Metabolites from the Dark Septate Endophyte Drechslera Sp

View metadata,citationandsimilarpapersatcore.ac.uk This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. 10.1002/cbdv.201800133 doi: lead article this todifferences as version between this andthe ofPleasecite Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer a Godeas SilessGastón histone culture extracts deacetylasewith inhibitors Evaluation by andLC/MS component principal analysis of endophyteMetabolites from dark the septate [email protected] id: mail author Type:Article Paper Full compounds intheculture medium or in the mycelium compared.was by means supervisedof principal component analysis of (PCA) LC/MS data. The presence of the evaluated by LC/MS and LC/MS/MS. Several differencesin the metabolite production weredetected and other histonedeacetylase inhibitors as epigenetic modifiersin the culture medium was conditions, are describedfor here thefirst time. Theuse ofhydroxamic suberoylanilide acid (SAHA) isolated from the roots ryegrassof ( Secondary metabolitesfrom thecultures of the dark septate fungal endophyte (DSE) Buenos Aires,Argentina. CONICET-Universidad de Buenos Aires, INBA.Ciudad Universitaria, Pab. II, 4° piso, C1428EHA, c Orgánica (UMYMFOR). Ciudad Universitaria, Pabellón piso, II,3° C1428EHA, BuenosArgentina. Aires, Universidad de Buenos Aires,Unidad de Microanálisis y Métodos Físicos aplicadosla Química a Departamento de Biodiversidad Biología y Experimental, dFacultad de Ciencias Exactas Naturales,y AcceptedDepartamentoQuímica de Orgánica, Article c,d , GabrielaCabrera M. a,b , Gabriela L. Gallardo a,b* a,b* a Lolliumandsp.) culturedunder different experimental , AlejandraM. Rodriguez b Facultad de Ciencias Exactas Naturales,y CONICET- c,d , Yuliet A.Rincón sp. Drechslera a,b , Alicia M. Drechslerasp. brought toyouby provided byCONICETDigital , CORE This article isprotected rights by All copyright. reserved. Accepted anthraquinones isolated from isolated from Drechslera produced by sesquiterpenes the common,most but also with the finding of macrocyclic estersand alkaloids. rareThe isolated metabolites from these genera belong plants,animals and humans, endophytes genusThe metabolitesin plant ecological rolenot is fully understood, and their metabolome oractivity the of their secondary other typeof endophytes. Althoughhas been it established that canDSE improve plantgrow increasing interest in fungiDSE butmany aspects theseof speciesare not as well understood as with toleranceagainst harsh climati melanized hyphaethat colonize intracellularly and intercellularlyroots, the improving plant´s orders of the phylum Ascomycota. These distributedwidely fungi areseptate and generallyhave septateDark endophytes (DSE) area group of root ArticleIntroduction Keywords xenobiotics. the to addition in observed was produc inhibitors the of biotransformation The medium. culture the to the induced also cultures the of additive an as SAHA of use The com these that phytopatogens. suggesting activity, antifungal macrosphelides. each of one compounds, culture. scale larger a fromThese isolation by confirmed was metabolites the of many of structure The

in f niugl eaoie, hwn te blt o ti edpyi sri t control to strain endophytic this of ability the showing metabolites, antifungal of tion eaoie were metabolites :

Drechslera spp Dreschler [5]

D. giganteaD.

D. avenaeD. Pyrenophora or pathogens. [9,10]

drechslerines [16]

oe f h compounds the of Some

have alsohave been reported, sh andrelated a - hostinteraction is mostly unknown. a

is a vastcomplex speciesof commonly associated to algae • . Othernon asperpentyn .

Thesesterterpene class were previously undescribed, prenylquinoids, diketopiperazines and diketopiperazines prenylquinoids, undescribed, previously were class

avenae [6] [7] hrceie a prenylhydroxybe as characterized [3]

Many Many metabolitesproduced by c conditions, as well as pathogen resistance. Therehas been an suggesting anecological role for thesecompounds. Interestingly, the

wereproduced by

Cochliobolussp. , a teleomorph, a of - related metabolitesalkaloids, as

• macrosphelides

mycotoxins hc wr rlae t te eim hwd good showed medium the to released were which owing thegreat metabolicvariability thisof genus. to several metabolicroutes, with [11,12] D. dematoideaD. - colonizing Drechslera ons ol protect could pounds

The macrocyclic

•

[1,2] opiobolins lysosphingolipid

fungi thatbelong mostlyfew to a Drechslera , whilerelated a pyrenopho

release of hexosylphytosphyngosine of release , zi ais n crmn, two chromans, and acids nzoic

while

were producedseveral by [14]

ester

spirocyclic

eremophilanes

spp

•

SAHA pyrenophorin haveshown toxicity to Lollium [3,4]

terpenoids among



- and lactams

[8]

plantsas rm fungal from

were [13] rol [15] , was , was th, their [6]

and

was

This article isprotected rights by All copyright. reserved. their possible ecologic bioautography on TLC platesfor someabundant metabolites inorderto obtain insightssome on and(NMR) MS. Antifungal activity against common phytopathogens was also assessed by direct isolation and structural elucidation theof co Aiming to identify the secondarymetabolites, large scale cultures were carried out,followed by the runs. dimensionality andgraphical a comparison was performed between different culture replicates and Principal component analysis (PCA) LC/MSof data was performed in orderto reduc SpectrometryMass (LC/MS)performedwas on all the culture andmedia the extracts theof mycelia. octanoylhydroxami additives. Suberoylanilidehydroxamic acid(SAHA), sodium valproate(VPA), and a SAHA analog, performed using two different culture media and epigenetic modifiers, HDAC inhibitors (HDACIs), as colonizer. For thispurpose gainand to knowledg from the roots of Inpresent the work, the study of the secondary metabolome of a fungal DSE, pathogenicity processesand adaptation to harsh climaticcondition metabolome, which is essential to understand chemical biology plantof Rational randomor variation of culture conditions could thenleadto a betterinsight tofungal the structure. productionThe o hyperacetylation histoneof residues leads to anopen, and transcriptionally moreactive chromatin deacetylases (HDACs).had It been noticed that inhibition of HDAC activity and subsequent genes. Histoneacetylation is controlledthe by antagoni histonemainly acetylation and methylation, arefactors involved inthe silencing or expression of t mechanisms, which affect thechromatin structure,restricting the access to the genetic material and repressionThe of secondary metabolite gene clusters has beenattributed to different epigenetic irradiation and even water composition conditions. to the selection particular a of metabolite what hasbeen called the OSMAC approach (onestrain, many compounds). This st metabolites, commo pathway genesare expressed and therefore a fraction only potentialof secondary metabolites are factors and nutrients. Understandard laboratory cultureconditions, onlysubset a biosyntheticof productionThe of fungalmost secondary Accepted Articlehus resultin reversible genesilencing. Post

nly described. Aiming increase to production the of previously unreported secondary [17]

many culture techniqueshad been developed in order to inducegene expression, in

Medium composition, solid or liquid media, vessel configuration, shaking, aeration, Lollium sp. c acid(OHA)were used asHDACIs al role. f new metaboliteswas assessed by this approach. , was conducted, was inorder understandto role the of this straina as plant

[18] - metabolitesis known to be influenced by environmental

producerstrain among hundreds of others understandard are some common parameters that had been explored. - mpounds by means Nuclearof Magnetic Resonance traslational modifications theof chromatin structure, e theof metabolome, small scalecultures were

( Figure1 c histonec acetyltransferases(HATs) and

) .

Liquid Chromatography coupled to s.

- hostinteraction, [19 Drechslera sp. - 21] isrategy opposed e thee , isolated

This article isprotected rights by All copyright. reserved. Accepted with anauthenticsample. hydroxamic were only present intheculture broths treated with SAHA and OHA, itis probablethat the (neutral losses of hydroxylamineand aniline respectively from SAHA). As these hexosyl protonated ion, inaddition to the charac spectrumsignal a at m/z 265.1547, corresponding withloss the anhexoseof (loss of 162u)from the [M+H] deprotonated moleculeswhen using straightforward by analysis of the andMS MS/MS spectra theof protonated molecules or hydroxyvalproic acids ( octanamide ( cultures whereSAHA usedwas additive. as In turn, Material).The inLC/MS the runs ( Several compounds originatedbiotransformation by ofemployed the HDACs wereeasily recognized Figure medium extracts are shown (Figure2). build andthe interpret PCA model inlater stages. LC/MS BasePeak Chromatograms (BPCs) of MS/MS spectra. Apeakto inspectionpeak was made discriminateto common impurities and also to casessome for compound identification sinceitionizes moleculesas [M+H] standardas the ion Articlewell resolved were obtained and analyzed separately by LC/MS. LC conditions wereselected in order to obtain a culturesThe werefiltered to separate medium and mycelium; differentepigenetic modifiers. Threereplicates of each culturecondition wereperformed. METhe culture was usedascontrol a culture andthe as base medium for theaddition of the differentculture media: malt extract liquid culture(ME) and a minimal medium liquid c inoculation. Once theseconditions were obtained, small scale cultures wereperformed using two Dreschlera Sincethe three HDCAs inhibitors used in this workcan inhibitgrowth the of the studied strain of Screening of cultureconditions Results and Discussion 1. Figure 2 .

BPC chromatograms of medium extracts. of chromatograms BPC modifiers. epigenetic as used HDACIs +

at m/z 427.2070 (consistentwith a molecular formula C , itwas fir acid functionality is the glycosylation site. Octanamide 23 - secompounds wereidentified asdihexosyl and hexosyl SAHA (

well spread profile signalsof in the chromatogram. ESI, inpositive ion mode, usedwas ) were identified inthe cul Table 1 ization method. APCI, in both positive and negativeion mode, also was usedin st necessary to determinethe optimal concentration HDCAof and timefor 13

and

) basedon their MS/MS spectra(

) in) thosecases n egativeion mode. For example, he

teristic fragments SAHAof at m/z 232.1327 and 172.0979 tures where OHA was anadditive

whereVPA used. was The identifications were

dihexosyl and hexosyl OHA ( Table ST1

their corresponding organic extracts 20 H 31

( 23 N , Supplementary 2 ) O

was identified 8 + xosyl ), showed), in the MS/MS

+ and twodif

12 ions, which givebetter - SAHA ( and 20

and 16 16

) in) the by comparison

21 ulture(MM). conjugates ferent )

with and)

This article isprotected rights by All copyright. reserved. 24.2 23.7 23.4 22.1 21.2 20.8 20.7 20.6 19.3 19.0 17.4 17.2 16.5 16.4 14.9 14.4 14.0 13.9 13.9 13.6 11.8 11.3 10.7 10.0 9.4 8.9 8.1 7.3 6.4 3.6 RT Table 1.

Accepted Article

Chemical composition of the extracts of of extracts the of composition Chemical 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 N°

4 Siccayne hexosyl 4 Harveynone 3 di(hexosyl) (L cyclo (L cyclo cyclo (Phe cyclo (Phe cyclo Asperpentyn (Leu/Ile cyclo (Pro cyclo 5 cochlione B (Gly cyclo Compound 14 Benzophenone acid anofinic 4 isomer C macrosphelide C macrosphelide inyl) 4 Octanamide isomer or B macrosphelide hexosyl di(hexosyl) isomer or A macrosphelide 3’ ------hydroxy acid hydroxyvalproic acid hydroxyvalproic methoxy hydroxy hydroxy - buten) deoxy - benzoic acid benzoic (Leu/Ile - - - - Phe Leu SAHA OHA c -

- - - macrosphelide C macrosphelide - - - benzoic acid benzoic - - Pro) Val) 3 3 3 - - - Hyp) Hyp)

cochlione B cochlione OHA SAHA - - - - - c L

L (1’ (3’ prenyl

- - - -

Pro) Hyp) Pro) Pro)

- -

methyl hydroxy b

- benzoic acid benzoic

- 3’

- 3’ -

buten

methyl Dreschlera sp. Dreschlera

- 1’ - - MF C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 8 16 14 20 16 12 11 20 8 11 8 26 14 11 11 14 14 11 11 10 12 11 7 16 13 12 12 16 16 12

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

17 16 16 10 40 16 18 18 16 16 12 18 16 16 14 22 10 12 14 22 22 10 20 27 37 22 14 10 30 10 N NO O O N N N N N N O N N O O O O O O O O O O NO NO O O O N O 2 3 3 2 2 2 2 2 2 2 2 2 3 5 5 O 6 3 3 7 7 3 8 8 4 2 3

O O O O O O O O O

2 7 12 13 2 2 2 3 3 3 2 8

589.2611 245.1280 211.1445 211.1443 261.1234 261.1233 193.0867 227.1397 197.1283 241.1061 227.0920 155.0816 m/z [M+H] 311.1488 183.0805 205.0864 207.1018 327.1446 327.1448 203.0709 144.1390 341.1226 322.1857 484.2380 343.1402 223.0965 175.0754 427.2070 161.1171 191.0693 161.1160

(ppm accuracy Mass )

------1.7 2.9 1.0 4.0 0.2 0.4 1.6 1.1 1.7 0.3 1.7 0.8 4.9 7.7 1.4 2.0 1.0 4.3 2.2 2.5 0.5 2.4 1.1 2.4 3.0 3.1 5.2 4.4 4.4 0

611.2444 267.1094 233.1259 233.1260 283.1048 283.1038 215.0692 249.1203 219.1108 263.0892 249.0729 177.0633 m/z [M+Na] 333.1311 205.0620 227.0688 229.0835 349.1254 349.1275 225.0531 363.1044 344.1699 506.2222 365.1223 245.0780 449.1897 183.0985 213.0512 183.0987

(ppm) Mass accuracy

------3.7 0.8 0.4 1.9 5.5 2.6 0.9 1.9 1.1 1.8 1.5 1.1 1.7 3.7 4.7 2.4 3.5 6.3 1.7 0.9 4.4 4.9 3.8 5.6 2.8 4.4 1.6 0.5

This article isprotected rights by All copyright. reserved. replicates of each experiment clustered well theon scores chart, and the signals strucof analysis theof raw data runsof LCMS of medium extracts performed,was but although the detect the signals with Principal component analysis allowed thereduction ofdimensionality the of the raw datain order to modelPCA and differences observed in differentculture conditions differentroutes. mycelium, their presenceshows the Independently of whether these metabolized products werepresent in the extracts of medium or 23 C 21 B 15 13 A 16 12 N° Table they would interact with a biomoleculein the mycelium. proteins. hydroxyThe This fact wouldrelated be with the known high aff were distributed between medium and mycelium with some tendency to be higherin the mycelium. that the compounds. butthe distribution theof corresponding metabolized products notwas in distributionThe ofHDACIs the and its metabolized products from mycelium and medium are listed observed a 27.6 26.4 SAHA: suberoyl SAHA:

Table 2 Accepted Article 2 .

Relative abundances of HDACIs and its derivativ its and HDACIs of abundances Relative

20.6 19.7 19.0 23.8 14.4 13.9 15.2 14.9 13.9 RT 32 31

biotransformation OHA is of

Itevident is that SAHA, OHA and VPA were mostly presentin the medium as expected, - anilide hydroxamic acid, acid, hydroxamic anilide

hexosyl hexosyl Hexosyl

octanamide OHA hexosyl v 4 3 SAHA hexosyl di(hexosyl) Compound - - alproic - - sphingosine phytosphingosine hydroxyvalproic acid hydroxyvalproic acid hydroxyvalproic

- OHA - -

- SAHA OHA acid valproic acids werefound to be most abundantin the mycelium highervariability between runs. Initially,unsupervised principal component -

SAHA

and

c b

OHA: octanoyl OHA:

octanamide werefound exclusively inthe medium,

es in mycelium and medium extracts. medium and mycelium in es plasticity of this

an efficient detoxification process. SAHA andderivatives its C C 24 24 - hydroxamic acid, acid, hydroxamic H H 47 49 NO NO 7 8

inity of SAHA for several membraneZn 462.3424 480.3526

c fungal strain to metabolize xenobiotics by [M+Na]

not not

% mycelium 0 18 0 12 88 71 27 55 32

0.2 1.1

the samefor allthe 484.3217

thu % medium 100 82 100 88 12 29 73 45 68

indicating indicating turally 2+

binding 5.7

This article isprotected rights by All copyright. reserved. structures of the compoundsexhibited are in in shown confirmation of secondary metabolites which couldn´tidentified be by means of MS and MS/MS chromatographic processing large of as tentativecandidates or probable structures bydatabase a search, A detailed analysis wa Secondary metabolites produced byDreschlera sp. Figure same family fragmentsmany and mass spectra of the metabolites which wereproduced exclusively inthe MM experimentshowed while produced significant changesin the metabolite profil other compounds than thoseobserved controlon experiments. The use of minimal medium which producedgreatest the effect in the culture, althoughdid it nottrigger the biosynthesis of resultsThese indicated thatall the experimentsgave different as SAHAother. signals showed be to correlated with compounds DKPs were also attenuated, probab attenuated, which theof diketopiperazines (DKPs) correlated, that is, they vary inacoordinated fashion among runs. Asshown the differe As grouped together a at higherlevel. a lowat level, inthesame asway for the MM data, whilethe OHA, VPA and control data were dendrogram chart ( clustered separately. Coherencebetween replicates of eachculturerun could be appreciated in the which clustered toget variance. scoresThe biplot model and clusteranalysis. In thisimproved model, thefirst 3 accountedPCs for 96%of the total metabolites theon chromatograms theof medium extracts variance.For this reason, the88 bucketscorresponding the to signals theof relevantmost related metabolites showed degree some of correlation, the first 5 representedPCs 75% of the total h

Accepted Articleit exosyl 3 is known, signals belonging buckets to largestof variability between runs larger have values on .

a) Scores and b) loadings biplots of supervised PCA PCA supervised of biplots b) and loadings Scores a) atsame the time the signals of T - ntPCs theof loadings biplot,and bucketscircumscribed to the samedirection are able 1 phytosphingosine .

tentativecandidates. The chemical composition of the extracts of

and complementary dataarep

was observed HDACIs when were added to the cultures, all othersignals of the Figure 1S, neutral lossesin common, indicatingthese that compounds belonged to the herwith OHA and VPA, being similar,more and SAHA and MM signals, which s performed theof MS and MS/MS spectra in order to identify thecompounds (Figure3a)

and benzophenonerespectively. Supplementary Material); alltheSAHA LC/MS data groupe

1 , 4 ly becausely these metabolitesare ,

- 5 other scale cultures allowedisolation the and

, showed a clear differencebetween the control experiments, analysis 7 - 11 meta .

(Figure 4) Figure 4 resented bolite

e: the production of DKPs decayed significantly, .

s are correlate

started to appear in

were selected for the supervised PCA Table ST1

profiles, and SAHA

and d; when one DKP signal was

biosynthetically or byor 31

( Suplementary material). The , which werelater identified

(Figure 2). de novo in identification of the Figure3b

and the also

was thewas additive Dreschlera sp. analysis. The

The MS/MS related to each d together , the

s ignals is This article isprotected rights by All copyright. reserved. formula C Compound ( biosyntheticallyOther asperpentyns. configuration of Curvularia differentenvironments like soil the fungus functionalized prenylquinoid previously was isolated from several strains obtained from very HR Compound Pestalotiopsis sp. Eutypa Related deoxygenated chromanoneswere previously reported from a common grapevinepathogen, C localization of this methoxy group atC related to an NMR spectrashowed most of the signals in common with compound showed signals corre indicatingmolecular a formula C of Compound as Dreschlera. from the endophyte Compound variety receptors,of showingbroad a range biological of activities. metabolites with structural characteristics that enable them to bind with high affinitylarge to a ( Pro) based on their andMS MS/MS spectra medium and mycelium extracts. The identification of DKPs Diketopiperazines (DKPs) werethe compounds with greater relative Figure 14 - 5 at5 ppm. 73.7 Thus, the structure of ymmetric centers(Supplementary Material). -

Accepted additional methoxy group at Article ), ESI 4 [32] . 11

Compounds produced by produced Compounds -

siccayne MS spectraMS and optical rotation coincident with those ( of lata ), werealso isolated and 12

2 inaequalis

H [25] 3 2 18 6

Molecular modeling was employed to confirmthe relative configuration of the with anadditional methoxy group.HMBC The experiment allowed theunambiguous

12 , the most abundantmetabolite theof ME is described herefor thefirst time. Its mass spectrum showed a signal at m/z 241.1061, showed NM

[30,31 O and some also chlorinated derivatives were isolated from a mangrove is described forhere the first time. Its mass spectrum consistentwas with a molecular

3 ( 6 ; the ; 17 [26]

] was confirmed by comparison with data ofsynthesized the (+) and (

There are no reportsabout any biological activity for theserelated compounds. Cochliobolussp. [33, 34] sponding the to loss C of [29] 1 Drechslera sp. Drechslera related compounds werealso isolated and identify like harveynone H and .

Both genera belong also to R and spectra MS identical with those cochlioneof B, previously isolated

the 3 13 C and 2D NMRC and 2D spectra were consistent with a trisubstituted benzene

-  prenyl identified by theirNMR and spectra.MS DKPs arecommon fungal

3.42 in compound 12 H , [24] 16 - - . [22] 5, as 5, the signal C of 4 3 O

a member of the -

was confirmedwas hydroxybenzoic acids 5

. The MS/MS. Thespectrum ofprecursor the ion m/z 241 Two of them, cyclo( Aspergillus 4 H

insame the wayin as compound the 3 , suggesting that thiscompound is

duricaulis

big scale

Pleosporaceae as 5

Pleosporaceae H 1 3 , - O

methoxy 4

L - - - 18 protons at 3.42 ppm correlated with 5 Leu [27,28] -

- extract,showed and , )

[23] - 24 asperpentyn. - L abundancein the LC/MS runs of

- andmarine the fungus , 2 7 Pro)( - 27

cochlione B. , exceptfor the presence - family. Theabsolute 11

family, thesame as

and the chromene

was straightforward 10 ) and) cyclo(

This highly 1 H, 2

. The - derived fungus 13 - C, 2D NMR,C, 2D )

– closely 1 L

H and - Phe

28 - . L

of of 13 - C

This article isprotected rights by All copyright. reserved. positivecontrols carbendazim and benomyl. Compounds with inhibition halos of 20, 12, 20 and 20 the fungal pathogen of soybean Macrosphelides bioactivities, havedriven to extensivebiological studiesand their total synthesis. adhesion HL of B werefirst isolated from the soil fungi macrosphelides 14 macrosphelides A,B diastereoisomeror these,of while same family a characteristic pattern which in turn 3 of macrosphelide C, 16 30 scale culture MMon alsowas performed. From the extract thisof culture, two compounds, C yielded fragmentation patterns for compounds belonging the to same family. Losses C of signals on MM extracts, notpresent in loadingsThe plot of the unsupervise involved in thegrowth and virulence in kinaseC and disturbing cytokinesis. likediseases Krabbedisease. signaling, andgalactosyl the organisms. transduction, found inessentially allanimals, plants, and fungi, as aswell insome important class of outer membrane compounds involved in structure, recognition and signal the loss hexose a of ( the corresponding [M+H] respectively by means theirof MS and MS/MS spectra. Compounds standard of commercial benzophenone. MSThe and MS/MS spectra andretention the timeof compound substituent inthebenzene ring, leadingstructure to the shown in as substituents. Further analysis theof HMBC and NOESYspectra allowedlocation the eaof ring,prenyl a with chain with aterminal double bond, a carboxylic acid andhydroxylatedmethine a 6 H Accepted- , Article deox 8 wereisolated and identifyby NMR and analysisMS as macrospheli - O hydroxybutyric acid (C 2

in theirMS/MS y - macrosphelideC ( [36]

31 directly from theLCMS runs without need isolation. for Lyso -

60 cells to human umbilical vein endothelial cells, among many other intriguing and [41]

2 5 -

glycosphingolipidsbeen have implicated ina variety regulatoryof rolesin cell andreported is hereas natural a productfor thefirst time. Macrosphelides Aand and 32 - 162 u) inaddition to thosetypical of membered macrolideshaving two subunits of sorbic acid(C

wereidentified hexosylas a spectrawere characteristic for these compounds. For this reason,larger a 30 +

, compound ions m/z 480.3526 and 462.3424respectively yielded productions with 4 ). Careful interpretation of the MS/MS spectraallowed recognition the of 30 - [37, 38] sphingosine ) hasbeen previously obtained asan intermediate inthe synthesis of Fusarium Psychosine [39] d PCA analysis, led to the characterization of the minor compounds theof In particular, sphingolipidsalso have been reported to be

27 MEextracts. Closer inspection LCMS andof MS/MSruns Microsphaeropsis Fusarium

psychosine, linkedwas to the pathogenesis severalof

(± 1) mm respectively, comparable with those theof

and tucumaniae

was alreadywas known to becytotoxic, inhibiting protein

anofinic acid (

graminearum - p Figure5, hytosphingosineand hexosyl ,

[35]

responsiblefor the sudden death syndrome, 26

27 sp sphingosines. (Glyco)sphingolipidsan are Collisional induced decomposition (CID) of

is a .

and [ 42] showed several aligned (correlated)

diastereoisomer of macrosphelide C. )

Their ability to inhibitcell the [40] showed antifungal activity against 28 29 Figure 4

also showed antifungal activity

Compounds matched with those of a deC and 14 .

[43, 44] 6 19

) andsubunit) one - prokaryotic - sphingosine deoxy

and

22 4 - H

are ch 25 O - 2 cell

and and This article isprotected rights by All copyright. reserved. released to the medium, allowing it to exert particularlyIt is remarkablethe that cochlioneB ( anofinic acid ( than mycelium extracts. In general, thesecompounds wereabundant more in th 18 biomarkergood for the strain. Asperpentyn( related phytotoxicity,to which hasbeenpreviously additives, and always presented the minimum RA valuesin MM experiments. Thesef relativeabundances in the medium extracts than mycelium extracts, higher inMEthan MEwith and mycelia relativeThe abundances (RA) of the identified metabolites produced by relevantto havea clueabout their possible biological role. organism. metabolites associated with the combative relationship of the organism with its environment, whilst the generallyIt is accepted thatthe extra Metabolite distribution correlate 5 Figure drechslerines. work: sesterpeneslike ophiobolines from Other metabolitespreviously described from other possible ecological rolefor these 1)(± againstthe fungal pathogen soybean of

Accepted, Article 24 mm re in presence .

Scores and loadings plot of unsupervised PCA analysis of LC of analysis PCA unsupervised of plot loadings and Scores

, each other. each 27 [45] and spectively, also comparable with those of thepositive controls. These resultssuggest a

present in the mycelium extract may havea biological related to the protection of the in all the experiments are shown in 2 Fo 6

28 [3,4] R ) was especially abundantin MEwith VPA. 28 ) presentwas in allthe experiments with minimal ed: ME, Blue: MM, circle: no HDACI, circle: MM, Blue: ME, ed: r this reason ).

of theof HDACs inhibitors, and chromanes Harveynone

, the distribution, the theof metabolitesbetween medium and mycelium is

Prenylquinoids(PQs) ( 14

compounds in the defense theof plant against 2 ) , was

3 most -

exhibited higher cellular : SAHA, x: OHA, +: VPA. +: OHA, x: SAHA, :

at least 15 times more abundantin MM experiments and Macrophomina

with the highest differencesfor cochlioneB ( Dreschlera antifung -

MS dataset. MS efficiently metabolitespresent in Table 3 ic compound, Dreschlera 14 associated with DPKs.

Arrow on loadings plot points were data from MM experiments which which experiments MM from data were points plot loadings on Arrow

gigantea

and abundances inthe medium than in the its biological role.

. phaseolina

DKPs showed a tendency have to higher

17 , pre

[10]

strains haven

anofinic acid( changesin RA, becoming then a or sesquiterpenesor like nylhydroxybenzoic acids(PHBAs) , with inhibition, with halos of and8 12

a culture filtrate may be Dreschlera sp.

[45,46] e controle experiments ot been ot detected in this

28 pathogens. ), was), mostly acts may be

2 in both media ) and)

This article isprotected rights by All copyright. reserved. Chromanes PHBAs PQs DKPs class compound Table protect medium and myc Macrosphelides wereproduced almost exclusively in MM, and werehighly abundantin both extracts theof SAHA experiments. factor. Benzophenone ( to the presence of the inh transducers, cytotoxic and are related to pathogenesis. ( the overproduction of extracts controlof experiments( hexosyl Medium extracts of culture broths treated with SAHA showedprominent a peak 32 a Accepted Article ) was

Relative abundances of the identified compounds in the different experiments. experiments. different the in compounds the identified of abundances Relative - Lolliumsp.

phytosphingosine (

observed inmainly the mycelium extracts. These compounds areknown be to signal 7 5 4 1 N° 19 28 3 2 27 24 18 17 14 6 11 10 9 8

macrosphelide macrosphelide acid anofinic B cochlione 5 B cochlione siccayne harveynone asperpentyn Pro) (L cyclo Pro) (L cyclo (Leu/Ile cyclo (Phe cyclo (Phe cyclo (Leu/Ile cyclo (Pro cyclo (Gly cyclo Compound - methoxy elium extracts. Considering its antifungal activity, these compounds could also

from common phytopathogens. - - Phe Leu

- -

- - - Pro) Val) Hyp) Hyp) 31

29 L L - - Pro) Hyp) - -

andrelease its to the medium. In the opposite sense,hexosyl

. ) alsowas produced with higherabundances inthe medium and mycelium ibitor may indicate that compound 31

) at 26.4min 1.8 9.3 0.0 8.1 2.9 5.2 5.0 1.0 1.5 3.6 14.8 4.8 4.1 10.1 8.3 7.4 7.7 30.7 ME

Table 3).

1.2 1.2 8.1 2.9 2.3 6.6 6.6 5.1 5.0 2.0 SAHA 5.8 1.0 0.0 1.0 2.6 1.0 1.0 0.0

This factsuggests thatthe HDAC inhibitor SAHA triggers (Figure2) extract Mycelium 1.2 6.9 0.0 5.0 5.0 4.6 0.0 0.0 1.0 1.0 3.1 1.2 1.0 2.4 2.0 2.1 1.8 1.6 OHA

which almost was absent in themedium

1.0 3.2 0.0 8.1 1.2 1.0 3.7 0.0 2.1 2.6 17.3 4.0 5.0 4.9 4.1 7.9 8.3 2.9 VPA [47]

Their releaseto the medium asresponse a

391.0 3.3 1.0 4.6 1.0 3.0 1.2 0.0 5.2 2.5 1.0 1.0 0.0 1.0 1.0 1.0 1.0 1.0 MM

may play

0.0 107.4 1.6 64.9 6.2 11.5 10.5 11.7 2.6 2.3 23.1 16.1 131.2 9.0 18.2 23.1 26.6 6.2 ME

a rolean as activation

1.0 3.7 0.0 1.0 1.0 3.0 1.0 1.5 1.0 1.0 2.4 1.6 14.2 1.8 2.7 5.4 2.7 2.8 SAHA

corresponding to extract Medium 0.0 1.0 1.0 1.7 4.9 19.8 6.1 1.8 4.5 1.3 21.0 15.2 107.9 3.9 12.2 1.0 22.0 6.3 OHA

- sphyngosine

0.0 8.5 4.3 80.4 1.0 2.1 4.4 1.0 18.7 1.9 16.3 8.0 80.4 7.7 15.7 20.8 15.6 7.4 VPA

249.6 17.1 1.2 15.8 1.0 1.0 1.4 4.2 79.6 2.1 1.0 1.0 1.0 1.0 1.0 2.5 1.0 1.0 MM

This article isprotected rights by All copyright. reserved. . that be may useful for the fungal protection from the environment. Additionally, this known cellular responseinducers. medium and mycelium extracts,and therelease of lyso the greatest changes, with a noticeableincrement in theproduction of produced changes inthe concentration of the metabolites. Particularly,use the SAHAof produced It was establis forrole these compounds as chemical defenses intheplant host plausible. is 27 firstdescribed for 14 belon prenylhydroxybenzoic acid grass From the cultures aof strain of Conclusions ME: control sphingolipids, SphL: Macrospherelides, MSP: acids, prenylhydroxybenzoic a SphL MSP

DKPs: diketopiperazines, PQs:prenylquinoids, PHBAs: PHBAs: PQs:prenylquinoids, diketopiperazines, DKPs: , ,

Accepted17 Article

28 ,prenylhyd the ging to different compound classes, the chromanes Lollium sp.

and 32 31 29 30 26 25 22 30

hed that the addition of different HDACIsepigenetic as modifiers toculture the broths displayed good antifungal activity againstthe phytopathogen sphingosine (hex phytosphingosine (hex benzophenone 14 isomer C macrosphelide C macrosphelide B/isomer macrosphelide A/isomer , twenty fivemetabolites wereidentified. Two them,of the chromanone - deoxy Drechslera osyl osyl

Drechslera

) ) roxybenzoic acids

- - - MSP C MSP C

strain presented a high ability to metabolize xenobiotics,capability a , being , wereisolated and identified for the firsttime. Othermetabolites 1.0 3.8 2.9 2.9 1.0 1.2 1.0 Drechslera sp

30 3.2 2.1 17.6 1.2 0.0 1.0 1.0

18 firstdescribed asalso natural a product. Metabolites

, 24 1.0 1.0 1.0 1.0 0.0 1.3 0.0 ., adarkseptate endophyte (DSE)isolated fromrye the , 27

, and the macrosphelides 1.2 0.0 0.0 0.0 0.0 15.6 1.5 -

glycosphingolipids to the medium, which are

2 , 3 , the prenylquinoids derivatives 3.2 1.7 12.0 101.0 45.2 72.1 68.4

0.0 1.2 0.0 1.8 3.5 18.0 31.9

benzophenone( 19 1.0 65.4 73.9 1.0 3.5 4.5 8.6 F. ,

tucumaniae , 25 0.0 1.2 1.3 4.0 1.8 1.0 3.4 ,

and 29 3 0.0 0.0 1.0 0.0 1 3.4 1.0 ) inthe

, and a

and the 30

6 18 ,

were , 0.0 1.0 1.4 92.7 577.4 452.6 499.3 25

This article isprotected rights by All copyright. reserved. KNO Accepted(10g) andsolution a of salts (100mL)perliter. solutionThe saltsof contained MgSO Insame the way,large a carried under out static conditions at 25°C for 7 days. five 4L Erlenmeyerflasks containing 1L each of the sameliquid medium. Finally, fermentation was liter. Fermentation was carried out for days 7 at25°C, andferment the flaskscontaining 75mL of malt extract medium composed of 30g maltextract and peptone 5g per Solid Fermentation large of cultures scale under static conditions at25°C. All the experiments were made leastat by triplicate. inhibitors were spiked with 100μL DMSO. Finally, fermentation was carried out for further days12 final concentration of 500 ControlμM. cultures (3 controlx and 3 x M carried at out 25°C for 72hs with orbital shaking, when inhibitors were added in 100 μL DMSOto a medium 3mL cultureof broth were used to inoculate 125 mL Erlemeyer flasks containing 50mL maltextract medium (ME). Fermentation carried was at out 25°C for 48hs with shaking.orbital Afterthis time, sterile corkbore and used to inoculate 125 5g peptoneand agar15g perliter. A 5 diameter mm agar diskof cultured fungus cutwas with a strainDSE was cultured and maintained in maltextract agar (MEA), composed of 30g maltextract, Fermentation small of scale Articlethe sequence ITS was deposited GenBankin the with accession number FJ868975. culture Collection (FCEN strainDSE Drechslera sp. was isolated from the roots ryeof grass (Lollium as sp.) previously reported[49] and was deposited Fungal material chloridetreated with aqueous conc. ammonium hydroxide. by known procedures hydroxylaminehydrochloride were purchased from Sigma Aldrich. SAHA and OHA weresynthesized methanol and water were purchased from Carlo Erba (Milan, Italy). DMSO,sodium valproate and software. residualThe non Two recorded on a Bruker Optical rotations were recorded a on Perkin General Experimental - 3 dimensional NMR spectraCOSY, HSQC

medium culture cutwas into 1x1cm plugs and used inoculation for threeof 250mL Erlenmeyer (0.8g), KCl (0.65g), KH

(3 x control, 3 SAHA,x x 3 OHA, x3 VPA) or minimal medium (3 x MM). Fermentation was

Section - UBA, CONICET) under the Accession Number 3419. BAFC This strain was previously characterized molecularly and

AvanceII spectrometer operating 500.13 at and 125.77 MHz, respectively. 8 cultures withcultures HDACIs ] . - scale culture was performed on minimal medium, Octanamide was - deuterated solventsignal usedwas 2 PO

(48 mg), Ca(NO

synthesized from octanoic acid through the octanoyl

ml ml Erlenmeyerflasks containing 50 ml of malt extract - - DEPT andDEPT HMBC were performed using standard Bruker Elmer 343 polarimeter. 3 ) 2 .4H

2 O (2.88g),O NaFeEDTA (0.08g), (7.5mg), KI

for calibration. LC/MS grade 1 M)without the addition of H and ation broth inoculated was to composed by sacharose 13 C NMR sp C NMR 4 .7H

ectra were 2 O (7.31g),O

in th e BAFC e - This article isprotected rights by All copyright. reserved. AcceptedMathWorks Inc., Natick, MA). were applied to simplified data matrix of relevantmost data using MATLAB v.7.6 (R2008a, which was converted ProfileAnalysis 2.0 (BrukerDaltonics), obtaining[31x9300] a matrixdata of [analysesx (tR; m/z)], Extraction and bucketing ofraw the LC/MS data and unsupervised PCA model was performed using Data instrument were carried using out and anAgilent tuning mix APCI/APPI was employed as calibrant. Data acquisition and processing positiveion mode. APCI For corona the was setat 4 point mass calibration was carried using out sodium a formate solution from m/z 50 to temperature of °C 200 using N instrument was operated ata capillary voltage 4.5of kV with an end plate offset of (BrukerDaltonics, Billerica, USA),MA, equipped with electrospray and ion APCI sources. The spectrMass Mass spectrometry times. from 190 to 950 nm coupled a to mass spectrometer. Allruns the were repeated leastat three min), 75 temperature was set at 30 °C. Lineargradientelution performwas Articlecontaining 0.1% formic acid and (A) methanol (B). Theflow rate was 0.3 mL/min andcolumn the μm, 2.0 × 100 mm; Phenomenex,Torrance, CA, USA). The mobilephase consisted of water analysesLCMS of each extractwere performed on a RRLC Agilent 1200 using a Luna C Liquid Chromatography nylon membrane. pressure;residue the was dissolved i 3x50mL of EtOAc. In both cases, mycelia and media, theorganic solvents wereremoved at reduced filtered and extracted with EtOAc (3x50mL); the media of the culturebroths were extracted with cultureThe broths (50mL) were filtered. The Extract preparation acid (5mg)per liter. (0.2mg),glycine (30mg), thiamine chlorhydrate (1mg), pyridoxine chlorhydrate (1mg)and nicotinic MnCl analysis Analysis Principal Component and 2 .4H

- 100 B (25% 2 O (60mg),O ZnSO .

ometricanalyses were performed using a Bruker

26 100 min), B % (26 to .csv datafor further analysis. Supervised PCA, clustering and dendrogram the 4 .7H software Bruker Compass AnalysisData version supplied 4.0 with the

2 2 O (26.5mg)O

as dry gasas dry at6.0 l/min andnebulizer a pressure bar.of 3.0 Multi n 2mL of methanol and filtered

- , H 46 min). Detection was performed withDAD a detector 3 mycelia were sonicate BO 3

(15mg),CuSO 000 nA and the vaporizer temperature at250°C

icrOTOF 4 .5H ed as follows: 10 d in forEtOH 30 min, then

2 through a 0.22μm poresize O (1.3mg),O Na - Q IIQ mass spectrometer 2 - MoO 500 V,a dry 75 B75 % (0 18

12 column (3column 4 .2H 00 in 2 - O 25 - This article isprotected rights by All copyright. reserved. abundance): 240 [M] 6), 58.3 (O 9); OC Hz, H 1H, Amorphous yellowsolid; 5 0.9 (5.5mg),anofinic acid 22.5x2.5cm, eluant MeOH 4.8 andmg) silicagel and mixtures hexane:of EtOAc as eluants, yielding asperpentyn ( asperpentyn as its main component. Fraction II subjectedwas to column chromatography using and mixtures water:of methanol as eluants, yielding fraction I to IV. Fract extractThe was subjected to dry column flash chromatography using reversephase silicagel (C 5x1LThe MM cul compound 21.1mm)x column, using methanol:water (40:60) under isocratic conditions (UV 215nm), obtaining 3 by column chromatography using silicagel and mixturesof dichloromethane: EtOAc yielding 1 mg of preparativesilicagel TLC (dichloromethane: EtOAc (7:3)),yielding 3 dicloromethane: EtOAc as eluants,yielding 45 antifungal assay.Fraction II subjected was to column chromatography using silicagel using diclorometane: EtOAc as eluan extractsThe were subjected dry to column flash chromatography using silicagel, with mixtures of 5x1LThe Extracti which wereused aspositivecontrol, showed inhibition zones of and22±2 concentration level Directbioautography on TLC was employed a Antifungal assay - . Fract methoxy H

Accepted Article 13 mg) 3 C NMR (CDCl ), 2.68), (d, on and isolation of metaboliteson and of isolation ion IV subjectedwas preparative to HPLC a on PhenomenexSynergi 4μm fusion .

- - cochlione 5 B. ME 5), 3.75 (dt, C 24 H 18

3 culturebroths werefiltered

), 57.0 (C and the diketopiperazines

J (2.2

= 16.5 Hz, 1H, H ture broths werefiltered and thecombined filtrates werepartitioned with AcOEt. 3 , 125MHz):

of of 20 μg/ mg). Fraction III w - + Methoxy . J (

(5), 225 [M 28 - 7 7), 48.7 (C - 8 1 , =3.7,J H(CDCl NMR - 4.7 H 2 - spot of each assayed compound used.was Carbendazim and b O 6

δ mg), macrosphelideC - ts, yieldingts, fractions Ito V.Fractions II and III resulted positivein the

hydroxy

(6:4), 5 mL/min; detection: UV 254 nm, RI), yielding compound 6 190.7 (C

- - 7 3a), 2.51 (d, - = -

8), 8), 47.2 (C CH 4 J 5 - 3 as subjected to preparativeHPLC (YMC column C18, 5 - 2,2 7 ] 3 + = 2.5 Hz, 2.5 1H, = H , 500MHz): -

- (1), (1), 182 (54), 83 (58). 4), 167.4 (C 10 dimethyl and thecombined filtrates werepartitioned with EtOAc.

and - s the method for detecting the antifungal activity 3), 3), 26.9 (C J

mg of = 16.5 Hz, H 1H, 11 - 5,6,7,8 . δ - ( 6

8a), 107.5 (C 25 4.51 (brt,

- andfraction, a which was further purified by - 7), 3.46 7), (brd,

tetrah - ,

10), 25.4 (C 1.6

ydro mg) andmg) 14

- J 3b), 1.53 (s, 3H,

- = Hz, 2.1 1H, H 7,8 - 4a), 4a), 83.4(C

mg of - - epoxychroman J 9).

= 3.7 Hz, 1H, H EIMS (70 eV) (relativem/z - deoxy 6 2 ion I(180mg) contained 20 , 16 . Fraction III was purified ±1 mm respectively. -

2) - mg),harveynone ( - macrosphelide C

6), 6), 4.44 (brt, H - , 73.7 (C 4 - - 10), 10), 1.43 (s, 3H, H one - 8), 8), 3.42 (s, 3H, (3) - RP

5), 5), 65.6 (C - 80A 80A (150 enomyl  J m, m, = 2.1 [50] - 27 18) (

. A 14,

30 , -

, -

This article isprotected rights by All copyright. reserved. [5] [4] [3] [2] [1] R A. Rodriguez and A.Godeas isolated, characterized and classified thefungal strain. theof project, and also performed and analyzed analyses. G. Silessand G. Gallardo isolated and identified the compounds. G. Cabrerathe is director G. Siless made the fungal culturesin different conditions, analyzed LCMS runs and performed PCA Author Contribution financialsupport. The authors thank Universidad Buenos de Aires and(W670 100352), CONICET (PIP 100516) and ANPCyT (PICT 2008 Acknowledgements http://dx.doi.org/10.1002/MS Supporting information for this article is Supplementary Material closelyon related (C120.5 125MHz, 10.8Hz, H 1H, (brs, H 1H, 1H, OH), 7.93 (dd, Amorphous yellowsolid; (8R) eferences Accepted Article - 4 hawaiiensis El A. A. M. M. 161 Abdel A. Fungu 306 Algicolous the from Sesquiterpenes ‘Rare Wright, D A. Höller, U. König, M. G. Osterhage, C. Change’, to Global Responses Plant Alter Symbionts ‘Fungal Rudgers, A. J. Emery, M. S. N. Kivlin, S. ‘A Meta Newsham, K. K. - Hydroxy - - 165. 313. - 1), 117.6 (C  -

- Lateff, T. Okino, W. M. Alarif, S. S. Al S. S. Alarif, M. W. Okino, T. Lateff, 11a), 5.03 (dd,

in ppm ): 169.6 (C

- of Morusalba L. Derived from Heavy Metals Habitats’, Habitats’, Heavy Metals from L. Derived Morusalba of

3 - - Gendy, N Gendy, - 9a), 9a), 2.49 (dd, (8 - hydroxy structures J =8.5Hz, 2.1Hz, 1H, H . M Hassanein, H. A. E. A. H. Hassanein, M . -

- Analysis of Plant Responses to Dark Septate Root Endophytes’, Endophytes’, Root Septate to Dark Plant Responses of Analysis 5), 5), (C115.6 Statement - 10

- J met =10.8Hz, 3.4Hz, 1H, H

J .

=13.8Hz, 3.3Hz, 1H, H [ - 51 7), 7), 161.0 (C hyl -

number - (MeOH, c=0.22): +16.5°; 53

- - 10 ] 11), 72.6 (C

- - Lihaibi, ‘Sesquiterpenes from the Marine Algicolous Fungus Fungus Algicolous Marine the from ‘Sesquiterpenes Lihaibi, H. Ibrahim, D. H. A. El A. H. Ibrahim,D. H. - buten) .

- 6), 7.76 (d, available theon WWW under - - benzoic acid 1), 141.3 (C - 8), 8), 46.6 (C Mycobiol.

- - LCMS runs. Y.Rincón performed the bioassays. M. Baky, ‘Heavy Metals Biosorption from Aqueous Solution by Endophytic Endophytic by Solution Aqueous from Biosorption Metals ‘Heavy Baky, 8), 4.93 (brs, 1H, H -

9b),1.86 (s, 3H, H J (18) =2.1, 1H, H

- 2017 10), 131.6 (C -

9), 9), 22.1 (C H NMR (CDCl ,

45 , 73, New Phytol. New -

83. -

2), 2), 6.93 (d, - - 12) Am. J. Bot J. Am. 6), 6), 129.8 (C -

11b), 2.60 (dd, s - 2011 12). 3 Drechslera dematioidea Drechslera . Configuration C of , 500MHz, , Drechslera sp. Drechslera 190 . 13 2013 , 783 , J C NMR (CDC NMR =8.5Hz, H 1H, , - 100 793. 793. - 2),125.9 (C , 1445 ,  ’,

J. Saudi Chem. Soc. Chem. Saudi J. in ppm): 8.88 (s, - J 1394) for partial =13.8Hz, – ’, 1457. 1457. J. Nat. Prod. Nat. J. 3 OD,

- - 5), 5.05 8 based 8 - 3) , 3) Drechslera Drechslera

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)

nalogs in the the in nalogs , 3641 , - -

Asperpentyn Asperpentyn , 3853, Harveynone, Harveynone, Aspergillus Aspergillus Drechslera Drechslera Drechslera Drechslera

having a a having 2002 – -

3716. 3855. 2000

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