Antifungal Activity and Action Mode of Cuminic Acid from the Seeds of Cuminum cyminum L

13 Pages·2017·4.39 MB·English

molecules
Article
Antifungal Activity and Action Mode of Cuminic
Acid from the Seeds of Cuminum cyminum L. against
Fusarium oxysporum f. sp. Niveum (FON) Causing
Fusarium Wilt on Watermelon
YangSun1,†,YongWang1,†,LiRongHan1,XingZhang1,2andJunTaoFeng1,2,*
1 ResearchandDevelopmentCenterofBiorationalPesticide,NorthwestA&FUniversity,
Yangling712100,China;[email protected](Y.S.);[email protected](Y.W.);
[email protected](L.R.H.);[email protected](X.Z.)
2 EngineeringandResearchCenterofBiologicalPesticideofShaanxiProvince,Yangling712100,China
* Correspondence:[email protected];Tel.:[phone]
† Jointfirstauthorship.
Received:31October2017;Accepted:21November2017;Published:30November2017
Abstract:Inordertodevelopanovelbiofungicide,theantifungalactivityandactionmodeofcuminic
acid from the seed of Cuminum cyminum L. against Fusarium oxysporum f. sp. niveum (FON) on
watermelonwasdeterminedsystematically. Inthisstudy,themedianeffectiveconcentration(EC )
50
value for cuminic acid in inhibiting mycelial growth of FON was 22.53 µg/mL. After treatment
withcuminicacid,themycelialmorphologywasseriouslyinfluenced;cellmembranepermeability
and glycerol content were increased markedly, but pigment and mycotoxin (mainly fusaric acid)
weresignificantlydecreased. Synthesisgenesofbikaverin(Bike1,Bike2andBike3)andfusaricacid
(FUB1,FUB2,FUB3andFUB4)bothweredownregulatedcomparedwiththecontrol,asconfirmed
byquantitativeRT-PCR.Ingreenhouseexperiments,cuminicacidatallconcentrationsdisplayed
significantbioactivitiesagainstFON.Importantly,significantenhancementofactivitiesofSOD,POD,
CATanddecreaseofMDAcontentwereobservedafterinvivocuminicacidtreatmentonwatermelon
leaves. Theseindicatedthatcuminicacidnotonlyshowedhighantifungalactivity,butalsocould
enhancetheself-defensesystemofthehostplant. Aboveall,cuminicacidshowedthepotentialasa
biofungicidetocontrolFON.
Keywords: watermelonfusariumwilt;p-isopropylbenzoicacid;biofungicide;diseasemanagement
1. Introduction
Watermelonisoneofthemostimportantfruitsworldwide. InChina,watermeloncultivationhas
beenincreasingyearbyyearduetoitscomparativelyhigheconomicvalueandincreasingconsumption,
but it is susceptible to fusarium wilt disease in continuous monocropping cultivation systems [1].
WatermelonfusariumwiltcausedbyFusariumoxysporumf.sp.niveum(FON)isadestructivesoil-borne
disease leading to serious economic losses and limiting watermelon production throughout the
world[2].
Importantly,FONisdifficulttoeliminatefromsoil. Laboratorystudieshasreportedthatthree
biologicalformsofF.oxysporumcouldsurvivemorphologicallyunchangedfor11ormoreyears[3].
Morethan50%ofFusariumspeciesaretoxigenicandproduceharmfulsecondarymetabolites(SM),
suchasthepigmentsfusarubinsandbikaverin[3],aswellasthemycotoxins,fumonisins,fusarins[4],
andfusaricacid[5,6]. Intheprogressionoftheinfection,fusariumspeciesdamagehostplantsthrough
intrusionofhyphaeintohostvascularsystem,secretionofhydrolyticenzymesandmycotoxinswhich
Molecules2017,22,2053;doi:10.3390/molecules22122053 www.mdpi.com/journal/molecules
Molecules2017,22,2053 2of13
leadtowatermelonrootandstemnecrosis,cellularapoptosis,foliarwiltingandthendeathwithina
fewweeks[7,8].
Due to the fact FON can survive for several years in soil as chlamydospores and many hosts
aresymptomless[9],fusariumwiltisdifficulttocontrol,althoughtraditionalcroprotationsarean
effectivestrategytocontrolFON[10]. Formanyotherpathogens,theapplicationoffungicidehasbeen
acommonandsuccessfulmethodfordiseasemanagement,however,theapplicationoffungicides
shouldbephasedoutbecauseoftheincreasingattentiontoenvironmentalandhumanhealthand
thedevelopmentoffungicideresistance[9,11]. Someexperimentshavedocumentedthatfungicides
have drastic effects on the soil biota and most cause a decline in soil fertility [12]. Consequently,
alternativecontrolstrategiesforthisdiseasewouldbeusefulandurgentinreducinghealthhazards,
environmentaldamageandthepollutionpotential[13]. Biofungicidesmaybeanattractivealternative
methodforcontrollingthisdisease.
Biofungicides are living organisms (plants, microscopic animals such as nematodes, and
microorganisms, including bacteria, fungi and viruses) or natural products derived from these
organisms,thatareusedtosuppresspestpopulationsandpathogens[14]. Firstly,manystudieshave
reportedthatusingnonpathogenicFusariumspp. fusariumwiltscouldbecontrolled[15]. Secondly,
someantagonisticstrainsshowedhighbioactivitiesagainstfusariumwilt,suchasTrichodermaspp.[16],
Bacillusspp.[17]andAspergillusspp.[18].Thirdly,plantextractsorphytochemicals,suchasessentialoils,
steroids,phenolicacidsandalkaloidshadgoodantifungalactivities. Forexample,ithasbeenreported
thatessentialoilsfrompepper,cassiatree,mustardandclovecouldsuppressdiseasedevelopment
causedbyF.oxysporumf. sp.melonisonmuskmelonandreducethepopulationdensityofpathogenin
greenhouseexperiments[19].Wuetal. foundmanybenzoicacidanaloguessuchasgallicacid,ferulic
acidandp-hydroxybenzoicacidstronglyinhibitedFONgrowth[20–22].
Cuminic acid (p-isopropylbenzoic acid), isolated from the seed of Cuminum cyminum. L. [23],
belongstothebenzoicacidchemicalgroup[24]. Inapreviousstudy,ithasbeenreportedthatcuminic
acid possessed good inhibition towards several plant pathogens, such as Sclerotinia sclerotiorum,
Phytophthoracapsici,Rhizoctoniacerealis,andFusariumoxysporum. EC valuesofcuminicacidagainst
50
mycelialgrowthofP.capsicandS.sclerotiorumwereonly19.7µg/mLand7.3µg/mL,respectively[25],
whichwerelowerthantheEC valueofotherbenzoicacidderivativesinpreviousreports[20–22].
50
In pot experiments, after the application of cuminic acid at 1000 µg/mL, control efficacies of over
60%againstP.capsicandS.sclerotiorumwereobtained,whichwascomparablewiththeefficacyof
metalaxyl(250µg/mL)[24]andprocymidone(100µg/mL)[25].
Consideringthebroadspectrumandsignificantantifungalactivityofcuminicacidandthedifficult
managementoffusariumwilt,it’snecessarytoevaluatecuminicacidasapotentialbiopesticideto
controlfusariumwiltonwatermelon. Theobjectivesofthisresearchwereto:(a)determinetheeffectof
cuminicacidonFONcolonygrowth;(b)evaluatetheeffectofcuminicacidonthemorphologicaland
physiologicalcharacteristicsofFON;(c)testtheefficacyofcuminicacidagainstFONinwatermelon
plantingreenhouseexperiments,andstudytheeffectofcuminicacidontheantioxidantdefensive
enzymesinwatermelonplantsubjectedtofusariumwilt;(d)examinetheeffectofcuminicacidon
differencesinthetranscriptionlevelsforFONgenesassociatedwiththebiosynthesisoffusaricacid
andbikaverinbyaquantitativeRT-PCRmethod.
2. Results
2.1. EffectofCuminicAcidonFONColonyGrowth
TheeffectsofvariousconcentrationsofcuminicacidonthemycelialgrowthofFONareshownin
Table1,andcuminicacidwerefoundtoinhibitthemycelialgrowthofcuminicacidinadose-dependent
manner.MycelialgrowthofFONwasstronglyinhibitedbycuminicacidatarelativelowconcentration
of25µg/mL.Basedonlog-transformationanalysis,EC EC andEC valueswerecalculatedas5.6,
30, 50 70
22.53and91.3µg/mL,respectively.
Molecules2017,22,2053 3of13
Molecules 2016, 21, 2053 3 of 12
TaTbalbel e1.1 T.hTeh eefeffefcetc toof fccuummiinniicc aacciidd oonn FFOONN ccoolloonnyyg grroowwthth..
Confidence Interval of EC50
CompoCuonmdps oundsRegreRsseigorne sEsqiounaEtiqouna t ion ECE50C (5μ0g(·µmgL·m−1L) −1) Confiden(pc(pe <<I 0n0.t0e.05r5)v )alofEC50 χ2χ2
Cuminic acid Y = 3.83 + 0.86X 22.53 17.85–25.96 4.83
Cuminicacid Y=3.83+0.86X 22.53 17.85–25.96 4.83
Note: Data represents the mean value of triplication. The EC50 was assessed based on
logN-toraten:sDfaotramreaptrieosnen atsntahleymsiesa. nYv: aPluroeboiftt-riinphlicibatiitoion.nT (h%e)E;C X50: lwoags-dasosesses.e dbasedonlog-transformationanalysis.
Y:Probit-inhibition(%);X:log-dose.
2.2. Effect of Cuminic Acid on Mycelial Morphology of FON
2.2. EffectofCuminicAcidonMycelialMorphologyofFON
A cAlecalre aerffeefcfte cotf otfhteh ceucmuminiinci cacaicdid oonn mmyycceeliliaa mmoorrpphhoollooggyy ooff FFOONN wwaasso obbsseervrveded( F(iFgiugruere1) 1.)A. Afteftrer
7 d7aydsa’y isn’ciuncbuabtiaotnio, ntr,etraetamtmenetn wtwitihth cucummininicic aacciidd aatt tthhee EECC5500,, tthhee ccoolloorr ooff mmyycceelilaiaw waassv ivsiisbilbeleli glihgtherter
thatnh acnonctornotlr o(Fli(gFuigrue r1ea1,da,)d i)ni nPPDDAA pplalatetess, ,wwhhililee tthhee mmyycceelliiaa ooff tthhee ccoonnttrroollw weereren natautruarla,lu, nuinseisreiartiaete
anda nudnuifnoirfmor m(Fi(gFuigruer 1eb1,bc),c b)yb ySESEMM. .FFoorr sstrtraaiinnss aammeennddeedd wwiitthh ccuummiinnicica accidida attt htheeE CEC505,0m, mycyecleialiaw wereere
sevseerveelrye ldyedfoerfmoremde,d t,wtwiniinnign ganandd clculustseterreedd ((FFiigguurree 11ee,,ff))..

Figure 1. Effect of cuminic acid on mycelia morphology of FON. (d–f) Untreated plates; (a–c) Plates
Figure1.EffectofcuminicacidonmyceliamorphologyofFON.(d–f)Untreatedplates;(a–c)Plates
treatrteeadt ewdiwthi tchucmuimniicn iaccaidci data EtCEC50 vavlauleu e(2(22.25.353 μµgg/m/mLL).) V.Vaalulueess aarree mmeeaannss aanndd ssttaannddaarrdde errrororsr.s.
50
2.3. EffectofCuminicAcidonCellMembranePermeabilityofFON
To confirm the membrane-disruption effects of cuminic acid on the hyphal cells, the relative
conductivity of the mycelia treated with cuminic acid were determined. As shown Figure 2, the
relativeconductivityofthemyceliatreatedwithcuminicacidincreasedgraduallyduringincubation,
beingabout45.78%higherthanthatofcontrolafter120minincubation.

Figure 2. Mycelial relative conductivity of FON with or without cuminic acid treatment at the
concentration of EC50 value (22.53 μg/mL). Values are means and standard errors.
Molecules 2016, 21, 2053 3 of 12
Table 1. The effect of cuminic acid on FON colony growth.
Confidence Interval of EC50
Compounds Regression Equation EC50 (μg·mL−1) χ2
(p < 0.05)
Cuminic acid Y = 3.83 + 0.86X 22.53 17.85–25.96 4.83
Note: Data represents the mean value of triplication. The EC50 was assessed based on
log-transformation analysis. Y: Probit-inhibition (%); X: log-dose.
2.2. Effect of Cuminic Acid on Mycelial Morphology of FON
A clear effect of the cuminic acid on mycelia morphology of FON was observed (Figure 1). After
7 days’ incubation, treatment with cuminic acid at the EC , the color of mycelia was visible lighter
50
than control (Figure 1a,d) in PDA plates, while the mycelia of the control were natural, uniseriate
and uniform (Figure 1b,c) by SEM. For strains amended with cuminic acid at the EC , mycelia were
50
severely deformed, twining and clustered (Figure 1e,f).

Figure 1. Effect of cuminic acid on mycelia morphology of FON. (d–f) Untreated plates; (a–c) Plates
Molecules2017,22,2053 4of13
treated with cuminic acid at EC50 value (22.53 μg/mL). Values are means and standard errors.
Molecules 2016, 21, 2053 4 of 12
2.3. Effect of Cuminic Acid on Cell Membrane Permeability of FON
To confirm the membrane-disruption effects of cuminic acid on the hyphal cells, the relative
conductivity of the mycelia treated with cuminic acid were determined. As sh own Figure 2, the
relative conductivity of the mycelia treated with cuminic acid increased gradually during
FigFuirgeu r2e. 2M. yMceylicaell iarellraetliavteiv ceocnodnudcuticvtiivtyit yofo fFFOONN wwitihth oorr wwiitthhoouutt ccuummiinniicc aaccidid trtereaatmtmenentta tatt htehe
incucboantcciooennnct,er nbatteriioantnigo o naf obEfoCEu5C0t v540a5vlu.a7el8u (%e22( 2h.52i3.g5 μh3geµ/rgm /thLma)L.n V) .taVhlaualetu seo saf racero emnmtereaoannls s aaafntneddr s s1ttaa2nn0dd maarriddn ee irrnrroocrruss.b. ation.
2.4. Glycerol Content of Mycelia
2.4. GlycerolContentofMycelia

After treatment with cuminic acid, the content of glycerol was always significantly higher than
Aftertreatmentwithcuminicacid,thecontentofglycerolwasalwayssignificantlyhigherthan
theth ceocnotrnotrl owlwithitohuotu ctucummininici caaccidid ttrreeaattmmeenntt ((FFiigguurree 33)).. AAss tthheec coonncceenntrtaratitoionnw wasasin icnrceraesaesde,dth, ethgel ygcleyrcoelrol
concotenntet notf othfeth meymceylcieal iinacirnecarseeadse odvoevr etirmtiem. Te.hTeh gelygcleyrcoelr ocolncotenntetsn ftsorf othrrteher eceocnocnencetrnattriaotniosn osf ocfucmuimniicn iaccid
(ECac30i,d E(CEC50 3a0n,dE CE5C07a0)n sdigEnCif7i0c)ansitglyn iifinccarneatlsyedin bcrye 7a9se.3d%b,y 31739..536%%,3 a1n3d.5 663%1.a5n7d%6, 3re1s.5p7e%ct,ivreeslyp ectively

Figure 3. Glycerol content of mycelia of FON with or without cuminic acid treatment at
Figure3.GlycerolcontentofmyceliaofFONwithorwithoutcuminicacidtreatmentatconcentrations
coonfceEnCtra(t5io.6nµs go/f mECL)3,0 E(5C.6 μ(2g2/.m53Lµ),g E/Cm5L0 )(2a2n.d53E μCg/m(9L1.)3 aµngd/ EmCL7)0. (B9a1r.s3 dμegn/omteLt)h. eBsatrasn ddeenrrootre othfeth srteaend
30 50 70
error of three experiments. Data represents means of three replicates with standard deviation. Data
experiments.Datarepresentsmeansofthreereplicateswithstandarddeviation.Data(means±SD,
(means ± SD, n = 3) followed by the same letters in the row show no significant differences (small
n=3)followedbythesamelettersintherowshownosignificantdifferences(smallletters,p<0.05).
letters, p < 0.05).
2.5. MycotoxinConcentrationofFONinLiquidCulture
2.5. Mycotoxin Concentration of FON in Liquid Culture
Mycotoxin(mainlyfusaricacid)concentrationofFONinPDBwassuppressedbycuminicacid
Mycotoxin (mainly fusaric acid) concentration of FON in PDB was suppressed by cuminic acid
treatmentinaconcentrationdependentmanner. Significantsuppressionwasfoundevenatthelower
treEaCtmevnat luine cao ncocenncternattrioanti.oTnh deempyecnodteonxtin mcoanncneenrt.r aStiigonnifwicaasndt escurepapsreedssbiyon2 4w.5a7s– 6f6o.2u2n%d ceovmenp aaret dthe
50
lowweitrh EcoCn5t0r ovla(lFuigeu rceon4)c.entration. The mycotoxin concentration was decreased by 24.57–66.22%
compared with control (Figure 4).

Figure 4. Mycotoxin production (mainly fusaric acid) concentration in FON with cuminic acid
treatments at concentrations of EC30 (5.6 μg/mL), EC50 (22.53 μg/mL) and EC70 (91.3 μg/mL) in liquid
culture. Bars denote the stand error of three experiments. Data represents means of three replicates
with standard deviation. Data (means ± SD, n = 3) followed by the same letters in the row show no
significant differences (small letters, p < 0.05).
Molecules 2016, 21, 2053 4 of 12
2.3. Effect of Cuminic Acid on Cell Membrane Permeability of FON
To confirm the membrane-disruption effects of cuminic acid on the hyphal cells, the relative
conductivity of the mycelia treated with cuminic acid were determined. As shown Figure 2, the
relative conductivity of the mycelia treated with cuminic acid increased gradually during
incubation, being about 45.78% higher than that of control after 120 min incubation.
2.4. Glycerol Content of Mycelia
After treatment with cuminic acid, the content of glycerol was always significantly higher than
the control without cuminic acid treatment (Figure 3). As the concentration was increased, the glycerol
content of the mycelia increased over time. The glycerol contents for three concentrations of cuminic acid
(EC , EC and EC ) significantly increased by 79.3%, 313.56% and 631.57%, respectively
30 50 70

Figure 3. Glycerol content of mycelia of FON with or without cuminic acid treatment at
concentrations of EC30 (5.6 μg/mL), EC50 (22.53 μg/mL) and EC70 (91.3 μg/mL). Bars denote the stand
error of three experiments. Data represents means of three replicates with standard deviation. Data
(means ± SD, n = 3) followed by the same letters in the row show no significant differences (small
letters, p < 0.05).
2.5. Mycotoxin Concentration of FON in Liquid Culture
Mycotoxin (mainly fusaric acid) concentration of FON in PDB was suppressed by cuminic acid
treatment in a concentration dependent manner. Significant suppression was found even at the
lower EC value concentration. The mycotoxin concentration was decreased by 24.57–66.22%
50
comMopleacureleds2 w017it,h22 c,2o0n53trol (Figure 4). 5of13

FigFuigrue re4. 4M. yMcyoctooxtoinx inprpordoudcutciotino n(m(maaininlyly ffuussaarriicc aacciidd)) ccoonncceennttrraattiioonn inin FFOONNw witihthc ucmuminiincica ciadcid
tretartematemnetsn tast actocnocnencetnratrtiaotinosn osfo EfCE3C0 3(05.(65 μ.6gµ/mg/Lm), LE)C,E50C (5202.(5232 .μ53g/µmgL/)m aLn)da EnCd7E0 (C9710.3( 9μ1g.3/mµgL/) minL l)iqinuid
culltiquureid. Bcaurlstu dree.noBtaer tshdee sntoatnedt heerrsotra nodf tehrrreoer eoxfptehrriemeeenxtpse. rDimatean trse.prDeasteantrse pmreesaennst somf teharnese orefpthlirceaetes
wirtehp slitcaantdesarwdi tdhesvtiaantdioanrd. Ddaevtaia (tmioena.nDsa ±ta S(Dm,e nan =s 3±) SfoDll,onw=e3d) bfoyl ltohwee sdambyet lheettsearms einl etthteer sroinwt hsheorwow no
sigsnhiofiwcannot dsiigfnfeifirecnacnetsd (isffmeraelnl cleestt(esrms,a pll <le 0tt.0er5s),. p<0.05).
2.6. GreenhouseExperiments
The effect of cuminic acid on FON was evaluated under greenhouse conditions (Table 2).
Ourexperimentsdemonstratedthatcuminicacidatallconcentrationshasasignificantsuppression
effectonFON.Inplantsundercuminicacidat2000µg/mLweobtaineda21.5%diseaseindexand
74.5%efficacy,whichwasnotsignificantlydifferencefromcarbendazimat1000µg/mL.However,in
plantsundercuminicacidat1000µg/mL,thediseaseindexandefficacywere38.8%and54.5%,which
waslowerthanundercarbendazimat1000µg/mL.
Table2.Effectofcuminicacidoncontroloffusariumwiltonwatermelon.
Compound Concentration DiseaseIndex(%) Efficacy(%)
1000µg/mL 38.8±2.5b 54.5±2.3b
Cuminicacid 2000µg/mL 21.4±1.51c 74.5±1.5a
3000µg/mL 24.8±1.15c 71.9±1.22a
Carbendazim 1000µg/mL 23.2±1.18c 72.8±1.4a
Water ___ 85.5±3.5a ___
Note:Resultsarethemeansof10watermelonplantsandfromtwoindependentexperiments.Meansfollowedby
thesameletterswerenotsignificantdifferentaccordingtoLSD(α=0.05).
2.7. AssayofDefenseEnzymeActivitiesandMalondialdehyde(MDA)Content
Theactivitiesofsuperoxidedismutase(SOD),peroxidase(POD)andcatalase(CAT)areshown
inFigure5a–candthecontentofMDAisshowninFigure5d. ActivitiesofSOD,POD,CATunder
cuminicacidtreatmentinwatermelonleaveswereenhancedincomparisonwithcontrol,exceptfor
cuminicacidtreatmentat4000µg/mLinPODactivities. SODandPODactivitiesexperiencedthe
trendinalltheplants,andthehighestenzymeactivity(43.65%)wasfoundintreatmentwithcuminic
acidat1000µg/mL(Figure5a)anda27.87%increasewasobservedcomparedwithcontrol(Figure5b).
As for CAT activity, the highest enzyme activity was found after cuminic acid treatment at
2000µg/mL,whichcorrespondedtoa59.55%(Figure5c)increasecomparedtocontrol. However,
MDAcontentdecreasedsteadilyinallthesamplesduringthewholeexperimentalperiodwiththe
increasedconcentrationofcuminicacid.
Molecules 2016, 21, 2053 5 of 12
2.6. Greenhouse Experiments
The effect of cuminic acid on FON was evaluated under greenhouse conditions (Table 2). Our
experiments demonstrated that cuminic acid at all concentrations has a significant suppression effect
on FON. In plants under cuminic acid at 2000 μg/mL we obtained a 21.5% disease index and 74.5%
efficacy, which was not significantly difference from carbendazim at 1000 μg/mL. However, in
plants under cuminic acid at 1000 μg/mL, the disease index and efficacy were 38.8% and 54.5%,
which was lower than under carbendazim at 1000 μg/mL.
Table 2. Effect of cuminic acid on control of fusarium wilt on watermelon.
Compound Concentration Disease Index (%) Efficacy (%)
1000 μg/mL 38.8 ± 2.5b 54.5 ± 2.3b
Cuminic acid 2000 μg/mL 21.4 ± 1.51c 74.5 ± 1.5a
3000 μg/mL 24.8 ± 1.15c 71.9 ± 1.22a
Carbendazim 1000 μg/mL 23.2 ± 1.18c 72.8 ± 1.4a
Water ___ 85.5 ± 3.5a ___
Note: Results are the means of 10 watermelon plants and from two independent experiments. Means
followed by the same letters were not significant different according to LSD (α = 0.05).
2.7. Assay of Defense Enzyme Activities and Malondialdehyde (MDA) Content
The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) are shown
in Figure 5a–c and the content of MDA is shown in Figure 5d. Activities of SOD, POD, CAT under
cuminic acid treatment in watermelon leaves were enhanced in comparison with control, except for
cuminic acid treatment at 4000 μg/mL in POD activities. SOD and POD activities experienced the trend
in all the plants, and the highest enzyme activity (43.65%) was found in treatment with cuminic acid at
Molecules2017,22,2053 6of13
1000 μg/mL (Figure 5a) and a 27.87% increase was observed compared with control (Figure 5b).
Molecules 2016, 21, 2053 6 of 12
2.8. Quantitative RT-PCR
To confirm whether the biosynthesis of fusaric acid and pigment in FON would be affected by
cuminic acid, expression of genes including the ones involved in the synthesis of bikaverin (Bike1,
Bike2 and Bike3), fusaric acid (FUB1, FUB2, FUB3 and FUB4) and components of a velvet-like
complex (Lae1 and Vel1) were quantified (Table 3). Relative to expression in the wild-type strain
(Figure 6), synthesis genes of bikaverin (Bike1, Bike2 and Bike3) and fusaric acid (FUB1, FUB2, FUB3
and FUB4) both exhibited decreased expression compared with the internal control.
Table 3. qRT-PCR primers applied in this study.
Gene Name Accession Number Primer Sequence (5′-3′)
Forward CGGTATCTGTGGTGGTGTC
Bike1 AJ278141
Reverse TCGGGAGGTGATGTTGTG
Forward TGCCTGCTCCACAGTCTACG
Bike2 AM229668
Reverse GCCAATCTTGACCGCCAC
Forward CGCCAAAGTCATCAAGGA
Bike3 AM229667
Reverse AGGCTCAGGCACCACAAA
Figure 5. SOD (a); POD (b) and CAT (c) activFiotriewsa ardn d MDA AacCtiTvTitCyG (CdC) ToCf GthTeC wAaTtCerTmCe lon leaves
Figure5.FSUOBD1 (a); POD(b)FFanUdJ_C02A1T05( c)activitiesandMDAactivity(d)ofthewatermelonleaves
treated with cuminic acid at 0, 1000, 2000 and 30R0e0v μegrs/em L, respGeActAivCeClyC. ADGatCa AreTpCreAsAenAtCs mTTeAanTs of three
treatedwithcuminicacidat0,1000,2000and3000µg/mL,respectively. Datarepresentsmeansof
repthlirceaetiroenpslFi UcwaBitti4ho n sstawnidtharsdta dnedFvaFirUadtJi_do0en2v.1 iD0a8tai otan (.mDeaFataonrs(wm ±a eSradDn ,s n± =S 3D) ,fConlA=loC3wC)efCodTl lbToyGw tCehdTeC bsAyamTthCee AlseCattmAereGsl ientt ethrse irnow
shothwe rnoow sisghnoiwficnaonst idginfiffiecreanntcedsi f(fsemreanlcle lse(tstemrsa,l lplR e<et tv0e.er0rs5s,e)p. <0C.0G5T).AAAAATATCCTTCCGAATAATC
Forward GCCAACTGCTGTCACTAT
FUB2 FFUJ_02106
Reverse TTCCGAGGTGGAGATTAG
As for CAT activity, the highest enzyme activity was found after cuminic acid treatment at
2.8. QuantitativeRT-PCR
Forward CCCGATACACCATACCCT
2000 μg/mL, wFhUiBch3 correspondFFeUdJ _to02 1a0 75 9.55% (Figure 5c) increase compared to control. However,
ToconfirmwhetherthebiosynthesisoffuRseavreicrsaec idandpCiCgAmAeCnTtTinCTFTOGNCCwGoTuGldAGbe affectedby
MDA content decreased steadily in all the samples during the whole experimental period with the
Forward TATTGGTACGGGCACAGG
cuminicacid,Leaxe1p ressionofgFeVnEeGs_i0n0c5l3u9d ingtheonesinvolvedinthesynthesisofbikaverin(Bike1,
increased concentration of cuminic acid. Reverse GGCATAAAGCCAGGAGGA
Bike2andBike3),fusaricacid(FUB1,FUB2,FUB3andFUB4)andcomponentsofavelvet-likecomplex
Forward CTACTAAGGAGGAAAGGGACT
(Lae1andVel1V)elw1 erequantifieFdN(5T4a8b1l4e2 3). Relativetoexpressioninthewild-typestrain(Figure6),
Reverse TCCATCAAACCAGGAAACT
synthesisgenesofbikaverin(Bike1,Bike2andBike3)andfusaricacid(FUB1,FUB2,FUB3andFUB4)
Forward GAGGGACCGCTCTCGTCGT
Related actin gene Foxq13729
bothexhibiteddecreasedexpressioncomparedRwevitehrsteh eintGeGrnAaGlcAoTnCtrCoAl.GACTGCCGCTCAG

Figure 6. Gene expression level of synthesis of fusaric acid (FUB1, FUB2, FUB3 and FUB4) and bikaverin
Figure6.Geneexpressionlevelofsynthesisoffusaricacid(FUB1,FUB2,FUB3andFUB4)andbikaverin
(Bike1, Bike2 and Bike3), and components of a velvet-like complex (Lae1 and Vel1) relative to without
(Bike1,Bike2andBike3),andcomponentsofavelvet-likecomplex(Lae1andVel1)relativetowithout
treatment cuminic acid. Values are the means ± standard error (SE) of three repeated experiments.
treatmentcuminicacid.Valuesarethemeans±standarderror(SE)ofthreerepeatedexperiments.
Expressions of FUB3, FUB4 and Bike2 were about 0.88, 0.77 and 0.46 fold lower relative to the
internal control. However, genes of components of a velvet-like complex (Lae1 and Vel1) exhibited
significantly increased expression (1.95 and 1.37-fold).
Molecules2017,22,2053 7of13
Table3.qRT-PCRprimersappliedinthisstudy.
GeneName AccessionNumber Primer Sequence(5(cid:48)-3(cid:48))
Forward CGGTATCTGTGGTGGTGTC
Bike1 AJ278141
Reverse TCGGGAGGTGATGTTGTG
Forward TGCCTGCTCCACAGTCTACG
Bike2 AM229668
Reverse GCCAATCTTGACCGCCAC
Forward CGCCAAAGTCATCAAGGA
Bike3 AM229667
Reverse AGGCTCAGGCACCACAAA
Forward ACTTCGCCTCGTCATCTC
FUB1 FFUJ_02105
Reverse GAACCCAGCATCAAACTTAT
Forward CACCCTTGCTCATCACAG
FUB4 FFUJ_02108
Reverse CGTAAAAATATCCTTCCGAATAATC
Forward GCCAACTGCTGTCACTAT
FUB2 FFUJ_02106
Reverse TTCCGAGGTGGAGATTAG
Forward CCCGATACACCATACCCT
FUB3 FFUJ_02107
Reverse CCAACTTCTTGCCGTGAG
Forward TATTGGTACGGGCACAGG
Lae1 FVEG_00539
Reverse GGCATAAAGCCAGGAGGA
Forward CTACTAAGGAGGAAAGGGACT
Vel1 FN548142
Reverse TCCATCAAACCAGGAAACT
Forward GAGGGACCGCTCTCGTCGT
Relatedactingene Foxq13729
Reverse GGAGATCCAGACTGCCGCTCAG
ExpressionsofFUB3,FUB4andBike2wereabout0.88,0.77and0.46foldlowerrelativetothe
internalcontrol. However,genesofcomponentsofavelvet-likecomplex(Lae1andVel1)exhibited
significantlyincreasedexpression(1.95and1.37-fold).
3. Discussion
In previous study, cuminic acid and cuminic aldehyde, as the major bioactive constituents of
C. cyminum seed, were reported to possess broad spectrum antifungal activities [23,25]. Cuminic
acidasarepresentativechemicalofthebenzoicacidgroup, alsoexhibitedasignificantantifungal
activityandenhancedthedefensecapacityofplantsagainstPhytophthoracapsic[24]. Thisstudywas
focusedonthebiochemistryandphysiologyalterationsinFusariumoxysporumf. sp. niveummediated
bycumunicacid,andconfirmsthatthischemicalhasavaluefordevelopmentandutilizationasa
potentialbiofungicide.
Inthecurrentstudy,resultsshowedthatthegrowthofFONwasstronglyinhibitedbycuminic
acidinaconcentration-dependentmanner,withanEC valueof22.53µg/mL.Cuminicacidexhibited
50
asignificanthigherantifungalactivityinPDAplatescomparedwithotherchemicalsofthebenzoic
acidgroup,suchascinnamicacid[26],gallicacid[20]andsinapicacid[8]. Interestingly,wefoundthat
thecolorofmyceliainthestrainstreatedwithcuminicacidattheEC inPDAplateswasvisiblylighter
50
thanthatincontrol,andmyceliawereabnormalbySEM.Inaddition,thecellmembranepermeability
andglycerolcontentweresignificantlyenhanced,whichwasconsistentwiththeactivityofcuminic
acidagainstPhytophthoracapsic[24],whichindicatedthatthemechanismofactionofcuminicacid
againstplantpathogensmightbethroughdamagingthemycelialstructureandinducingintracellular
plasmaleakage.
Mycotoxin (mainly fusaric acid) production is widely distributed among the whole Fusarium
species [27], particularly pathogenic strains of F. oxysporum. It is an important pathogenic factor
causingwiltdiseasesinvariousplants,suchaswatermelon,tomatoandcucumber. Importantly,there
isanincreasedvirulencetothehostwiththeincreaseofmycotoxinproductionbyF.oxysporum[28].
Intheinitiationofinfectionandsymptomdevelopment,thetoxinsproducedbypathogenswerea
Molecules2017,22,2053 8of13
pathogenicitydeterminantinFON[29]. Inthecurrentstudy,asignificantreductionofmycotoxinwas
observedaftertreatmentwithcuminicacid,indicatingthatcuminicacidcouldreducethepathogenicity
ofFONbyinhibitingthesecretionofmycotoxins(mainlyfusaricacid). Accordingtothereductionof
pigmentsandfusaricacidproduction,weselectedninegenesassociatedwiththebiosynthesisoffusaric
acid[5,30]andpigments[3]todeterminewhetherFONtreatmentwithcuminicacidwouldaffectthe
biosynthesisoffusaricacidandpigmentbyquantitativeRT-PCR.Synthesisgenesofbikaverin(Bike1,
Bike2andBike3)andfusaricacid(FUB1,FUB2,FUB3andFUB4)werebothdownregulatedcompared
withthecontrol,whichwasconsistentwithpreviousresults. Previousstudieshavedocumentedthat
genesofcomponentsofavelvet-likecomplex(Lae1andVel1)participatedinthebiosynthesisofand
modulatetheexpressionoffusaricacid[3,5]. However,thesesignificantlyoverexpressedgenesinthis
studystillneedtobefurtherstudied.
Ingreenhouseexperimenta,cuminicacidatallconcentrationshadasignificantsuppressiveeffect
onFON.Aftertreatmentwithcuminicacidat2000µg/mL,thediseaseindexandefficacywerenot
significantlydifferentfromthoseaftertreatmentwithcarbendazimat1000µg/mL,indicatingthat
cuminicacidhassignificantantifungalactivitiesagainstFONandpossessespotentialasabiofungicide.
ReactiveOxygenSpecies(ROS)areharmfultoseveralcellularcomponentsandtheycancause
lipid peroxidation and induce membrane injury, then resulting in cell senescence [31]. Moreover,
antioxidantenzymessuchasSOD,POD,CATplaycrucialrolesinsuppressingoxidativestress. When
ROS increases, SOD directly catalyzes the conversion of O2− into H O , which is then converted
2 2
into water and oxygen by CAT [32], while POD decomposes H O into H O and O . Meanwhile,
2 2 2 2
theenzymePODparticipatesintheconstruction,rigidificationandlignificationofcellwalls,which
protectsplanttissuesfromdamage[33]. Inaddition,thehighMDAreflectsthehigherproductionof
H O andROS[34]. Inthepresentstudy,activitiesofSOD,POD,CATinwatermelonleavesunder
2 2
cuminicacidtreatmentweresignificantlyenhancedincomparisonwithcontrol. Correspondingly,
adecreasedMDAcontentincuminicacidtreatedwasobserved. Thesedataclearlysuggestedthat
cuminicacidcouldpreventFONdevelopmentandreducetheleveloflipidperoxidationthrougha
mechanisminvolvingactivationofantioxidantdefensiveenzymes.
In conclusion, cuminic acid has a high inhibition effect in the mycelial growth of FON on
watermelon plants. Although further work is needed to entirely understand the mode of action
ofcuminicacidagainstFON,wecanconcludethatcuminicacidusedinthisstudycouldbedeveloped
asapromisingbiofungicide.
4. MaterialsandMethods
4.1. PathogenStrainsandFungicides
Fusariumoxysporumf. sp. niveumwerecollectedfrominfectedwatermelonplantandmaintained
onpotatodextroseagar(PDA)[24]mediumprovidedbytheLaboratoryofResearchandDevelopment
CenterofBiorationalPesticide,NorthwestA&FUniversity. Cuminicacid(98%)andcarbendazim
(98.0%)usedintheexperimentwerepurchasedfromtheSigmaCo. (St. Louis,MO,USA).
4.2. EffectofCuminicAcidonFONColonyGrowth
Theeffectofcuminicacidoncolonygrowthwasdeterminedasfollows:PDAmediawasamended
with a series of cuminic acid addtions at the final concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and
100µg/mL.A5-mmmycelialplugtakenfromtheleadingedgeof7-day-oldcolonieswasinoculated
intothecenteroftheamendedPDAmedium. Platewasincubatedinagrowthchamberat28◦Cfor
7days,colonydiameterwasdeterminedbymeasuringtheaverageoftwoperpendiculardirections
on each plate. According to previous studies [24], the EC values were calculated by regressing
50
percentagegrowthinhibitionagainstthelogofcuminicacidconcentration. Eachconcentrationwas
testedthricewiththreereplicates.
Molecules2017,22,2053 9of13
4.3. EffectofCuminicAcidonMycelialMorphologyofFON
Myceliaplugscutfromthemarginof7-day-oldcolonywereplacedonPDAplatescontaining
cuminicacidattheEC (22.5µg/mL)forinhibitionofmycelialgrowth. Controlwasplateswithout
50
cuminicacid. After7daysat28◦C,themarginofmediumarea(10mm × 10mm)wasplacedon
slideglass. High-resolutionimagesofmycelialmorphologychangesincuminicacidtreatedsamples
wereobtainedbyscanningelectronmicroscope(SEM,JSM-6360LV,JEOL,Tokyo,Japan)[35]. Three
replicateswereprocessedandtheexperimentwasrepeatedtwice.
4.4. EffectofCuminicAcidonCellMembranePermeabilityofFON
Mycelialcellmembranepermeabilitywasexpressedastherelativeconductivity. Tenmycelial
plugswereaddedinto250-mLflaskscontaining100mLofpotatodextrosebroth(PDB).Theflasks
wereshakenat180rpmand28◦Cfor5days,partialflaskswereamendedwithcuminicacidatthe
EC (22.5 µg/mL). Control was flasks without cuminic acid. The flasks were shaken for 2 days,
50
myceliasamples(0.5gofmycelia)werecollectedbyfiltrationthroughfilterpaper,andsuspended
in20mLofdistilledwater. Conductivityofthetreatedwaterwasmeasuredafter5,30,60,120,180,
240minwithaconductivitymeter(CON510Eutech/Oakton,BukitBatok,Singapore),after240min,
finalconductivitywasdeterminedbymyceliawereboiledfor5mintocompletelykillthetissuesand
releaseallelectrolytesandcooledto25◦C.Theexperimentwiththreereplicateswasrepeatedthree
times. Therelativeconductivitywascalculatedasfollowingformula[36]:
Conductivityatdifferenttimes
Relativeconductivity= ×100% (1)
Finalconductivity
4.5. GlycerolContentofMycelia
Glycerol content was determined using the described method [37] with minor modifications.
Astandardcurveforglycerolwasobtainedaccordingtothedescribedmethod. ThemyceliaofFON
strainwaspreparedasdescribedabove. Inaddition,partialflaskswereamendedwithcuminicacidat
theEC (5.6µg/mL),EC (22.5µg/mL)andEC (91.3µg/mL).Mycelia(0.5gpersample)were
30 50 70
groundwithafrozenpestleandamortar. Thesamplewaswashedthricewithautoclaveddistilled
waterandtransferredto10-mLcentrifugetubes. Thevolumeforeachsamplewasadjustedto10mL
withwater. Accordingtothestandardcurve, glycerolcontentofthesamplewascalculated. Each
treatmentwasprocessedwiththreereplicates,andthetestwasrepeatedthreetimes.
4.6. MycotoxinConerntrationofMycelia
Mycotoxin(mainlyfusaricacid)concentrationwasdeterminedasdescribedbyWuetal.[26].
Astandardcurvewaspreparedwithstandardoffusaricacid(SigmaCo.). Tenmycelialplugswere
addedinto250-mLflaskscontaining100mLPDB.Theflaskswereshakenat150rpmand28◦Cfor
10 days, partial flasks were amended with cuminic acid at the EC , EC and EC , Control was
30 50 70
flaskswithoutcuminicacid. Theflaskswerecontinuedtoshakefor4days,andtheculturefiltrate
wascollectedafterfiltration. TheculturefiltratewasacidifiedtopH2with2MHCLandanequal
volume of ethyl acetate was added, followed by vigorous shaking for 1 min, and held for 30 min.
Thecollectedorganicphasewasthenplacedinanewtube. Theaboveprocedurewasrepeatedthree
times. Theorganicphasewascentrifugedat4000rpmfor15minandthesupernatantwascollected
anddried. Thedriedresiduewasredissolvedwithethylacetateto5mL.ByUVspectrophotometry
(UV-5100spectrophotometer,YuanXi,Shanghai,China),theOD wasmeasured. Eachtreatment
268
wasprocessedwiththreereplicates,andthetestwasrepeatedthreetimes.
Molecules2017,22,2053 10of13
4.7. PreparationofFONInoculumandtheWatermelonSeedlings
Tenmycelialplugswereaddedinto250-mLflaskscontaining100mLPDB.Theflaskswereshaken
at150rpmand28◦Cfor7–10days,dependingonexperiments.Thesporesuspensionswerefilteredand
adjustedto1×106cfu/mLwithahemacytometer(ThermalFisherScientific,Hennigsdorf,Germany).
The watermelon seeds were surface disinfected in sodium hypochlorite (5%, w/v) for 5 min,
washed twice with sterile water and then germinated in a 9 cm diameter sterile plates containing
wetfilterpaper. Thegerminatedseedsweresownintoeachnurserycups(4cmdiameter,6cmhigh)
containingasterilizedmixtureofnurserysoil,organicmanureandsand(2:1:1,w/w). Theseedlings
weregrowningreenhouse(naturallightat32/18◦C(day/night)and50–70%humiditywith). Seeding
werewateredwhenneeded.Watermelonseedlings(twocotyledonperiodstage)weretransplantedinto
pots(10cmdiameter,15cmhigh)containingenoughsterilizedmixtureofnurserysoil. Theseedings
(twotrueleavesstage)wereusedforallexperiments.
4.8. GreenhouseExperiments
Experimentswerecompletelyrandomizeddesignswithfivetreatments. Thefivetreatmentswere
asfollows: water,cuminicacidat1000,2000and4000µg/mL,andcarbendazimat1000µg/mL.10mL
oftreatmentwerepouredintotheplantrootwhen10mLofFONsporesuspension(106cfu/mL)was
inoculated. Duringtheprocedureoftreatment,plantrootswereinjuredbyminorvulnerablecuts.
After3weeksoftreatment,10watermelonplantspertreatmentwereexaminedanddiseaseseverity
wasmeasuredaccordingtoRojanetal.[38]. Thediseaseindexandefficacywerecalculatedaccording
toZhaoetal.[39]. Tenplantspertreatmentwereappliedandtheexperimentwasrepeatedtwice.
4.9. AssayofDefenseEnzymesActivitiesandMalondialdehyde(MDA)Content
Watermelon leaves cut from the plants treated with cuminic acid in the above section were
collectedonice. Threegofleafpersamplewerehomogenizedandsuspendedin8mLof0.5mM
phosphatebuffer, pH7.8, containing0.2mMEDTAand2%PVPPandcentrifugedat100,000rpm
for20at4◦Candtheresultingsupernatantsweredirectlyusedforassay. PODandSODactivities
weredeterminedbythemethodsofGarcia-Limonesetal.[40]. CATactivitywasassyedfollowing
theproceduresdescribedbySunetal.[41]. AsforMADcontent,theassaymixtureconsistedof5%
trichloroaceticacid(TCA)and0.6%thiobarbituricacid(TBA).MDAconcentrationwasdetermined
accordingtothemethodsdescribedbyHeathandPacker[2]. Fiveleavespertreatmentwereusedand
theexperimentwasconductedtwice.
4.10. QuantitativeRT-PCR
QuantitativeRT-PCRwascarriedoutinFONtoexaminedifferencesintranscriptlevelsforgenes
associatedwiththebiosynthesisoffusaricacid[5,30]andpigment[3]. ThemyceliaofFONstrain
waspreparedasdescribedin2.4. TotalRNAwasisolatedfrommyceliaofFONstrainusingaRNA
extractionkit(Takara,Dalian,China)accordingtothemanufacturer’sprotocol. First-strandcDNAwas
generatedfromRNAusingthePrimeScriptRTreagentkit(Takara). Inthisstudy,actingenewassetas
theinternalcontrol,andallappliedprimersforqRT-PCRwerelistedinTable3. qRT-PCRwascarried
outina20µLreactionmixturescontaining12µLSYBRPremixExTaqII(Takara),0.8µLofeachprimer
and1.6µLtemplatedDNA.AllquantitativeRT-PCRswereperformedwithanCFX96TMreal-time
detectionsystem(Bio-Rad, Hercules, CA,USA).Eachsamplewasruntwiceinthreeindependent
biologicalexperiments. Witharelatedactingene(Foxq13729)asthereferencegene,relativeexpression
levelsoftargetgeneswerecalculatedaccordingtothe2−(cid:52)(cid:52)Ctmethod[42].
4.11. StatisticalAnalysis
Inthisstudy,datafromrepeatedexperimentswerecombinedforanalysis,owningtothefactthe
variancesbetweenexperimentswerehomogeneous. AlldatawereprocessedandanalyzedusingSPSS