Characterization of Antifungal Natural Products Isolated from Endophytic Fungi of Finger Millet

14 Pages·2016·9.26 MB·English

molecules
Article
Characterization of Antifungal Natural Products
Isolated from Endophytic Fungi of Finger Millet
(Eleusine coracana)
WalaaKamelMousa1,2,AdrianL.Schwan3andManishN.Raizada1,*
1 DepartmentofPlantAgriculture,UniversityofGuelph,Guelph,ONN1G2W1,Canada;
[email protected]
2 DepartmentofPharmacognosy,MansouraUniversity,Mansoura35516,Egypt
3 DepartmentofChemistry,UniversityofGuelph,Guelph,ONN1G2W1,Canada;[email protected]
* Correspondence:[email protected];Tel.:[phone](ext.53396);Fax:[phone]
AcademicEditor:DerekJ.McPhee
Received:13July2016;Accepted:27August2016;Published:3September2016
Abstract:FingermilletisanancientAfrican-Indiancropthatisresistanttomanypathogensincluding
the fungus, Fusarium graminearum. We previously reported the first isolation of putative fungal
endophytesfromfingermilletandshowedthatthecrudeextractsoffourstrainshadanti-Fusarium
activity. However,activecompoundswereisolatedfromonlyonestrain. Theobjectivesofthisstudy
weretoconfirmtheendophyticlifestyleofthethreeremaininganti-Fusariumisolates, toidentify
themajorunderlyingantifungalcompounds,andtoinitiallycharacterizethemode(s)ofactionof
eachcompound. Resultsofconfocalmicroscopyandaplantdiseaseassaywereconsistentwiththe
threefungalstrainsbehavingasendophytes. Usingbio-assayguidedfractionationandspectroscopic
structuralelucidation,threeanti-Fusariumsecondarymetaboliteswerepurifiedandcharacterized.
These molecules were not previously reported to derive from fungi nor have antifungal activity.
Thepurifiedantifungalcompoundswere: 5-hydroxy2(3H)-benzofuranone,dehydrocostuslactone
(guaianolide sesquiterpene lactone), and harpagoside (an iridoide glycoside). Light microscopy
andvitalitystainingwereusedtovisualizetheinvitrointeractionsbetweeneachcompoundand
Fusarium; theresultssuggestedamixedfungicidal/fungistaticmodeofaction. Weconcludethat
fingermilletpossessesfungalendophytesthatcansynthesizeanti-fungalcompoundsnotpreviously
reportedasbio-fungicidesagainstF.graminearum.
Keywords: finger millet; Fusarium sp.; endophyte; fungus; Penicillium sp.; 5-hydroxy
2(3H)-benzofuranone;dehydrocostuslactone;harpagoside
1. Introduction
Fingermillet(Eleusinecoracana)isanancientcerealcropwidelygrownbysubsistencefarmersin
AfricaandIndia. FingermilletwasdomesticatedinEthiopiaandUgandaat5000BCandthenreached
Indiaby3000BC[1]. Unlikeotherrelatedcereals,fingermilletiswellknownforitsabilitytotolerate
stressconditionsandtoresistmanypathogens,includingFusariumgraminearum[2–5]. F.graminearum
isaseriousfungalpathogenthatcausesFusariumHeadBlight(FHB)inwheatandGibberellaEarRot
(GER)incorn[6]. Bothdiseasesresultincatastrophiclossesingrainyieldandareassociatedwith
mycotoxinaccumulationingrains[7]. Fusariumsp. areancientfungithathavebeendatedto8.8mya
inAfrica[8],thesamecontinentwherefingermilletwasdomesticated.
Wehypothesizedthatthepotentiallong-termco-evolutionbetweenfingermilletandFusariumsp.
maynotonlyhaveplacedselectionpressureonthegenomeoffingermilletbutalsoonitsassociated
microbiome. Specifically, we hypothesized that finger millet may host endophytes that confer
Molecules2016,21,1171;doi:10.3390/molecules21091171 www.mdpi.com/journal/molecules
MoMlecoulelecsul2e0s 1260,1261, ,211,1 171171 2 of2 1o4f 14
associated microbiome. Specifically, we hypothesized that finger millet may host endophytes that
resistance to F. graminearum. Endophytes have been reported to help their host plants to combat
confer resistance to F. graminearum. Endophytes have been reported to help their host plants to
pathogens[2,9,10].
combat pathogens [2,9,10].

Figure 1. Characterization of the putative fungal endophytes previously isolated from finger millet
Figure1. Characterizationoftheputativefungalendophytespreviouslyisolatedfromfingermillet
and the antifungal activity of their extracts. (a) Picture of a finger millet plant; (b–d) Pictures of
and the antifungal activity of their extracts. (a) Picture of a finger millet plant; (b–d) Pictures of
fungal endophytes WF4, WF5 and WF7 isolated from finger millet, respectively; (e) Predicted fungal
fungalendophytesWF4,WF5andWF7isolatedfromfingermillet,respectively;(e)Predictedfungal
endophyte nomenclatures, alongside the corresponding plant tissue from which they were isolated
endophytenomenclatures,alongsidethecorrespondingplanttissuefromwhichtheywereisolated
and best BLAST match taxonomic identification based on 18S rDNA sequencing; (f–h) Example of
andbestBLASTmatchtaxonomicidentificationbasedon18SrDNAsequencing;(f–h)Exampleofeach
each endophyte fermented on rice medium as indicated; (i) Quantification of the effects of each
endophytefermentedonricemediumasindicated;(i)Quantificationoftheeffectsofeachendophyte
endophyte extract (250 mg/mL) on the growth of F. graminearum compared to the fungicide nystatin
extract(250mg/mL)onthegrowthofF.graminearumcomparedtothefungicidenystatin(10µg/mL)
(10 µg/mL) (diameter of inhibition zone in mm, n = 3); (j–l) Representative images illustrating the
(diameter of inhibition zone in mm, n = 3); (j–l) Representative images illustrating the results of
results of co-incubation of each endophytic fungus with F. graminearum in vitro.
co-incubationofeachendophyticfunguswithF.graminearuminvitro.
Consistent with our hypothesis, in a previous study, we reported the isolation of four putative
funCgoanl seinsdteonpthwytietsh (osturraihnys pWoFth4e-7s)i sf,roinma fipnrgeevri omuilslestt uanddy, swhoewreepd othrtaetd ththeier iesxotlraatcitosn haodf faonutir-fpuuntgaatli ve
fungalendophytes(strainsWF4-7)fromfingermilletandshowedthattheirextractshadanti-fungal
Molecules2016,21,1171 3of14
Molecules 2016, 21, 1171 3 of 14
activity against F. graminearum [2]. Only one of these endophytes, Phoma sp. strain WF4, was
activityagainstF.graminearum[2]. Onlyoneoftheseendophytes,Phomasp. strainWF4,wasanalyzed
analyzed in detail including isolation of the underlying anti-Fusarium compounds. Of the remaining
indetailincludingisolationoftheunderlyinganti-Fusariumcompounds. Oftheremainingstrains,
strains, WF6 and WF7 (Penicillium sp.) were isolated from finger millet roots, while WF5 (Fusarium
WF6andWF7(Penicilliumsp.) wereisolatedfromfingermilletroots,whileWF5(Fusariumsp.) was
sp.) was isolated from shoots (Figure 1) [2]. The crude culture extracts of each strain inhibited the
isolatedfromshoots(Figure1)[2]. Thecrudecultureextractsofeachstraininhibitedthegrowthof
growth of F. graminearum (Figure 1) [2]. The objectives of this study were to confirm the endophytic
F.graminearum(Figure1)[2]. Theobjectivesofthisstudyweretoconfirmtheendophyticbehaviour
behaviour of the three remaining, putative, anti-Fusarium fungal endophytes, to identify the
ofthethreeremaining,putative,anti-Fusariumfungalendophytes,toidentifytheunderlyingmajor
underlying major antifungal compounds and to study the mode of action of each pure compound.
antifungalcompoundsandtostudythemodeofactionofeachpurecompound.
2. Results
2. Results
22..11.. CCoonnffiirrmmiinngg tthhee EEnnddoopphhyyttiicc BBeehhaavviioouurr ooff tthhee PPuuttaattiivvee FFuunnggaall EEnnddoopphhyytteess
TToo ccoonnffiirrmm tthhee eennddoopphhyytitci cbebheahvaivoiuoru orf othfet hiseoliasoteldat efidngfienr gmerillmet iflulentgfiu, ntwgio, etxwpoereimxpeenrtism wenertes
wunedreerutnakdeenrt.a ken.
22..11..11.. PPllaanntt PPaatthhooggeenniicciittyy AAssssaayy
SSeeeeddlliinnggss ooff fifinnggeerr mmiilllleett wweerree ccoo--iinnccuubbaatteedd wwiitthh eeaacchh ppuuttaattiivvee ffuunnggaall eennddoopphhyyttee oorr wwiitthh aa
kknnoowwnn ppaatthhooggeenn ooff fifinnggeerr mmiilllleett,, AAlltteerrnnaarriiaa aalltteerrnnaattaa.. TThhee sseeeeddlliinnggss iinnooccuullaatteedd wwiitthh tthhee ppaatthhooggeenn
ddeevveellooppeedd ddiisseeaassee ssyymmppttoommss iinncclluuddiinngg bbllaacckk rroooottss aanndd lleeaaff ssppoottss aanndd rreedduuccttiioonn iinn ppllaanntt lleennggtthh
((FFiigguurree 22aa)),, ccoommppaarreedd ttoo ccoonnttrrooll sseeeeddlliinnggss tthhaatt rreecceeiivveedd tthhee bbuuffffeerr oonnllyy ((FFiigguurree 22bb)).. HHoowweevveerr,, nnoonnee
ooff tthhee ppuuttaattiivvee ffuunnggaall eennddoopphhyytteess sshhoowweedd ssttaattiissttiiccaallllyy ssiiggnniiffiiccaanntt ddiisseeaassee ssyymmppttoommss oonn ffiinnggeerr mmiilllleett
sseeeeddlliinnggss ccoommppaarreedd ttoo tthhee ppaatthhooggeenn ccoonnttrrooll ((FFiigguurree 22cc––gg))..

FFiigguurree 22.. CCoonntt..
Molecules2016,21,1171 4of14
Molecules 2016, 21, 1171 4 of 14
Molecules 2016, 21, 1171 4 of 14

Figure2.Plantpathogenicityassayofeachputativeendophytefollowinginoculationontofingermillet.
Figure 2. Plant pathogenicity assay of each putative endophyte following inoculation onto finger
Fig(uar)eR e2p. rPelsaenntt aptiavtehopgicetnuirceitsyh oawssianyg othf eeeafcfhe cptuoftatthievek neonwdonpphaythteo gfoenlloAwltienrgna irniaoaclutelarntiaotna oonnfiton gfienrgmeril let
millet. (a) Representative picture showing the effect of the known pathogen Alternaria alternata on
milsleeet.d (lain) gRse(pproessietnivtaeticvoen tprioclt)u;r(eb )shRoewpriensge nthtaet ievfefepcti cotuf rtehes hkonwowinng pthaetheoffgeecnt oAfltbeurnffaerriao anltfiernngaetra monil let
finger millet seedlings (positive control); (b) Representative picture showing the effect of buffer on
fingseeerd mlinilglest (sneeegdaltiinvgesc o(pnotrsoitl)iv;(ec –ceo)nRtreoplr);e s(ben) tRateipvreepsiecntutarteisvesh poiwctiunrge tshheoewffeincgto tfhteh eefefaeccht opfu btautfivfeerf ounn gal
finger millet seedlings (negative control); (c–e) Representative pictures showing the effect of the each
fingenerd mopihllyett eseoendfilinnggesr (mneigllaettisveee cdolinntgrsola)s; (icn–dei)c aRteepdr;e(sf)enQtuatainvtei fipciacttiuorneso fshthoewpinergc ethneta egfefeocft loefs itohnes ecaacuhs ed
putative fungal endophyte on finger millet seedlings as indicated; (f) Quantification of the
putbaytiveaec hfufnugnaglu senodrotphehyptaet hoong efninAgletre rnmarililaeta ltseerendatliancgosm apsa riendditcoattehde; co(fn)t rQoluuasnitnifgicAatsisoens sosfo ftthwea re;
percentage of lesions caused by each fungus or the pathogen Alternaria alternata compared to the
per(cge)nQtaugaen toifif cleastiioonnso fctahuesetodt ablyl eenagcthh ofufnfignugse romr itlhleet pseaethdolignegns iAnlotceurnlaatreiad awltietrhneataac hcoemndpoaprhedy titco ftuhneg us
control using Assess software; (g) Quantification of the total length of finger millet seedlings
conotrrothl eupsainthgo gAesnsAeslste rsnoafrtiwaaalrtee;r n(agt)a cQoumapnatrifeidcattoiothne ocof ntthroe l.toFtoarl bloetnhg(tfh) aonfd f(ign)g,leert temrsiltlheta tsaereeddliinffgesre nt
inoculated with each endophytic fungus or the pathogen Alternaria alternata compared to the control.
inofcruolmateodn ewaitnho ethaecrh iennddicoaptheytthiac tfuthnegirusm oera tnhsea praetshtoagtiesnti cAalltleyrndairffiae raelntetrn(Mataa ncno-mWphairtende ytot -tthees tc,opn≤tro0l..0 5).
For both (f) and (g), letters that are different from one another indicate that their means are statistically
ForT bhodetihwff e(hfri)es naktne (drMs (iagnn)d,n li-ecWtatteheristtn hteheyas ttt -aatneresd tda, rpidf f≤e e0rre.r0no5tr) .fo rTofhmteh ewonmheies akanneor.st hinedr iicnadteic tahtee sthtaant dtharedir emrreoarn osf athree smtaetaisnt.i cally
different (Mann-Whitney t-test, p ≤ 0.05). The whiskers indicate the standard error of the mean.
2.1.2. RootColonizationAssay
2.1.2. Root Colonization Assay
2.1.2. RoToot tCesotltohneizaabtiiloitny Aofsseaaych endophytetocolonizetheinternaltissuesoffingermilletroots,confocal
To test the ability of each endophyte to colonize the internal tissues of finger millet roots,
miTccoorno ftsoeccsoatp l tyhmiemi caraobgsiilcniotgyp ywo ifam seacagocihnn dgeu nwcdtaeosdp c.hoSynetdeeud tcloitne dcgo.s lSioneneoidczulei nlatghtsee d iniwnotcieutrhlnatatheld et ibwsusiuftfhee srt hooenf lbyfuin(fcfgeoernr otrmnolilyl)l es(cht oornwotoreotdsl), no
conofbosscheaorlvw maebdicl ernofosu conobgpsaeylr vgimarobalwge itfnhugni ngwsailad sge rcotohwnetdhtui sicnstsueideds.e (StFheieegd utlirisnesug3esa s,i bn(F)o.icgAuultraett he3dea, ebwa).ri tlAhyt ptthhoees teb-iaunrflofyce urp looasntti-loiynn o(tccioumnlaettripoolon)i nt
shouwsteeidmd, enm poo oosintbtso eufrstvehadeb, flmeu nofusgtna oglfah lty hgperh ofauwentgwha eli nrhesyiopdbhesa teehr wev eetdirseso uonebssth e(reFvireghdui zroeon p 3tlaha,enb )er.h, Aihzoto wpthleaevn eeea,r rheloyaw cpheovosetfr-it nehaoecchfuu lonafgt itiohwne as
timvei sfpuuoanilgniizt ewudsaetsod v,i inmsuitoaiaslittz eoedcf o tthlooe n infiuzitanitagitoaeln chooylfoptnhhiaezeae twpioiendr eeor fmo tbhaslee arevnpedidds euormnb -atehlp eai dnrdehr imzsuoabpl-lealapnyiede, erhrsmoowaflefi vlnaeygree errsa mcohfi l lofeifnt tgrheoero ts,
funpgriom vwiildlaesitn rvgoisofuutsra,t lhpizereordvs iudtopin pigon rfiuttiratththeae trct oshuleopynpiaozrraett ietohnnadt o otphf ehtyhy etae rseep oeifnddfieonrpmghearylt measni lodlef tfsi(unFbgig-eerup mriedi3lelcre–mt h(aF)l.i gluaryee r3sc –ohf) .f inger
millet roots, providing further support that they are endophytes of finger millet (Figure 3c–h).

FFiigguurree 33.. CCoonntt..

Figure 3. Cont.
Molecules2016,21,1171 5of14
Molecules 2016, 21, 1171 5 of 14

FigFuigreur3e. 3T.e Tstesfto frotrh tehea baibliitliytyo foft htheep puutatattiivvee ffuunnggaall eennddoopphhyytteess ttoo ccoololonniziez efifingnegre mrmillieltl ertorootso tussuinsgin g
concofoncfoaclaslc sacnanninninggl alasseerr mmiiccrroossccooppyy.. (a(a––bb) )RReperperseesnetnattiavteiv peicptuicrteus roefs roooftr toisostuteiss siunoecsuilnaotecdu lwatitehd thwei th
thebubfuffefre rcocnotnrtorlo; l(;c(–ch–)h )RReperperseesnetnattiavteiv peipctiuctruesr eosf orforooto ttistsisuseuse isnioncouclautleadte wdiwthi tehaechac phuptauttiavtei vfeunfugnalg al
endenodpohpyhtyetae saisn idnidciactaetded..F Fuunnggii flfuluoorreessccee ppuurrppllee--bblluuee dduuee ttoo ssttaainininingg wwitihth caclaclocfoluflouro. rP.lPanlat nttistsiusseus es
appapeaprearer dreddu deutoe atou taou-tflou-folureosrceesncecne.ceW. Whitheitaer raorrwoswps opinoitntto tfou fnugnigini sinidsiedteh tehtei stsiusseuse.sA. Abbbrberveivaitaiotinosn:sP: R,
priPmRa, rpyrirmooatr;yR rHoo,tr; oRoHt,h raoior.t hair.
2.22..B2.i oB-iAo-sAsasysaGy uGiudieddedP uPruirfiicfiactaitoinona annddS Sttrruuccttuurraall EElluucciiddaattiioonn ooff AAnnttii--FFuussaarriuiumm CComompopuonudnsd s
Bio-assay guided purification was conducted to isolate the active anti-fungal compounds from
Bio-assayguidedpurificationwasconductedtoisolatetheactiveanti-fungalcompoundsfrom
each endophyte extract. Three active anti-Fusarium compounds were purified (Figure 4). The
eachendophyteextract. Threeactiveanti-Fusariumcompoundswerepurified(Figure4). Thediameter
diameter of zones of growth inhibition of F. graminearum were 2, 2.5 and 1.5 cm for compounds 1, 2
of zones of growth inhibition of F. graminearum were 2, 2.5 and 1.5 cm for compounds 1, 2 and 3,
and 3, respectively. The minimum inhibitory concentration (MIC) for compounds 1–3 were 31.25,
respectively. Theminimuminhibitoryconcentration(MIC)forcompounds1–3were31.25,250.00and
250.00 and 31.25 µg/mL, respectively. The three compounds were subjected to further spectroscopic
31.25µg/mL,respectively. Thethreecompoundsweresubjectedtofurtherspectroscopicstructure
structure elucidation. Results corresponding to each compound are shown separately below. 1D-
elucidation. Resultscorrespondingtoeachcompoundareshownseparatelybelow. 1D-and2D-NMR
and 2D-NMR spectra for these three compounds are presented (Figures S1–S15).
spectraforthesethreecompoundsarepresented(FiguresS1–S15).
2.3. Structural Elucidation of the Isolated Antifungal Compounds
2.3. StructuralElucidationoftheIsolatedAntifungalCompounds
2.3.1. Compound 1 (Isolated from Fungus WF5)
2.3.1. Compound1(IsolatedfromFungusWF5)
Compound 1 (molecular formula C8H6O3) was eluted with hexane-ethyl acetate (90:10) as a
Compound1(molecularformulaC H O )waselutedwithhexane-ethylacetate(90:10)asawhite
white amorphous powder, with an 8Rf 6va3lue of 0.46, and further moved as a single band in
amorphouspowder, withanRfvalueof0.46, andfurthermovedasasinglebandinhexane-ethyl
hexane-ethyl acetate (85:15), with an Rf of 0.58. The yield of compound 1 was 50 mg from 3 g of total
acetate(85:15),withanRfof0.58.Theyieldofcompound1was50mgfrom3goftotalextract.IR:3320,
extract. IR: 3320, 1762, 1604, 1241, 1077, 948 cm−1. 1H-NMR (600 MHz, DMSO): 3.83 (2H, s, H-3), 6.65
1762,1604,1241,1077,948cm−1. 1H-NMR(600MHz,DMSO):3.83(2H,s,H-3),6.65(1H,dd,J=8.6,
(1H, dd, J = 8.6, 1.6 Hz, H-6), 6.75 (1H, d, J = 1.6 Hz, H-4), 6.95 (1H, d, J = 8.6 Hz, H-7), 9.32 (1H, s,
1.6Hz,H-6),6.75(1H,d,J=1.6Hz,H-4),6.95(1H,d,J=8.6Hz,H-7),9.32(1H,s,Ar–OH).13C-NMR
Ar–OH). 13C-NMR (150 MHz, DMSO): 175 (C-2), 153.9 (C-8), 146.7 (C-5), 125.1 (C-9), 114.2 (C-4),
(15101M1.8H (zC,-D6)M, S1O10).:51 7(C5-(7C),- 23)3,.135 3(C.9-3(C). -8C)o,m14p6a.7rin(Cg- 5s)p,e1c2t5ra.1l (dCa-t9a) ,(1F1ig4u.2re(Cs -S41),–1S15)1 .8w(iCth- 6a), 1p1re0v.5io(uCs- 7),
33.r3ef(eCr-e3n)c.eC [o1m1]p, tahrein cgomsppeocutrnadl dwaatsa c(oFnigfiurrmesedS 1a–sS 55-)hwyditrhoxayp 2r(e3vHio)-ubsenrezfoefruernacneo[n1e1 (]F,itghuerceo 4ma)p. oundwas
confirmedas5-hydroxy2(3H)-benzofuranone(Figure4a).
2.3.2. Compound 2 (Isolated from Fungus WF6)
2.3.2. Compound2(IsolatedfromFungusWF6)
Compound 2 (molecular formula C15H18O2) was eluted from the hexane-ethyl acetate (50:50)
fraCctoiomnp aosu an cdol2o(rmlesosl escoulilda.r Tfhoer mcoumlapCou15nHd 1w8Oas2 )thweans peulruitfeiedd fbroym prtehpearhaetixvaen Te-LeCth uyslinagce ata stoelv(5e0n:t5 0)
framctiixotnuraes oaf hcoexloarnlee-sesthsyoll iadc.etTahtee (3co0:m70p),o wunitdh awna Rsft vhaelnuep oufr 0ifi.7e3d. Tbhye pyrieelpda orfa tciovmepToLuCndu 2si wngasa 1s0o mlvge nt
mixtureofhexane-ethylacetate(30:70),withanRfvalueof0.73. Theyieldofcompound2was10mg
Molecules2016,21,1171 6of14
from3gtotalextract. TheIRand1D-NMRdatawereasfollows: IR:2934,1765,1257,1146,998cm−1.
1H-NMR(600MHz,CDCl ): δ2.91(1H,m,H-1),1.91(1H,m,H-2α),1.85(1H,m,H-2β),2.14(1H,m,
3
H-3α),2.5(2H,m,H-3),2.87(1H,m,H-5),3.95(1H,t,J=10Hz,H6),2.90(1H,m,H-7),2.24(1H,m,
H-8α),1.42(1H,m,H-8β),2.48(1H,m,H-9α),2.19(1H,m,H-9β),6.22(1H,d,J=3.6Hz,H-13α),5.47
(1H,d,J=3.6,H-13β),4.87(1H,s,H-14α),4,79(1H,s,H-14β),5.25(1H,d,J=2.4Hz,H-15α),5.04
(1H,d,J=2.4Hz,H-15β). 13C-NMR(150MHz,CDCl ): δ30.2(C-3),30.9(C-8),32.4(C-2),36.2(C-9),
3
45.09(C-1),47.5(C-7),52.0(C-5),85.2(C-6),109.59(C-14),112.6(C-15),120.19(C-13),139.7(C-11),149.2
(C-10),151.2(C-4),170.27(C-12). Comparingspectraldata(FiguresS6–S10)withreferencedata[12],the
Molecules 2016, 21, 1171x 10 of 19
compoundwasconfirmedasdehydrocostuslactone,anguaianolidesesquiterpenelactone(Figure4c).

Figure 4. StruFicgtuurer e4s. Satnrudctuirnes vaintdro ina vcittirvo iatcytivoitfy tohf ethep upurriififieedd aantni-tFiu-sFaruiusmar icuommpocuonmds pfrooumn fdusngfarl om fungalCommented [t1]: Please replace Fig 4
endophyte streanidnosphWyteF s5tr,aiWns FW6F5a, WndF6 WandF W7.F7.( (aa)) SStrturcutucrteu orfe coomfpocuonmd 1p,5o-huynddrox1y ,b5e-nhzyofdurraonxoyne b(freonmz ofuranone
strain WF5); (b) Representative picture of the disc diffusion assay showing the anti-Fusarium activity
(fromstrainWF5);(b)Representativepictureofthediscdiffusionassayshowingtheanti-Fusarium
of compound 1; (c) Structure of compound 2, dehydrocostus lactone (from strain WF6); (d)
activity of comRepporeusenndtati1v;e p(cic)tuSret rouf tchteu driesc doifffucsoiomn apsosauy nshdow2i,ngd teheh ayndtir-Foucsoarsiutmu sactliavcittyo onf ecom(fprooumnd s2;t rain WF6);
(d)Representat(iev) eStrpuiccttuurer eofo cfomthpeoudnids c3,d hiafrfpuasgioosnidea s(fsraomy ssthraoiwn WinFg7)t; h(fe) Raenptrie-sFeuntsaatirvieu mpicatucrtei vofi ttyheo dfiscco mpound2;
diffusion assay showing the anti-Fusarium activity of compound 3. Numbers (1–9) denote a
(e) Structure of compound 3, harpagoside (from strain WF7); (f) Representative picture of the
concentration gradient of 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.62 and 7.8 µg/mL while C
disc diffusiondaesnsoateys thseh soowlveinntg cotnhtreol.a nti-Fusarium activity of compound 3. Numbers (1–9) denote a
concentrationgradientof4000,2000,1000,500,250,125,62.5,31.25,15.62and7.8µg/mLwhileC
2.3.3. Compound 3 (Isolated from Fungus WF7)
denotesthesolventcontrol.
Compound 3 (molecular formula C24H30O11) was eluted from the 100% methanol fraction as an
amorphous powder. The compound was further purified using preparative TLC with a mobile phase
2.3.3. Compound3(IsolatedfromFungusWF7)
mixture of methanol-water (95:5), with an Rf value of 0.12. The yield of compound 3 was 5 mg from
3 g total extract. The IR and 1D-NMR data were as follows: IR: 3392, 2884, 1334, 1283, 989, 770 cm−1.
Compound3(molecularformulaC H O )waselutedfromthe100%methanolfractionasan
1H-NMR (600 MHz, CDCl3): 1.33 (32H4, s,3 H0-101)1, 1.74 (1H, dd, J = 14.5 and 4 Hz, H-7α), 2.15 (1H, d, J = Formatted: Font: Not Italic
amorphouspo1w4,5d Herz., HT-h7βe),c 2o.9m3 (p1oHu, sn, Hd-w9), a3s.36f-u3r.8t4h (eGrlcp-2u-6r)i, fi4.e6 d(Gulcs-1in), g3.9p8 r(e1Hp,a dr,a Jt =iv =e4 HTzL, CH-6w), i4t.h83 a(1mH, obilepFhoramsaetted: Font: Not Italic
mixtureofmedth, Ja =n =o8 lH-wz, aHt-e4)r, 6(.915 (1:5H),, sw, Hi-t1h), 6a.3n6 R(1fHv, da, lJu =e =6o.4f H0z.1, H2.-3T), h6.e38y (1ieHl,d d, oJ f= =c1o6m Hpz, oHu-αn)d, 7.347w (3aHs, 5mgFforrommatted: Font: Not Italic
3gtotalextracmt., HT3h′-e5′)I,R 7.5a5n (2dH1, mD,- HN-2M′ aRndd 6a′),t a7.5w8 (e1rHe, da,s J f=o =l1l6o Hwzs, :HI-R β):. 3133C9-N2M,2R8 (81540,M1H33z,4 C,D1C2l83)3: 9,49(C89-,770cFmorm−a1tt.ed: Font: Not Italic
1), 143.4 (C-3), 104.7 (C-4), 72.7 (C- 5), 76.5 (C-6), 45.0 (C-7), 87.3 (C-8), 53.4 (C-9), 22.4 (C-10), 119 (C-
1H-NMR(600αM), 1H44z.9, (CCD- βC), l136)7:.31 (.C3=3O)(,3 1H34,.2s (,CH-1′-),1 102)8,.81 (.C7-42′)(, 11H28.,2 d(Cd-3,′)J, 1=301.34 (.C5-4a′)n, d1284.2H (Cz-5,′)H, 1-278α.8()C,-2.15(1FHor,mdatt,ed: Font: Not Italic
J=14.5Hz,H-67′)β, 9)9,.21 .(9G3lc-(11′)H,), ,7s2.,7H (G-l9c-)2,),3 7.53.69 -(3G.l8c-43)(, G71l.7c0- 2(G-6lc)-,4)4, .765.(9G (Glclc--15)),, 631..89 8(G(lc1-H6)., Cdo,mJp=ari4ngH spze,cHtra-l6 ),4.83Fo(r1mHatt,ed: Font: Not Italic
data (Figures S11–S15) results to the literature [13], the compound was identified as harpagoside, an Formatted: Font: Not Italic
d,J=8Hz,H-4),6.1(1H,s,H-1),6.36(1H,d,J=6.4Hz,H-3),6.38(1H,d,J=16Hz,H-α),7.47(3H,m,
iridoide glycoside (Figure 4e).
H3(cid:48)-5(cid:48)),7.55(2H,m,H-2(cid:48) and6(cid:48)),7.58(1H,d,J=16Hz,H-β). 13C-NMR(150MHz,CDCl ): 94(CCom-1m)e,nted [t2]:
3
143.4(C-3),10 4.7(C-4),72.7(C-5),76.5(C-6),45.0(C-7),87.3(C-8),53.4(C-9),22.4(C-10),119(C-α),
144.9(C-β),167.3(C=O),134.2(C-1(cid:48)),128.8(C-2(cid:48)),128.2(C-3(cid:48)),130.3(C-4(cid:48)),128.2(C-5(cid:48)),128.8(C-6(cid:48)),
Molecules 2016, 21, 1171 7 of 14
(1H, d, J = 8 Hz, H-4), 6.1 (1H, s, H-1), 6.36 (1H, d, J = 6.4 Hz, H-3), 6.38 (1H, d, J = 16 Hz, H-α), 7.47
Mol(e3cuHle,s m20,1 6H,231′-,51′1)7, 17.55 (2H, m, H-2′ and 6′), 7.58 (1H, d, J = 16 Hz, H- β). 13C-NMR (150MHz, CDC7l3o):f 14
94(C-1), 143.4 (C-3), 104.7 (C-4), 72.7 (C- 5), 76.5 (C-6), 45.0 (C-7), 87.3 (C-8), 53.4 (C-9), 22.4 (C-10), 119
(C-α), 144.9 (C-β), 167.3 (C=O), 134.2 (C-1′), 128.8 (C-2′), 128.2 (C-3′), 130.3 (C-4′), 128.2 (C-5′),
99.112(8G.8l(cC-1-(cid:48)6)′,),7 929.7.1( G(Glclc--21)′,),7 752.9.7( G(Glclc-3-2)),,7 715.7.90 ((GGlclc-3-4),) ,7715.7.90 ((GGllcc--45)),, 7651..98 ((GGllcc--56)),. 6C1.o8m (Gplacr-i6n)g. Cspomecptraarlindga ta
(FigspuercetsraSl1 1d–aSt1a5 )(Freigsuurltess toS1th1–eSl1it5e)r arteusruelt[s1 3t]o, ththeec olimtepraotuunred w[1a3s], idtheen ticfioemdpaosuhnadr pwagaos siiddeen,atinfieirdid aosid e
glyhcaorspidageo(sFiidgeu, raen4 ieri)d.oide glycoside (Figure 4e).
2.42..M4. iMcroicsrcoospciocpEicx Eamxaimnaintiaotnioonf otfh tehIen ItnetrearcatciotinonB BetewtweeenenE EacahchP PuurereA AnntitfiufunnggaallC Coommppoouunndd aanndd FF.. graminearum
inVgriatrmoinearum in Vitro
ToTvoi svuiasuliazleizteh tehein itnetrearcatcitoionnb beetwtweeeenn eeaacchh ppuurree ccoommppoouunndd aanndd FF. .ggrarmaminienaeraurmu min invivtriotr, ol,iglhigt ht
micmroicsrcoospcoypwy awsauss uesded(F (iFgiugruere5 )5.).

Figure 5. The effects of the purified endophyte-derived anti-fungal compounds on F. graminearum in
Figure5. Theeffectsofthepurifiedendophyte-derivedanti-fungalcompoundsonF.graminearum
vitro using neutral red staining. (a) Cartoon of the experimental methodology used to examine the in
invitrousingneutralredstaining.(a)Cartoonoftheexperimentalmethodologyusedtoexaminethe
vitro interactions between F. graminearum (pink) and each compound (orange) or the buffer control
invitrointeractionsbetweenF.graminearum(pink)andeachcompound(orange)orthebuffercontrol
(respective compound solvent). Microscope slides were pre-coated with PDA and incubated for 24 h.
(respectivecompoundsolvent).Microscopeslideswerepre-coatedwithPDAandincubatedfor24h.
F. graminearum hyphae were then stained with neutral red. Shown are representative microscope
F.graminearumhyphaewerethenstainedwithneutralred.Shownarerepresentativemicroscopeslide
slide pictures (n = 3) of the interactions of F. graminearum with: (b) 5-hydroxy benzofuranone (5
pictures(n=3)oftheinteractionsofF.graminearumwith:(b)5-hydroxybenzofuranone(5mg/mL)
mg/mL) compared to (c) the buffer control; (d) dehydrocostus lactone (5 mg/mL) compared to (e) the
comparedto(c)thebuffercontrol;(d)dehydrocostuslactone(5mg/mL)comparedto(e)thebuffer
buffer control; (f) harpagoside (5 mg/mL) compared to (g) the buffer control. The blue arrows point
control;(f)harpagoside(5mg/mL)comparedto(g)thebuffercontrol.Thebluearrowspointtoareas
to areas of apparent breakage of F. graminearum hyphae. Each scale bar equals 25 µm.
ofapparentbreakageofF.graminearumhyphae.Eachscalebarequals25µm.
Allcompoundsresultedinreducedhyphalgrowthassociatedwithfrequenthyphalbreakage
(Figure5b,d,f),comparedtothecontrol(Figure5c,e,g),suggestiveofamixedfungistatic/fungicidal
modeofaction. Interestingly,thehyphaeofF.graminearumappearedtobendawayfromthecontact
zone with compounds 2 and 3 (Figure 5d,f). However, the effect of all purified compounds on
Molecules2016,21,1171 8of14
F.graminearumhyphaewasweak/moderatewhencomparedtootherantifungalcompoundspreviously
isolatedfromfingermillerfungalendophytes[2].
3. Discussion
Endophytesaredefinedasmicrobesthatareabletocolonizetheinternaltissuesoftheirhost
plantswithoutcausingdisease[14]. Wehypothesizedthatfingermilletmayhostendophyteswith
anti-fungalactivity,includingagainstthepathogenFusariumgraminearum,becausethiscropisknown
to be resistant to many pathogens including F. graminearum [2,3,5], and its endophytes may have
co-evolvedwithFusariumpathogensinAfrica[15,16]andSouthAsia[17,18]. Inapreviousstudy[2],
weidentifiedfourdistinct,putative,fungalendophytespeciesfromfingermillet(WF4-7). Theputative
endophyteswereisolatedfromplantsoffirstgenerationseeds,andthengrownonTurfaceclayrock
usinghydroponicsratherthanonsoil.Thisgrowthsystemmayhavecontributedtothelowabundance
andbiodiversityoftheisolatedendophytes.
In our earlier study [2], we showed that extracts of the putative endophytes have antifungal
activitiesincludingagainstF.graminearum. OnlytheextractfromWF4,aPhomasp. wascharacterized
in detail, and revealed four anti-Fusarium compounds (viridicatol, tenuazonic acid, alteraniol and
alteraniol methyl ether). Here, we confirmed that these fungal strains are able to re-colonize the
internal tissues of the host without causing pathogenic symptoms, when compared to the known
pathogen Alternaria alternate, consistent with their classification as endophytes. We identified the
compounds underlying the antifungal activity of these endophytes alongside a suggested mode
ofaction. Thecurrentanti-Fusariumcompoundsaredifferentthanthosepreviouslyidentifiedand
broadentherangeofbio-fungicidesidentifiedfromthefingermilletmicrobiomeagainstthisimportant
croppathogen.
3.1. PreviousReportsofNon-PathogenicFusariumandPenicilliumsp. asEndophytes
One fungal strain reported in this study was predicted to be a Fusarium sp. (WF5). Previous
studiesinvolvingFusariumandAspergillussp. haveshownthatnon-pathogenicfungalstrainsthat
belong to the same species as a pathogen can, in some cases, control the ability of the pathogen
tocausediseaseandproducemycotoxins[19,20]. Severalcompetitiveexclusionmechanismshave
beensuggestedthatexplainthebiologicalcontrolability[21]. Thesemechanismsincludeblockage
ofinfectionsites,competitionforlimitednutrientsinthesoil,inhibitionofsporegerminationand
inductionofhostresistance[22,23]. Inthecurrentstudy,weshowedthattheabilityofnon-pathogenic
Fusariumsp. tocontrolpathogenicFusariumsp. mayalsobemediatedbyproductionofantifungal
secondarymetabolites.
Thetwootherfungalstrains(WF6andWF7)characterizedinthisstudywerecloselyrelatedto
Penicilliumsp.,includingonethatmostcloselyresembledPenicilliumchrysogenum(WF6)taxonomically.
P. chrysogenum was previously isolated as an endophyte of marine red algal species of the genus
Laurencia, and was reported to have antifungal activity against the fungus Alternaria brassicae.
The antifungal metabolite was identified to be a mono-terpene derivative [24]. This observation
isofinterestbecausetheanti-FusariumcompoundthatwepurifiedfromtheapparentP.chrysogenum
isolate from finger millet is a sesquiterpene lactone, suggesting that the terpenoid pathway may
playanimportantroleintheanti-pathogenesisofP.chrysogenumendophytes. OtherPenicilliumsp.
wereisolatedfromdiversehostsincludingtheSouthAsianmedicinalplantOcimumtenuiflorum[25],
theMoroccanplantCeratoniasiliqua[26], CannabissativaL.[27]andPanaxginseng[28]. Anextract
fromthePenicilliumisolateofPanaxginsengshowedanti-fungalactivityagainstPyriculariaoryzae[28].
WeemphasizethislastobservationbecauseP.oryzaeisthemostimportantfungalpathogenoffinger
millet,thecausalagentofblastdisease[29].
Molecules2016,21,1171 9of14
3.2. Anti-FusariumCompoundsPurifiedfromFingerMilletEndophytes
Here, we have elucidated the structures of three active anti-Fusarium compounds: 5-hydroxy
benzofuranone,dehydrocostuslactone,andaniridoideglycoside(harpagoside)(Figure4).
3.2.1. 4-Hydroxybenzofuranone(IsolatedfromFusariumsp. StrainWF5)
Simple benzofuranone derivatives have previously been isolated from fungi including
Coniothyriumminitans[30]. Otherconjugatedbenzofuranoneshavebeenreportedfromendophytic
fungisuchasPestalotiopsisphotiniae,anendophytethatinhabitstheChineseplant,Roystonearegia[31].
Somebenzofuranonederivativeswerereportedtohavealgicidal,hypotensiveandanti-inflammatory
activities[32,33]. Tothebestofourknowledge,thisisthefirstreportof4-hydroxy-benzofuranone
havinganti-Fusariumactivity.
3.2.2. DehydrocostusLactone(IsolatedfromPenicilliumsp. StrainWF6)
Dehydrocostus lactone derivatives were previously isolated from plant species including
Inularacemosa,CentaureapannonicaandSaussureacostus[34–36]. Thecompoundwasreportedtohave
antimicrobialactivityincludingagainstMycobacteriumsp.,Propionibacteriumacnes,Staphylococcusaureus,
andMalasseziafurfur[36,37]. Dehydrocostuslactonewasreportedtohavemultiplebiologicalactivities
includingsuppressionofmelaninproduction,inductionofapoptosisinsofttissuesarcomacelllines,
inhibitionofmigrationofprostatecancercellsandanti-trypanosomaactivity[35,36,38,39]. Tothebest
ofourknowledge,thisisthefirstreportofdehydrocostuslactonefromafungalsourceandthefirst
reportofithavinganti-Fusariumactivity.
3.2.3. Harpagoside(IsolatedfromPenicilliumsp. StrainWF7)
HarpagosidewasfirstisolatedfromHarpagophytumprocumbens(Devil’sclaw),aplantwidelyused
infolkmedicineforitsanti-microbial,anti-inflammatoryandanalgesicproperties[40]. Subsequently,
harpagosidewasidentifiedindiverseplantsincludingRadixscrophulariae,aplantusedasanherbal
medicinefortreatmentofcongestion,constipationandsorethroats[41]. Harpagosidewasreported
tohaveanti-inflammatory,antioxidantandanalgesicproperties[42]Tothebestofourknowledge,
thisisthefirstreportofdehydrocostuslactonefromafungalsourceandthefirstreportofithaving
anti-Fusariumactivity.
4. ExperimentalSection
4.1. SourceofBiologicalMaterials
PutativefungalendophytestrainWF5(Fusariumsp.) waspreviouslyfromfingermilletshoots
(Genbank: KF957640)usingapreviouslydescribedprotocol(Figure1)[2]. Putativefungalendophyte
strainsWF6andWF7(Penicilliumsp.) werepreviouslyisolatedfromfingermilletroots(Genbank:
KF957641andKF957642)(Figure1)[2]. Inbrief,commercialfingermilletseedsoriginatingfromIndia
weregerminatedandgrowninpailsofTurfaceclayplacedinthefield(ArkellFieldStation,Arkell,
ON,Canada,GPS:43◦39(cid:48) N,80◦25(cid:48) W,and375mabovesealevel). Atthepre-floweringstage,five
sampleswerecollectedfromseeds,intactrootsandshoots. Theentiresamplingwasrepeatedthree
timesindependently. Samplesweresurfacesterilizedaccordingtoastandardprotocol[2]. Sterilized
tissuesweregroundinLBliquidmediumandtheextractswereplatedontoPotatoDextroseAgar
(PDA).Fungiwithuniquemorphologywerecollectedandre-culturedonfreshmediaforpurity.
TheF.graminearumstrainusedinthisstudy(15AcetylDONProducer)wasobtainedfromthe
Agriculture and Agrifood Canada Fungal Type Culture Collection (AAFC Food Research Centre,
Guelph, ON, Canada). The effect of the crude culture extract of each fungus on the growth of
F.graminearumwaspreviouslyshown(Figure1)[2].
Molecules2016,21,1171 10of14
4.2. CompetitiveInhibitionExperiment
TotesttheabilityofeachendophyticstraintocompetitivelyinhibitthegrowthofF.graminearum,
co-incubationexperimentswereconducted. EachfunguswasgrownonPDAplatesat25◦Cforone
week. Thereafter,5mmagarplugsfromeachendophyticfunguswereinoculatedneartheedgeof
freshPDAplates,and5mmagarplugsofF.graminearumwereinoculatedontheotheredge. Both
F.graminearumandeachendophyticfunguswereco-incubatedat25◦Cfortwoweeks. Therewere
threereplicatesforeachendophyticstrain.
4.3. PlantPathogenicityAssay
Toconfirmthattheputativefungalendophytesarenotpathogens,aplantpathogenesisassay
was conducted. Seeds of finger millet were surface sterilized by washing in 0.1% Triton X-100
detergent (10 min), followed by 3% sodium hypochlorite (20 min, twice). Seeds were planted on
sterile Phytagel based medium consisted of: 1 package Murashige and Skoog modified basal salt
mixture(Catalog#M571,PhytoTechnologyLaboratories,ShawneeMission,KS,USA),4gPhytagel,
1mLpyridoxineHCl(0.5mg/mL),500µLnicotinicacid(1mg/mL),0.332gCaCl , 1mLglycine
2
(2mg/mL),10mLthiamineHCl(100mg/L),and1mLMgSO (18g/100mL),perliter. Themedium
4
wasdistributedintosterileglasstubes. Sevenseedsweretransferredtoeachtubeandallowedto
germinate in the dark for seven days, then transferred to light shelves (25 ◦C, 16 h light). When
seedlingswereoneweekold,eachendophyte(orcontrol)wasappliedontothegelsurface(11mm
diameteragardiscs),intriplicate. Seedlingsthatwerenotinoculatedwithanyfungiservedasthe
negative control. Seedlings that were infected with the fungal pathogen Alternaria alternata were
thepositivecontrol[43]. Plantswerethenassessedvisuallyfordiseasesymptomsat10daysafter
inoculationwitheachendophyteorcontrolpathogen. Then,thepercentagesurfaceareathatshowed
lesionswasquantifiedusingAssessSoftware(Version2.0,AmericanPhytopathologicalSociety,St.Paul,
MN,USA)incomparisontocontrols.ResultswerestatisticallyanalyzedusingPrismsoftwareversion5
(GraphPadSoftwareInc.,LaJolla,CA,USA).
4.4. RootColonizationAssay
Totestiftheputativeendophytesareabletocolonizetherootsoffingermillet,confocalmicroscopy
imaging was conducted. Finger millet seeds were surface sterilized and planted in glass tubes
containingsterilePhytagelbasedmedium(asdescribedabove). Eachfungalendophytewasapplied,
intriplicate(100µLofa48holdculturegrowninpotatodextrosemedium)tofingermilletseedlings
(17daysaftergermination)andco-incubatedwiththeseedlingsatroomtemperature. Thecontrol
consistedoffingermilletseedlingsincubatedwithpotatodextrosemediumonly. Calcofluorwhite
stainwasusedtostainfungi(catalogue#18909,SigmaAldrich,St. Louis,MO,USA),followingthe
manufacturer’sprotocol. Thereafter,fingermilletrootswerescannedwithaLeicaTCSSP5confocal
laser scanning microscope (Leica Microsystems, Wetzlar, Germany) at the Molecular and Cellular
ImagingFacility,UniversityofGuelph.
4.5. Bio-GuidedPurificationofActiveAnti-FusariumCompounds
Forlargescalefermentation,eachendophytewasfermentedin2Lflasks(fiveflaskspereach
endophyte)containing50gwhitericeand100mLH O.Theflaskswereincubatedat25◦Cfor30days
2
withoutshaking(Figure1f–h). Then,eachculturewasextractedwithethylacetate,andwashedwith
water to remove salts and sugars. The extract was dried under vacuum at 45 ◦C and subjected to
fractionationbetweenmethanolandn-hexanephasestoremovelongchainfattyacids. Topurifythe
active compounds, the dry residues were separated by flash liquid chromatography. The column
wasfilledwithflashgradesilicagel(SiliaFlash® P60230-400, SiliCycle, QuebecCity, QC,Canada,
Catalog#R12030B)andsaturatedwiththedesiredmobilephasejustpriortosampleloading. Foreach
endophyte,3gofthedryresidueextractwasdissolvedinchloroform,mixedwithsilicaandevaporated