scaold. Additionally, several
known natural products including griseofulvin, dechlorogriseofulvin, epoxy/cytochalasin D, zygosporin
E, hirsutatin A, cyclic pentapeptides #1–2 and xylariotide A were also characterized from this species.
Vaccinium angustifolium (wild lowbush blueberries or commonly wild blueberries) were consumed fresh and
preserved for the winter by the Indigenous peoples of northeastern North America and rapidly incorporated into
the diets of European settlers in Canada from the early 17th century1,2. Today, blueberries comprise more than
half of all fruit production in Canada. Wild blueberries oen grow in forests where Pinus strobus (eastern white
pine) is the dominant tree species. Eastern white pine is an economically, ecologically, and culturally important
keystone tree species in eastern N. American forests, especially for bird species3,4.
Endophytes are an ecological category of phylogenetically diverse fungi that can asymptomatically colonize
healthy plant tissues. Ascomycetous endophytes of various species of Vaccinium have been reported over the
past three decades. is includes from surface-sterilized tissues of Vaccinium vitis-idaea (lingonberry, European
1Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, L8S 4M1, Canada.
2Department of Chemistry, Carleton University, Ottawa, Ontario, K1S 5B6, Canada. 3Pacic Forestry Centre, Canadian
Forest Service, Natural Resources Canada, Victoria, British Columbia, V8Z 1M5, Canada. 4Ottawa Research and
Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, K1A 0C6, Canada. 5Department of Plant,
Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS, B2N 5E3, Canada. 6London
Research and Development Centre, Agriculture and Agri-Food Canada, London, Ontario, N5V 4T3, Canada. 7Present
address: LifeMine Therapeutics, Cambridge, Massachusetts, 02140, USA. *email: mark.sumarah@canada.ca
OPEN
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blueberry) and V. myrtillus (bilberry, whortleberry) in Europe5, V. dunalianum var. urophyllum (South China
blueberry) in China6, as well as from stems of V. macrocarpon (cranberries) and V. corymbosum (northern high-
bush blueberry) in New Jersey7,8. ere is some evidence of the same endophyte species occurring in both conifer
and Vaccinium species, e.g. Nemania diusa (Xylariaceae)6,9,10 and Phacidiaceae species such as Allantophomopsis
lycopodina, Phacidium lacerum, and Strasseria geniculata11–15. Indirect evidence of a conifer-Vaccinium shared
endophyte includes the aquatic hyphomycete Dwayaangam colodena, a common needle endophyte of Picea spp.,
which was reported from rainwater collected from foliage of Picea abies, Pinus sylvestris, and Vaccinium myrtillus
in Europe16–19. Discovery of an aquatic hyphomycete conifer endophyte and reports of hardwood saprotrophs as
conifer endophytes (e.g. Phialocephala piceae20) are evocative of more complex interactions between endophytes
and their environment.
Endophytes belonging to the family Xylariaceae (Xylariales, Sordariomycetes) are ubiquitous and detected in
varying abundance in most studies involving woody plants, regardless of geographic location or host, whether
by isolation of cultures or by studies of DNA, oen exhibiting little host preference and including known sapro-
trophs21–24. Xylariaceae endophytes are common but dicult to identify to species because of a lack of reference
sequences and the limited taxonomic resolution of the asexual states (usually the only morphological characters
produced in vitro). However, careful eld observations can provide connections between the oen conspicu-
ous Xylariaceae stromata found in nature and the corresponding endophytes isolated in culture or detected by
DNA sequences from the same forests23–25. Taxonomically, Xylariaceae comprises at least 37 genera with likely
more than 1,000 species26. Many endophyte studies based on morphological identication of cultures report
geniculosporium-like morphs attributable to Anthostomella, Rosellinia and Xylaria species27–31. e classical
nature of most taxonomic studies of Xylariaceae is reected by the need for the sexual state to conrm identica-
tion, with a relative paucity of species-specic DNA barcodes and phylogenetic markers compared to many other
ascomycete groups. us, xylariaceous endophytes may include species and genera known to classical taxonomy
but not included in sequence databases (i.e.: named-but-unsequenced species).
Species of Xylariaceae are a rich source of secondary metabolites, and chemotaxonomy is oen part of taxo-
nomic studies. Species in this family can produce diverse metabolites from multiple biosynthetic families includ-
ing dihydroisocoumarins, punctaporonins, cytochalasins, butyrolactones, and succinic acid derivatives32,33.
Exploration of Xylaria metabolites using newer chemical methods led to discovery of a broad array of metabolites
from both tissues of stromata and culture extracts34.
Although there have been many studies of metabolites from fungal endophytes35,36, there are few reports from
endophytes of Vaccinium37,38. We previously described production of the antifungal compounds griseofulvin and
piliformic acid from an unknown Xylaria species isolated as a foliar endophyte from wild blueberry in natural and
commercial sites, and from white pine. Aer the Richardson et al. (2014) study, we continued to isolate the same
unidentied species of Xylaria as an endophyte of white pine needles and as an endophyte of leaves and stems
of both wild and highbush blueberry at three dierent locations in Nova Scotia, New Brunswick, and Ontario,
Canada38. We also conducted eld sampling to discover the putative sexual state of this unknown Xylaria species.
is would provide information on morphological characters of sexual structures, permitting its identication.
is previously unknown endophyte is described here as Xylaria ellisii based on morphological and genomic
evidence. Representative sequences in NCBI GenBank from other studies indicate that X. ellisii has been isolated
many times as an unidentied endophyte from a wide variety of plant hosts, allowing us to infer additional infor-
mation about its distribution, biology, and chemistry.
In our eort to discover novel natural products, we applied a LC-MS metabolomic-guided discovery approach
to these Xylaria strains from wild and highbush blueberry plants (Fig.1). is approach allows for a global survey
of small molecule metabolites from an extract and visual representation of metabolite variances between group-
ings or extracts. us, discriminating between like and dierent features allows extracts to be prioritized for fur-
ther investigation39,40. Fieen strains were grown on two media and the resulting ethyl acetate extracted ltrates
and mycelium were screened using standardized LC-UV/MS conditions. Multivariate statistical analysis was used
to organize resulting analytical data to reveal extracts that appeared to have dierences in their major secondary
metabolites. is approach led to the discovery of a family of eight new proline-containing cyclic nonribosomal
pentapeptides named ellisiiamides A–H. Ellisiiamide A is an alanine (Ala) substituted variant, a rst report for
this scaold, and demonstrated modest activity against Escherichia coli.
Results
Identication, biology and ecology of Xylaria sp. Approximately 30 strains of Xylaria sp. were isolated
from surface-sterilized blueberry tissues collected from highbush and wild blueberry elds within a ~300 ×
100 km triangular area. All elds were surrounded by forested lands. Preliminary phylogenetic analysis using the
internal transcribed spacer (ITS) barcode combined with morphological features conrmed conspecicity of iso-
lated endophytic Xylaria sp. strains. However, identication of the strains to species was not possible using molec-
ular or in vitro morphological data. Based on a BLAST query of the Xylaria sp. ITS and RPB2 sequences with
available GenBank sequences, the endophyte strains were closest related to sequences identied as Xylaria berteri,
X. castorea, X. cubensis, X. laevis, and X. longipes, species that form conspicuous sexual reproductive structures
(stromata) from decaying hardwood. Given the close phylogenetic relationship of the unknown Xylaria endo-
phyte to these species and evidence of prevalent endophytic-saprotrophic life histories within Xylariaceae23–25,41,42,
we inferred that the unknown Xylaria endophyte likely produces stromata from decaying hardwood in mixed-
wood stands in the Acadian forest. us, Xylaria stromata were selectively sampled during ongoing eld surveys
to collect the putative sexual state of the endophyte. is would provide material for identication and insight
into its life history20,43.
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A Xylaria sp. producing stroma reminiscent of X. corniformis and X. curta was collected from decaying, oen
partially buried, Acer saccharum branches or logs in late summer and autumn. Sequences (ITS, SSU, LSU, BenA,
EF1-α, RPB2) obtained from stromatal tissue and ascospore cultures were identical to those obtained from
the Xylaria sp. endophyte cultures, indicating they are conspecic and evincing a saprotrophic-endophytic life
history. Based on morphological study of the stromata, this species is equivalent to X. corniformis var. obovata
Sacc., Xylaria corniformis sensu Laessøe44, and Xylaria curta sensu Rogers45. From the RPB2 phylogeny, X. corni-
formis var. obovata is weakly supported (posterior probability value (PP) = 0.56) sister to X. laevis and other
species within the strongly-supported (PP = 1.0) X. cubensis aggregate clade. Xylaria is polyphyletic, including
Amphirosellinia nigrospora, Stilbohypoxylon quisquiliarum, and Nemania serpens, and the type species (X. hypox-
ylon) occurs in a basal clade sister to X. bambusicola. Additional RPB2 sequences for related Xylaria species are
needed to generate a more comprehensive phylogeny (Fig.2).
Several DAOMC herbarium specimens identied as X. corniformis from Acer spp. wood in Ontario and
Quebec were morphologically similar to X. laevis. e resulting ITS sequences from these specimens showed that
they formed a clade sister to X. longipes and X. primorskensis and were distinct from the griseofulvin-producing
X. corniformis var. obovata (Fig.3). We support the distinction of X. corniformis var. obovata from X. corni-
formis, and thus describe a new species, Xylaria ellisii, to accommodate its novelty and fulll the need to delineate
boundaries in species complexes with robust species concepts connected to authenticated reference sequences
and specimens.
LC-MS analysis of culture extracts and multivariate data analysis. Fieen strains of X. ellisii were
subject to further study: four from cultivated highbush blueberry plants and 11 from wild blueberry plants. Ethyl
acetate extracts of the culture ltrate and associated mycelium were screened using standardized LC-UV/MS
conditions.
In order to identify unique secondary metabolite dierences between extracts of Xylaria isolates of highbush
and wild blueberry plants we compared the extracted ltrates and mycelium with three dierent pair-wise com-
parisons. ese comparisons included: ethyl acetate extracts of Xylaria strains grown on 2% malt extract broth
(ML) versus those grown in potato dextrose broth (PDB) cultures; ML media cultures of highbush versus wild
varieties; and, PDB medium cultures of highbush versus wild isolates (Fig.1S). A supervised multivariant analysis
Figure 1. Discovery of new griseofulvin-producing fungal endophyte species Xylaria ellisii isolated from
highbush and wild blueberry leaves and stems. (A) Isolation and culturing of fungal endophyte, (B) LC-UV
comparative prole analysis of crude ltrate extracts at λ 210 nm, revealing dierences in metabolite
production, (C) Most likely tree from a RAxML analysis of ITS dataset containing representative endophytes.
Culture numbers precede the species name and RAxML bootstrap support percentages ≥50 from a summary
of 1000 replicates are presented at the branch nodes. is tree was rooted with Mucor ellipsoideus (ATCC MYA-
4767; NR_111683) and the scale bar represents the number of substitutions per site.
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method, Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), was used to identify outlier metab-
olites biosynthesized under the dierent culture conditions tested. OPLS-DA correlates dierences in secondary
metabolite feature abundances (X variables) to various treatment groups (Y variables) by identifying principle
components that describe dierences. R2X, R2Y, and Q2 parameters are important validation parameters used for
OPLS-DA, where R2X and R2Y describes the percentage of X and Y variables described by the model (Fig.4 and
Supplementary Fig.2S). A valid model is dened as having a prediction statistic of Q2 > 0.4, with values above 0.7
being highly signicant46. Metabolite features with a high Variable Importance in Projection (VIP) scores (>0.7)
are responsible for driving the dierences between treatment groups, and these values are considered signi-
cant47. eir metabolic features can be viewed at both ends of the OPLS-DA S-plot.
Fractions with VIP scores above 0.7 were selected for further study and compounds were identied where pos-
sible. OPLS-DA validation parameters for each of the extracted ltratesand myceliummetabolite models tested
are summarized in Table1 and Supplementary Table1S. In total, 3856 metabolite features were identiedfrom
Figure 2. Bayesian 50% majority rule RPB2 consensus tree containing Xylaria ellisii and related species. All
unlabeled branches have Bayesian posterior probability values of 1.0; values lower than 1.0 are presented at
nodes. e tree was rooted to Barrmaelia rhamnicola (CBS:142772) and the scalebar indicates the expected
number ofchanges per site. Strain numbers follow species names and type specimens are indicated in bold.
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Figure 3. Bayesian 50% majority rule ITS consensus tree containing Xylaria ellisii and related species. All unlabeled
branches have Bayesian posterior probability values of 1.0; values lower than 1.0 are presented at nodes. e tree was
rooted to Nemania serpens and the scalebar indicates the expected number ofchanges per site. GenBank accession
numbers and host information follow species names (when applicable). Type specimens are indicated in bold.
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the extracted ltrates, with a Q2 value of 0.615 for ML versus PDB, and Q2 values of 0.778 for ML and PDB, as well
as highbush versus wild varieties.
Metabolomic-guided discovery and metabolite identication of knowns 1–11. We rst evaluated
metabolites with the top VIP (30, 50, 100) scores for ethyl acetate extracts of the ltrates and methanol/acetone (1:)
extractedmycelia from Xylaria. e initial focus was on metabolites that displayed UV absorption maxima at ~210,
254, 275 or 350 nm (Table2 and Supplementary TablesS1–8). Compounds (100–2000 μg) were puried by reverse
phase semi-preparative HPLC and characterized by NMR (Bruker Advance III 700 MHz NMR with cryoprobe)
(Supplementary Fig.1S). Metabolites were dereplicated against natural product databases including Antibase
(https://www.wiley.com/en-us/AntiBase%3A+The+Natural+Compound+Identifier-p-9783527343591),
Dictionary of Natural Products (http://dnp.chemnetbase.com/faces/chemical/ChemicalSearch.xhtml) and
NORINE (https://bioinfo.li.fr/norine/) using molecular formulas dictated by HRMS data. In addition, a com-
parative analysis was conducted against known fungal metabolites48–51. Using this dereplication approach,
the previously reported compounds 1–11 were identified; (1) griseofulvin, (2) dechlorogriseofulvin, (3)
Figure 4. Supervised multivariate analyses of extracted ltrates from blueberry isolates of X. ellisii endophytes.
e OPLS-DA score plot (a) and S-plot (b) for comparison between X. ellisii endophytes cultured in ML or PDB
media. e OPLS-DA score plots and S-plots compared the X. ellisii endophytes isolates from highbush or wild
blueberries cultured in ML (c,d) or PDB (e,f) medium, respectively.
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cytochalasin D, (4) zygosporin E, (5) epoxycytochalasin D, (6) hirsutain A, (7) pilformic acid, (8) 2, 3-dihydro-2
,4-dimethylbenzofuran-7-carboxylic acid, (9) cyclic pentapeptide 1, (10) xylarotide A, and (11) cyclic pentapep-
tide 2 (Table2). LC-HRMS, NMR, and spectroscopic data for compounds 1–11 conrming their structures can
be found in the Supplementary Methods, Figs.2 and 3S, 32–49S and Tables1–9S).
Structure elucidation of ellisiiamides A-C (12–14). Ellisiiamides A–C (12–14) were identi-
ed by metabolomic analysis of the extracted ltrates and mycelium with high VIP scores (2.6–11.59; Fig.4,
Supplementary Tables1–8S). ese new cyclic pentapeptides are structurally similar to cyclic pentapeptide 1 (9),
with amino acid dierences at positions 2 (Ala/IsoLeu vs. Val) and 3 (Val vs. IsoLeu) within the peptide scaold
(Fig.5 and Supplementary Fig5S, Tables9–12S).
Ellisiiamide A (12) was isolated as a white powder and afforded a protonated molecular ion at m/z 556
(C30H45N5O5 with 11 double bond equivalents). Examination of the 1H and 13C NMR data revealed the
Model Variables*R2X(cum) R2Y(cum) Q2(cum) Conditions
1a 3856 0.15 0.939 0.615 ML, PDB
1b 100 0.313 0.935 0.737 ML, PDB (including top 100 VIP)
1c 3756 0.299 0.982 0.434 ML, PDB (excluding top 100 VIP)
1d 3556 0.207 0.954 0.394 ML, PDB (excluding top 300 VIP)
2a 3856 0.477 0.998 0.778 ML-H, ML-L
2b 30 0.861 0.984 0.864 ML-H, ML-L (including top 30 VIP)
2c 3826 0.544 1 0.718 ML-H, ML-L (excluding top 30 VIP)
2d 3156 0.121 0.73 -0.187 ML-H, ML-L (excluding top 700 VIP)
3a 3856 0.648 1 0.778 PDB-H, PDB-L
3b 50 0.589 0.995 0.885 PDB-H, PDB-L, (including top 50 VIP)
3c 3806 0.651 1 0.668 PDB-H, PDB-L (excluding top 50 VIP)
3d 2956 0.0851 0.894 -0.175 PDB-H, PDB-L (excluding top 900 VIP)
Table 1. A summary of validation parameters (R2X, R2Y, Q 2) of all calculated OPLS-DA models for extracted
ltrates of X. ellisii endophytes isolates from wild and highbush blueberries cultured in ML and PDB media.
ML-H, endophyte isolates from highbush blueberries cultured in ML medium; ML-L, endophyte isolated from
wild blueberries cultured in ML medium; PDB-H, endophyte isolates from highbush blueberries cultured
in PDB medium; PDB-L, endophyte isolates from wild blueberries cultured in PDB medium. *Number of
metabolomic features included in the OPLS-DA analysis.
#Compound Class Rt Molecular
Formula Measure and
Calculated [M+H]+ppm
error
Known
1Griseofulvin PKS 11.42 C17H18ClO6353.0793 353.0786 −1.98
2Dechlorogriseofulvin*PKS 10.01 C17H19O6319.1173 319.1176 0.94
3Cytochalasin D*PKS-NRPS 11.81 C30H38NO6508.2687 508.2694 1.38
4Zygosporin E*PKS-NRPS 13.94 C30H38NO5492.2742 492.2744 0.41
5Epoxycytochalasin D PKS-NRPS 10.87 C30H38N07524.2651 524.2661 1.91
6Hirsutatin A*NRPS 15.85 C34H53N4O10 677.3741 677.3756 2.21
7Piliformic acid PKS 10.84 C11H18O4Na 237.1094 237.1097 1.27
82,3-dihydro,2,4- dimethylbenzofuran
-7- carboxylic acid PKS 11.15 C11H13O3193.0857 193.0859 1.04
9Cyclic pentapeptide 1*NRPS 16.20 C32H50N5O5584.3816 584.3806 −1.71
10 Xylarotide A NRPS 15.97 C29H52N5O5550.3973 550.3963 −1.82
11 Cyclic pentapeptide 2 NRPS 14.28 C28H50N5O5536.3819 536.3806 −2.42
New
12 Ellisiiamide A*NRPS 14.76 C30H46N5O5556.3501 556.3493 −1.44
13 Ellisiiamide B*NRPS 15.19 C31H48N5O5570.3656 570.3650 −1.05
14 Ellisiiamide C*NRPS 17.04 C33H52N5O5598.3968 598.3963 −0.84
15 Ellisiiamide D NRPS 14.47 C27H48N5O5522.3662 522.3650 −2.30
16 Ellisiiamide E NRPS 16.89 C30H54N5O5564.4132 564.4119 −2.30
17 Ellisiiamide F NRPS 14.11 C31H48N5O6586.3616 586.3599 −1.72
18 Ellisiiamide G NRPS 14.00 C32H50N5O6600.3768 600.3756 −2.00
19 Ellisiiamide H NRPS 14.89 C33H52N5O6614.3936 614.3912 −2.41
Table 2. Identication of known and new secondary metabolites from X. ellisii via LC- UV/HRMS and LC-
HRMS/MS analysis. Select metabolites have been further isolated and characterized by 1D and 2D NMR.
*Structures elucidated by 1D and 2D NMR, HRMS and MS/MS analysis.
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presence of ve α protons (δ 5.08/55.8, 4.49/46.0, 4.17/55.7, 4.74/46.5, 5.10/58.9 ppm), and three key amide
N-H protons for Ala (δ 8.52), ΙsoLeu (δ 6.94) and Leu (δ 8.49) and the N-Methyl group at (δ 3.04/30.2 ppm).
Examination of the multiplicity edited 1H-13C HSQC, 1H-13C HMBC, and 1H-1H COSY NMR data revealed the
individual amino acid spin systems within the peptide scaold based on α proton correlations to the individual
carbonyl carbon, including amide protons to neighboring amino acid carbonyls, and α, β and γ proton corre-
lations (Fig.5, Supplementary Figs.4–13S and Tables9 and 10S). ese correlations supported the amino acid
sequence of cyclo-(NMePhe-Ala-IsoLeu-Leu-Pro). NOESY through-space correlations of αΗ(Ν−MePhe)/NH
(Ala), NH(Ala)/β Η (Ala), NH(IsoLeu)/αΗ (Ala), NH(Leu)/αΗ (IsoLeu) and H3-NMe (N-MePhe)/β Η (Pro)
further supported the amino acid sequence and relative stereochemistry. Analysis of the LC-MS/MS spectra of
ellisiiamide A revealed key diagnostic b-ion fragments of m/z 459.3 (-Pro), 346.2 (-Leu), 233.1 (-IsoLeu), 162.1
(-Ala) and the presence of two fragmentation pathways as seen in cyclic pentapeptide 1 with ring-opening cleav-
age events at the N-MePhe-Pro and Pro-Leu sites52.
Ellisiiamide B (13) was isolated as a white powder with a protonated molecular ion at m/z 570 aording a
molecular formula of C31 H47N5O5 with 11 double bond equivalents. Examination of 1H and 13C NMR data
revealed presence of ve α protons (δ 5.10/56.0, 3.95/57.6, 4.10/56.8, 4.72/46.6, 5.08/58.7 ppm), key amide N-H
protons for Val (δ 8.18), Val2 (δ 6.98), and Leu (δ 8.43), and the N-Methyl group at (δ 3.03/30.2 ppm). Ellisiiamide
B (13) diers from (9) with Val substituted for IsoLeuc at position # 3 (Fig.4, Supplementary Fig.4S and 14–21S,
Table9 and 11S). Examination of the MS/MS spectra revealed a similar fragmentation pattern as in (9) and
(12), with key diagnostic b-ion fragment ions at m/z 471.3 (-Pro), 360.2 (-Leu), 261.2 (-Val), and 162.1 (-Val).
e cyclo-(NMePhe-Val1-Val2-Leu-Pro) amino acid sequence was conrmed with key HMBC correlations of
αΗ(Ν−MePhe)/CO (N-MePhe), H3-NMe (N- MePhe)/CO (Pro), αΗ(Val1)/CO(Val1), αΗ(Val2)/CO(Val2), αΗ
(Leu)/CO (Leu), and αΗ (Pro)/CO (Pro). Key NOESY correlations of αΗ(Ν−MePhe)/NH(Val1), NH(Val1)/αΗ
(Val1), NH(Val2)/αΗ (Val1), NH(Val2)/αΗ (Val2), NH(Val2)/β Η (Val2), NH(Leu)/ΝΗ (Val2), and H3-NMe
(N-MePhe)/β Η (Pro) further supported the assignments.
Ellisiiamide C (14) was isolated as a white powder with a protonated molecular ion at m/z 598 aording
a molecular formula of C33H51N5O5 with 11 double bond equivalents. Examination of 1H and 13C NMR data
revealed the presence of ve α protons (δ 5.10/56.0, 4.05/55.8, 4.25/55.1, 4.72/46.5, 5.09/58.7 ppm), key amide
Figure 5. Ellisiiamides A–H (12–19), new cyclic nonribosomal peptides from Xylaria ellisii. (a) ellisiiamides
A–C (12–14) isolated and characterized by 1D and 2D NMR, LC-HRHMS and LC-HRMS/MS analysis with
new amino acid substituent highlighted. Corresponding COSY/TOCSY (1H -1H), HMBC (1H-13C) and long-
range through-space NOESY/ROESY correlations are shown. (b) Structures of ellisiiamides D–H (15–19)
based on LC-MS/MS, comparative LC-MS/MS analysis of cyclic pentapeptides 9, 11 and 12–14 (c) Amino
acid scaold of the cyclic pentapeptide family of compounds. Cyclic pentapeptide 1 (9) shown with established
amino acid substituents.
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N-H protons for IsoLeu1 (δ 8.12), IsoLeu2 (δ 6.94) and Leu (δ 8.45), and the N-Methyl group at (δ 3.04/30.2
ppm). Ellisiiamide C (14) diers from (9) with IsoLeu substituted for Val at position # 2 (Fig.4, Supplementary
Fig.4S and 22–31S, and Tables9 and 11S). Examination of the 1H-1H COSY, multiplicity edited 1H-13C HSQC
and HMBC NMR data revealed the individual spin system for the new IsoLeuc group with correlations of
β−3Η(IsoLeu)/αΗ (IsoLeu) and δΗ(IsoLeu)/βΗ (IsoLeu). Correlations of the remaining α protons to the indi-
vidual carbonyl carbons, amide protons to neighboring amino acid carbonyl, and HMBC α, β and γ proton
correlations for Leu, Pro and N-MePhe is consistent with the cyclic peptide scaold (Supplementary Table9S.).
NOESY through-space correlations of αΗ(Ν−MePhe)/NH (IsoLeu1), NH (IsoLeu1)/NΗ (IsoLeu2), NH (Leu)/
αΗ (IsoLeu2)) further supported the amino acid sequence. Analysis of the MS/MS spectra of (14) revealed key
diagnostic b-ion fragments of m/z 501.3 (-Pro), 388.3 (-Leu), 275.2 (-IsoLeu2), and 162.1 (-IsoLeu1) further con-
rming the amino acid sequence of cyclo-(N- MePhe-IsoLeu1-IsoLeu2-Leu-Pro).
e optical rotation for ellisiiamides A–C were measured at [α]21 -86.1 (0.06, MeOH), [α]21 -43.1 (0.04,
MeOH), and [α]20 -47.8 (0.06, MeOH) respectively, and were consistent with 9 at [α]21 -63.4 (0.18, MeOH)
(Supplementary Table9S).
LC-MS/MS analysis and putative identication of new cyclic pentapeptides. Ellisiiamide D–H
(15–19) was identied by metabolomic analysis of the extracted ltrates and mycelium models as unique outliers
with high VIP scores (1.92–6.46). Evaluation of the HRESIMS derived molecular formulas and MS/MS fragmen-
tation patterns of (15–19) indicated that the fragmentation sequence and ring-opening events were consistent
with ellisiiamide A–C and cyclic pentapeptide 1. We have therefor assigned putative identication and annotated
structures for ellisiiamides D–H. LC-HRMS/MS characterization data can be found in the Supporting Methods
and Figs.4S and Table 1S and 9S.
Bioactivity activity screening. Compounds 9 and 12–14 were screened for biological activity against
three species of microorganisms in accordance with the Clinical Laboratory Standards Institute (CLSI) proto-
cols (National Committee for Clinical Laboratory Standards, 2000, 1997). e microorganism included E. coli
BW25113 ΔbamBΔtolC, Saccharomyces cerevisiae B4741, and Candida albicans ATCC# 90028.
Ellisiiamide A (12) showed modest activity against E. coli with a minimum inhibitory concentration (MIC) of
100 μg/mL. Such activity against E. coli is a rst report for the cyclic pentapeptide scaold. Compound 9 showed
no antifungal activity against S. cerevisiae or C. albicans at 100 μg/mL, which is consistent with reported data52.
Similarly, compounds 13–14 showed no activity against any test microorganisms at concentrations between
50–200 μg/mL.
Taxonomy of Xylaria ellisii. Xylaria ellisii. J.B. Tanney, Seifert & Y.M. Ju, sp. nov. MycoBank MB832257
(Fig.6)
= Xylaria corniformis (Fr.: Fr.) Fr. var. obovata M.C. Cooke & J.B. Ellis, Grevillea 6: 92. 1878.
Etymology. Named for the prolic mycologist Job Bicknell Ellis who, with Mordecai Cubitt Cooke, described
Xylaria corniformis var. obovata Sacc., a synonym of X. ellisii.
Typus. Canada: New Brunswick, Alma, Fundy National Park, East Branch Trail, 45.6433-65.1156, stromata on
partially buried, mostly decorticated Acer saccharum branch, 28 Sep 2014, J.B. Tanney NB-623 (holotype DAOM
628556). Ex-type culture DAOMC 252031.
Colonies 32–38 mm diam aer 14 d in the dark at 20 °C on MEA; white, velvety, appressed, sometimes sec-
tored; margin diuse, hyaline; surface and reverse white. Exudates and soluble pigments absent. Mycelium con-
sisting of hyaline, smooth, septate, branched, hyphae 1.5–3 µm diam.
Conidiophores on MEA macronematous, arising vertically from mycelium, hyaline to pale brown, smooth,
cylindrical, thin-walled, dichotomously branched several times, septate, 30–60 × 3–4 µm, or occurring in
synnemata, grey to olive brown (4D2–4E3). Synnemata cylindrical to clavate, occurring singly, gregariously,
or in clusters joined at base, up to 10 mm high by 1–3 mm diam, surface appearing powdery due to conidia.
Conidiogenous cells intercalary and terminal, cylindrical, straight or undulating to geniculate, 7–16(−20) ×
3–4 µm, hyaline to pale brown, producing one or more conidia holoblastically from lateral or apical regions,
crater-shaped protruding secession scars 1–1.5 × 1–1.5 µm. Conidia pyriform to obovoid, subhyaline to pale
brown, (5−)5.5–7(−7.5) × (2.5−)3(−3.5) µm, attened basal scar indicating former site of attachment to conid-
iogenous cell.
Stromata upright, solitary, unbranched or occasionally branched once, cylindrical to spathulate or clavate, api-
ces broadly rounded, divided into fertile head and sterile stipe, (2−)2.5−4(−5) × 0.8–1.2 cm including stipes (0.4–
1.5 cm high); surface even to irregularly attened or wrinkled, frequently cracked into a network of light brown
to brownish orange (6D4–6D5) angular plates above black basal layer; stromatal interior white; stipes brownish
orange to light brown (6D4–6D5) frequently with black longitudinal cracks extending from fertile head; arising
from brown (7D7) to black pannose bases, basal mycelia oen appearing iridescent. Perithecia immersed, subglo-
bose to globose, 0.3–1 mm diam, lining the perimeter of the stromata. Ostioles conspicuous, papillate, 100–300 µm
diam. Asci 95–130 × 6–7 µm, partis sporiferae 50–80 µm, eight-spored, cylindrical, with ascospores arranged
uniseriately; apical apparatus inverted hat-shaped, amyloid, 1.5–2 µm long. Ascospores (8−)9–9.5(−10) ×
(4.5−)5–5.5(−6) µm, dark brown, smooth, unicellular, ellipsoid-inequilateral, narrowly or broadly rounded ends,
1–2 guttules frequently observed, inconspicuous long, straight germ slits which are more or less the spore length,
occurring on convex side; small ephemeral cellular appendage 1.5–2 × 1.5 µm, visible on less pigmented imma-
ture ascospores and disappearing as spores reach maturity.
Cardinal temperatures: Range 5–30 °C, optimum 20 °C, minimum slightly <5 °C, maximum slightly >30 °C.
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Host range: Stromata on decaying hardwood including Acer, Betula, Fagus, and other hardwood trees. Foliar
endophyte of Abies balsamea, Picea glauca, P. mariana, P. rubens, and Pinus strobus. Foliar and stem endophyte of
Vaccinium angustifolium and V. corymbosum. Closely related ITS sequences in GenBank suggest a broad endo-
phytic and endolichenic host range.
Figure 6. Xylaria ellisii morphology. (A,B) Stromata on partially buried, decaying Acer saccharum branches,
arrow pointing to longitudinal section of stroma with perithecia lining outer surface. (C) Base of stroma
showing ostioles and reticulations. (D) Ostioles on stroma surface. (E) Eight-week-old colony on oatmeal agar.
(F) Longitudinal section of perithecium. (G) Asci and paraphyses. (H) Conidiogenous cells. (I) Conidia. (J,K)
Asci with amyloid, inverted hat-shaped apical apparatuses. (L) Ascospores, arrow denoting germ slit. Scale bars:
(F,G) = 100 µm, (H,J–L) = 10 µm, I = 5 µm.
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Distribution: Eastern Canada and U.S.A.
Additional specimens and cultures examined: DAOM 696463, DAOM 696464, DAOM 696466, DAOM 696480,
DAOM 696488, DAOM 696489, DAOM 696492, DAOM 696493, DAOM 696503, NB-699, NB-701, NB-702,
NB-703, NB-708, NB-721, NB-722, NB-723, NB-727, NB-746, CH-12, CH-15, CH-16, CH-37, CH-38, CH-4,
CH-5, DT-181, DT-6, NB-236-1F, NB-236-2F, NB-236-2I, NB-285-10A, NB-285-10D, NB-285-1A, NB-285-3A,
NB-285-6B, NB-285-7A, NB-285-7B, NB-285-7C, NB-285-7D, NB-365-4E, NB-365-71G, NB-365-
8A, NB-366-1F, NB-366-2E, NB-366-3L, NB-366-4C, NB-382-1C, NB-382-3A, NB-382-3C, NB-382-3D, NB-382-
4B, NB-391-1E, NB-391-2C, NB-391-4C, NB-406-2A, NB-406-2B, NB-406-5A, NB-421-1B, NB-437-5E,
NB-464-10A, NB-487-5B, NB-487-5C, NB-487-6A, NB-487-6H, NB-488-6L, NB-505-4D, NB-746, RS9-10E,
RS9-12C, T1-3B-2, T1-4B-1, T2-4A-2, T3-2A-2, T3-2B-1, T3-3A-3, T4-3A-1, T5-1A-1, T5-3B-1-1, T6-4B-1,
T6-5A-1-2.
Notes: Xylaria ellisii is equivalent to X. corniformis var. obovata, e.g.: Ju et al. (2016) recorded blackish-brown
ascospores, 8–10.5 × 4.5–5.5(−6) µm from the X. corniformis var. obovata holotype53. e X. corniformis species
concept is unresolved and consequently the name has been misapplied to various species within the X. corni-
formis and X. polymorpha aggregates45. e Xylaria corniformis aggregate is a polyphyletic morphotaxonomic
concept comprising species characterized by stromata with a wrinkled surface and a thin outer layer that gradu-
ally cracks into ne scales with maturation, including X. bipindensis, X. cuneata, X. curta, X. divisa, X. feejeensis,
X. humosa, X. luteostromata, X. maumeei, X. montagnei, X. plebeja, and X. rhytidophloea45,53–55. Rogers (1983)
noted the taxonomic confusion surrounding X. corniformis and its misapplication to X. bulbosa, X. castorea, X.
curta, and other morphologically similar species, and recommended that Xylaria taxonomy would be best served
if the name X. corniformis were no longer used45. Xylaria corniformis s.s. is possibly a rare species known only
from Swedish and Polish collections and is characterized by delicate, horn-like stromata with attenuated or sterile
apices versus the robust stromata of X. ellisii, which also have darker coloured ascospores44,55,56. Ju et al. (2009)
concluded that X. corniformis var. obovata was an equivalent of X. corniformis sensu Læssøe (1987)55. Læssøe
(1987) noted that X. corniformis var. obovata was probably the most frequently encountered member of the X.
corniformis complex in northern temperate regions44. Ju et al. (2009) considered X. corniformis and X. corniformis
var. obovata as distinct species but refrained from making a formal taxonomic decision pending additional evi-
dence55. Xylaria ellisii is common on decaying fallen Acer saccharum branches in New Brunswick during late
summer and autumn and is a frequently isolated endophyte of Picea, Pinus strobus, and Vaccinium angustifolium
in Eastern Canada38. Conspecic ITS sequences in GenBank suggest that X. ellisii is capable of endophytically
infecting a wide range of hosts.
Discussion
Xylaria ellisii was the most commonly isolated Xylariaceae endophyte from Picea and Pinus in Eastern Canada57.
Stromata of X. ellisii were commonly found on decaying Acer saccharum branches or stems in the same forest
stands where it was isolated as a Picea endophyte. Endophyte ITS sequences in GenBank corresponding to X.
ellisii originate from an exceptional diversity of hosts, including Tsuga canadensis, bryophytes (e.g.: Hypnum sp.),
liverworts (e.g.: Metzgeria furcata, Trichocolea tomentella), and lichens (e.g.: Flavoparmelia caperata, Sticta beau-
voisii, Xanthoparmelia conspersa) (Fig.3). In New Brunswick, corresponding X. ellisii stromata were commonly
found in late summer and early fall only on decaying Acer saccharum wood; however, the stromatal host range
is likely broad. For example, Læssøe (1987) examined European specimens of Xylaria corniformis (probably X.
ellisii) from Carpinus and Fagus44 and Rogers (1983) examined North American collections from Betula, Fagus,
Malus, and Tsuga45.
Xylaria ellisii is a common Picea and Pinus endophyte even in conifer-dominated stands lacking Acer saccha-
rum or any other hardwood hosts possibly suitable for the production of stromata. is indicates that the fungus
is capable of persisting in the environment in the prolonged absence of a suitable primary host. e method of
transmission between foliage is currently unknown. It is conceivable that the dry, powdery masses of conidia
produced from conidiomata in vitro are also produced on dead foliage and capable of infecting new foliage by
means of air currents or insect vectors58,59. Abscised foliage infected with X. ellisii is probably capable of sapro-
trophically colonizing hosts by means of direct contact (viaphytism), as demonstrated in other Xylaria species25.
e known range of hosts that X. ellisii can endophytically infect includes lichens and various understory and
overstory plant species with dierent successional statuses, allowing for its persistence across forest succession
pathways and disturbances (e.g.: as an endophyte of the re-adapted seral species Vaccinium angustifolium). A
proposed endophytic-saprotrophic life history is described and illustrated for Xylaria ellisii (as Xylaria sp.) by
Tanney et al.60.
e production of the potently antifungal compound griseofulvin by X. ellisii, an apparently ubiquitous endo-
phyte with a broad host range, is signicant. Griseofulvin is toxic to a wide variety of plant pathogens61–64 and
is systemically translocated within plants65, suggesting that X. ellisii endophyte infections could increase host
resistance to plant pathogens. For example, Park et al. (2005) described griseofulvinproduction in an unidentied
Xylaria endophyte of Abies holophylla and showed its ability to control the development of plant diseases such
as barley powdery mildew (Blumeria graminis f. sp. hordei), rice sheath blight (Corticium sasaki), wheat leaf rust
(Puccinia recondita), and rice blast (Magnaporthe grisea)64. Griseofulvin and related compounds are reported
from Xylaria endophytes of Asimina triloba, Chrysobalanus icaco, and Garcinia hombroniana66–68. Richardson
et al. (2014) reported the production of the antifungal compound griseofulvin by Xylaria ellisii (as Xylaria sp.)
isolated as a foliar endophyte of Pinus strobus and Vaccinium angustifolium38. ese isolates produced griseof-
ulvin and its de-halogenated analogue (Fig.1), along with piliformic acid38. Subsequent investigations of white
pine seedlings infected with this Xylaria species found griseofulvin at biologically eective concentrations in the
needles69.
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Nonribosomal peptides (NRPS) are of great interest as they represent a unique class of natural products with
diverse therapeutic applications such as antimicrobial agents (caspofungin, penicillin, vancomycin), antican-
cer compounds (bleomycin, daptomycin), immunosuppressants (cyclosporine, rapamycin) and as insect toxins
(beauvercin, enniatin)70–73. is complex structural diversity of linear, cyclic, and cyclic branched architectures is
synthesized through a modular enzymatic assembly line process70,73. In principle, this enzyme complex is capable
of incorporating >500 proteinogenic and nonproteinogenic building blocks, including polyketide and terpene
hybrid moieties.
In this study, we have applied a LC-MS metabolomic guided discovery approach to prole the chemical space
of a novel endophytic species described here as Xylaria ellisii. Our collections of isolates have identical ITS DNA
sequences yet dier in their LC-MS metabolite proles and bioactivity. OPLS-DA and S-plot analysis identi-
ed features separated by a statistical toll, Variable Importance in Projection (VIP) scores. VIP scores from the
extracted ltrates and mycelium extracts were calculated and extracts dierentiated by this method were targeted
for compound isolation and structural characterization. is approach resulted in the discovery of three new
cyclic pentapeptides given the trivial names ellisiiamides A–C (12–14) and the putative identication and anno-
tation of ellisiiamides D–H by LC-HRMS and LC-HRMS/MS analysis. Additionally, 11 known compounds are
reported to be produced by these strains. Ellisiiamide A (12) was active against Gram-negative bacteria and is
a rst report for this scaold. ese ndings are of interest as the isolates were also reported from eastern white
pine needles in a pine-blueberry forest ecotype. Endophytes from wild Vaccinium species may be an interesting
source of novel bioactive compounds. is information provides a better understanding of the chemical ecology
of plant-fungi microbiomes. In the long term, opportunities may present to employ this information for inte-
grated pest management crop protection strategies.
Methods
Sampling, isolation, and culturing. Plant material, including leaves and stems from highbush and wild
blueberries, were collected from three dierent locations within the Acadian forest region of Nova Scotia, Canada.
Highbush blueberry endophyte isolates were obtained from a commercial eld in Rawdon, Nova Scotia and wild
blueberry endophytes isolates were collected from commercial elds in Mount om, Debert, and Portapique,
Nova Scotia. Specimens were collected in labelled bags and stored at -20 °C prior to fungal isolation. Plant tissues
were rst washed with sterile deionized water to remove any loose debris and surface contaminants, followed by
a chemical surface-sterilization process using sodium hypochlorite bleach (6%) and ethanol (70%). Small seg-
ments were then cut and/or incised and placed in Petri plates containing 2% malt extract agar (MEA; 20 g Bacto
malt extract, Difco Laboratories, Sparks, USA; 15 g agar, EMD Chemicals Inc., Gibbstown, USA; 1 L deionized
H2O). Inoculated plates were incubated at 25 °C for 4–8 weeks, depending on the presence of lamentous hyphae.
Endophytic fungi that grew from cut ends were then transferred to potato dextrose agar (PDA, Sigma-Aldrich,
Canada) plates and incubated at 25 °C.
Field specimens of stromata were collected and stored in paper bags. Single-ascospore isolates were made
by axing with petroleum jelly a small (ca. 5 mm2) piece of stroma containing mature perithecia to the lid of
a Petri dish containing water agar (WA; 15 g agar, EMD Chemicals Inc., Gibbstown, USA; 1 L deionized H2O).
Germination of ejected ascospores on the agar surface was conrmed by stereo microscope (Olympus SZX12,
Olympus, Tokyo, Japan) and germinating ascospores were transferred to individual Petri plates containing
2% MEA and incubated at 20 °C. Dried specimens were accessioned in the Canadian National Mycological
Herbarium (Ottawa, Ont.; DAOM). Living cultures were deposited in the Canadian Collection of Fungal Cultures
(Ottawa, Ont.; DAOMC). Additional specimens used for morphological comparison and phylogenetic analyses
were also obtained from DAOM, DAOMC, and the personal culture collection of J.B. Tanney.
Xylaria strains from highbush blueberry and wild blueberry were cultured in PDB (24 g/L potato dextrose
broth) and ML (30 g/L malt) fermentation media. Each strain was grown in 1 L Roux bottles containing 200 mL
of media and grown statically for 4–6 weeks at 25 °C. e culture broth was then separated from the mycelium by
vacuum ltration using a Whatman #4 lter paper. e ltrate was extracted with equal volumes of ethyl acetate,
while the mycelium was rst lyophilized for 24 h and then extracted with equivalent volumes of methanol and
acetone (1:1). Organic fractions were then dried under reduced pressure by rotary vacuum. Extracts were then
re-suspended in 600 μL of HPLC grade acetonitrile with minimal amounts of DMSO added for solubility. e
ltrates were then centrifuged at 13,000 rpm for 15 min and Acro-disk (13 mm, 0.45 μm GHP) ltered prior to
LC-MS analysis.
Morphological study. Sections of stromata were cut by hand using a safety razor blade or with a freezing
microtome (ca. 15–30 µm thick) and mounted in either water, 5% KOH, 85% lactic acid, or Lugol’s solution with
or without 5% KOH pretreatment to test amyloid reactions74. Stromata and colony colours were described using
alphanumeric codes75. Observations of the asexual morph were made from living cultures grown on oatmeal agar
(OA)76. Microscopic measurements were taken from living material mounted in deionized water and are pre-
sented as ranges calculated from the mean ± standard deviation of each measured value with outliers in brackets.
Observations were made using an Olympus BX50F4 light microscope and an Olympus SZX12 stereo microscope
(Olympus, Tokyo, Japan). Images were captured with an InnityX-32 camera (Lumenera Corp., Ottawa, Canada)
using Innity Analyze v. 6.5.2 (Lumenera Corp.) soware. Photographic plates were assembled using Adobe
Photoshop CC 2017.1.1 (Adobe Systems, San Jose, California, USA). Cardinal temperatures were assessed for the
type strain (DAOMC 252031) by incubating single-point inoculated Petri dishes containing MEA at 5 °C inter-
vals from 5–40 °C. Each treatment was conducted in triplicate and colony diameters were measured two weeks
aer inoculation.
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DNA extraction, sequencing, and phylogenetic analyses. DNA was extracted from cultures and
stromata using the Ultraclean Microbial DNA Isolation Kit (Mo Bio, Carlsbad, CA) or NucleoSpin Plant II Kit
(Macherey-Nagel, Düren, Germany). Stromatal tissue from fresh collections and herbarium specimens under-
went an initial grinding stage in liquid nitrogen using an Axygen polypropylene pestle (PES-15-B-SI, Union City,
CA, USA).
Loci chosen for sequencing included the internal transcribed spacer rDNA region (ITS), β-tubulin (BenA),
translation elongation factor 1-alpha (EF1-α), the second largest subunit of RNA polymerase II (RPB2), 18 s nuc
rDNA (SSU), and 28 S nuc rDNA (LSU). Primer pairs used for PCR amplication and sequencing included: ITS1
and ITS477 or ITS4A and ITS578 for ITS; Bt2a and Bt2b for BenA79; RPB2-5f2 and RPB2-7CR80 for RPB2; and
EF1-728F and EF1-986R81 for EF1-α. LSU was amplied using LR0R and LR5 and sequenced using the primers
LR0R, LR3, LR3R, and LR582. SSU was amplied using the primers NS1 and NS4, and sequenced using the prim-
ers NS1, NS2, NS3, and NS477. PCR and sequencing were performed as described by Tanney and Seifert (2017)57.
To improve ITS amplication in herbarium specimens, 0.5 μm of 20 mg/ml bovine serum albumin (BSA) was
added per reaction.
For all analyses, sequences were aligned using MAFFT v783 and visually inspected and manually aligned
when necessary in Geneious R8 v8.1.5 (Biomatters, Auckland, New Zealand). e most suitable sequence evolu-
tion model was determined based on the optimal Akaike information criterion scores in MrModeltest v2.2.684.
Consensus trees were visualized in FigTree 1.4.2 (available at http://tree.bio.ed.ac.uk/soware/gtree/) and
exported as SVG vector graphics for assembly in Adobe Illustrator v10 (Adobe Systems, San Jose, CA, USA).
Three separate phylogenetic analyses were performed. The first phylogeny included ITS sequences of
diverse representative endophytes isolated from highbush and wild blueberry leaves and stem. e ex-type
of Mucor ellipsoideus (ATCC MYA-4767; NR_111683) was selected as outgroup because of its basal position
(Mucoromycotina). Maximum likelihood (ML) analysis was performed using RAxML v8.2.4 in PAUP v4.0b10
starting from a random starting tree with 1000 bootstrap replicates85,86.
e second phylogenetic analysis included RPB2 sequences from related Xylaria species. e resulting align-
ment was 1058 bp long and consisted of 47 taxa, including the outgroup Barrmaelia rhamnicola (CBS 142772).
Bayesian analysis was performed using MrBayes v3.2.687. Three independent Markov Chain Monte Carlo
(MCMC) samplings were performed with 12 chains (11 heated and one cold) with sampling every 500 genera-
tions until the standard deviation of split frequencies was <0.01. e rst 25% of trees were discarded as burn-in
and the remaining trees were kept and combined into one consensus tree with 50% majority rule consensus.
Convergence was assessed from the three independent runs using Tracer v1.688. e third phylogenetic analysis
included ITS sequences from related endophytic Xylaria isolates. e alignment was 593 bp long and included
sequences from 107 isolates or samples. e resulting phylogenetic analysis was performed in the same manner
as described above, with Nemania serpens (GU292820) as the outgroup.
All novel sequences used in this study were accessioned in GenBank (Supplementary Table13S) and taxo-
nomic novelties and associated metadata were deposited in MycoBank (www.MycoBank.org).
LC-UV/HRMS and LC-UV/HRMS/MS screening. Extracts of endophytic cultures were screened using a
Dionex Ultimate 3000 HPLC-UV system coupled to a Bruker maXis 4 G ultra-high-resolution-qTOF mass spec-
trometer operated in positive electrospray ionization (ESI) with calibrations done using sodium formate ion clus-
ters. LC-MS data were collected using a scan range of 150–1100 m/z, with the nebulizer gas (nitrogen) at 3 bar, dry
gas ow at 8 L/min, dry gas temperature at 240 °C, and capillary voltage at 4500 V. Chromatographic separations
were performed using a standardized HPLC-UV method with a Supelco Ascentis Express C18 reverse-phase
core-shell column (150 × 4.6 mm, 2.7 μm, Sigma Aldrich, USA) operating at 750 μL/min and at 40 °C. UV/vis
data were acquired from 190–600 nm and monitored at four wavelengths (210, 254, 275 and 350 nm). Mobile
phase composition was linear with a gradient of 5% organic from 0 to 1 min, 5–95% from 1 to 24 min, 95–100%
from 24 to 25 min, and 100% from 25 to 31 min. Solvent A was H2O + 0.1% formic acid and solvent B was ace-
tonitrile with 0.1% formic acid (v/v). HR-MS/MS analysis was performed on a ermo Q-Exactive Orbitrap mass
spectrometer operated in positive electrospray ionization (ESI+) and coupled to an Agilent 1290 HPLC system.
Data processing and multivariate statistical analysis. Data processing and analyses were modied
from a previously published protocol (Fei et al., 2014). Post-acquisition internal calibration using sodium for-
mate clusters in both ESI+ and ESI- were performed with Bruker’s Data Analysis 4.0 SP4. LC-MS data les were
converted to.mzXML format using Bruker CompassXport. Metabolic features were extracted and aligned using
open source XCMS with centWave algorithm89; adducts, isotopic ions, and in-source fragments were identied
using CAMERA90,91. To acquire the nal metabolite feature list, isotopic ions and features with integrated peak
area under 10,000 were removed. For myceliummetabolome, metabolite features that eluted aer 25 min were
eliminated.
Both extracted ltrates and mycelium were analyzed using supervised multivariate OPLS-DA aer pareto
scaling by SIMCA-P+ 12.0.1 (Umetrics, Kinnelon, NJ). e statistical parameters R2X(cum), R2Y(cum), and
Q2(cum) of OPLS-DA were used to assess the tness of the model. R2X (R2Y) indicated the fraction in which
metabolite features (X) and group (Y) matrix was were explained by the model. A prediction statistic (Q2) above
0.4 was indicative of a statistically robust model, i.e. true dierences between the comparing groups, and Q2
between 0.7–1.0 indicated the model was statistically signicant46. Both R2 and Q2 followed an upward trend from
0 to 1. For an over-t model, R2 approached 1, and Q2 fell toward 092. Signicant features between classes were
identied based on OPLS-DA S-plot and their Variable Importance in Projection (VIP) score. To ensure the iden-
tied metabolites are the sole important markers, the two OPLS-DA analyses were conducted in parallel by only
including the signicant features or by removing the signicant features from the raw data92. A useful metabolite
subset was produced if the rst model was successful and the later model failed.
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Metabolite Isolation and characterization. NMR experiments for 1D and 2D measurements were per-
formed on a Bruker Advance III 700 MHz NMR spectrometer equipped with a 5 mm QNP cryoprobe, operating
at 700.17 MHz for 1H NMR and 176.08 MHz for 13C NMR or a Bruker Advance III HD 850 MhZ NMR spectrom-
eter equipped with a 5 mm TXI probe operating at 850.21 MHz for 1 H NMR and 213.81 MHz for 13 C NMR,
with chemical shis referenced to the residual solvent signal93. Nitrogen dried compounds were re-suspended
in 200 μL of deuterated solvent (C6D6, CD3OD, or DMSO-d6) and transferred to 3 mm NMR tubes (Wilmad
335-pp-7) for NMR measurements. NMR data processing was done using MNOVA NMR soware ver. 10.0.1
by Mestrelab Research. Optical rotation measurements were done using an Autopol IV Polarimeter (Rudolph
Research Analytical).
Purication of metabolomic targeted metabolites was performed on a semi-preparative HPLC system consist-
ing of an Agilent 1100 series HPLC with a G1311A Quaternary Pump, a G1379A Degasser, a G1367A Wellplate
Autosampler, a G1316A Column ermostat, a G1315B Diode Array Detector (DAD), and a G1364C Automatic
Fraction Collector controlled by Agilent ChemStation soware (Rev. B.03.02-SR2). Metabolites were isolated
using a Phenomenex Synergi-Max reverse-phase C-12 column (250 × 10 mm, 4 μm) (Torrence, CA, USA) oper-
ating at 5 mL/min and 40 °C. Mobile phase composition was a linear gradient of 5% organic from 0 to 3 min,
5–30% from 3 to 16 min, 30% from 16 to 20 min, and 30–85% from 20–37 min with fractions collected every 20 s.
Known isolated compounds (mg/L): dechlorogriseofulvin (2) eluted at 27.1 min (4 mg); griseofulvin (1) eluted at
29.1 min (2.8 mg); cytochalasin D (3) eluted at 30.2 min (2.5 mg); zygosporin E (4) eluted at 32.5 min (2 mg); hir-
sutatin A (6) eluted at 33.9 min (2 mg); and cyclic pentapeptide #1 (9) eluted at 34.9 min (4 mg) (Supplementary
Figs.3S and 32–49S)
Newly-isolated compounds (mg/L): ellisiiamide G (18) eluted at 31.6 min (0.3 mg); ellisiiamide A (12) eluted
at 32.8 min (2.0 mg); ellisiiamide B (13) eluted at 33.4 min (1.3 mg); and ellisiiamide C (14) eluted at 35.6 min
(2.3 mg). Compound fractions, from multiple HPLC runs, were pooled together and dried under N2 gas in
pre-weighed vials prior to NMR and optical rotation measurements (Supplementary Figs.5–31S, Tables10–12S).
Ellisiiamide A (12) C30H45N5O5; white powder; [α]21 −86.1 (0.18, MeOH); For 1H and 13C NMR (DMSOd6)
spectroscopic data see Supporting Table9S: HRESIMS (m/z) 556.3501 [M+H]+ (calcd for C30H46N5O5, 556.3493).
Ellisiiamide B (13) C31H47N5O5; white powder; [α]21 −43.1 (0.04, MeOH); For 1H and 13C NMR (DMSOd6)
spectroscopic data see Supporting Table10S: HRESIMS (m/z) 570.3656 [M+H]+ (calcd for C31H48N5O5,
570.3650).
Ellisiiamide C (14) C33H51N5O5; white powder; [α]21 −47.8 (0.06, MeOH); For 1H and 13C NMR (DMSOd6)
spectroscopic data see Supporting Table11S: HRESIMS (m/z) 598.3968 [M+H]+ (calcd for C33H52N5O5,
598.3963).
Biological activity screening. Compounds were tested for their minimum inhibitory concentration
(MIC) according to the Clinical Laboratory Standards Institute (CLSI) protocols M7-A5 and M27-A (National
Committee for Clinical Laboratory Standards, 2000, 1997). Stock working solutions were made to 5, 10, and
20 mg/mL and tested at a maximum concentration of 200 μg/mL in 96-well liquid culture (National Committee
for Clinical Laboratory Standards, 1997, 2003) as previously described37. Preliminary evaluation of biologi-
cal activity was against E. coli BW25113 ΔbamBΔtolC, a membrane and eux pump compromised strain,
Staphylococcus aureus ATCC# 29213, Bacillus subtilis 1A1, Micrococcus luteus, Saccharomyces cerevisiae B4741,
and Candida albicans ATCC# 90028. A cut-o of <25% growth was used for inhibition, with the trend across
dilutions also considered37.
Received: 2 October 2019; Accepted: 28 January 2020;
Published: xx xx xxxx
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Acknowledgements
The authors would like to thank M. Kelman (AAFC), T. McDowell (AAFC) and D. Sorensen (McMaster
University) for technical assistance. We would like to thank Linda Ejim (McMaster University) for antimicrobial
testing of purified compounds. We thank The Center for Microbial Chemical Biology (CMCB), and the
Biointerfaces Institutes (BI) at McMaster University for access to state-of-the-art instrumentation. J.B. Tanney
thanks Jacques Fournier, Ju Yu-Ming, and Marc Stadler for insightful discussion on Xylaria taxonomy. A.I was
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funded through an Ontario Graduate Scholarship (OGS) Doctoral Research Award. is project was funded by
an AAFC grant to MWS and KAS. Additional support was provided by the Natural Sciences and Engineering
Research Council of Canada (NSERC SYN 479724-15) to J.D. Miller and by J.D. Irving Ltd.
Author contributions
Ashraf Ibrahim, Joey Tanney, and Mark Sumarah conducted the primary research. Fan Fei performed the
statistical analysis. Ashraf Ibrahim, Mark Sumarah, J. David Miller and Keith Seifert conceived the experiments.
Alfredo Capretta discussed research and structural characterization. Chris Cutler provided blueberry samples for
endophyte isolation. All authors contributed to the manuscript writing and review.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-61088-x.
Correspondence and requests for materials should be addressed to M.W.S.
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