mic oo ganisms
A icle
Phylogene ic Assignmen o he Fungicolous
Hypoxylon in adens (Ascomyco a, Xyla iales) and
In es iga ion o i s Seconda y Me aboli es
Ke in Becke 1,2 , Ch is ophe Lambe 1,2,3 , Jö g Wieschhaus 1and Ma c S adle 1,2,*
1Depa men o Mic obial D ugs, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI), Inho ens aße 7,
38124 B aunschweig, Ge many; [email p o ec ed] (K.B.);
ch is ophe [email p o ec ed] (C.L.); [email p o ec ed] (J.W.)
2Ge man Cen e o In ec ion Resea ch Associa ion (DZIF), Pa ne si e Hanno e -B aunschweig,
Inho ens aße 7, 38124 B aunschweig, Ge many
3Depa men o Molecula Cell Biology, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI)
Inho ens aße 7, 38124 B aunschweig, Ge many
*Co espondence: [email p o ec ed]; Tel.: +49-531-6181-4240; Fax: +49-531-6181-9499
Recei ed: 23 July 2020; Accep ed: 8 Sep embe 2020; Published: 11 Sep embe 2020


Abs ac :
The ascomyce e Hypoxylon in adens was desc ibed in 2014 as a ungicolous species g owing
on a membe o i s own genus, H. agi o me, which is conside ed a a e li es yle in he Hypoxylaceae.
This ende sH. in adens an in e es ing a ge inou e o s o ind new bioac i e seconda yme aboli es om
membe s o he Xyla iales. So a , only ola ile o ganic compounds ha e been epo ed om H. in adens,
bu no in es iga ion o non- ola ile compounds had been conduc ed. Fu he mo e, a phylogene ic
assignmen ollowing ecen ends in ungal axonomy ia a mul iple sequence alignmen seemed
p ac ical. A cul u e o H. in adens was hus subjec ed o subme ged cul i a ion o in es iga e he p oduced
seconda y me aboli es, ollowed by isola ion ia p epa a i e ch oma og aphy and subsequen s uc u e
elucida ion by means o nuclea magne ic esonance (NMR) spec oscopy and high- esolu ion mass
spec ome y (HR-MS). This app oach led o he iden i ica ion o he known la iolin (
1
) and 3,3-bi la iolin
(
2
) as he main componen s, which had ne e been epo ed om he o de Xyla iales be o e. Assessmen
o hei an imic obial and cy o oxic e ec s ia a panel o commonly used mic oo ganisms and cell
lines in ou labo a o y did no yield any e ec s o ele ance. Concu en ly, genomic DNA om he
ungus was used o cons uc a mul igene phylogeny using ibosomal sequence in o ma ion om he
in e nal ansc ibed space egion (ITS), he 28S la ge subuni o ibosomal DNA (LSU), and p o einogenic
nucleo ide sequences om he second la ges subuni o he DNA-di ec ed RNA polyme ase II (RPB2) and
β
- ubulin (TUB2) genes. A placemen in a newly o med clade wi h H. ugodes was s ongly suppo ed in
a maximum-likelihood (ML) phylogeny using sequences de i ed om well cha ac e ized s ains, bu he
exac posi ion o said clade emains unclea . Bo h, he chemical and he phylogene ic esul s sugges
u he inqui ies in o he li es yle o his unique ungus o ge a be e unde s anding o bo h, i s ecological
ole and unc ion o i s p oduced seconda y me aboli es hi he o unique o he Xyla iales.
Keywo ds:
Ascomyco a; ch oma og aphy; mul igenephylogeny; naph hoquinones; axonomy; Xyla iales
1. In oduc ion
Hypoxylon is he la ges genus wi hin he ungal amily Hypoxylaceae (o de Xyla iales,
Ascomyco a) wi h mo e han 230 desc ibed species [
1
]. The s oma a ( ui ing bodies) o Hypoxylon
a e o en a he conspicuous and can equen ly be ound on dead wood in all o es ed a eas o
he wo ld, bu he mycelia o hese ungi a e also among he mos equen ly isola ed endophy es
o seed plan s. This unde lines he ecological impo ance o he genus [
2
,
3
]. Recen axonomical
Mic oo ganisms 2020,8, 1397; doi:10.3390/mic oo ganisms8091397 www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2020,8, 1397 2 o 14
ad ances e ol ed on he eo ganiza ion o Hypoxylon and allies, using a polyphasic concep combining
mo phological, molecula phylogene ics wi h special emphasis on mul i-locus genealogies and
chemo axonomic app oaches. This ul ima ely led o he esu ec ion o he Hypoxylaceae i sel and he
seg ega ion o gene a like Hypomon agnella and Jack oge sella [
2
,
3
], while inc easing chemo axonomical
knowledge suppo ed he ea men o gene a like Annulohypoxylon as an own genus whe e sequence
in o ma ion de i ed om he widely used In e nal ansc ibed space egion (ITS) alone did no
gi e a conclusi e esolu ion [
4
,
5
]. Membe s o he Hypoxylacaeae a e also known o p oduce an
as onishing amoun o di e se seconda y me aboli es, making hem aluable a ge s o na u al p oduc
esea che s [
6
]. This esea ch b anch is u he suppo ed by he inc easing numbe o high-quali y
genomes a ailable [
7
], expanding he classical sc eening app oaches o na u al p oduc chemis y by
he possibili y o genome mining and biosyn hesis s udies o p omising bioac i e compounds.
The mono ypic species Hypoxylon in adens was ecen ly epo ed om F ance as a ungicolous
species g owing on s oma a o a membe o i s own genus, H. agi o me [
8
]. Only a ew cases o
his appa en ly mycophilic li es yle a e known om he genus Hypoxylon and o he gene a ha now
belong o he Hypoxylaceae (i.e., Chlo os oma and Thuemenella [
9
], o which no mycelial cul u es a e
p esen ly a ailable in he public domain), and i was no possible o e alua e whe he he s oma a jus
g ow coinciden ally on he s oma a o hei ela i es, o whe he he e is some o m o pa asi ism
o mu ualism in ol ed. We we e able o ob ain a cul u e om he ascospo es o he holo ype
specimen o H. in adens ha can now be s udied in-dep h o i s physiological and ecological ai s,
including seconda y me aboli e p oduc ion. In a p e ious s udy we ha e checked he cul u es o
H. in adens and H. mac oca pum [
8
], and he only epo ed me aboli es om hose species a e se e al
ola ile o ganic compounds (VOC) [
10
,
11
] ha we e in es iga ed by GC-MS analysis and o al syn hesis
a e obse a ion o a s ong odo o igina ing om mycelia g own on oa meal aga [8].
The cu en s udy is dedica ed o assessing he phylogene ic a ini ies o H. in adens ia a ou -locus
phylogeny, as well as he in es iga ion o i s seconda y me aboli es, ollowed by e alua ion o he
cy o oxici y and an imic obial ac i i y o he newly isola ed compounds.
2. Ma e ials and Me hods
2.1. Gene al
Elec osp ay mass (ESI-MS) spec a we e eco ded wi h an Ul iMa e
®
3000 Se ies UHPLC
(The mo ishe Scien i ic, Wal man, MA, USA) u ilizing a C18 Acqui y
®
UPLC BEH column
(2.1
×
50 mm, 1.7
µ
m; Wa e s, Mil o d, MA, USA), connec ed o an amaZon speed
®
ESI-Ion ap-MS
(B uke , Bille ica, MA, USA). HPLC pa ame e s we e se as ollows: sol en A: H
2
O+0.1% o mic acid,
sol en B: ace oni ile (ACN)+0.1% o mic acid; g adien : 5% B o 0.5 min, inc easing o 100% B in 19.5 min,
keeping 100% B o u he 5 min; low a e 0.6 mL/min, DAD de ec ion 200−600 nm.
High esolu ion elec osp ay mass (HR-ESI-MS) spec a we e ob ained wi h an Agilen 1200 In ini y
Se iesHPLC (Agilen Technologies, San aCla a, CA,USA) connec ed o amaXis
®
elec osp ay ime-o - ligh
mass spec ome e (ESI-TOF-MS; B uke ; HPLC condi ions same as o ESI-MS measu emen s).
Nuclea magne ic esonance (NMR) spec a we e eco ded wi h an A ance III 500 spec ome e
(B uke ,
1
H NMR: 500 MHz,
13
C NMR: 125 MHz). UV/ is spec a we e aken wi h a UV-2450
spec opho ome e (Shimadzu, Kyo o, Japan).
2.2. Fungal Ma e ial and Molecula Phylogeny
A cul u e om he holo ype specimen o Hypoxylon in adens was used in his s udy. A well-g own
cul u e o H. in adens on YM 6.3 aga was used o genomic DNA (gDNA) ex ac ion using a
comme cially a ailable ki as desc ibed by Kuhne e al. [
12
]. The ob ained gDNA was used o ampli y
ou di e en loci: he in e nal ansc ibed space egion (ITS), he 28S la ge subuni o ibosomal
DNA (LSU), he second la ges subuni o he DNA-di ec ed RNA polyme ase II (RPB2) and
β
- ubulin
Mic oo ganisms 2020,8, 1397 3 o 14
(TUB2) wi h p ime s as desc ibed elsewhe e [
13
–
17
]. PCR p og ams, subsequen amplicon pu i ica ion
and sequencing we e conduc ed as desc ibed p e iously [9,18].
Sequence assembly and analysis was done ollowing Wend e al. [
2
] wi h he p og am
package Geneious
®
7.1.9 (h p://www.geneious.com, [
19
]). Taxon selec ion and complemen a ion o
sequences was based on he da ase o Si e al. [
20
], which comp ised many ype s ains, as well as
au hen ic ep esen a i es o all phylogene ic lineages o he genus Hypoxylon and allied Hypoxylaceae.
The GenBank accession numbe s o he sequences used a e gi en in Table 1. Loci we e aligned
using he in-buil e sion o he p og am MAFFT 7.017 wi h se ings o he G-INS-I algo i hm se
o de aul [
21
]. The esul ing alignmen was il e ed o phylogene ic in o ma i e posi ions ia he
se e e sion o Gblocks (Cas esana Labs, [
22
]), low s ingency se ings wi h op ions o smalle inal
blocks and gap posi ions wi hin inal blocks, see Tala e a and Cas esana [
23
] and Wend e al. [
2
].
Molecula phylogene ic in e ence ollowed using he clien e sion o IQT ee (Ve sion 2.0-RC2, [
24
])
wi h a pa i ioned da ase [
25
] and ModelFinde o model selec ion [
26
]. Non-pa ame ic boo s ap
(BS,
≥
50%, majo i y consensus [
27
]) and Shimodai a-Hasegawa app oxima e likelihood- a io- es
(SH-aLRT, ≥85% [28,29]) alues we e assigned o he bes sco ing maximum-likelihood (ML) ee.
Mic oo ganisms 2020,8, 1397 4 o 14
Table 1.
Sequence accession numbe s, co esponding isola es, and li e a u e e e ence o sequences used o he molecula phylogene ic in e ence. Species- ype
specimen a e gi en he co esponding label (HT =holo ype; ET =Epi ype). The newly sequenced H. in adens isola e is highligh ed in bold.
Species S a us S ain O igin GenBank Acc. No. o DNA Loci Re e ences
ITS LSU RPB2 TUB2
Annulohypoxylon
annula um ET CBS 140775 Texas KY610418 KY610418 KY624263 KX376353 [2,30]
A. annula um DSM 107931 Texas MK287534
AJ390397 MK287546 MK287559 MK287572 [20]
A. mo i o me CBS 123579 Ma inique KX376321 KY610425 KY624289 KX271261 [2,31]
A. ni ens MFLUCC 12.0832 Thailand KJ934991 KJ934992 KJ934994 KJ934993 [32]
A. s ygium MUCL 54601 F ench Guinea KY610409 KY610475 KY624292 KX271263 [2]
A. unca um ET CBS 140778 Texas KY610419 KY610419 KY624277 KX376352 [2,30]
A. unca um DSM 107925 Texas MK287531 MK287543 MK287556 MK287569 [20]
Daldinia concen ica CBS 113277 Ge many AY616683 KY610434 KY624243 KC977274 [2,12,33]
D. eschschol zii MUCL 45435 Benin JX658484 KY610437 KY624246 KC977266 [2,12,34]
D. pe iniae ET MUCL 49214 Aus ia AM749937 KY610439 KY624248 KC977261 [2,12,35]
D. placen i o mis MUCL 47603 Mexico AM749921 KY610440 KY624249 KC977278 [2,12,35]
En onaema liquescens ATCC 46302 USA KY610389 KY610443 KY624253 KX271248 [2]
G aphos oma pla ys oma
HT CBS 270.87 F ance JX658535 DQ836906 KY624296 HG934108 [2,34,36,37]
Hypom. ba ba ensis HT STMA 14081 A gen ina MK131720 MK131718 MK135891 MK135893 [3]
Hypom. mon iculosa ET MUCL 54604 F ench Guiana KY610404 KY610487 KY624305 KX271273 [2]
Hypom. submon iculosa CBS 115280 F ance KC968923 KY610457 KY624226 KC977267 [2,12]
Hypoxylon ca neum MUCL 54177 F ance KY610400 KY610480 KY624297 KX271270 [2]
H. ce cidicola CBS 119009 F ance KC968908 KY610444 KY624254 KU684189 [2,12]
H. c oceum DSM 107926 Texas MK287535 MK287547 MK287560 MK287573 [20]
H. c oceum DSM 107932 Texas MK287532 MK287544 MK287557 MK287570 [20]
H. c ocopeplum CBS 119004 F ance KC968907 KY610445 KY624255 KC977268 [2,12]
H. endle i MUCL 54792 F ench Guinea KF234421 KY610481 KY624298 KF300547 [2,12]
H. agi o me ET MUCL 51264 Ge many KC477229 KM186295 MK887342 KX271282 [2,20,32,38]
H. uscum ET CBS 113049 F ance KY610401 KY610482 KY624299 KX271271 [2]
H. g iseob unneum T CBS 331.73 India KY610402 KY610483 KY624300 KC977303 [2,12]
H. guilanense HT MUCL 57726 I an MT214997 MT214992 MT212235 MT212239 [39]
H. haema os oma ET MUCL 53301 Ma inique KC968911 KY610484 KY624301 KC977291 [2,12]
H. howeanum MUCL 47599 Ge many AM749928 KY610448 KY624258 KC977277 [2,12,35]
H. hypomil um MUCL 51845 Guadeloupe KY610403 KY610449 KY624302 KX271249 [2]
Mic oo ganisms 2020,8, 1397 5 o 14
Table 1. Con .
Species S a us S ain O igin GenBank Acc. No. o DNA Loci Re e ences
ITS LSU RPB2 TUB2
H. in adens HT MUCL 51475 F ance MT809133 MT809132 MT813037 MT813038 This S udy
H. in es iens CBS 118183 Malaysia KC968925 KY610450 KY624259 KC977270 [2,12]
H. la e ipigmen um HT MUCL 53304 Ma inique KC968933 KY610486 KY624304 KC977290 [2,12]
H. leno mandii CBS 119003 Ecuado KC968943 KY610452 KY624261 KC977273 [2,12]
H. lienhwacheense MFLUCC 14-1231 Thailand KU604558 MK287550 MK287563 KU159522 [20,40]
H. musceum MUCL 53765 Guadeloupe KC968926 KY610488 KY624306 KC977280 [2,12]
H. oli aceopigmen um T DSM 107924 Texas MK287530 MK287542 MK287555 MK287568 [20]
H. papilla um HT ATCC 58729 USA KC968919 KY610454 KY624223 KC977258 [2,12]
H. pe o a um CBS 115281 F ance KY610391 KY610455 KY624224 KX271250 [2]
H. pe iniae HT CBS 114746 F ance KY610405 KY610491 KY624279 KX271274 [2,30]
H. pilge ianum STMA 13455 Ma inique KY610412 KY610412 KY624308 KY624315 [2]
H. po phy eum CBS 119022 F ance KC968921 KY610456 KY624225 KC977264 [2,41]
H. pulicicidum HT CBS 122622 Ma inique JX183075 KY610492 KY624280 JX183072 [2,42]
H. ickii ET MUCL 53309 Ma inique KC968932 KY610416 KY624281 KC977288 [2,12]
H. ubiginosum ET MUCL 52887 Ge many KC477232 KY610469 KY624266 KY624311 [2,38]
H. samuelsii ET MUCL 51843 Guadeloupe KC968916 KY610466 KY624269 KC977286 [2,12]
H. exense T DSM 107933 Texas MK287536 MK287548 MK287561 MK287574 [20]
H. icinense CBS 115273 F ance JQ009317 KY610471 KY624272 AY951757 [2,4]
H. ugodes ET MUCL 54794 S i Lanka KF234422 KY610493 KY624282 KF300548 [2,12]
H. ogesiacum CBS 115273 F ance KC968920 KY610417 KY624283 KX271275 [2,12]
Jack oge sella cohae ens CBS 119126 Ge many KY610396 KY610497 KY624270 KY624314 [2]
J. minu ella CBS 119015 Po ugal KY610381 KY610424 KY624235 KX271240 [2]
J. mul i o mis ET CBS 119016 Ge many KC477234 KY610473 KY624290 KX271262 [2,12,30]
Py enopolypo us hun e i ET MUCL 52673 I o y Coas KY610421 KY610472 KY624309 KU159530 [2,30]
P. laminosus HT MUCL 53305 Ma inique KC968934 KY610485 KY624303 KC977292 [2,12]
P.nica aguensis CBS 117739 Bu kina Faso AM749922 KY610489 KY624307 KC977272 [2,12,35]
Rhopalos oma angolense CBS 126414 I o y Coas KY610420 KY610459 KY624228 KX271277 [2]
Thamnomyces dend oidea HT CBS 123578 F ench Guinea FN428831 KY610467 KY624232 KY624313 [2,43]
Xyla ia hypoxylon ET CBS 122620 Sweden KY610407 KY610495 KY624231 KX271279 [2,44]

Mic oo ganisms 2020,8, 1397 6 o 14
2.3. Cul i a ion, Ex ac ion, and Isola ion
Fo cul i a ion, seed cul u es we e p epa ed by adding h ee mycelial plugs o ca. 0.25 mm
2
size
each om well-g own yeas -mal aga pla es (YM 6.3 [
45
], supplemen ed wi h 20 g/L aga ) in o 250 mL
shaking lasks con aining 100 mL o liquid YM 6.3 medium. Seed cul u es we e inocula ed on a o a y
shake (In o s, Bo mingen, Swi ze land) o 13 d (23
◦
C, 140 pm). Fo p epa a ion o he subme ged
cul u es, 20
×
500 mL shaking lasks con aining 200 mL o ZM-
1
2
medium [
45
] we e inocula ed
wi h 3 mL o seed cul u e. The lasks we e incuba ed on a o a y shake unde he a o emen ioned
condi ions and consump ion o glucose was moni o ed by using glucose es s ipes (Mache ey-Nagel,
Dü en, Ge many) daily. Besides glucose, a isual es ima ion o he ed colo o he cul u e b o h was
aken in o accoun o de e mine he e mina ion o e men a ion. Acco dingly, h ee days a e he
glucose es s ipes indica ed ha he glucose had been consumed (and he colo was o an in ense ed),
he cul u es we e ha es ed and ex ac ed.
A e e mina ion o e men a ions, supe na an and mycelium we e sepa a ed by cen i uga ion
and subsequen gauze il a ion. The mycelium was ex ac ed wo imes, i s using 1 L o e hyl
ace a e (E OAc) and hen 1 L o ace one as o ganic phases a 40
◦
C in an ul a-sonic ba h o one
hou . Bo h o ganic phases we e indi idually ex ac ed wice wi h ca. 1 L o deionized wa e using a
sepa a o y unnel and he aqueous phases disca ded.
The o ganic phases o he wo ex ac ions o he ZM-
1
2
medium we e combined and d ied in
acuo o yield he c ude ex ac (526 mg). An aliquo o his ex ac was dissol ed in DMSO and
subjec ed o HPLC-DAD/MS analysis (see Figu e S1). The c ude ex ac o he cul u e p epa ed in
ZM-
1
2
medium was g adually sepa a ed using wo subsequen s eps o ch oma og aphy o yield he
pu e compounds
1
and
2
. As a i s s ep, he c ude ex ac was dissol ed in 4 mL ACN:H
2
O (75:25)
and ans e ed o an open solid phase ca idge (S a a
®
X 33
µ
m Polyme ic Re e sed Phase Tube,
1 g/12 mL, SN 8B-S100-JDG; Phenomenex, Ascha enbu g, Ge many). Elu ion was achie ed by using
low acuum (ca. 800 mba ) and a s ep-g adien o H
2
O:ACN (90:10, 60:40, 30:70, 100:0; 4 mL pe s ep).
The e luen s o he g adien s eps 90:10 and 60:40 we e combined acco ding o ESI-MS esul s and d ied
in acuo, yielding 221 mg o ex ac . The esul ing c ude ex ac was u he sepa a ed by dissolu ion in
ca. 5 mL o ACN:H
2
O (60:40) and subjec ion o a PLC 2250 HPLC sys em (Gilson, Middle on, WI, USA),
u ilizing a Nucleodu
®
C18ec column (125
×
40 mm, 7
µ
m; Mache ey-Nagel, Dü en, Ge many) and
sol en s: A: H
2
O+0.1% o mic acid, B: ACN+0.1% o mic acid, low: 45 mL
×
min
−1
, ac iona ion:
15 mL, g adien : isoc a ic condi ions a 10% B o 10 min, ollowed by an inc ease o 70% B in 60 min,
hen inc ease om 70% B o 100% B in 5 min, ollowed by isoc a ic condi ions o 100% B o 5 min.
The ac ions we e combined acco ding o he DAD ch oma og am o he HPLC sys em: la iolin (
1
):
R
=29.5
−
31.0 min, 18.2 mg; 3,3
0
-bi la iolin (
2
):
R
=38.0
−
40.0 min, 19.2 mg. Ch oma og ams, DAD and
MS aces o he pu e 1and 2a e depic ed in Figu e S2.
Fu he mo e, o he g ow h media we e checked o p esence o he isola ed compounds
1
–
2
,
as well as o he compounds o a chemo axonomic in e es . H. in adens was hus addi ionally g own in
subme ged Q6-
1
2
, YM 6.3 (bo h [
45
]), MMK2 [
46
], and HLX [
47
], as well as he solid medium BRFT [
45
].
Fo his pu pose, cul u e condi ions as desc ibed abo e we e applied, wi h he di e ence o only one
500 mL shaking lask being incuba ed pe medium ( he solid BRFT medium was also no agi a ed
du ing cul i a ion). Ex ac ion o mycelia was conduc ed as men ioned abo e, bu wi h only 200 mL
o E OAc being used o ul a-sonica ion and he ace one ul a-sonica ion s ep omi ed. A e wa ds,
he o ganic E OAc phase was ex ac ed wi h ca. 200 mL o wa e as desc ibed abo e. The supe na an ,
in u n, was di ec ly ex ac ed wi h ca. 200 mL o E OAc each. In he case o BRFT, no sepa a ion o
supe na an and mycelium was possible due o he solid na u e o he medium, hence i was ea ed like
he o he mycelia. HPLC-DAD/MS esul s o bo h supe na an s and mycelia a e depic ed in Figu e S3.
2.4. An imic obial Ac ici y and Cy o oxici y Assay
Compounds
1
–
2
we e dissol ed in MeOH (1 mg/mL) o he bioac i i y assays. The sol en was
also used as nega i e con ol.
Mic oo ganisms 2020,8, 1397 7 o 14
Minimum inhibi o y concen a ions (MIC) we e de e mined in a se ial dilu ion assay as desc ibed
p e iously [
48
]. Va ious es o ganisms o ungal and bac e ial o igin we e es ed o co e a b oad ange
o mic oo ganisms: bac e ia: Bacillus sub ilis,S aphylococcus au eus,Mic ococcus lu eus,Ch omobac e ium
iolaceum,Esche ichia coli, Pseudomonas ae uginosa; mycobac e ia: Mycolicibac e ium smegma is; ungi:
Candida albicans,Schizosaccha omyces pombe,Muco hiemalis,Pichia anomala and Rhodo o ula glu inis.
The MTT cy o oxici y assay was pe o med agains he cell lines L929 (mouse ib oblas s) and KB
3.1 (human papilloma i us- ela ed endoce ical adenoca cinoma) as desc ibed p e iously [49].
2.5. Spec al Da a
2.5.1. Fla iolin
(
1
): Red solid. MW =206.16 Da, MF =C
10
H
6
O
5
. NMR (ace one-d
6
,
1
H NMR: 500 MHz,
13
C NMR:
125 MHz): see Table S1; UV/ is (c =0.01 mg/mL, ACN):
λmax
(
ε
)=214 (4.41), 262 (3.94), 301 (3.97)
nm; ESI
−
MS: m/z206.92 [M+H]
+
, 204.79 [M
−
H]
−
; HR
−
ESI
−
MS: m/z207.0284 [M+H]
+
(calcula ed o
C10H7O5, 207.0288); R=4.8 min.
2.5.2. 3,30-Bi la iolin
(2): Red solid. MW =410.29 Da, MF =C
20
H
10
O
10
. NMR (ace one-d
6
,
1
H NMR: 500 MHz,
13
C NMR:
125 MHz): see Table S1; UV/ is (c =0.01 mg/mL, ACN):
λmax
(
ε
)=211 (4.56), 266 (4.39), 300 (4.15)
nm; ESI
−
MS: m/z411.02 [M+H]
+
, 408.92 [M
−
H]
−
; HR
−
ESI
−
MS: m/z411.0345 [M+H]
+
(calcula ed o
C20H11O10, 411.0347); R=6.8 min.
3. Resul s
3.1. Molecula Phylogeny o H. in adens
The newly gene a ed sequences o H. in adens and he da ase p esen ed in Figu e 1we e aligned
as desc ibed abo e and cu a ed ia Gblocks, which lead o ou alignmen s spanning 347, 1113, 815 and
1047 posi ions (ITS, LSU, RPB2, and TUB2, espec i ely; c . Table S3). In o al, 1015 posi ions we e
shown as pa simony in o ma i e, while 2049 posi ions ep esen ed cons an si es. The pa i ioned analysis
(see Table S3 o a lis o selec ed bes - i models ollowing Bayesian-in o ma ion c i e ion, BIC) in e ed a
ML- ee wi h a log-likelihood (lLn) sco e o
−
40,100.7533 (Figu e 1). The ee was oo ed o he ou g oup
consis ing o ep esen a i es o he G aphos oma aceae and Xyla iaceae (O) wi h he Hypoxylaceae
ecei ing maximum BS and 99% SH-aLRT suppo , wi h H. papilla um appea ing basal o all o he clades
(Hx1). The Hypoxylaceae u he mo e show a pa aphyly o Hypoxylon, spli in o one s ongly suppo ed
clade (Hx2, 98% SH-aLRT /95% BS) and one s ongly suppo ed sis e clade (78% BS, Hx7) (85%/56%) pu
nex o Annulohypoxylon (A, 100%) and Jack oge sella (J, 98%/100%). Fu he mo e, one non-suppo ed clade
con aining H. agi o me and allies (Hx3) was placed as sis e g oup nex o a clade con aining mos ly o he
gene a like Py enopolypo us (P, 100% suppo ), Daldinia and allies (99%/100%; E, D, R, T), Hypomon agnella
(Hy, 100%) and o he nes ed sequences wi h non- o low suppo ed posi ions o Hypoxylon and En onaema
(E, Hx4, Hx5, Hx6) wi h mode a e BS and high likelihood- a io suppo (95%/58%). These pa e ns we e
al eady desc ibed by Wend e al. [
2
], Si e al. [
20
], and Pou moghaddam e al. [
39
]. The H. in adens-de i ed
sequences we e placed nex o H. ugodes wi h maximum suppo (Hx5).
Mic oo ganisms 2020,8, 1397 8 o 14
Figu e 1.
In e ed maximum-likelihood (lLn =
−
40,100.7533) ee o a molecula phylogeny o
sequenced loci o ibosomal o igin (ITS, LSU) and p o einogenic genes (RPB2, TUB2), wi h pa i ion-wise
assigned e olu iona y models. SH-aLRT (>85%) and BS (>50%) suppo is gi en a i s and second
posi ion a espec i e b anches. The labeled clades co espond o he gene a Annulohypoxylon (A),
Daldinia (D), En onaema (E), Hypoxylon (Hx 1
−
7), Hypomon agnella (Hy), Jack oge sella (J), Py enopolypo us
(P), Rhopalos oma (R), Thamnomyces (T) and he oo ed ou g oup (O). The clades ha e been highligh ed
by al e na i e g ey and ligh blue backg ound o allow o easie dis inc ion. The posi ion o H. in adens
is highligh ed in bold. No e: The b anch leng h o H. lienhwacheense has been unca ed, as indica ed.
B anch leng h equals subs i u ions pe si e.
3.2. S uc u e Elucida ion o Fla iolin (
1
) and 3,3
0
-Bi la iolin (
2
) and Analysis o Ex ac s om o he Cul u e Media
Two seconda y me aboli es we e isola ed om an ex ac (c . Figu e S1) o cul u es o H. in adens
in ZM-
1
2
medium, which we e ul ima ely iden i ied as he known naph hoquinones la iolin (
1
) and
i s dime , 3,3
0
-bi la iolin (
2
) (see Figu e 2). A combina ion o 1D and 2D NMR, as well as a di ec
compa ison o UV/ is spec al da a wi h e e ence da a [50] was applied.
Mic oo ganisms 2020,8, 1397 9 o 14
Figu e 2.
Chemical s uc u es o la iolin (
1
) and 3,3
0
-bi la iolin (
2
), seconda y me aboli es isola ed
om cul u es o Hypoxylon in adens.
Fla iolin (
1
) was isola ed as a ed solid and shown o possess a molecula o mula (MF) o C
10
H
6
O
5
by HR-ESI-MS. Analysis o 1D and 2D NMR da a led o he s uc u e p oposal o
1
. The comple e
s uc u e could be con i med when di e en long ange coupling cons an s in
1
H/
13
C he e onuclea
mul iple-bond co ela ion spec oscopy (
1
H/
13
C HMBC) expe imen s (CNST 13 =2 Hz, 6 Hz) we e
used, which allowed o
3
J, as well as
4
Jco ela ions o be obse ed. Taken oge he , hese co ela ions
allowed he con i ma ion o he isola ed compound as la iolin (1).
Fo he ed solid 3,3
0
-bi la iolin (
2
), a MF C
20
H
10
O
10
was calcula ed. I s s uc u e was con i med
ia NMR spec oscopy o be a symme ical dime o
1
. This was deduced by i s molecula o mula,
which ep esen ed a duplica ion o a oms compa ed o
1
wi h a o mal loss o wo hyd ogens,
highly simila UV/ is spec a (Figu e S2), and he simila i y o p o on and ca bon signals in NMR da a
o
1
–
2
. The b idging a om in 3,3
0
-bi la iolin was de i ed o be C-3/C-3
0
, as he
1
H NMR signals o 3-H
we e missing in 2as compa ed o 1.
NMR da a, key co ela ions, as well as 1D and 2D spec a can be ound in Table S1, Figu e S4 and
Figu es S5–S16, espec i ely.
The HPLC-DAD/MS ch oma og ams de i ed om i e sc eening cul u es in six di e en media
in which H. in adens had been e men ed we e checked o he p esence o compounds
1
and
2
, as well
o he chemo axonomically ele an seconda y me aboli es. Aside om he ZM-
1
2
medium, whe e
1
and
2
we e majo componen s, we we e only able o de ec hem in HLX medium (c . Figu e S3B).
No ably, compounds
1
and
2
we e no ound in ex ac s om he YM 6.3 medium, which was he
s anda d medium in a comp ehensi e chemo axonomic s udy by Bi ze e al. [
35
] (Figu e S3C).
Fu he mo e, hey we e nei he p esen in he ex ac s om subme ged MMK2 and Q6-
1
2
media, no in
he solid, ice-con aining BRFT medium (Figu e S3D–F). No known compounds om ela ed species
(including mellein de i a i es, which we e p esen in ex ac s om subme ged cul u es in YM medium
o almos all Hypoxylon sp. p e iously s udied [
35
], could be assigned o he la ge numbe o peaks
p esen in he ex ac s.
3.3. An imic obial and Cy o oxic Ac i i ies o Fla iolin (1) and 3,30-Bi la iolin (2)
The an imic obial and cy o oxic ac i i ies o la iolin (
1
) and 3,3
0
-bi la iolin (
2
) we e e alua ed as
desc ibed in he me hods sec ion and a e summa ized in Table S2.
An ibac e ial ac i i y o
1
agains S aphylococcus au eus wi h a minimum inhibi o y concen a ion
(MIC) o 66.7
µ
g
×
mL
−1
was de e mined, which was e y weak in compa ison o he e e ence
oxy e acycline (MIC =0.4
µ
g
×
mL
−1
). No ac i i y o
1
–
2
agains any o he o he mic obial es
o ganisms was obse ed. E alua ion o he cy o oxic e ec s o
1
–
2
on mammalian cell lines esul ed in
1
exhibi ing a hal -maximal inhibi o y concen a ion (IC
50
) o 92.2
µ
M agains L929 mouse ib oblas s.
The e e ence epo hilone B, in u n, exhibi ed an IC
50
o 0.00006
µ
M agains L929. O he cy o oxic
e ec s we e no obse ed agains he chosen cell lines.