Diketopiperazines from Batnamyces globulariicola, gen. & sp. nov. (Chaetomiaceae), a fungus associated with roots of the medicinal plant Globularia alypum in Algeria

Abstract

Eight diketopiperazines including five previously unreported derivatives were isolated from an endophytic fungus cultured from the medicinal plant Globularia alypum collected in Algeria. The strain was characterised by means of morphological studies and molecular phylogenetic methods and was found to represent a species of a new genus in the Chaetomiaceae, for which we propose the name Batnamyces globulariicola. The taxonomic position of the new genus, which appears phylogenetically related to Stolonocarpus and Madurella, was evaluated by a multi-locus genealogy and by morphological studies in comparison to DNA sequence data reported in the recent monographs of the family. The culture remained sterile on several culture media despite repeated attempts to induce sporulation, and only some chlamydospores were formed. After fermentation in submerged culture and extraction of the cultures with organic solvents, the major secondary metabolites of B. globulariicola were isolated and their chemical structures were elucidated by extensive spectral analysis including nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionisation mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) measurements. The isolated compounds were tested for their biological activities against various bacteria, fungi, and two mammalian cell lines, but only three of them exhibited weak cytotoxicity against KB3.1 cells, but no antimicrobial effects were observed.

Full text

ORIGINAL ARTICLE
Diketopiperazines from Batnamyces globulariicola,gen.&sp.nov.
(Chaetomiaceae), a fungus associated with roots of the medicinal
plant Globularia alypum in Algeria
Sara R. Noumeur
1,2
&Rémy B. Teponno
1,3
&Soleiman E. Helaly
1,4
&Xue-Wei Wang
5
&Daoud Harzallah
6
&
Jos Houbraken
7
&Pedro W. Crous
7
&Marc Stadler
1
Received: 13 October 2019 / Revised: 26 March 2020 /Accepted: 27 March 2020
Abstract
Eight diketopiperazines including five previously unreported derivatives were isolated from an endophytic fungus
cultured from the medicinal plant Globularia alypum collected in Algeria. The strain was characterised by means of
morphological studies and molecular phylogenetic methods and was found to represent a species of a new genus in the
Chaetomiaceae, for which we propose the name Batnamyces globulariicola. The taxonomic position of the new genus,
which appears phylogenetically related to Stolonocarpus and Madurella, was evaluated by a multi-locus genealogy and
by morphological studies in comparison to DNA sequence data reported in the recent monographs of the family. The
culture remained sterile on several culture media despite repeated attempts to induce sporulation, and only some
chlamydospores were formed. After fermentation in submerged culture and extraction of the cultures with organic
solvents, the major secondary metabolites of B. globulariicola were isolated and their chemical structures were eluci-
dated by extensive spectral analysis including nuclear magnetic resonance (NMR) spectroscopy, high-resolution
electrospray ionisation mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) measurements. The
isolated compounds were tested for their biological activities against various bacteria, fungi, and two mammalian cell
lines, but only three of them exhibited weak cytotoxicity against KB3.1 cells, but no antimicrobial effects were
observed.
Keywords Diketopiperazines .New genus .New species .Phylogenetic methods .Sordariomycetes
Sara R. Noumeur and Rémy B. Teponno contributed equally tothis work.
Section Editor: Hans-Josef Schroers
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11557-020-01581-9) contains supplementary
material, which is available to authorized users.
*Marc Stadler
marc.stadler@helmholtz-hzi.de
1
Department of Microbial Drugs, Helmholtz Centre for Infection
Research and German Centre for Infection Research (DZIF), partner
site Hannover/Braunschweig, Inhoffenstrasse 7,
38124 Braunschweig, Germany
2
Department of Microbiology and Biochemistry, Faculty of Natural
and Life Sciences, University of Batna 2, 05000 Batna, Algeria
3
Department of Chemistry, Faculty of Science, University of
Dschang, P.O. Box 67, Dschang, Cameroon
4
Department of Chemistry, Faculty of Science, Aswan University,
Aswan 81528, Egypt
5
State Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, No. 3, 1st Beichen West Road,
Chaoyang District, Beijing 100101, China
6
Laboratory of Applied Microbiology, Department of Microbiology,
Faculty of Natural and Life Sciences, University Sétif 1 Ferhat
Abbas, 19000 Sétif, Algeria
7
Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508
AD Utrecht, The Netherlands
Mycological Progress (2020) 19:589–603
https://doi.org/10.1007/s11557-020-01581-9
#The Author(s) 2020

Introduction
In recent years, it has been demonstrated that fungal endo-
phytes are playing an important role in most ecosystems of
the world. These fungi, which colonise their host plants with-
out causing any disease symptoms, have been shown to rep-
resent various known and new phylogenetic lineages
(Blackwell and Vega 2018).
Moreover, several studies have led to the isolation and
structure elucidation of important secondary metabolites from
endophytic fungi, thus raising the prospect of using such or-
ganisms as alternative sources of lead compounds for devel-
opment of new drugs or agrochemical pesticides
(Suryanarayanan et al. 2009; Bills and Gloer 2016).
Moreover, it is now also being attempted to use these organ-
isms as biocontrol agents and biofertilisers (Hyde et al. 2019;
White et al. 2019).
In our ongoing search for bioactive fungal metabolites
from fungi associated with Algerian medicinal plants
(Noumeur et al. 2017; Teponno et al. 2017), 12 secondary
metabolites including five previously unreported
diketopiperazines (1–5) were isolated from the culture of a
fungus that was obtained from Globularia alypum
(Plantaginaceae) using a well-established protocol that has
been used for the isolation of endophytic fungi for several
decades. The producer organism turned out to be new to
Science and is formally described in the present paper.
Moreover, the isolation of its secondary metabolites, and their
structure elucidation and their preliminary biological charac-
terisation are reported.
Material and methods
Origin and isolation of the strain
Fresh healthy roots of the medicinal plant Globularia alypum
(Plantaginaceae) were collected in June 2015 from Ain Touta
(Batna, Algeria). Along with other cultures, strain CBS
144474 was isolated according to established protocols in-
volving surface disinfection (Noumeur et al. 2017; Teponno
et al. 2017) and was maintained in liquid nitrogen at the HZI
culture collection, Braunschweig, Germany. Morphology and
macroscopic features of the culture were determined on sev-
eral different culture media including yeast-malt-glucose
(YMG; Fig. 1), see Richter et al. (2016), and all other the
standard media that are currently in use for induction of spor-
ulation of Sordariomycetes, i.e. potato dextrose agar (PDA),
oatmeal agar (OA), synthetic nutrient-poor agar (SNA), and
malt extract agar (MEA) (cf. Crous et al. 2009;Wangetal.
2019b). Cardinal temperature requirements for growth were
only checked on YMG at temperatures ranging from 24 to
43 °C.
Molecular analysis and sequencing
Genomic DNA extraction from the fungal colonies growing
on YMG was performed using an EZ-10 Spin Column
Genomic DNA Miniprep kit (Bio Basic Canada Inc.,
Markham, Ontario, Canada) as specified by the manufacturer.
The internaltranscribed spacer 1 and 2 including the interven-
ing 5.8S nrDNA (ITS), the 28S nrDNA (LSU) including the
D1/D2 domains, a part of the DNA-directed RNA polymerase
II second largest subunit gene (RPB2), and the β-tubulin gene
(TUB2) were selected for phylogenetic inference. PCR and
generation of DNA sequences followed the procedure
outlined by Wendt et al. (2018).
After a BLAST result based on GenBank data, which allo-
cated the new fungus to the Chaetomiaceae, the phylogenetic
affinities were studied based on an analysis of a combined
ITS, LSU, RPB2,andTUB2 dataset in comparison with
DNA sequence data reported in the recent monographs of
the family (Wang et al. 2016a,b,2019a,b). Alignments were
madeusingthewebinterfaceMAFFTv.7(Katohand
Standley 2013), followed by manual adjustments with
MEGA v. 6 (Tamura et al. 2013). Phylogenetic analysis was
performed using maximum likelihood (ML) and Bayesian in-
ference (BI) approaches under RAxML-HPC2 on XSEDE
8.2.10 (Stamatakis 2014) using the Cipres Science gateway
portal (Miller et al. 2010) and MrBayes v. 3.2.6 (Ronquist
et al. 2012), respectively. For BI, the best evolutionary model
for each locus was determined using MrModeltest v. 2.0
(Nylander 2004). The maximum likelihood analysis used the
GTRGAMMA model. Obtained trees were viewed in FigTree
v. 1.1.2 (Rambaut 2009) and subsequently visually prepared
and edited in Adobe
®
Illustrator
®
CS6. Confident branch sup-
port is defined as Bayesian posterior probabilities (PP) ≥0.95
and maximum likelihood bootstrap values (ML-BS) ≥70%.
Instrumentation
Optical rotations were determined with a Perkin Elmer
(Überlingen, Germany) 241 MC polarimeter (using the sodi-
um D line and a quartz cuvette with a 10-cm path length and
0.5-mL volume). Circular dichroism (CD) spectra were re-
corded on a JASCO spectropolarimeter, model J-815
(JASCO, Pfungstadt, Germany). Nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker (Bremen,
Germany) 500 MHz Avance III spectrometer with a BBFO
(plus) SmartProbe (
1
H 500 MHz,
13
C 125 MHz) and a Bruker
700 MHz Avance III spectrometer with a 5-mm TCI cryo-
probe (
1
H 700 MHz,
13
C 175 MHz), locked to the deuterium
signal of the solvent. Chemical shifts are given in parts per
million (ppm) and coupling constants in Hertz (Hz). Spectra
were measured at 24.8 °C in CD
3
OD, acetone-d
6
, and deuter-
ated chloroform; chemical shifts were referenced to residual
solvent signals with resonances at δ
H
/
C
3.31/49.15 for
590 Mycol Progress (2020) 19:589–603

CD
3
OD, δ
H
/
C
2.05/29.92 for acetone-d
6
,andδ
H
/
C
7.24/77.23
for deuterated chloroform. High-performance liquid chroma-
tography coupled with diode array detector and mass spectro-
metric detection (HPLC-DAD/MS) analysis was performed
usinganamaZonspeedETDiontrapmassspectrometer
(Bruker Daltonics) in positive and negative ionisation modes.
The mass spectrometer was coupled to an Agilent 1260 series
HPLC-UV system (Agilent Technologies) (Santa Clara, CA,
USA) (column 2.1 × 50 mm, 1.7 μm, C18 Acquity uPLC
BEH (Waters); solvent A: H
2
O + 0.1% formic acid; solvent
B: acetonitrile (ACN) + 0.1% formic acid; gradient: 5% B for
0.5 min, increasing to 100% B in 20 min, maintaining
isocratic conditions at 100% B for 10 min, flow = 0.6 mL/
min, UV−vis detection 200–600 nm). High-resolution
electrospray (HR-ESI) MS spectra were recorded on a
maXis ESI TOF mass spectrometer (Bruker Daltonics) (scan
range m/z100–2500, rate 2 Hz, capillary voltage 4500 V, dry
temperature 200 °C), coupled to an Agilent 1200 series
HPLC-UV system (column 2.1 × 50 mm, 1.7 μm, C18
Acquity uPLC BEH (Waters); solvent A: H
2
O + 0.1% formic
acid; solvent B: ACN + 0.1% formic acid; gradient: 5% B for
0.5 min, increasing to 100% B in 19.5 min, maintaining 100%
Bfor5min,FR=0.6mL/min,UV−vis detection 200–
600 nm). The molecular formulas were calculated including
the isotopic pattern (Smart Formula algorithm). Preparative
HPLC purification was performed at room temperature on
an Agilent 1100 series preparative HPLC system
(ChemStation software (Rev. B.04.03 SP1); binary pump sys-
tem; column: Kinetex 5u RP C18 100 Å, dimensions 250×
21.20 mm; mobile phase: ACN + 0.05% trifluoroacetic acid
(TFA) (solvent B) and water + 0.05% TFA (solvent A); flow
rate 20 mL/min; diode-array UV detector; 226 fraction
collector).
Fermentation, extraction, and isolation
Pieces of well-colonised agar of strain CBS 144474 from
YMG plates were inoculated in Q61/2 medium (Chepkirui
et al. 2019) in a 500-mL Erlenmeyer flask containing
200 mL of media and incubated at 23 °C for 7 days. This seed
culture was used to inoculate 25 other flasks (5 L) of the same
medium composition after homogenisation with a Heidolph
Silent Crusher. The flasks were incubated at 23 °C under con-
stant shaking at 140 rpm on a rotary shaker for 12 days. After
separationof the mycelia byvacuum filtration, the supernatant
was extracted with ethyl acetate (EtOAc).The EtOAc fraction
was dried over anhydrous Na
2
SO
4
, filtered, and concentrated
under vacuum to yield 590 mg of extract. The wet mycelia
were extracted twice with acetone, then with methanol in an
ultrasonic bath at 40 °C for 30 min. The resulting solution was
evaporated to yield an aqueous phase, which was further ex-
tracted with EtOAc (3 × 500 mL). After drying over anhy-
drous Na
2
SO
4
, the EtOAc fraction was concentrated under
vacuum to yield 416.7 mg of crude extract. In the meantime,
pieces of a well-grown YMG agar plate of strain CBS 144474
were inoculated in a YMG medium (Richter et al. 2016), in a
500-mL Erlenmeyer flask containing 200 mL of media, and
incubated at 23 °C for 8 days. The homogenised seed culture
was used to inoculate 25 other flasks (5 L) of the same medi-
um composition. The flasks were incubated at 23 °C under
constant shaking at 140 rpm on a rotary shaker for 11 days.
After separation, the supernatant and the mycelia were extract-
ed as described above to give 408.5 and 512.8 mg of extracts,
respectively.
The crude supernatant extract from the fermentation in
Q61/2 medium (ca. 400 mg) was purified by preparative
HPLC using a gradient of 25–50% solvent B for 40 min,
50–100% B for 10 min, and 100% B for 10 min. The fractions
were combined according to UVabsorption at 220, 280, and
325 nm and concurrent HPLC-MS analyses. Compounds 5
(0.9 mg; Rt = 11.12 min), 1(1 mg; Rt = 13.80 min), 2
(0.8 mg; Rt = 15.40 min), 8(0.8 mg; Rt = 21.34 min), 3
(1.2 mg; Rt = 25.42 min), 6(1.4 mg; Rt = 29.84 min), 7
(0.7 mg; Rt = 32.16 min), 4(0.7 mg; Rt = 36.12 min), and 9
(0.5 mg; Rt = 38.69 min) were eluted.
The supernatant extract from the fermentation in YMG
medium(ca.400mg)wasalsopurifiedbypreparative
HPLC. The gradient used was 5–35% solvent B for 40 min,
35–100% solvent B for 20 min, and 100% B for 10 min. The
fractions were combined according to UV absorption at 220,
280, and 325 nm to yield compounds 10 (8.7 mg; Rt =
18.17 min), 11 (9.3 mg; Rt = 19.69 min), and 12 (3.7 mg;
Rt = 23.11 min).
Mycol Progress (2020) 19:589–603 591
Summary of spectral data for the new compounds (1–5)
(3R,6Z)-3-Thiomethyl-6-[4-O-[(2E)-4-hydroxy-3-methylbut-
2-enyl]benzylidene]piperazine-2,5-dione (1): yellowish gum;
α½
25
D+22.0(c0.05, MeOH); UV (MeOH) λ
max
(log ε): 204
(4.37), 229 (4.26), 323 nm (4.34); CD (c0.5 mg/mL, EtOH)
λ
max
309 (+), 235 nm (+);
1
H NMR (CD
3
OD, 700 MHz) and
13
C NMR (CD
3
OD, 175 MHz) data (see Table 1); HRESIMS:
m/z 349.1217 [M + H]
+
(calcd for C
17
H
21
N
2
O
4
S
+
, 349.1217).
(3R,6Z)-3-Thiomethyl-6-[4-O-[(2Z)-4-hydroxy-3-methylbut-2-
enyl]benzylidene]piperazine-2,5-dione (2): yellowish gum; α½
25
D+37.5(c
0.04, MeOH); UV (MeOH) λ
max
(log ε): 203 (4.13), 229
(3.97), 323 nm (3.96); CD (c0.5 mg/mL, EtOH) λ
max
316
(+), 237 nm (+);
1
H NMR (CD
3
OD, 700 MHz) and
13
CNMR
(CD
3
OD, 175 MHz) data (see Table 1); HRESIMS: m/z
349.1217 [M + H]
+
(calcd for C
17
H
21
N
2
O
4
S
+
, 349.1217).
(3R,6Z)-3-Hydroxy-6-[4-O-(3-methylbut-2-
enyl)benzylidene]piperazine-2,5-dione (3): yellowish gum;
α½
25
D+26.7(c0.06, MeOH); UV (MeOH) λ
max
(log ε):
206 (4.22), 224 (4.26), 321 nm (4.15); CD (c0.5 mg/mL,
EtOH) λ
max
309 (+), 228 nm (+);
1
HNMR(CD
3
OD,

Table 2
13
Cand
1
H NMR spectroscopic data of compounds 4(MeOH-
d
4
)and5(Acetone d
6
)
Position 4 5
δ
13
C, type δ
1
Hδ
13
C, type δ
1
H
2 166.1, C / 165.2, C /
3 59.6, CH 4.97 s 59.0, CH 5.01 s
5 163.5, C / 165.1, C /
6 125.2, C / 69.0, C /
7 119.4, CH 6.87 s 43.0, CH
2
2.94 d (13.5)
3.60 d (13.5)
8 126.7, C / 128.1, C /
9/9′132.1, CH 7.44 d (8.6) 133.1, CH 7.26 d (9.0)
10/10′116.5, CH 6.99 d (8.6) 115.4, CH 6.83 d (9.0)
11 161.0, C / 159.3, C /
12 66.1, CH
2
4.58 br d (6.6) 64.8, CH
2
4.61 br d (6.3)
13 121.1, CH 5.46 m 122.7, CH 5.47 m
14 139.1, C / 141.3, C /
15 26.0, CH
3
1.79 s 61.5, CH
2
4.15 s
16 18.3, CH
3
1.76 s 21.4, CH
3
1.81 s
17 13.1, CH
3
2.26 s 10.6, CH
3
1.36 s
18 13.4, CH
3
2.23 s
Table 1
13
Cand
1
HNMR
spectroscopic data of compounds
1–3in methanol-d
4
Position 1 2 3
δ
C
, type δ
H
δ
C
,type δ
H
δ
C
,type δ
H
1 10.17 s
a
10.17 s
a
9.92 s
a
2 166.0, C / 166.0, C / 167.0, C /
3 59.6, CH 4.97 s 59.6, CH 4.97 s 76.1, CH 5.09 s
49.03brs
a
9.02 br s
a
8.92 d 3.9
a
5 163.5, C / 163.5, C / 163.8, C /
6 125.3, C / 125.3, C / 125.4, C /
7 119.3, CH 6.87 s 119.3, CH 6.86 s 119.5, CH 6.80 s
8 126.8, C / 126.8, C / 126.8, C /
9/9′132.1, CH 7.45 d (8.6) 132.1, CH 7.45 d (8.6) 132.1, CH 7.46 d (8.7)
10/10′116.4, CH 7.00 d (8.6) 116.5, CH 7.00 d (8.6) 116.4, CH 6.98 d (8.7)
11 160.9, C / 160.0, C / 160.9, C /
12 65.8, CH
2
4.68 br d (6.4) 65.3, CH
2
4.67 br d (6.4) 66.1, CH
2
4.58 br d (6.6)
13 120.8, CH 5.73 m 123.5, CH 5.57 m 121.1, CH 5.47 m
14 141.5, C / 141.8, C / 139.1, C /
15 67.9, CH
2
3.99 s 21.5, CH
3
1.85 br d (1.1) 26.0, CH
3
1.79 s
16 14.2, CH
3
1.76 s 61.8, CH
2
4,16 s 18.3, CH
3
1.76 s
17 13.1, CH
3
2.26 s 13.1, CH
3
2.26 s
a
Measured in DMSO-d
6
592 Mycol Progress (2020) 19:589–603
700 MHz) and
13
CNMR(CD
3
OD, 175 MHz) data (see
Tab le 1); HRESIMS: m/z 303.1334 [M + H]
+
(calcd for
C
16
H
19
N
2
O
4
+
, 303.1339).
(3R,6Z)-3-Thiomethyl-6-[4-O-(3-methylbut-2-
enyl)benzylidene]piperazine-2,5-dione (4): yellowish gum;
α½
25
D+ 48.6 (c0.035, MeOH); UV (MeOH) λ
max
(log ε):
202 (4.19), 229 (4.02), 324 nm (4.00); CD (c0.5 mg/mL,
EtOH) λ
max
310 (+), 230 nm (+);
1
HNMR(CD
3
OD,
700 MHz) and
13
CNMR(CD
3
OD, 175 MHz) data (see
Tab le 2); HRESIMS: m/z 355.1088 [M + Na]
+
(calcd for
C
17
H
20
N
2
NaO
3
S
+
, 355.1092), 687.2282 [2 M + Na]
+
.
(3S,6R)-3,6-Bisthiomethyl-6-[4-O-[(2Z)-4-hydroxy-3-
methylbut-2-enyl]phenylmethyl]piperazine-2,5-dione (5):
yellowish gum; α½
25
D+ 33.3 (c0.045, MeOH); UV
(MeOH) λ
max
(log ε): 208 (4.16), 226 (4.06), 275 nm
(3.38); CD (c0.5 mg/mL, EtOH) λ
max
315 (+), 248 (−),
220 nm (−);
1
H NMR (CD
3
OD, 700 MHz) and
13
C NMR
(CD
3
OD, 175 MHz) data (see Table 2); HRESIMS: m/z
397.1249 [M + H]
+
(calcd for C
18
H
25
N
2
O
4
S
2
+
, 397.1250),
419.1068 [M + Na]
+
(calcd for C
18
H
24
N
2
O
4
S
2
Na
+
,
419.1075).
Screening for biological activities
The antimicrobial activity and the in vitro cytotoxicity (IC
50
)
were evaluated according to our previously reported proce-
dures (Sandargo et al. 2018; Teponno et al. 2017). Briefly,
minimum inhibitory concentrations (MICs) in μg/mL of the
isolated compounds were determined by serial dilution assays
against Schizosaccharomyces pombe DSM 70572, Pichia
anomala DSM 6766, Mucor hiemalis DSM 2656, Candida
albicans DSM 1665, Rhodotorula glutinis DSM 10134,

Mycol Progress (2020) 19:589–603 593
Micrococcus luteus DSM 1790, Bacillus subtilis DSM 10,
Escherichia coli DSM 1116, Staphylococcus aureus DSM
346, Mycobacterium smegmatis ATCC 700084,
Chromobacterium violaceum DSM 30191, and
Pseudomonas aeruginosa DSM PA14. The assays were car-
ried out in 96-well microtiter plates in YMG media for fila-
mentous fungi and yeast and EBS for bacteria. Gentamycin,
kanamycin, nystatin, and oxytetracycline were used as posi-
tive control, and the negative control was methanol. The cy-
totoxicity against HeLa cells KB3.1 and mouse fibroblasts
L929 cells was determined by using the MTT (2-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method in
96-well microplates. The cell lines were cultured in DMEM
(Gibco). Briefly, 60-μL aliquots of serial dilutions from an
initial stock of 1 mg/mL in MeOH of the test compounds were
added to 120-μL aliquots of a cell suspension (5 × 10
4
cells/
mL) in 96-well microplates. After 5 days of incubation, an
MTT assay was performed, and the absorbance was measured
at 590 nm using an ELISA plate reader (Victor). The concen-
tration at which the growth ofcells was inhibited to 50% of the
control (IC
50
) was obtained from the dose-response curves.
Epothilone B was used as a positive control, while methanol
was used as a negative control.
Results and discussion
Taxonomy
The fungus was isolated as sterile mycelia without any repro-
ductive structures, and its identification by phenotypic char-
acters was impossible by conventional methods. The mor-
phology and macroscopic features of the culture on the plate
were determined on YMG, PDA, and OA.
Molecular phylogeny
The concatenated alignment consisted of 65 taxa including
representatives of 35 genera in the Chaetomiaceae (cf.
Tab le 3). Five isolates representing four species of the family
Podosporaceae were selected as outgroups. The alignment
contained 3055 characters (including gaps) and is composed
of four partitions: 858 characters for RPB2, 961 characters for
TUB2, 664 characters for ITS, and 572 characters for LSU. Of
the total characters, 1573 were constant, 1220 were parsimo-
ny-informative, and 262 were parsimony-uninformative. For
the Bayesian inference, the GTR+I+G model was selected as
optimal for RPB2,TUB2, ITS, and LSU based on the result of
the MrModeltest. Isolate CBS 144474 was located in a sepa-
rate clade along with representatives of the genera
Stolonocarpus,Madurella,andCanariomyces (ML-BS =
100%, PP = 0.99), but could not be accommodated in either
of these genera (Fig. 2). Therefore, a novel genus Batnamyces
is proposed.
Batnamyces Noumeur, gen. nov. MB 832844
Etymology—in reference to the town in Algeria where the
type was collected.
Diagnosis—Differs from the genera Canariomyces,
Stolonocarpus,andMadurella, to which it appears phyloge-
netically most closely related, in the absence of sexual features
and conidiogenous structures, except for producing terminal
chains of hyphal chlamydospores.
Type species: Batnamyces globulariicola Noumeur, sp.
nov. MB 832845 (Fig. 1)
Typus:Algeria,Batna,AinToutafromrootsofGlobularia
alypum Plantaginaceae, June 2015, S. R. Noumeur (holotype
CBS H-23624), ex-type culture in CBS 144474; GenBank
Acc nos of DNA sequences: MT075917 (ITS/LSU),
MT075918 (RPB2), MT075919 (TUB2).
Colonies on YMG and OMA at 23 °C spread over the
whole 9-cm Petri dish after 14 days while they attain a diam-
eter of 53 mm on PDA, initially appearing dark brown, then
becoming covered with white patches with age (Fig. 1b).
Mycelium on SNA, with brown, thick-walled, smooth,
branched, septate, 5–6μm diam hyphae, giving rise to hya-
line, thin-walled, smooth, branched, septate, 1.5–2μmdiam
hyphae. Colonies remained sterile on SNA, PDA, OA, and
MEA (see images in Supplementary information). After sev-
eral transfers onto new OA agar plates, oval, chlamydospores
(5–12 × 8 μm) were formed in short chains, arising from the
hyphal tips (Fig. 1e). Even after several months of
subcultivation, no other conidiogenous structures or sexual
morph were observed either in the old or the newly inoculated
plates. The growth maximum was determined to be 28–30 °C,
and no growth was observed at 37 °C.
Notes—
The new genus Batnamyces is primarily defined
based on its molecular phylogeny, since we neither observed
the characteristic structures of the asexual nor sexual morph of
the species in this genus. Its classification in the family was
inferred from the molecular phylogeny that was established on
the basis of a multi-locus genealogy comprising representa-
tives of all important genera of Chaetomiaceae. The genera
Batnamyces,Canariomyces,Madurella, and Stolonocarpus
formed a single lineage (Fig. 2). Canariomyces species typi-
cally produce non-ostiolate ascomata together with single-
celled conidia arising from reduced conidiophores that are
reduced to a hyphal cell (cf. figs. 19–22 in Wang et al.
2019b). Madurella species usually produce only sterile (non-
sporulating) hyphae and sparse aerial mycelium, growing re-
strictedly in culture and often producing buff, cinnamon, si-
enna, or orange exudates diffusing into the agar (cf. fig. 18 in
Wang et al. 2019b). On the other hand, Stolonocarpus is
characterised by non-ostiolate ascomata arising from a

Table 3 Details of strains used in this study
Current name Culture accession number
1
GenBank accession numbers
2
References
ITS LSU RPB2 TUB2
Chaetomiaceae
Achaetomium globosum CBS 332.67 TKX976570 KX976695 KX976793 KX976911 Wang et al. 2016a
Achaetomium strumarium CBS 333.67 TAY681204 AY681170 KC503254 AY681238 Cai et al. 2006,
Wang et al. 2016a
Acrophialophora nainiana CBS 100.60 TMK926793 MK926793 MK876755 MK926893 Wang et al. 2019b
CBS 417.67 MK926794 MK926794 MK876756 MK926894 Wang et al. 2019b
Amesia atrobrunnea CBS 379.66 TJX280771 JX280666 KX976798 KX976916 de Hoog et al. 2013,
Wang et al. 2016a
Amesia nigricolor CBS 600.66 TKX976578 KX976703 KX976802 KX976920 Wang et al. 2016a
Arcopilus aureus CBS 153.52 KX976582 KX976707 KX976806 KX976924 Wang et al. 2016a
Arcopilus flavigenus CBS 337.67 TKX976587 KX976712 KX976811 KX976929 Wang et al. 2016a
Batnamyces globulariicola CBS 144474 TPresent study
Botryotrichum murorum CBS 163.52 KX976591 KX976716 KX976815 KX976933 Wang et al. 2016a
Botryotrichum piluliferum CBS 654.79 KX976597 KX976722 KX976821 KX976939 Wang et al. 2016a
Brachychaeta variospora CBS 414.73 TMK926797 MK926797 MK876759 MK926897 Wang et al. 2019b
Canariomyces microsporus CBS 276.74 TMK926799 MK926799 MK876760 MK926899 Wang et al. 2019b
CBS 161.80 MK926800 MK926800 MK876761 MK926900 Wang et al. 2019b
Canariomyces notabilis CBS 548.83 TMK926802 MK926802 MK876763 MK926902 Wang et al. 2019b
Carteria arctostaphyli CBS 229.82 TMK926807 MK926807 MK876767 MK926907 Wang et al. 2019b
Chaetomium elatum CBS 142034 TKX976612 KX976733 KX976832 KX976954 Wang et al. 2016a
Chaetomium globosum CBS 160.62 TKT214565 KT214596 KT214666 KT214742 Wang et al. 2016b
Collariella bostrychodes CBS 163.73 KX976641 KX976738 KX976837 KX976983 Wang et al. 2016a
Collariella robusta CBS 551.83 TKX976652 KX976747 KX976846 KX976994 Wang et al. 2016a
Collariella virescens CBS 148.68 TKX976654 KX976749 KX976848 KX976996 Wang et al. 2016a
Condenascus tortuosus CBS 610.97 MK926817 MK926817 MK876777 MK926917 Wang et al. 2019b
Corynascus sepedonium CBS 111.69 THQ871751 KX976777 HQ871827 KX977027 van den Brink et al. 2012,
Wang et al. 2016a
Chrysanthotrichum allolentum CBS 644.83 TMK926808 MK926808 MK876768 MK926908 Wang et al. 2019b
Chrysanthotrichum lentum CBS 339.67 TMK926809 MK926809 MK876769 MK926909 Wang et al. 2019b
Chrysocorona lucknowensis CBS 727.71 eT MK926813 MK926813 MK876773 MK926913 Wang et al. 2019b
CBS 385.66 MK926816 MK926816 MK876776 MK926916 Wang et al. 2019b
Corynascella humicola CBS 337.72 TKX976656 KX976751 KX976850 KX976998 Wang et al. 2016a
CBS 379.74 KX976657 KX976752 KX976851 KX976999 Wang et al. 2016a
Dichotomopilus funicola CBS 159.52 TGU563369 GU563354 KX976856 JF772461 Wang et al. 2016a
Dichotomopilus indicus CGMCC 3.14184 TGU563367 GU563360 KX976861 JF772453 Wang et al. 2016a
Floropilus chiversii CBS 558.80 TMK926818 MK926818 MK876778 MK926918 Wang et al. 2019b
Humicola fuscoatra CBS 118.14 TLT993579 LT993579 LT993498 LT993660 Wang et al. 2019a
Hyalosphaerella fragilis CBS 456.73 TKX976693 KX976791 MK876779 KX977042 Wang et al. 2019b
Madurella fahalii CBS 129176 TMK926819 MK926819 MK876780 MK926919 Wang et al. 2019b
Madurella mycetomatis CBS 109801 TMK926820 MK926820 MK876781 MK926920 Wang et al. 2019b
Madurella pseudomycetomatis CBS 129177 TMK926821 MK926821 MK876782 MK926921 Wang et al. 2019b
Madurella tropicana CBS 201.38 TMK926824 MK926824 MK876785 MK926924 Wang et al. 2019b
CBS 206.47 MK926825 MK926825 MK876786 MK926925 Wang et al. 2019b
Melanocarpus albomyces CBS 638.94 TKX976679 KX976773 KX976886 KX977021 Wang et al. 2016a
CBS 747.70 KX976680 KX976774 KX976887 KX977022
594 Mycol Progress (2020) 19:589–603

stolon-like mycelium and covered by pigmented hypha-like
hairs (fig. 41 in Wang et al. 2019b). The genus Batnamyces is
more similar to Canariomyces and Stolonocarpus than to
Madurella with respect to the morphology of the colonies
and mycelia but can be easily distinguished from them by
the lack of reproductive structures. Since the ex-type strain
of Batnamyces was obtained by using an isolation procedure,
which is well established for endophytes, from an endemic
plant in an area that has never been studied intensively for
the biodiversity of its mycobiota, it did not come as a surprise
that no reproductive structures are produced. After all, it is
pretty well known that endophytic fungi often do not produce
any propagules. However, we isolated the fungus only one
time and can therefore not be sure about its actual lifestyle.
Poor statistic support (PP < 0.95; ML-BS = 85%) also implied
that B. globulariicola was not a member of either Madurella
or Stolonocarpus.
Isolation and structure elucidation of compounds 1–5
Fractionation of crude ethyl acetate extracts from the culture
of Batnamyces globulariicola in Q61/2 and YMG media led
to the isolation and structure elucidation of 5 new 2,5-
diketopiperazines (1–5) together with seven known metabo-
lites identified by spectroscopic analysis and comparison with
literature data, such as Sch 54796 6(Chu et al. 1993;Usami
Tabl e 3 (continued)
Current name Culture accession number
1
GenBank accession numbers
2
References
ITS LSU RPB2 TUB2
Wang et al. 2016a
Microthielavia ovispora CBS 165.75 TMK926826 MK926826 MK876787 MK926926 Wang et al. 2019b
Myceliophthora lutea CBS 145.77 THQ871775 KM655351 HQ871816 KX977026 van den Brink et al. 2012,
Wang et al. 2016a
Mycothermus thermophilus CBS 625.91 TLT993604 LT993604 LT993523 LT993685 Wang et al. 2019a
Ovatospora medusarum CBS 148.67 TKX976684 KX976782 KX976897 KX977032 Wang et al. 2016a
Ovatospora mollicella CBS 583.83 TKX976685 KX976783 KX976898 KX977033 Wang et al. 2016a
Parathielavia hyrcaniae CBS 353.62 TKM655329 KM655368 KM655401 KX977043 van den Brink et al. 2015,
Wang et al. 2016a
Parathielavia kuwaitensis CBS 945.72 TKM655332 KM655371 KM655404 KX977044 van den Brink et al. 2015,
Wang et al. 2016a
Pseudothielavia terricola CBS 165.88 TKX976694 KX976792 MK876795 KX977045 van den Brink et al. 2015,
Wang et al. 2016a
CBS 487.74 MK926834 MK926834 MK876796 MK926934 Wang et al. 2019b
Remersonia thermophila CBS 645.91 LT993611 LT993611 LT993530 LT993692 Wang et al. 2019a
Staphylotrichum coccosporum CBS 364.58 TLT993620 LT993620 LT993539 LT993701 Wang et al. 2019a
Stellatospora terricola CBS 811.95 TMK926835 MK926835 MK876797 MK926935 Wang et al. 2019b
Stolonocarpus gigasporus CBS 112062 TMK926836 MK926836 MK876798 MK926936 Wang et al. 2019b
Subramaniula thielavioides CBS 122.78 TKP862597 KP970654 KP900670 KP900708 Wang et al. 2016a
Thermothielavioides terrestris CBS 117535 TMK926837 MK926837 MK876799 MK926937 Wang et al. 2019b
CBS 492.74 MK926838 MK926838 MK876800 MK926938 Wang et al. 2019b
Thermothelomyces heterothallica CBS 202.75 THQ871771 KM655354 HQ871798 KX977025 van den Brink et al. 2012,
Wang et al. 2016a
Trichocladium asperum CBS 903.85 TLT993632 LT993632 LT993551 LT993713 Wang et al. 2019a
Trichocladium griseum CBS 119.14 TLT993639 LT993639 LT993558 LT993720 Wang et al. 2019a
Podosporaceae
Cladorrhinum foecundissimum CBS 180.66 TMK926856 MK926856 MK876818 MK926956 Wang et al. 2019b
Podospora fimicola CBS 482.64 TMK926862 MK926862 MK876824 MK926962 Wang et al. 2019b
CBS 990.96 MK926863 MK926863 MK876825 MK926963 Wang et al. 2019b
Triangularia anserina CBS 433.50 MK926864 MK926864 MK876826 MK926964 Wang et al. 2019b
Triangularia bambusae CBS 352.33 TMK926868 MK926868 MK876830 MK926968 Wang et al. 2019b
Mycol Progress (2020) 19:589–603 595

et al. 2002), Sch 54794 7(Chu et al. 1993; Usami et al. 2002),
cyclo-(glycyl-
L
-tyrosyl)-3,3-dimethylallyl ether 8(Koolen
et al. 2012), 4-O-(3-methylbut-2-enyl)benzoic acid 9
(Nozawa et al. 1989),
L
-Pro-
L
-Ile 10 (Ren et al. 2010),
L
-
Pro-
L
-Leu 11 (Ren et al. 2010; Sansinenea et al. 2016), and
L
-Pro-
L
-Phe) 12 (Sansinenea et al. 2016)(Fig.3).
Compound 1was isolated as a yellowish gum. Its molecu-
lar formula C
17
H
20
N
2
O
4
S was deduced from the HRESIMS
which exhibited the pseudomolecular ion peak at m/z
349.1217 [M + H]
+
(calcd for C
17
H
21
N
2
O
4
S
+
, 349.1217).
This was confirmed by the ESIMS ion cluster at m/z371.11
[M + Na]
+
and a prominent ion fragment and 301 [M + H–
48]
+
revealing the loss of a methanethiol (CH
3
SH) unit (Chu
et al. 1993). Its
1
H NMR spectrum displayed resonances for an
AA′BB′spin system at δ
H
7.45 (d, J= 8.6 Hz, H-9, and H-9′)
and 7.00 (d, J= 8.6 Hz, H-10, and H-10′) suggesting the pres-
ence of a 1,4-disubstituted benzene ring in the molecule
(Table 1). It also showed a signal assigned to a vinyl proton
δ
H
6.87 (H-7) and a set of resonances depicted at δ
H
4.68 (brd,
J= 6.4 Hz, H-12), 5.73 (m, H-12), 3.99 (s), 3.99 (s, H-15), and
1.76 (s, H-16) attributed to an O-isoprenol group. Other sig-
nals were those of a thiomethyl singlet at δ
H
2.26 (s, H-17) and
a methine singlet at δ
H
4.97 (s, H-3). The downfield shift of
the latter revealed its bis-heteroatom connectivity (Chu et al.
1997)(Table1). The
13
C NMR spectrum showed two
amidocarbonyl carbon signals characteristic of a
diketopiperazine core at δ
C
166.0 (C-2) and 163.5 (C-5)
(Chu et al. 1993; Guimarães et al. 2010;Fuetal.2011;Fan
et al. 2017). It also displayed resonances at δ
C
125.3 (C-6),
119.3 (C-7), 126.8 (C-8), 132.1 (C-9, C-9′), 116.4 (C-10,
C-10′), and 160.9 (C-11) evidencing the presence of an
oxybenzylidene moiety. In addition, the signals of two meth-
ylenes at δ
C
66.8 (C-12) and 67.9 (C-15), a methine at δ
C
120.8 (C-13), a methyl at δ
C
14.2 (C-13), and a quaternary
carbon at δ
C
141.5 (C-14) were assigned to the O-isoprenol
group. The remaining signals were those of a methine at δ
C
Fig. 1 Morphological characteristics of the new genus Batnamyces.a
Colonies obverse and reverse on PDA at 23 °C after 21 days. b
Colonies obverse and reverse on OM at 23 °C after 21 days. cColonies
obverse and reverse on YMG at 23 °C after 21 days. dand e
chlamydospore-like structures formed at hyphal tips. Scale bars d, e =
10 μm

Fig. 2 Phylogenetic tree resulting from a maximum likelihood analysis of
the concatenated RPB2,TUB2, ITS, and LSU sequence data sets. The
confidence values are indicated at the notes: bootstrap proportions from
the ML analysis above branches, and the posterior probabilities from the
Bayesian analysis below branches. “-”means lacking statistical support
(< 70% for bootstrap proportions from ML or MP analyses; < 0.95 for
posterior probabilities from Bayesian analyses). The branches with full
statistical support (MP-BS = 100%; ML-BS = 100%; PP = 1.0) are
indicated by thickened branches. Genus clades are discriminated with
boxes of different colours. The scale bar shows the expected number of
changes per site. The tree is rooted with members of the Podosporaceae.
“T”after the respective DNA sequences are derived from ex-type (includ-
ing ex-epitype and ex-neotype) specimens
596 Mycol Progress (2020) 19:589–603

0.2
Trichocladium griseum CBS 119.14 T
Humicola fuscoatra CBS 118.14 T
Stolonocarpus gigasporus CBS 112062 T
Subramaniula thielavioides CBS 122.78 T
Chrysanthotrichum lentum CBS 339.67 T
Ovatospora medusarum CBS 148.67 T
Parathielavia hyrcaniae CBS 353.62 T
Thermothielavioides terrestris CBS 492.74
Pseudothielavia terricola CBS 165.88 T
Mycothermus thermophilus CBS 625.91 T
Collariella robusta CBS 551.83 T
Pseudothielavia terricola CBS 487.74
Myceliophthora lutea CBS 145.77 T
Collariella virescens CBS 148.68 T
Podospora fimicola CBS 482.64 T
Carteria arctostaphyli CBS 229.82 T
Canariomyces microsporus CBS 161.80
Triangularia anserina CBS 433.50
Madurella tropicana CBS 201.38 T
Podospora fimicola CBS 990.96
Microthielavia ovispora CBS 165.75 T
Cladorrhinum foecundissimum CBS 180.66 T
Melanocarpus albomyces CBS 638.94 T
Chrysocorona lucknowensis CBS 385.66
Trichocladium asperum CBS 903.85 T
Melanocarpus albomyces CBS 747.70
Arcopilus aureus CBS 153.52
Triangularia bambusae CBS 352.33 T
Brachychaeta variospora CBS 414.73 T
Floropilus chiversii CBS 558.80 T
Chrysanthotrichum allolentum CBS 644.83 T
Acrophialophora nainiana CBS 100.60 T
Chaetomium globosum CBS 160.62 T
Condenascus tortuosus CBS 610.97
Amesia atrobrunnea CBS 379.66 T
Achaetomium globosum CBS 332.67 T
Dichotomopilus indicus CGMCC 3.14184 T
Botryotrichum murorum CBS 163.52
Madurella pseudomycetomatis CBS 129177 T
Dichotomopilus funicola CBS 159.52 T
Chrysocorona lucknowensis CBS 727.71 T
Corynascella humicola CBS 337.72 T
Botryotrichum piluliferum CBS 654.79
Canariomyces microsporus CBS 276.74 T
Thermothelomyces heterothallica CBS 202.75 T
Stellatospora terricola CBS 811.95 T
Amesia nigricolor CBS 600.66 T
Corynascus sepedonium CBS 111.69 T
Achaetomium strumarium CBS 333.67 T
Remersonia thermophila CBS 645.91
Ovatospora mollicella CBS 583.83 T
Madurella fahalii CBS 129176 T
Canariomyces notabilis CBS 548.83 T
Madurella mycetomatis CBS 109801 T
Chaetomium elatum CBS 142034 T
Corynascella humicola CBS 379.74
Thermothielavioides terrestris CBS 117535 T
Staphylotrichum coccosporum CBS 364.58 T
Parathielavia kuwaitensis CBS 945.72 T
Arcopilus flavigenus CBS 337.67 T
Hyalosphaerella fragilis CBS 456.73 T
Batnamyces globularicola CBS 144474 T
Madurella tropicana CBS 206.47
Collariella bostrychodes CBS 163.73
Acrophialophora nainiana CBS 417.67
0.99/100
0.99/85
0.99/100
0.98/100
-/93
0.98/100
0.95/-
0.99/100
0.99/100
0.96/96
0.98/91
0.99/100
0.99/100
0.99/100
0.99/100
0.96/95
0.99/100
0.99/100
0.95/92
0.98/94
0.99/97
0.99/100
0.96/92
0.97/82
0.99/100
0.99/100
0.97/100
0.95/71
0.99/90
0.99/100
0.99/100
0.99/100
-/85
0.99/100
0.99/100
0.99/100
0.99/100
0.96/81 0.99/100
0.99/100
0.99/100
0.99/100
0.97/100
-/86
0.98/100
0.99/100
Mycol Progress (2020) 19:589–603 597

59.6 (C-3) and the thiomethyl group at δ
C
13.1 (C-17)
(Table 1). The location of the thiomethyl group at C-3 was
further evidenced by the HMBC correlation observed between
thiomethyl protons signal at δ
H
2.26 (s, H-17) and the carbon
at δ
C
59.6 (C-3). Furthermore, the HMBC correlation from H-
12 (δ
H
4.68) to carbon C-11 (δ
C
160.9) confirmed that the O-
isoprenol group was linked at C-11 (Fig. 4). Careful examina-
tion of the
1
H-
1
H COSY, HSQC, and HMBC spectra proved
that 1was related to Sch 56396, a metabolite produced by the
fungus Tolypocladium sp. (Chu et al. 1997), the main differ-
ence was the hydroxylation of one methyl of the isopentenyl
group to form 1.TheEgeometry for the Δ
13,14
double bond
was determined from the NOESY correlation depicted be-
tween the olefinic proton H-13 (δ
H
5.73) and the methylene
protons H-15 (δ
H
3.99). To solve the stereochemistry of Δ
6,7
double bond, a NOESY spectrum was measured in DMSO-d
6
.
The lack of NOESY correlation between NH-1 depicted at δ
H
10.17 (s) and the vinyl proton H-7 (δ
H
6.87) was in favour of
the Zconfiguration. This was further confirmed by the
NOESY correlation depicted between NH-1 (δ
H
10.17 s)
and H-9/H-9′(δ
H
7.45, d, 8.6). The absolute configuration
at C-3 was determined to be Rby comparison of the experi-
mental ECD of 1(Fig. S9, Supporting information) with the
calculated ECD spectra for the four stereoisomers (3R,6E;
3S,6E;3R,6Z;3S,6Z) of a related compound (Guimarães
et al. 2010). Although the absorption on the experimental
spectrum was not intense, the Cotton effects of 1were in
accordance with the experimental Cotton effects of 3R,6Zste-
reoisomer especially with positive absorption in the regions of
200–240 nm and 275–350 nm, respectively. The structure of
compound 1was unambiguously elucidated as (3R,6Z)-3-
thiomethyl-6-[4-O-[(2E)-4-hydroxy-3-methylbut-2-
enyl]benzylidene]piperazine-2,5-dione.
Metabolite 2was obtained as a yellowish gum. It possessed
the same molecular formula as 1as evidenced by the
HRESIMS which showed a protonated molecular ion peak
at m/z349.1217 [M + H]
+
(calcd for C
17
H
21
N
2
O
4
S
+
,
349.1217) despite the fact that both compounds had different
retention times. The
1
Hand
13
C NMR spectra of 2(Table 1)
were closely related to those of 1, especially for signals of the
diketopiperazine core and the oxybenzylidene moiety. The
only difference was the downfield or upfield shifts of some
1
Hand
13
C signals probably due to the change of configura-
tion at the Δ
13,14
double bond. This configuration was de-
duced to be Zfrom careful analysis of the NOESY spectrum
which exhibited a cross-peak correlation between the methy-
lene protons at δ
H
4.67 (brd, J= 6.4 Hz, H-12) and 4,16 (s,
H-16). The NOESY correlation between the olefinic proton
H-13 (δ
H
, 5.57, m) and the methyl protons H-15 (δ
H
1.85) was
also depicted. In view of determining the configuration of the
Δ
6,7
double bond, the NOESY spectrum of 2was also mea-
suredinDMSO-d
6
. The NOESY correlation observed
N
H
H
NO
O
O
R
3
R
2
R
1
1
2
345
6
7
8
9
10
11
12
13
14
9'
10'
R
1
R
2
R
3
SMe CH
2
OH CH
3
SMe CH
3
CH
2
OH
OH CH
3
CH
3
SMe CH
3
CH
3
1:
2:
3:
4:HN
N
O
O
HN
N
O
O
HN
N
O
O
10
11 12
O
CH
2
OH
N
H
H
NO
O
MeS
SMe
O
N
H
H
NO
O
MeS
SMe
O
N
H
H
NO
O
MeS
SMe
O
O
HO
O
N
H
H
NO
O
5
6
7
8
9
1
2
345
678
9
10
11
12
13
14
9'
10' 15
16
17
18
Fig. 3 Structures of compounds 1–12 isolated from the culture of Batnamyces globulariicola
598 Mycol Progress (2020) 19:589–603

between the NH-1 proton signal (δ
H
10.17) and H-9/H-9′(δ
H
7.45, d, J=8.6)showedthat Δ
6,7
has the same geometry in
metabolites 1and 2. The experimental ECD spectrum of 2
(Fig. S18, Supporting information) was nearly identical to that
of 1, leading to the deduction of the 3Rabsolute configuration
for 2. The structure of metabolite 2was thus concluded as
(3R,6Z)-3-thiomethyl-6-[4-O-[(2Z)-4-hydroxy-3-methylbut-
2-enyl]benzylidene]piperazine-2,5-dione.
Compound 3was obtained as a yellowish gum. Its molecular
formula was determined as C
16
H
18
N
2
O
4
from the HRESIMS
analysis which showed the pseudomolecular ion at m/z
303.1334 [M + H]
+
(calcd for C
16
H
19
N
2
O
4+
, 303.1339). Its
1
H
NMR spectrum exhibited resonances for an AA′BB′spin system
at δ
H
7.46 (d, J=8.7Hz,H-9,andH-9′) and 6.98 (d, J=8.7Hz,
H-10, and H-10′) suggesting a para-disubstituted aromatic ring
in the structure. It also showed signals for a vinyl proton at δ
H
5.47 (m, H-13), an oxymethylene at δ
H
4.58 (brd, J=6.6 Hz,
H-12), and two vinyl connected methyl singlets at δ
H
1.79 (H-15)
and1.76(H-16)characteristicofanO-isoprenyl moiety. Signals
of the vinyl proton H-7 and the methine proton H-3 were ob-
served at δ
H
6.80 (s) and 5.09 (s), respectively. The
13
CNMR
spectrum exhibited signals of two amidocarbonyl carbons at δ
C
167.0 (C-2) and 163.8 (C-5). Those characteristic of the
oxybenzylidene moiety were observed at δ
C
125.4 (C-6), 119.5
(C-7), 126.8 (C-8), 132.1 (C-9, C-9′), 116.4 (C-10, C-10′), and
160.9 (C-11). The remaining resonances depicted at δ
C
66.1
(C-12), 121.1 (C-13), 139.1 (C-14), δ
C
26.0 (C-15), and 18.3
(C-16) confirmed the presence of the O-prenyl group in the mol-
ecule (Table 1). The downfield shift of C-3 in metabolite 3(δ
C
76.1) with respect to compounds 1and 2(δ
C
59.6) suggested the
presence of an OH group at C-3 in 3instead of the thiomethyl
moiety. This was further supported by mass analysis and the
absence of the thiomethyl signal on the
1
Hand
13
CNMRspectra.
The structure was confirmed by a comprehensive analysis of the
2D NMR data, particularly
1
H-
1
H COSY, HSQC, and HMBC
spectra (Fig. 4). The configuration of the Δ
6,7
double bond was
determined to be Zby the NOESY spectrum measured in
DMSO-d
6
on which correlations were observed between NH-1
and H-9 (or H-9′) but not between NH-1 (δ
H
9.92, s) and H-7 (δ
H
6.80, s). The ECD spectrum of 3(Fig. S27, Supporting
information) indicated the same absolute configuration at C-3
(R) as for metabolites 1and 2. The structure was finally conclud-
ed as (3R,6Z)-3-hydroxy-6-[4-O-(3-methylbut-2-
enyl)benzylidene]piperazine-2,5-dione.
The molecular formula of 4also obtained as a yellowish
gum was deduced to be C
17
H
20
N
2
O
3
S from the HRESIMS
which showed ion clusters [M + Na]
+
at m/z 355.1088 (Calcd
355.1087) and [2M + Na]
+
at m/z 687.2283 (Calcd 687.2281).
The NMR data (Table 2) showed similarities with the previ-
ously described metabolites 1–3.Its
1
H-NMR spectrum ex-
hibited in addition to signals of the diketopiperazine and the
oxybenzylidene moieties two olefinic methyl resonances at δ
H
1.76 and 1.79, an oxygenated methylene doublet at δ
H
4.58
(J= 6.6 Hz), and a vinyl proton signal at δ
H
5.46 (m) charac-
teristic of a γ,γ-dimethylallyloxy moiety (Sritularak and
Likhitwitayawuid 2006). Careful examination of the
1
H-
1
H
COSY, HSQC, and HMBC spectra proved that 4was similar
to Sch 56396 previously isolated from the fermentation broth
of the fungus Tolypocladium sp. (Chu et al. 1997), but the only
difference was on the sign of their optical rotations.
Compound 4showed a positive optical rotation, while the
optical rotation of Sch 56396 was negative, confirming that
they are stereoisomers. The Zconfiguration of the Δ
6,7
was
deduced from the chemical shift of H-7 (δ
H
6.87) in compar-
ison with those of the same proton in compounds 1–3, since it
was reported that the (Z)-vinyl proton of the 6-benzylidene-
substituted piperazine-2,5-diones is farther downfield than the
(E)-vinyl proton because of the deshielding effect of the 5-
ketone (Fu et al. 2011). Since the configuration of the chiral
centre C-3 of Sch 56396 was not determined, we measured the
ECD spectrum of 4(Fig. S36, Supporting information) and its
comparison with those of compounds 1–3allowed us to as-
sign the 3Rconfiguration. Compound 4was then elucidated as
(3R,6Z)-3-thiomethyl-6-[4-O-(3-methylbut-2-
enyl)benzylidene]piperazine-2,5-dione, the stereoisomer of
Sch 56396.
Compound 5was obtained as a yellow gum from metha-
nol. Its HRESIMS showed ion clusters at m/z 397.1249 [M +
N
H
H
NO
O
O
CH
3
CH
2
OH
H
3
CS
O
CH
2
OH
CH
3
N
H
H
NO
O
H
3
CS
SCH
3
N
H
H
NO
O
O
CH
2
OH
CH
3
H
3
CS
N
H
H
NO
O
O
CH
3
CH
3
HO
1
H-
1
HCOSY
HMBC
123
5
Fig. 4 Selected
1
H-
1
H COSY and HMBC correlations for compounds 1–3and 5
Mycol Progress (2020) 19:589–603 599

H]
+
and 419.1068 [M + Na]
+
consistent with the molecular
formula of C
18
H
24
N
2
O
4
S
2
(calcd for C
18
H
25
N
2
O
4
S
2+
,
397.1250; calcd for C
18
H
24
N
2
O
4
S
2
Na
+
, 419.1075). The pres-
ence of two thiomethyl groups was confirmed by the ion frag-
ments depicted at m/z 349.1215 [M + H–48]
+
and 301.1177
[M + H–2×48]
+
revealing the loss of two methanethiol
(CH
3
SH) units (Chu et al. 1993). Its
1
HNMRspectrum
showed in addition to the signals of the O-isoprenoltyrosine
moiety at δ
H
7.26 (d, J= 9.0 Hz, H-9, and H-9′), 6.83 (d, J=
9.0 Hz, H-10, and H-10′), 4.61 (brd, J= 6.3 Hz, H-12), 5.47
(m, H-12), 4.15 (s, H-15), and 1.81 (s, H-16) those of two
thiomethyl groups at δ
H
1.36 (s, H-18) and 2.23 (s, H-17) as
well as a methylene group observed as an AX spin system at
δ
H
2.94 (d, J= 13.5, H-7A) and 3.60 (d, J= 13.5, H-7X)
(Table 2). Careful examination of the
13
C,
1
H-
1
HCOSY,
HSQC, and HMBC spectra proved metabolite 5to have the
same planar structure as meromutides A and B recently iso-
lated after pleiotropic activation of natural products in
Metarhizium robertsii by deletion of a histone acetyltransfer-
ase (Fan et al. 2017). The Zgeometry for the Δ
13,14
double
bond was determined from the NOESY correlation depicted
between the methylene protons at δ
H
4.61 (brd, J= 6.4 Hz,
H-12) and 4.15 (s, H-16). Compound 5with the Zgeometry of
the olefinic double bond can possess 4 stereoisomers (3S,6S),
(3R,6S), (3S,6R), and (3R,6R). Up to now, only two of them,
namely meromutide A (3S,6S) and meromutide B (3R,6S)
were isolated and characterised. Its absolute configuration
was determined by careful comparison of the chemical shifts
for both protons and carbons of the thiomethyl groups linked
at C-3 and C-6 of its known stereoisomers. For meromutide A,
these chemical shifts were as follows: C-3 (δ
H
2.24; δ
C
13.9)
and C-6 (δ
H
2.30; δ
C
13.4), while for meromutide B, they
were C-3 (δ
H
2.21; δ
C
10.4) and C-6 (δ
H
1.20; δ
C
12.9) (Fan
et al. 2017). The chemical shifts of the corresponding protons
and carbons of the thiomethyl groups at C-3 and C-6 in com-
pound 5(also measured in methanol-d
6
) were different from
those of the known stereoisomers ((δ
H
1.36; δ
C
10.6) and (δ
H
2.23; δ
C
13.4), respectively) indicating a change of configu-
ration in one of the chiral centres. On the other hand, since
metabolite 5is the C-15 hydroxyl derivative of Sch54796 (6)
and Sch54794 (7) possessing the known (3R,6S)and(3S,6S)
configurations, its absolute configuration at C-6 must be Ras
supported by the positive optical rotation (+ 33.3, MeOH) in
comparison with those of the known congeners (Sch54796 (−
25, DMSO) and Sch54794 (−70, DMSO); see Chu et al.
(1993). Furthermore, the NOESY correlation depicted be-
tween H-3 (δ
H
5.01, s) and CH
3
-18 (2.23, s) revealed the
trans-orientation of the two thiomethyl groups on the
diketopiperazine ring. To further confirm the 3S,6Rconfigu-
ration, we compared the ECD spectrum of metabolite 5with
that of fusaperazine, a related thiodiketopiperazine possessing
a3R,6Rconfiguration obtained from a marine algae-derived
fungus Penicillium sp. KMM 4672 (Yurchenko et al. 2019).
Both compounds exhibited a negative Cotton effect between
220 and 240 nm, probably due to the common 6Rconfigura-
tion. However, a negative Cotton effect was observed on the
ECD spectrum of compound 5between 240 and 290 nm while
a positive Cotton effect was depicted in the same zone of the
ECD spectrum of fusaperazine. The structure of 5was finally
elucidated as (3S,6R)-3,6-bisthiomethyl-6-[4-O-[(2Z)-4-hy-
droxy-3-methylbut-2-enyl]phenylmethyl]piperazine-2,5-
dione.
Diketopiperazines are cyclic peptides produced by bacteria
and fungi arising from the cyclisation of two or more amino
acids catalysed either by two-modular non-ribosomal peptide
synthetases or by cyclodipeptide synthases (Huang et al.
2014; Brockmeyer and Li 2017). Since the isolated com-
pounds are biogenetically related, their biosynthetic relation-
ships were proposed. Compounds 10,11, and 12 could be
obtained from the cyclisation of L-proline and
L
-isoleucine,
L-proline and L-leucine, and L-proline and L-phenylalanine,
respectively. While working on the biosynthesis of the
epidithiodiketopiperazine gliotoxin, Scharf et al. 2011 discov-
ered that a specialised glutathione S-transferase (GliG) plays a
key role in C-S bond formation (sulfurisation) and that
bishydroxylation of the diketopiperazine by oxygenase
(GliC) is a prerequisite for glutathione adduct formation.
Cyclisation of glycine and L-tyrosine followed by O-
prenylation affords cyclo-(glycyl-L-tyrosyl)-3,3-dimethylallyl
ether 8which could undergo bishydroxylation by oxygenase
GliC to yield the intermediate 13 (not isolated). Thiolation of
13 in the presence of GliG could afford Sch 54796 (6) and Sch
54794 (7). The C-6 epimerisation of Sch 54794 (7)followed
by hydroxylation at C-15 could lead compound 5. The inter-
mediate 13 could also undergo dehydration to afford metabo-
lite 3which could give compound 9after an oxidative cleav-
age of the C-6–C-7 double bond. Thiolation of metabolite 3
could also lead compound 4, which could undergo hydroxyl-
ation of one of the prenylmethyl groups to give 1and 2(Fig.
5).
Biological activities
Since some derivatives of diketopiperazines were reported to
display significant antibiotic, antitumor, and immunosuppres-
sant properties (Ameur et al. 2004), while others show a wide
range of biological effects in cell cycle progression (Cui et al.
1996), the isolated compounds were tested for their antimicro-
bial and cytotoxic activities against various bacteria, fungi,
and two mammalian cell lines, but only weak cytotoxic activ-
ity was observed for metabolites 1,4,and9against KB3.1
cells. The evaluation of these compounds in additional bioas-
says is presently underway.
In general, Batnamyces globulariicola belongs to a group
of Chaetomiaceae that has been poorly studied for secondary
metabolites, suggesting that it will be worthwhile to examine
600 Mycol Progress (2020) 19:589–603

further strains that appear phylogenetically related for the pro-
duction of diketopiperazines and other secondary metabolites.
The lack of suitable morphological features for the classifica-
tion of these fungi using a polythetic approach could thus be
compensated by chemotaxonomic methodology, as recently
accomplished for some genera of the Xylariales (cf.
Samarakoon et al. 2020; Wittstein et al. 2020).
Conclusion
Batnamyces globulariicola was only isolated once among
several hundreds of cultures that inhabited the rhizosphere of
the host plant during the course of a PhD thesis (Noumeur
2018). We selected this strain out of many others that were
isolated concurrently because it turned out to represent a hith-
erto unknown phylogenetic lineage among the Sordariales.
We were at first unsure whether to report the fungus as an
“unknown member”of the Chaetomiaceae, along with the
secondary metabolites, but finally decided to name the taxon,
and place it in its phylogenetic context, as part of an ongoing
taxonomic study (Wang et al. 2019b). It was rather surprising
to see that it still did not fit in any of the known genera of the
Chaetomiaceae even by comparison with the latest mono-
graphic work.
We are aware of the potential pitfalls regarding the classi-
fication of such a single strain as a member of a monotypic
genus, and in particular the requests by some members of the
mycological community that more than one culture needs to
be deposited to justify the formal description of a new taxon.
However, such arguments mostly come from scientists who
are working with ubiquitous fungal genera that make up the
bulk of new species in the large classes of Eurotiomycetes,
Sordariomycetes, and Dothideomycetes, where ex-type
strains as well as large numbers of congeneric isolates are
readily available.
We cannot be absolutely certain whether our new fungus
really represents an endophyte, because it was only isolated
once. However, a sterile mycelium could hardly have survived
the applied, rather harsh root disinfection procedure unless it
forms persistent propagules which may have survived the
chemical treatment. Nevertheless, we cannot exclude that it
is heterothallic and that a sexual state may exist in nature.
Even though we did not observe sporulation of the culture,
O
N
H
H
NO
O
MeS
SMe
O
N
H
H
NO
O
MeS
SMe
O
O
HO
6
7
8
9
O
N
H
H
NO
O
1
2
345
678
9
10
11
12
13
14
9'
10' 15
16
HO
O
OH
NH
2
O
OH
H
2
N
Glycine
L-Tyrosine Cyclisation
Prenylation
Bishydroxylation
O
N
H
H
NO
O
HO
OH
Thiolation
13
C-6 Epimerization
C-15 Hydroxylation
O
CH
2
OH
N
H
H
NO
O
MeS
SMe
N
H
H
NO
O
O
HO
Dehydration
Oxidative
cleavage
N
H
H
NO
O
O
MeS
N
H
H
NO
O
O
R
2
R
1
MeS
R
1
R
2
CH
2
OH CH
3
CH
3
CH
2
OH
1:
2:
3
Thiolation
5
Hydroxylation
4
Fig. 5 Proposed biogenetic pathway for the formation of compounds 1–9from glycine and
L
-tyrosine
Mycol Progress (2020) 19:589–603 601

aside from the production of chlamydospores, we cannot ex-
clude that it forms persistent propagules in its natural habitat
that we have not been able to induce in the laboratory. There
remains a chance that a yet unknown stage of the fungus has
persisted in a soil particle that was attached to the roots, and it
should be attempted to re-isolate Batnamyces from other parts
of the plant. The taxonomy of other fungi that were already
frequently isolated from similar biotopes, such as the so-called
dark septate root endophytes, which mostly belong to the
Dothideomycetes (Knapp et al. 2015; Bonfim et al. 2016),
poses a similar challenge. Their taxonomy cannot be resolved
by using a morphocentric approach based on conidiogenous
structures but must rely on molecular methods. Even here, a
chemotaxonomic approach would be interesting to pursue to
attain complementary phenotype-derived data.
Acknowledgements The technical assistance of Nadine Wurzler and
Vanessa Stiller is appreciated. We thank Wera Collisi for conducting the
bioassays, Christel Kakoschke for recording NMR spectra and Sabrina
Karwehl for HRESIMS measurements.
Funding information Open Access funding provided by Projekt DEAL.
RBT and SEH are grateful for financial support from the Alexander von
Humboldt Foundation. SRN acknowledges the Ministry of Higher
Education and Scientific Research (MESRS) of Algeria for the financial
support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article
are included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in the
article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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