Antifungal Activity of an Abundant Thaumatin-Like Protein from Banana against Penicillium

16 Pages·2017·3.29 MB·English

molecules
Article
Antifungal Activity of an Abundant Thaumatin-Like
Protein from Banana against Penicillium expansum,
and Its Possible Mechanisms of Action
WenxiaoJiao,XiangxinLi,HandongZhao,JiankangCaoandWeiboJiang*
CollegeofFoodScienceandNutritionalEngineering,ChinaAgriculturalUniversity,17QinghuadongluRoad,
Beijing100083,China;[email protected](W.J.);[email protected](X.L.);
[email protected](H.Z.);[email protected](J.C.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:StevenL.Cobb (cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:7May2018;Accepted:29May2018;Published:14June2018
Abstract: Thaumatin-likeproteinfrombanana(designatedBanTLP)hasbeenpurifiedbyemploying
asimpleprotocolconsistingofdiethylaminoethylSephadex(DEAE–Sephadex)chromatography,
gel filtration on Sephadex G50, and reversed-phase chromatography. The purified protein was
identified by MALDI-TOF mass spectrometry, with an estimated molecular weight of 22.1 kDa.
BanTLP effectively inhibited invitro spore germination of Penicillium expansum, one of the main
postharvest pathogens in fruits. This study further investigated the antifungal properties and
underlyingmechanismsofBanTLPagainstP.expansum. ResultsdemonstratedthatBanTLPexhibited
antifungal activity in a wide pH range (4.0–10.0) at 20–50 ◦C. Propidium iodide (PI) influx and
potassium release confirmed that BanTLP induced membrane disruption of the test pathogen,
increasing the membrane permeability and disintegration of the cell. This led to cell death,
asevidencedbytheassaysofthiobarbituricacid-reactivespecies(TBARS)content,theproduction
of reactive oxygen species (ROS), and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence integrity.
UltrastructuralalterationsinP.expansumconidiaafterBanTLPtreatmentrevealedseveredamage
tothecellwall. TheseresultssuggestthatBanTLPpurifiedfrombananaexertsantifungalactivity
againstP.expansumbyinducingplasmamembranedisturbanceandcellwalldisorganization.
Keywords: banana;thaumatin-likeprotein;antifungalactivity;Penicilliumexpansum;mechanisms
ofaction
1. Introduction
Banana(Musaacuminate)isconsideredtobeoneofthemostimportantandpopularfruitsinthe
humandietduetoitspleasanttasteanditshighnutritionalvalue,inadditiontoitsanti-hypertension,
anti-diabetic,anti-ulcerogenic,anti-cancer,anti-proliferative,andantioxidantfunctions[1]. Research
hasshownthatthepulpofripebananacontainsagreatquantityofthaumatin-likeprotein(TLP)[2].
TLP,whichbelongstopathogenesis-related(PR)proteinfamily5(PR5protein),sharesanamino
acidsequencewiththaumatin(asweet-tastingproteinoriginallyfoundinthearilsoftheripeWest
AfricanrainforestshrubThaumatococcusdanielli),andisstructurallysimilar[3].TLPsaretheproductsof
alarge,highlycomplexgenefamilycontainingfungal,plant,andanimalTLPs. TLPsappeartopossess
biologicalfunctionssuchasantifungal,glucanase,andxylanaseinhibitionactivities,andareconsidered
tobeinvolvedinhostdefense,stresstolerance,andcellsignaling[4–6]. Recently,manytransgenic
plantsoverexpressingTLPshavebeendevelopedforenhancinghostresistanceagainstpathogens,
indicatingthatTLPsholdagreatprospectiveforthemolecularbreedingofresistantplants[3].
TLPs isolated from banana, peach, and kiwifruit have been proven to be potent antifungal
proteins [7–9]. However, this activity is reportedly lacking in other TLPs in plants [2,4]. An early
Molecules2018,23,1442;doi:10.3390/molecules23061442 www.mdpi.com/journal/molecules
Molecules2018,23,1442 2of16
studybyKoiwaexplainedthattheefficacyofTLPsdependsonthecombinationoffungalspecies
and TLP isoforms [10]. During the last two decades, much attention has been drawn to revealing
the underlying mechanism of antifungal activity of TLPs, but to date it is still poorly understood.
Ithasbeenreportedpreviouslythatthereisadirectrelationshipbetweentheantifungalactivities
ofTLPsandtheirabilitytobindtoseveralwater-insolubleβ-1,3-glucans, cellwallcomponentsof
pathogenicfungi[4]. AmoleculardockingassayconductedbyLiufurtherconfirmedthatAspand
GluresiduesfoundinthecleftofsoybeanhullsTLPinteractedwithlinearβ-1,3-glucansthroughan
invertingorretainingmechanismofglucanhydrolysis[11]. However,thesepreviousstudiesfocused
ontheanalysisofthestructurefeaturesunderlyingtheseantifungalactivities,withemphasisonthe
molecularmechanism. Fewstudieshaveinvestigatedthephysiologicalandultrastructuralchanges
infungiexposedtoTLPs. Thisworkwillleadtonewinsightsintothemechanismofactionofthe
antifungalproteinagainstfungi.
P.expansumisadevastatingpathogeninfruit,leadingtoconsiderableeconomicallossesduring
the postharvest storage and handling processes [12]. It has been reported that TLP purified from
basraibananacanbeusedasapreservativeinthefoodprocessingindustryduetoitsinhibitoryeffect
on the growth of Aspergillus phoenicis and Aspergillus flavus on white bread [7]. Another example
for application of TLP would be the protection of rye bread against Penicillium requeforti, a major
contaminatingstraininthefoodprocessingindustry[13]. Toourknowledge,thisworkinvestigatesfor
thefirsttimetheeffectofBanTLPonthepostharvestpathogenfungi(P.expansum)inordertopromote
theapplicationofTLPfordirectcropprotectionandgeneengineering.
Thisstudyaimedto: (1)validatetheantifungalactivityofBanTLPisolatedandpurifiedfrom
thebananafruitagainstP.expansum;and(2)observethephysiologicalandultrastructuralchangesin
P.expansumexposedtoBanTLPandelucidateitsmechanismsofaction.
2. ResultsandDiscussion
2.1. PurificationandIdentificationofThaumatin-LikeProteinfromBanana(BanTLP)
BanTLPhasbeenpurifiedusingathree-stepchromatographicpurificationprocedureconsisting
ofanion-exchangechromatography,gelfiltration,andreversed-phasechromatography,wherebythe
elutesweremonitoredbydeterminingtheabsorbanceat280nm. Acrudeextractofbananafruitwas
applied to a DEAE–Sephadex A50 column eluted with a gradient of NaCl in the buffer, and three
major fractions (A1, A2, and A3) were obtained (Figure 1A). A broad absorbance peak (A1) with
immunocompetenceagainsttheBanTLPantibodywasfurtherpurifiedbySephadexG50onthebasis
of molecularmass. Aftergel filtration, peakB1 was devoidof immunocompetence, and peaksB2
andB3withimmunocompetencewereobtained(Figure1B).Theactivefractionswerepooledand
subjectedtoreversed-phasechromatographyonaC5column,andpeakC2withimmunocompetence
wasobtained(Figure1C).ThepurityofBanTLPwasconfirmedbySDS-PAGE,whichrevealedthatthe
purifiedproteinconsistedexclusivelyofasinglepolypeptide(Figure1C,insert). Thepurifiedprotein
wasidentifiedasthaumatin-likeproteinwithamolecularweightof22.1kDabymassspectrometry
analysis(seeSupplementaryMaterialsFigureS1),andtheMascotscorewas414(greaterthan77is
significant, p < 0.05). A yield of 58 mg of pure BanTLP was obtained from 1.5 kg of banana pulp
(Table1). Thiscombinationofdifferentchromatographtechniquesformsanefficientisolationmethod
for bioactive compounds from plants, and is used extensively in protein purification. This work
proposedathree-stepchromatographicpurificationproceduretopurifyanabundantprotein(BanTLP)
frombanana,whichhadsimilarchromatographicbehaviorstothaumatinproteinspurifiedfromplants
inpreviousstudies[2,9].
Molecules 2018, 23, x FOR PEER REVIEW 3 of 15
Table 1. Protein yields at different stages of purification of thaumatin-like protein from banana
(BanTLP).
Fraction Total Protein from 1.5 kg Flesh (mg)
Crude extract 1038
Unbound fraction from DEAE–Sephadex A50 648
MoleculMes2a0j1o8r, 2p3e,a14k4 2from Sephadex G50 chromatography 261 3of16
After reversed-phase chromatography 58

Figure 1. Purification of BanTLP. (A) Ion-exchange chromatography of the fraction of banana fruit
Figure1. PurificationofBanTLP.(A)Ion-exchangechromatographyofthefractionofbananafruit
extract (crude banana extract) with DEAE–Sephadex chromatography A50. The target protein was
extract(crudebananaextract)withDEAE–SephadexchromatographyA50.Thetargetproteinwasonly
only observed in fraction A1. (B) Gel filtration of fraction A1 by fast protein liquid chromatography
observedinfractionA1.(B)GelfiltrationoffractionA1byfastproteinliquidchromatographyona
on a Sephadex G50 column. The target protein was observed in fractions B2 and B3. (C) The active
SephadexG50column.ThetargetproteinwasobservedinfractionsB2andB3.(C)Theactivefractions
fractions from gel filtration were pooled and used in a reverse-phase Supelco C5 column. The target
fromgelfiltrationwerepooledandusedinareverse-phaseSupelcoC5column.Thetargetproteinwas
protein was observed in fraction C2. (Insert) SDS-PAGE profiles of purified antifungal protein from
observedinfractionC2.(Insert)SDS-PAGEprofilesofpurifiedantifungalproteinfrombananafruit
banana fruit visualized using a silver-staining technique. Lane 1: the target protein; Lane 2: crude
visualizedusingasilver-stainingtechnique.Lane1:thetargetprotein;Lane2:crudebananaextract.
banana extract. Numbers on the right refer to molecular mass markers. MW represents the molecular
Numbersontherightrefertomolecularmassmarkers.MWrepresentsthemolecularweightmarker.
weight marker.
Table1.Proteinyieldsatdifferentstagesofpurificationofthaumatin-likeproteinfrombanana(BanTLP).
2.2. Antifungal Properties of BanTLP against P. expansum
TLPs are classified as Ffroarcmtioinng part of the pathogeneTsoitsa-lrPelraotteedin (fProRm) p1.r5okteginFl efsahm(imlyg )5 because
they can be induced byC bruiodteice xatrnadct abiotic stresses such as microbial 1i0n3f8ection, plant hormones,
osmotic stUrensbso, uannddf rwacotiuonndfrionmg. DIEnA pEr–eSveipohuasd esxtuAd5y0, TLPs were isolated 6a4n8d purified from various
MajorpeakfromSephadexG50chromatography 261
fruits. The antifungal protein isolated from kiwifruit showed a remarkable inhibitory effect on
Afterreversed-phasechromatography 58
Botrytis cinerea and some suppressive effects on Mycosphaerella arachidicola and Coprinus comatus [9].
In another study, antifungal activity was observed in Verticillium albo-atrum, which exhibited
2.2. AntifungalPropertiesofBanTLPagainstP.expansum
sensitivity to BanTLP purified from banana [4]. However, so far, there are few detailed reports on the
antifuTnLgPasla prerocplaesrstiiefise dofa TsLfoPr. mHienngcpe,a trhtiosf wthoerkp aetxhaomgienneedsi ist-sr eelfafteecdts( aPnRd) pthroe tmeinecfhaamniilsym5sb inecvaoulvseedth. eIny
cthane pbereisnednut csetdudbyy wbiiotthic BaanndTaLbPi oatti c20st μreMss,e tshseu scphoarsem geicrrmobiniaaltiionnfe octfi oPn. ,expplaanntsuhmor wmaosn eresd,ouscmedo ttioc satbroesust,
ahnadlf wthoaut nodfi nthge. Icnopntrreovli,o wusitsht uad gy,eTrmLPinsawtieorne irsaotlea toefd 4a7n%d. pBuarnifiTeLdP farot m60v μarMio uwsafsr uaibtsl.e Ttho ecaonmtipfulentgelayl
pinrhoitbeiint tihseo lsapteodref groemrmkiniwatiifornu iotfs hPo. wexepdanasurmem (Fairgkuarbel e2Ain)h aibt i2to8 r°yCe. fOfeucrt roensuBlot tcraynti sbcei nreerlaeateadn tdo seoamrlye
srueppoprrtess sdievsecerifbfeecdts boyn YMasymcoisnp h[a7e]r,e wllahaor afcohuindidco tlhaaatn TdLCPo pisroinlautsecdo mfraotmus b[9a]n.aInnaa ninohthibeirtesdtu tdhye, agnrotiwfutnhg oafl
aFcutsiavriituymw oaxsyospbosreurmve, dAisnpeVrgeriltliucisl lniuigmera, lAbosp-aetrrguilmlu,sw fuhmicihgaetxuhs,i baintedd Tsreicnhsoidtievrimtya tvoirBidaen, TwLitPh pICur50i fiveadluferso mof
b9.a7n μanMa, [141]..8H3 oμwMe,v 4e.r6,1s oμfMar,, athnedr e21a.r4e3 fμewM,d reetsapileecdtirveeplyo.r tsontheantifungalpropertiesofTLP.Hence,
thiswSoodrkiuemxa mhyinpeodchiltosreitfefe citss aann detfhfiecimenetc haanndi smqusicinkv oanlvteifdu.nIgnalt haegpernets efnotr sctuodmymweritchiaBl aunsTeL. PWaet
2in0vµeMsti,gtahteeds ptohree egfefremct inoaf tihoynpoofchP.loerxiptean (s0u.m05w%a sofr eadcuticveed ctholoarbionue)t haanldf thBaatnTofLPth e(6c0o nμtMro)l ,owni ththae
ggeerrmmiinnaattiioonn oraf tPe. oefxp4a7n%su.mBa cnoTnLidPiaa ta6ft0erµ 1M2 hw oafs ianbcluebtaoticoonm apt l2e8te °lCy,i unhsiinbgit hthypeoscpholroerigtee rams ian patoisointivoef
Pc.oenxtproanl.s Tuhme( rFeisguulrtes 2shAo)wated28 th◦Cat. BOaunrTrLePsu wltacsa nefbfeectrievlea taetd rteodeuacrinlygr tehpeo rstpsodree sgcerribmeidnabtyioYna bsmy iunp[ 7to],
w10h0o%f oauftnedr 8th ha toTf LePxpisoosulartee dtrferaotmmebnatn oafn caoinnihdiibai,t ebdutt htheeg eroffwectth wofasF ulessasr ituhmano xtyhsapto oruf mh,yApospcherlgoirliltues,
nwigheirc,hA respperregsislleuds 1fu00m%ig tahtue ss,paonrde gTerircmhoindeartmioan vaifrtiedre ,juwsti t2h hI Cof trveaaltumeesnot f(F9i.7guµrMe 2,B1)1.. 83 µM, 4.61 µM,
50
and2T1h.4e3 bµioMlo,greicsaple fcutinvcetliyo.ns of TLP can be directly influenced by pH and temperature, although its
strucStuordei uism cohnysipdoecrhedlo rtoit ebei sstaabnilizeefdfi cdieunet toa nthde hqiugihck nuamntbiefur nogf adlisauglfeindte bfoorndcso [m14m,1e5r]c. iRalesuusltes.
Wfroemin tvheiss tsigtuadteyd inthdeicaetffeedc tthoaft hthyep ogcehrmloirnitaetio(0n.0 o5f% P.o efxpaacntisvuemc cholonridiniea) waansd stBraonnTgLlyP in(6h0ibµitMed) boontht hine
gcoenrmtrionl aatniodn troefaPt.mexenpat ngsruomupcso wnihdeian apfHte rv1a2luheso offi nthcue bcautlitounreast m28ed◦Cia, wuseirneg lohwypero tchhalonr 2it eora hsiaghpeors itthivane
c ontrol. TheresultsshowedthatBanTLPwaseffectiveatreducingthesporegerminationbyupto
100% after 8 h of exposure treatment of conidia, but the effect was less than that of hypochlorite,
whichrepressed100%thesporegerminationafterjust2hoftreatment(Figure2B).
Molecules 2018, 23, x FOR PEER REVIEW 4 of 15
12, in agreement with the findings of Li (Figure 2C) [16]. After 12 h of exposure of the conidia, BanTLP
significantly suppressed the germination of P. expansum conidia when pH values ranged from 4 to
10, with an optimal antifungal activity at pH 6. Additionally, a decrease in the antifungal activity was
observed at pH 10, and completely loss of antifungal activity occurred at higher pH values. The
thermal stability of BanTLP was examined and the antifungal activity was stable at temperatures
ranging from 20 to 50 °C, with optimal antifungal activity at 30 °C (Figure 2D). A comparison of the
observations in this work with those of Ho indicates that antifungal activity of TLP isolated from
emperor banana is more stable in a wider pH range (pH 1 to 11) and up to 70 °C [17]. These differences
may be ascribed to different fungal species and analysis methods. Similarly, Menu found that BanTLP
iMsoolleacuteleds2 0f1ro8,m23 ,b14a4n2ana exhibited endo-β(1,3)-glucanase activity in a pH-dependent manner, 4wofit1h6
optimal activity at pH 5 [4].

Figure 2. The effects of concentration (A), contact time (B), pH (C), and temperature (D) on the spore
Figure 2. The effects of concentration (A), contact time (B), pH (C), and temperature (D) on the
germination of Penicillium expansum exposed to BanTLP in potato dextrose broth (PDB). The
spore germination of Penicillium expansum exposed to BanTLP in potato dextrose broth (PDB).
Tgehremgienramtiionna trioante roaft ePo. fePx.peaxnpsaunmsu wmaws adsetdeertmerimneinde adftaefrt e1r21 h2 hofo ifnicnucubabtaitoionn aat t2288 °◦CC. .EEaacchh vvaalluuee iiss tthhee
mean of three replicates for two independent experiments. Vertical bars represent standard
meanofthreereplicatesfortwoindependentexperiments.Verticalbarsrepresentstandarddeviations
deviations of means. Those data marked with different letters are significantly different (p < 0.05)
ofmeans.Thosedatamarkedwithdifferentlettersaresignificantlydifferent(p<0.05)accordingto
according to Duncan’s multiple range test.
Duncan’smultiplerangetest.
2.3. ETffhecet boifo BloagniTcLaPlf ounn MctieomnbsroanfeT PLePrmcaenabbileitdy iarnecdt lDyisintuflrubaennccee dinb Py. pexHpaannsdumte mperature,althoughits
structureisconsideredtobestabilizedduetothehighnumberofdisulfidebonds[14,15]. Resultsfrom
The integrity of mycelia membrane of P. expansum was investigated after exposure to 60 μM
thisstudyindicatedthatthegerminationofP.expansumconidiawasstronglyinhibitedbothincontrol
BanTLP by monitoring the propidium iodide (PI) influx. PI is a DNA-staining fluorescent probe that
andtreatmentgroupswhenpHvaluesoftheculturesmediawerelowerthan2orhigherthan12,
can enter the cell if the plasma membrane of cell is disrupted, resulting in a nucleus stained with a
inagreementwiththefindingsofLi(Figure2C)[16]. After12hofexposureoftheconidia,BanTLP
red fluorescent color [18]. Results in Figure 3A indicate that fluorescent intensity increased in conidia
significantlysuppressedthegerminationofP.expansumconidiawhenpHvaluesrangedfrom4to10,
of P. expansum exposed to BanTLP as the incubation times extended. After 4 h of incubation with
withanoptimalantifungalactivityatpH6. Additionally,adecreaseintheantifungalactivitywas
BanTLP, the percentage of PI fluorescent-stained conidia was 87%, while that of the control was
observedatpH10,andcompletelylossofantifungalactivityoccurredathigherpHvalues.Thethermal
14.2%. After 6 h of incubation, 100% of conidia of P. expansum were stained in BanTLP-treated group,
stabilityofBanTLPwasexaminedandtheantifungalactivitywasstableattemperaturesrangingfrom
while only 18.3% were stained in the control group. The result of PI influx indicated that BanTLP is
20to50◦C,withoptimalantifungalactivityat30◦C(Figure2D).Acomparisonoftheobservationsin
able to induce the plasma membrane disruption. The corresponding fluorescence images of PI influx
thisworkwiththoseofHoindicatesthatantifungalactivityofTLPisolatedfromemperorbananais
is displayed in Supplementary Materials Figure S2.
morestableinawiderpHrange(pH1to11)andupto70◦C[17]. Thesedifferencesmaybeascribed
todifferentfungalspeciesandanalysismethods. Similarly,MenufoundthatBanTLPisolatedfrom
bananaexhibitedendo-β(1,3)-glucanaseactivityinapH-dependentmanner,withoptimalactivity
atpH5[4].
2.3. EffectofBanTLPonMembranePermeabilityandDisturbanceinP.expansum
The integrity of mycelia membrane of P. expansum was investigated after exposure to 60 µM
BanTLPbymonitoringthepropidiumiodide(PI)influx. PIisaDNA-stainingfluorescentprobethat
canenterthecelliftheplasmamembraneofcellisdisrupted,resultinginanucleusstainedwithared
fluorescentcolor[18]. ResultsinFigure3Aindicatethatfluorescentintensityincreasedinconidiaof
Molecules 2018, 23, x FOR PEER REVIEW 5 of 15
The potassium ion release assay was conducted to estimate the permeability of the plasma
membrane. Compelling evidence from early studies indicated that the plasma membrane of toxin-
damaged cells became permeable for monovalent ions, with subsequent potassium ions release from
the cell [19]. K+ homeostasis may play a critical role in the maintenance of the membrane potential.
Results in this study showed that the concentration of extracellular K+ significantly increased in the
initial 10 h of incubation with BanTLP. In contrast, for the control treatment, the concentration of
extracellular K+ was maintained at a low level throughout the experiment, with little K+ release
(Figure 3B).
A 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence assay was performed to estimate the plasma
membrane perturbation in terms of lipid bilayer. The DPH probe can be buried completely in the
Mbiollaecyuelers c2o01r8e, 2w3,i1th44 2no effect on the membrane structure. Any disorder in the membrane lipid bi5laoyfe16r
was a result of DPH parting from the membrane, with a subsequent decrease in the DPH fluorescent
polarization [20]. The results in Figure 3C show that the DPH fluorescent intensity descended
P.expansumexposedtoBanTLPastheincubationtimesextended. After4hofincubationwithBanTLP,
remarkably in conidia of P. expansum exposed to BanTLP as the incubation times extended. After 6 h
thepercentageofPIfluorescent-stainedconidiawas87%,whilethatofthecontrolwas14.2%. After6h
of incubation, the fluorescent intensity of conidia exposed to BanTLP was 48.7% lower than that of
ofincubation,100%ofconidiaofP.expansumwerestainedinBanTLP-treatedgroup,whileonly18.3%
the control (p < 0.05). The result indicated that BanTLP treatment perturbed the membrane lipid
werestainedinthecontrolgroup. TheresultofPIinfluxindicatedthatBanTLPisabletoinducethe
bilayer of the test fungus, resulting in DPH detachment and a decrease in fluorescent intensity.
plasmamembranedisruption. ThecorrespondingfluorescenceimagesofPIinfluxisdisplayedin
However, for the control treatment, the fluorescent intensity was maintained as the incubation times
SupplementaryMaterialsFigureS2.
increased, indicating the membrane lipid bilayer was stable and well-organized.

Figure 3. Effects of BanTLP at 60 μM on the plasma membrane of P. expansum conidia as the exposure
Figure3.EffectsofBanTLPat60µMontheplasmamembraneofP.expansumconidiaastheexposure
times extended. (A) Spores stained with propidium iodide (PI); (B) the leakage of intracellular
times extended. (A) Spores stained with propidium iodide (PI); (B) the leakage of intracellular
potassium ions; (C) 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence intensity of P. expansum. Each
potassiumions;(C)1,6-diphenyl-1,3,5-hexatriene(DPH)fluorescenceintensityofP.expansum.Each

valueismeanofthreereplicatesfortwoindependentexperiments.Verticalbarsrepresentstandard
deviationsofmeans. Thosedatamarkedwithdifferentlettersaresignificantlydifferent(p<0.05)
betweencontrolandtreatmentgroupsatthesametimeaccordingtoDuncan’smultiplerangetest.
The potassium ion release assay was conducted to estimate the permeability of the plasma
membrane. Compelling evidence from early studies indicated that the plasma membrane of
toxin-damagedcellsbecamepermeableformonovalentions,withsubsequentpotassiumionsrelease
fromthecell[19].K+homeostasismayplayacriticalroleinthemaintenanceofthemembranepotential.
ResultsinthisstudyshowedthattheconcentrationofextracellularK+ significantlyincreasedinthe
initial 10 h of incubation with BanTLP. In contrast, for the control treatment, the concentration of
Molecules2018,23,1442 6of16
extracellular K+ was maintained at a low level throughout the experiment, with little K+ release
(Figure3B).
A1,6-diphenyl-1,3,5-hexatriene(DPH)fluorescenceassaywasperformedtoestimatetheplasma
membraneperturbationintermsoflipidbilayer. TheDPHprobecanbeburiedcompletelyinthe
bilayercorewithnoeffectonthemembranestructure. Anydisorderinthemembranelipidbilayerwas
a result of DPH parting from the membrane, with a subsequent decrease in the DPH fluorescent
polarization [20]. The results in Figure 3C show that the DPH fluorescent intensity descended
remarkablyinconidiaofP.expansumexposedtoBanTLPastheincubationtimesextended. After6hof
incubation,thefluorescentintensityofconidiaexposedtoBanTLPwas48.7%lowerthanthatofthe
control(p<0.05). TheresultindicatedthatBanTLPtreatmentperturbedthemembranelipidbilayer
ofthetestfungus, resultinginDPHdetachmentandadecreaseinfluorescentintensity. However,
forthecontroltreatment,thefluorescentintensitywasmaintainedastheincubationtimesincreased,
indicatingthemembranelipidbilayerwasstableandwell-organized.
Previousstudiesinvestigatedanumberofantifungalagents(suchasisoquercitrin,monocaprin,
citral,andsoon)thatdirectlyorindirectlyattacktheplasmamembranesofpathogenicfungi,providing
much insight into membrane permeabilization [21,22]. Through PI influx, potassium ion leakage,
and DPH assays, this study confirmed the hypothesis that BanTLP caused the damage of cell by
inducingmembranepermeabilizationanddisturbances. SomestudiesonthestructureofTLPsisolated
fromplantssupportedtheideathatthemembrane-permeabilizingactivityofTLPmightbeascribed
toitsacidiccleft[10]. Ithasbeendemonstratedthatwhenpositively-chargedTLPisolatedfromsoy
hullsandthenegatively-chargedmicrobialmembranesarecombinedsuperficiallyandelectrostatically,
theTLPpartiallyentersthehydrophobicpartofcytoplasmicmembranes[11].Therefore,muchresearch
is needed to investigate the deeper mechanisms of BanTLP acting as a membrane-permeabilizing
proteinagainstP.expansum.
2.4. OxidativeStressinP.expansumInducedbyBanTLP
Reactiveoxygenspecies(ROS)andthiobarbituricacid-reactivespecies(TBARS)areconsidered
oxidativestressbiomarkers. TheaccumulationofROSmolecules,includingsuperoxide,hydrogen
peroxide, and hydroxyl radicals, can lead to detrimental stress responses in fungi, disrupting
the cell physiological status and causing oxidative death [23]. ROS were determined using a
2(cid:48),7(cid:48)-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe, which has proven efficiency for
characterizingtheoverallROSgenerationinmanybiologicalmaterials. AsshowninFigure4A,B,
after6hofincubationwithBanTLP,thepercentageofcellsmarkedwithintracellularROSinP.expansum
increased9.8-foldcomparedtothecontrol. Moreover,whentheincubationtimewasincreasedupto
12h,a6.3-foldincreaseintheintracellularROSgenerationwasobservedintheBanTLPtreatment
groupcomparedtothatofthecontrolgroup(p<0.05).
TBARS is the primary product of polyunsaturated fatty acids peroxidation, and can be used
asanindicatorofoxidativedamageofthefungalplasmamembrane. Thevariationsinamountsof
TBARSfurtherconfirmedtheresultsfromtheROSassay. WhenconidiaofP.expansumwereincubated
withBanTLPat28◦Cfor6hor12h,TBARScontentswere2.58-and4.54-foldcomparedtothatof
thecontrol,respectively(p<0.05). Figure4CshowsthatTBARScontentsinconidiaofP.expansum
preparedinBanTLPcontinuouslyincreasedasthecontacttimesincreased,whereasthatofthecontrol
showednovariationthroughouttheexperiment.
Similarly,thepreviousstudydemonstratedthatosmotin,atobaccoPR-5familyprotein,activated
theRAS2/cAMPpathwayandinducedhyperproductionofintracellularROStopromoteprogrammed
cell death of the yeast Saccharomyces cerevisiae [24]. Additionally, Hayea described a promising
antifungal defensin, NaD1, with activity against Candida albicans because NaD1 induced ROS
generation, leading to cell death as the result of oxidative damage [25]. Hence, BanTLP in this
workpromotedtheaccumulationofintracellularROS,resultinginoxidativestressinP.expansum,
whichcanbeexplainedasanotherpossiblefungicidalactionofBanTLP.
Molecules2018,23,1442 7of16
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Figure 4. Effects of BanTLP at 60 μM on oxidative stress of P. expansum. Figures panels show images
Figure 4. Effects of BanTLP at 60 µM on oxidative stress of P. expansum. Figures panels show
of (A) the generation of intracellular reactive oxygen species (ROS), (B) the percentage of 2′,7′-
images of (A) the generation of intracellular reactive oxygen species (ROS), (B) the percentage of
2d(cid:48)i,c7h(cid:48)-ldoircohfllouroorflesucoeriens dceiaincedtaiatec e(tDaCteFH(D-CDFAH) -pDoAsi)tivpeo scietlilvse, acnedll s(,Ca)n tdhio(Cba)rtbhiitoubriacr baictiudr-irceaacctiidv-er esapcetciviees
(TBARS) contents. The DCFH-DA fluorescent probe was visualized with the aid of fluorescence
species(TBARS)contents.TheDCFH-DAfluorescentprobewasvisualizedwiththeaidoffluorescence
microscopy in P. expansum conidia treated with distilled water for 6 h (a,b) and 12 h (e,f) or treated
microscopyinP.expansumconidiatreatedwithdistilledwaterfor6h(a,b)and12h(e,f)ortreatedwith
with BanTLP for 6 h (c,d) and 12 h (g,h). Upper panels display the bright field images, while lower
BanTLPfor6h(c,d)and12h(g,h).Upperpanelsdisplaythebrightfieldimages,whilelowerpanels
dpiasnpelalsy dthisepcloaryr ethspe ocnodrrinesgpflounodriensgc efnluceoriemsacegnecsef oimrtahgeessa fmore tfiheel ds.amThee fsiecladle. Tbhare rsecparlees benarts r1e0prµemse.nEtsa c1h0
μm. Each value is mean of three replicates for two independent experiments. Vertical bars represent
valueismeanofthreereplicatesfortwoindependentexperiments.Verticalbarsrepresentstandard
dsteavniadtairodn sdeovfimatieoannss .ofT mhoesaensd. aTthaomsea drkaetad mwairthkeddi fwfeirtehn dtilfefettreernst alertetesrisg narifie csaignntliyficdainfftelyre dnitff(epre<n0t .(0p5 <)
b0e.0tw5)e beentwcoenetnro cloanntdrotlr eaantdm terneattgmroeunpt sgarotuthpes saatm theet ismameaec tciomrde iancgcotordDinugn ctaon D’sumncualnti’psl emrualntigpelete rsatn.ge
test.
2.5. EffectofBanTLPonCellWallIntegrityinP.expansum
2.5. Effect of BanTLP on Cell Wall Integrity in P. expansum
TheeffectofBanTLPonP.expansumcellwallwasevaluatedusingaCalcofluorWhite(CFW)
The effect of BanTLP on P. expansum cell wall was evaluated using a Calcofluor White (CFW)
fluorescent probe. CFW can monitor abnormal chitin alterations located in the cell walls of fungi
fluorescent probe. CFW can monitor abnormal chitin alterations located in the cell walls of fungi
throughaspecificcombinationwithchitin[26]. Inthisstudy,afterbeingtreatedwithBanTLPat60µM
through a specific combination with chitin [26]. In this study, after being treated with BanTLP at 60
for 6 h, a bright blue fluorescent signal was detected. When the incubation time was increased to
μM for 6 h, a bright blue fluorescent signal was detected. When the incubation time was increased to
12h,thebrightbluefluorescencewasenhancedinconidiacellsinthetreatmentgroup. Therewasno
12 h, the bright blue fluorescence was enhanced in conidia cells in the treatment group. There was no
apparentfluorescentsignalinconidiacellsinthecontrolgroupthroughouttheexperiment(Figure5).
apparent fluorescent signal in conidia cells in the control group throughout the experiment (Figure
TheresultfromthepresentstudyindicatedthatBanTLPtreatmentseverelydamagedthecellwall
5). The result from the present study indicated that BanTLP treatment severely damaged the cell wall
ofP.expansumconidia. Similarly,barleyTLPsexertedtheirantifungalactivitybybindingtonascent
of P. expansum conidia. Similarly, barley TLPs exerted their antifungal activity by binding to nascent
β-1,3-glucansduringfungalwallsynthesis,preventingthegrowthoffungi[27]. Thepreviousstudy
β-1,3-glucans during fungal wall synthesis, preventing the growth of fungi [27]. The previous study
confirmedthatTLPsfromplantcouldinducedisruptionofthefungalcellwall,leadingtocelldeath.
confirmed that TLPs from plant could induce disruption of the fungal cell wall, leading to cell death.
Molecules2018,23,1442 8of16
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Molecules 2018, 23, x FOR PEER REVIEW 8 of 15

FigFuigruer5e. 5E. fEfeffcetcot foBf aBnaTnTLLPPa att6 600µ μMMo onnt thhee cceellll wwaallll iinntteeggrriittyy ooff PP.. eexxppaannssuumm ccoonnididiaia. .TThhe eCCalaclocfoluflouro r

White (CFW) fluorescent probe was visualized with the aid of fluorescence microscopy in P. expansum
White(CFW)fluorescentprobewasvisualizedwiththeaidoffluorescencemicroscopyinP.expansum
conFidigiua rter e5a. tEefdfe wcti tohf dBaisntTilLlePd a wt 6a0te μr Mfo ro n6 hth (ea c,bel)l awnadl l1 i2n the g(eri,tfy) oorf Ptr.e eaxtpeadn swuimth c oBnaindTiaL. PT hfoer C 6a hlc o(cf,ldu)o ra nd
conidiatreatedwithdistilledwaterfor6h(a,b)and12h(e,f)ortreatedwithBanTLPfor6h(c,d)and
12 hW (gh,ihte) .( CUFpWpe) rfl puaonreeslcse dnits pprloabye t whea sb rviigsuhat lfiizeeldd wimitahg tehse, awidh iolfe f llouworeers cpeanncee lms dicirsopslcaoyp tyh ien c Po.r erxepsapnosunmdi ng
12h(g,h).Upperpanelsdisplaythebrightfieldimages,whilelowerpanelsdisplaythecorresponding
fluocroensicdeina ctree iamteadg wesit fho dr itshtiell esadm wea tfeier lfdo.r T6h he ( sac,ba)l ea nbda r1 2re hp (ree,sfe) notrs t r1e0a tμemd w. ith BanTLP for 6 h (c,d) and
fluorescenceimagesforthesamefield.Thescalebarrepresents10µm.
12 h (g,h). Upper panels display the bright field images, while lower panels display the corresponding
2.6. Morfpluhoorleosgciecnacl ea inmda Ugelst rfoasr ttrhuec staumrael fCiehlda.n Tghees socfa lPe. b eaxrp raenpsreusmen Cts o1n0i μdima .
2.6. MorphologicalandUltrastructuralChangesofP.expansumConidia
2.6. Morphological and Ultrastructural Changes of P. expansum Conidia
2.6.1. Scanning Electron Microscope (SEM)
2.6.1. ScanningElectronMicroscope(SEM)
2.6In.1 .o Sucra wnnoirnkg, Eoltehcetrro snig Mnsic orof sdcaompea (gSeE tMo )t he cell wall of P. expansum conidia were found according
Inourwork,othersignsofdamagetothecellwallofP.expansumconidiawerefoundaccordingto
to analysis with the aid of a scanning electron microscope. After 12 h of incubation at 28 °C, the
analysiswInit houtrh weoarikd, ootfhaers csaignnnsi onfg dealmecatgreo nto mthiec rcoelslc wopalel .oAf Pft. eerxp1a2nshumof cionnciudbiaa wtioenre afotu2n8d◦ Cac,ctohredicnogn idia
contiod iaan atrlyesaitse dw iwthi tthh ed aisidti lolef da wscaatnenri ndgi seplleacytreodn am nicorormscoalp ea.n Adf tceor m12p ahc to fm inocrupbhaotlioong ya t( F2i8g u°Cre, t6hae, b),
treatedwithdistilledwaterdisplayedanormalandcompactmorphology(Figure6a,b),whereasthe
whceorenaids iath ter ecaotendid wiai tthr edaitsetdil lewdi twh aBtearn TdiLsPpl aayt e6d0 aμ Mno rsmhaolw aendd icroremgpualactr mmoordpihfioclaotgiyo n(sF.i gAusr es h6oa,wb)n, in
conidia treated with BanTLP at 60 µM showed irregular modifications. As shown in Figure 6c,d,
Figwurhee 6reca,ds , tcheel lc sounridfaicae tsr eoaft nedu mweitrho uBsa ncTonLPid aiat i6n0 tμhMe t rsehaotwmeedn ti rgreroguulpa ra pmpoedairfiecda ttioo nssu. fAfesr sfhroomw ns eivn ere
cellsurfacesofnumerousconidiainthetreatmentgroupappearedtosufferfromseverecontraction
conFtrigauctrieo 6nc ,adn, dce dlli ssruurfpatcieosn o. f numerous conidia in the treatment group appeared to suffer from severe
anddciosnrutrpactitoionn. and disruption.

Figure 6. SEM of P. expansum. P. expansum conidia treated with distilled water (a,b), or BanTLP at 60
FigFuigruer6e. 6S. ESEMMo offP P..e exxppaannssuumm.. PP.. eexxppaannssuumm ccoonnididiai atrteraetaetde dwiwthi tdhisdtiilslteidll ewdawtera t(ear,b()a, ,obr) ,BoarnTBLaPn TaLt 6P0a t
μM (c,d) for 12 h. Arrows refer to the morphologic alterations in conidia, such as collapsed cell wall
60μµMM ((cc,d,d)) ffoorr 1122 hh. .AArrrorowws srerefefer rtoto ththee mmoorprphhoolologgici calatletreartaitoinosn sini ncocnoindiidai,a s,uscuhc hasa csoclolallpaspesde dceclle lwlwalla ll
and severe contraction (magnification ×5000, and 20,000).
anadnsde sveevreerceo cnotnratrcatciotinon(m (magangnifiifciacatitoionn× ×55000000,, aanndd 2200,,000000))..
Molecules2018,23,1442 9of16
Molecules 2018, 23, x FOR PEER REVIEW 9 of 15
2.6.2. TransmissionElectronMicroscope(TEM)
2.6.2. Transmission Electron Microscope (TEM)
TheeffectofBanTLPontheultrastructuralchangesofP.expansumconidiawasestimatedusinga
The effect of BanTLP on the ultrastructural changes of P. expansum conidia was estimated using
transmissionmicroscope. After12hofexposuretreatment,theconidiatreatedwithdistilledwater
a transmission microscope. After 12 h of exposure treatment, the conidia treated with distilled water
showedwell-organizedcellswithahomogeneouscytoplasm,undamagedcellwalls,integratedcell
showed well-organized cells with a homogeneous cytoplasm, undamaged cell walls, integrated cell
membranes,andnormalorganelles(Figure7a). Incontrast,forthetreatmentgroup,thevacuolesof
membranes, and normal organelles (Figure 7a). In contrast, for the treatment group, the vacuoles of
thecellsexpandedandformedinlargenumbers,revealinganosmoticstressresponseinP.expansum
the cells expanded and formed in large numbers, revealing an osmotic stress response in P. expansum
conidiainducedbyBanTLP(Figure7b,c).Additionally,thedisorganizationofcytoplasmandorganelles
conidia induced by BanTLP (Figure 7b,c). Additionally, the disorganization of cytoplasm and
wasevidentafterBanTLPtreatment(Figure7d,e). AcollapsedcellwallcanbeobservedinFigure7f,g.
organelles was evident after BanTLP treatment (Figure 7d,e). A collapsed cell wall can be observed
Theseultrastructuralchangesinconidiamaybeattributedtoenzymaticdigestionofthecellwalland
in Figure 7f,g. These ultrastructural changes in conidia may be attributed to enzymatic digestion of
increasingmembranepermeabilization,followedbytheleakageofsmallmolecularsubstancesand
the cell wall and increasing membrane permeabilization, followed by the leakage of small molecular
ions,andcellmetabolismdisorder[22].
substances and ions, and cell metabolism disorder [22].

Figure 7. Ultrastructural changes of P. expansum observed by TEM. P. expansum conidia treated with
Figure7.UltrastructuralchangesofP.expansumobservedbyTEM.P.expansumconidiatreatedwith
distilled water (a), or BanTLP at 60 μM (b–g) for 12 h. The scale bar represents 1 μm. Each treatment
distilledwater(a),orBanTLPat60µM(b–g)for12h.Thescalebarrepresents1µm.Eachtreatment
was repeated three times, and at least two blocks were detected for each replicate. Va, vacuole; CW,
wasrepeatedthreetimes,andatleasttwoblocksweredetectedforeachreplicate.Va,vacuole;CW,
cell wall; PM, plasma membrane; Cy, cytoplasm; N, nucleus; M, mitochondria. (Magnification
cellwall;PM,plasmamembrane;Cy,cytoplasm;N,nucleus;M,mitochondria.(Magnification×30,000).
×30,000).
3. MaterialsandMethods
3. Materials and Methods
3.1. FruitandFungalMaterials
3.1. Fruit and Fungal Materials
Banana(Musaacuminata)fruitweretransportedfromGuangzhoutoourlaboratoryattheChina
AgricBualntuarneaU (Mniuvsear saictuymiminmatae)d firauteitl ywaefrtee rtrhaanrsvpeosrt,teadn dfrostmor Geduaatn2g0zh◦oCuf otor ocoumr lpalbeoterartiopreyn aint gth.e China
Agriculture University immediately after harvest, and stored at 20 °C for complete ripening.
TheplantpathogenP.expansumwasobtainedfromthemicrobiologylaboftheChinaAgriculture
UnivTehrsei typ.laTnhte ppaatthhooggeenn sPt.r aeixnpwanassumcu lwtivaas teodbtianinpeodt atforodme xtthroes emaigcarorb(iPoDloAgy) alta2b8 o◦Cf tfhoer 7Cdhainyas.
Agriculture University. The pathogen strain was cultivated in potato dextrose agar (PDA) at 28 °C
Subsequently,thecultureswerewashedwithsteriledistilledwatercontaining0.05%(v/v)Tween-80.
for 7 days. Subsequently, the cultures were washed with sterile distilled water containing 0.05% (v/v)
Afterfiltrationthroughfourlayersofsterilecheesecloth,thesporesuspensionwasobtainedinthe
Tween-80. After filtration through four layers of sterile cheesecloth, the spore suspension was
absenceofadherentmycelia. Theconcentrationofsporeswasdeterminedusingahemocytometerand
ocabltiabirnaetded into thaefi nabalsecnocnec eonft raadthioenreantt1 m×y1ce0l6ias.p Tohrees cmonLc−e1n.tration of spores was determined using a
hemocytometer and calibrated to a final concentration at 1 × 106 spores mL−1.
3.2. PurificationandIdentificationofBanTLPfromBanana
3.2. Purification and Identification of BanTLP from Banana
ProteinextractionwasperformedaccordingtothemethodofXi[28]. Inbrief, peeledbanana
(1.5 kPgr)otweians ehxotrmacotgioenn iwzeads piner1fo80rmmeLd aocfc1orMdinTgri tso( tphHe m11e.t2h)o,dco onft Xaiin [i2n8g]. 1In5 %bripeof,l ypveeinleydlp byarnraonliad o(1n.5e
k(PgV) wPPa)s. hAofmteorgceennitzreifdu gina t1i8o0n m(1L0 ,o0f0 10 ×M gT,r3is0 (mpHin ,141.2◦C), )c,otnhteaisnuipnegr 1n5a%ta nptowlyavsincyollpleycrtreodlidanodned (iPaVlyPzPe)d.
A(MftWer CcOen8tr0i0fu0g-1a4ti0o0n0 ,(S1e0r,0v0a0d× iga,l y3s0i smtuinb,i n4 g°)Ca)g, athines stuTpriesr-nHaCtalnbt uwffaesr c(5olmleMcteTdr iasn-Hd Cdli,aplyHze7d.5 ()MaWt4C◦OC
8fo0r001-214h0.0S0u, bSeservqau edniatllyy,stihs etucbriundge) eaxgtariancstt wTraiss-cHoCncl ebnutfrfaetre d(5 fimvMe-f oTlrdis-wHiCthl,t phHea 7id.5)o afta 4f r°eCe zfoer-d 1r2y ehr..
STuhbespeqrouteenintlyc,o tnhtee ncrtuodfet hexetcraructd weaesx tcroancctewntarsateesdti mfivaet-efdoladc wcoirthd itnhge taoidB orfa da fforerdezue-sdinrygebro. Tvhinee psreortueimn
caolbnutemnitn o(fS tihgem caruAdled erxicthra,cStt .wLaosu eisst,imMaOte,dU aScAc)oardsisntagn tdo aBrrdad[2f9o]r.d using bovine serum albumin (Sigma
Aldrich, St. Louis, MO, USA) as standard [29].
Molecules2018,23,1442 10of16
The BanTLP was purified from the banana extract (300 mL) using a pre-equilibrated
anion-exchange column (DEAE–Sephadex A50, 5 × 5 cm) in 30-mM Tris-HCl buffer with pH 8.0,
andchromatographywasperformedinbatches. ThecolumnwaselutedwithagradientofNaClfrom
0 to 0.6 M in the same buffer at a flow rate of 1 mL min−1. After that, the immunocompetence of
everyfractionwasmeasuredthroughwesternblotassaywiththerabbitantiserumagainstBanTLPin
bananafruit[30]. Therefore,unboundproteinsandtheproteinselutedunderthelowconcentrationof
NaClfromtheion-exchangechromatography(fractionA1)werepooledandcollected. Subsequently,
thefractionA1wasdialyzedagainstTris-HClbuffer(5mMTris-HCl,pH7.5)at4◦Cfor12h,andthen
concentratedontoagelfiltrationcolumn(70×1.6cm)ofSephadexG50equilibratedwith30-mM
Tris-HClbufferwithpH8.0,containing0.1MNaCl. Thecolumnwaselutedwiththesamebufferata
flowrateof1mLmin−1. Theelutingpeaks(fractionsB2andB3)withtheprimaryantibodyreacting
bandswerepooledandcollected,andresolvedonaC5HPLCcolumn(250×4.6mm,5µm,Supelco,
Bellefonte,PA,USA)equilibratedwith1%trifluoroaceticacid(TFA)inwater. Proteinswereeluted
withagradientof0–70%acetonitrilein0.1%TFAin40min. Thepurifiedproteinswereobtainedby
collectingproteinfraction(C2)withimmunocompetence,anddialyzedagainstTris-HClbuffer(5mM
Tris-HCl, pH7.5)at4 ◦Cfor12h. Theproteinafterdialysiswasconcentratedandstoredinsmall
aliquotsat−20◦Cuntiluse.
Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)furtherconfirmedthat
the purified protein contained only a single polypeptide according to the method described by
Laemmli [31], using a miniprotean electrophoresis apparatus (Beijing Liuyi Instrument Factory
DYY-1517C,Beijing,China). Thegelwithproteinbandswasstainedusingasilver-stainingtechnique
accordingtoNesterenko[32].
Protein in-gel digestion assay was conducted according to Liu [11]. Identification of the
protein depended on Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
(4700MALDI-TOF/TOFProteomicsAnalyzer,AppliedBiosystems,Waltham,MA,USA).Mascotdata
baseprogram(in-houseMASCOTserverv2.1,MatrixScienceCo.,London,UK)wasemployedto
analyzeallspectraofproteinsagainsttheNCBInrdatabase.
3.3. EffectofBanTLPonSporeGerminationofP.expansum
Dose-dependentassaywasperformedbytheexposureofsporegerminationofP.expansumto
variousconcentrationsofBanTLPfollowingthemethoddescribedpreviously[33]. Fiveconcentrations
of BanTLP at 0, 5, 10, 15, 20, 40, or 60 µM were prepared in 5 mL of potato dextrose broth (PDB)
in10-mLglasstubesseparately. Afteradding100µLofP.expansum(1×106 sporesmL−1)toeach
tube, samples were cultivated at 200 rpm at 28 ◦C for 12 h and centrifuged (8000× g) at 4 ◦C for
5min. Afterwards,theconidiawererinsedtwicewithphosphate-bufferedsaline(PBS,pH7.0)and
resuspendedwiththesamebuffer. Approximately200sporesperreplicatewerescoredtocalculatethe
germinationrateofthetestpathogen.Theminimalinhibitoryconcentrations(MICs)ofBanTLPagainst
otherthreecommonpostharvestfungiRhizopusstolonifera,Botrytiscinerea,andAlternariaalternatawere
60,60,and30µM,respectively(SupplementaryMaterialsTableS1). Therewerethreereplicatesper
treatment,andeachexperimentwasrepeatedtwice.
Minimum contact time between BanTLP and spores of P. expansum was evaluated following
thedescriptionbyDawithslightmodifications[34]. Thesteriledistilledwater(asnegativecontrol),
0.05%(v/v)sodiumhypochlorite(aspositivecontrol),andBanTLPat60µMwerepreparedin5mL
ofPDBcontainingP.expansumsporesuspension(1×106 sporesmL−1)in10-mLglasstubes. After
0, 2, 4, 6, 8, 10, or 12 h incubation in a shaker of 200 rpm at 28 ◦C, the conidia were centrifuged
(8000×g,5min,4◦C),washedtwicewith50mMPBS(pH7.0)toremovetheextratreatmentsolutions,
andresuspendedin5mLofPDBtoafinalconcentrationof1×106sporesmL−1. Theconidiawere
incubatedinashakerof200rpmat28◦Cforanother12,10,8,6,4,2,or0hforthegerminationof
spores,respectively. Finally,thegerminationratewasdeterminedasdescribedabove. Therewere
threereplicatespertreatment,andeachexperimentwasrepeatedtwice.