Biocontrol of Aspergillus Species on Peanut Kernels by Antifungal Diketopiperazine Producing ...

14 Pages·2014·12.88 MB·English

Biocontrol of AspergillusSpecies on Peanut Kernels by
Antifungal Diketopiperazine Producing Bacilluscereus
Associated with Entomopathogenic Nematode
Sasidharan Nishanth Kumar1,Sreerag Ravikumar Sreekala1, DileepChandrasekaran2, Bala Nambisan1*,
Ruby John Anto3
1DivisionofCropProtection/DivisionofCropUtilization,CentralTuberCropsResearchInstitute,Sreekariyam,Thiruvananthapuram,India,2DepartmentofBotany,SD
College, Kalarcode, Thookkukulam, Alappuzha, India, 3Integrated Cancer Research Program, Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology,
Thiruvanathapuram,India
Abstract
The rhabditid entomopathogenic nematode associated Bacillus cereus and the antifungal compounds produced by this
bacteriumwereevaluatedfortheiractivityinreducingpostharvestdecayofpeanutkernelscausedbyAspergillusspeciesin
invitroandinvivotests.TheresultsshowedthatB.cereushadasignificanteffectonbiocontroleffectivenessininvitroand
invivo conditions. The antifungal compounds produced by the B. cereus were purified using silica gel column
chromatography and their structure was elucidated using extensive spectral analyses. The compounds were identified as
diketopiperazines (DKPs) [cyclo-(L-Pro-Gly), cyclo(L-Tyr-L-Tyr), cyclo-(L-Phe-Gly) and cyclo(4-hydroxy-L-Pro-L-Trp)]. The
antifungalactivitiesofdiketopiperazineswerestudiedagainstfiveAspergillusspeciesandbestMICof2 mg/mlwasrecorded
againstA.flavusbycyclo(4-hydroxy-L-Pro-L-Trp).Toinvestigatethepotentialapplicationofcyclo(4-hydroxy-L-Pro-L-Trp)to
eliminatefungalspoilageinfoodandfeed,peanutkernelswasusedasafoodmodelsystem.Whitemyceliaanddark/pale
greensporesofAspergillusspecieswereobservedinthecontrolpeanutkernelsafter2daysincubation.Howeverthefungal
growthwasnotobservedinpeanutkernelstreatedwithcyclo(4-hydroxy-L-Pro-L-Trp).Thecyclo(4-hydroxy-L-Pro-L-Trp)was
nontoxictotwonormalcelllines[foreskin(FS)normalfibroblastandAfricangreenmonkeykidney(VERO)]upto200 mg/ml
in MTT assay. Thus the cyclo(4-hydroxy-L-Pro-L-Trp) identified in this study may be a promising alternative to chemical
preservatives as a potential biopreservative agent which prevent fungal growth in food and feed. To the best of our
knowledge, this is the first report demonstrating that the entomopathogenicnematode associated B. cereus and cyclo(4-
hydroxy-L-Pro-L-Trp)couldbeusedasabiocontrolagentsagainstpostharvestfungaldiseasecausedbyAspergillusspecies.
Citation: Kumar SN, Sreekala SR, Chandrasekaran D, Nambisan B, Anto RJ (2014) Biocontrol of Aspergillus Species on Peanut Kernels by Antifungal
DiketopiperazineProducingBacilluscereusAssociatedwithEntomopathogenicNematode.PLoSONE9(8):e106041.doi:10.1371/journal.pone.0106041
Editor:IlseD.Jacobsen,LeibnizInstituteforNaturalProductsResearchandInfectionBiology-HansKnoellInstitute,Germany
ReceivedMay1,2014;AcceptedJuly27,2014;PublishedAugust26,2014
Copyright: (cid:1) 2014 Kumar etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfiles.
Funding:Thiswasselffinancedwork.TheonlyfundingreceivedwasthefellowshipofSRF.Thefundershadnoroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
Introduction suchasstressordamagetothecropduetodroughtbeforeharvest,
insect activity, soil type and inadequate storage conditions [6].
Aspergillusisafilamentousfungusthatproducesmycotoxinsin Among naturally occurring aflatoxins, AFB1 is considered as the
many food and feed crops. Aflatoxins are toxic, carcinogenic,
most potent hepatotoxic and hepatocarcinogenic chemical. AFs
immunosuppressive,mutagenic,andpolyketide-derivedsecondary
have been detected in numerous agricultural commodities [7].
metabolites produced mainly by certain strains of Aspergillus
Consumption of aflatoxins contaminated foods and feeds are a
flavusandA.parasiticusandinalessextentbyseveralstrainsof
seriousproblemfromtheviewpointofnotonlypublichealth,but
A. nomius, A. pseudotamarii, A. bombycis, and A. ochraceoroseus
also economiclosses.
[1].A.flavusandA.fumigatusarethemainsourceofaflatoxins,
Peanuts (Arachis hypogaea) is one of the most important food
the most important mycotoxins in the world’s food supplies.
andoilseedcropscultivatedandutilizedinmostpartsoftheworld.
Aflatoxin B1 (AFB1) and B2 (AFB2) are the most important
They are widely accepted as an excellent source of nutrition to
among 18 different types of aflatoxin in crops and agricultural
both human and animals due to their high protein content.
commodities [2–3]. Aflatoxins are produced on peanuts, corn,
Several investigations have listed a large number of fungi which
sorghum,cottonseed,pistachionuts,copra,cereals,fruits,oilseeds,
couldbeisolatedfrompeanutsduringstorage[8].A.flavusisthe
dried fruits, cocoa, and spices in the field and during storage.
dominant storage fungus colonizing peanuts, capable of causing
Aflatoxins occur mainly in hot and humid regions where high
seed rots, moulding of seeds, pre- and post-emergence damping
temperature and humidity are optimal for moulds growth and
off,andreducingseedviabilityandseedlinggrowthinpeanuts[9].
toxins production [4–5]. Their presence is enhanced by factors
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BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Table1. Effect ofB.cereusand cellfreeculturefiltrate onAspergillus species.
Testfungi Percentageofinhibition(%)
Modifiedliquidmediumalone Cellfreeculturefiltrate 16108CFU/mlB.cereuscellsuspension Watercontrol
A.flavus + 2661.72a 2061b +
A.niger + 2961a 2261.52b +
ValuesfollowedbydifferentlettersinsamerowweresignificantlydifferentaccordingtoDuncan’smultiplerangetestp=0.05.
+fullgrowthuptotheedgeofthePetriplate.
doi:10.1371/journal.pone.0106041.t001
World-wide occurrence of A. flavus and aflatoxins in a great structuralelucidationoftheantifungalcompoundsproducedbyB.
varietyoffoodcropsespeciallyinnutshastriggeredmuchresearch cereusinmodifiedliquidmediumandalsoaimedtodeterminethe
with regard toits causes,progress andprevention[3]. ability of the isolated compounds in preventing the growth of
The need for protection of food and feedstuffs against Aspergillus inpeanut foodmodel system.
Aspergillusspeciesisuniversallyrecognizedandseveralapproach-
es such as treatment of peanuts with fungicides, fumigants, and Materials and Methods
plant products have been suggested. Recently there has been an
extensivesearchforalternativestochemicalfungicidesthatwould Chemicals and media
provide satisfactory Aspergillus control with low impact on the All the chemicals used for extraction and purification were of
environment and human health [10]. Although the use of analytical grade. High performance liquid chromatography
synthetic fungicides is a most effective decay control treatment, (HPLC) grade methanol and thin layer chromatography (TLC)
there is an urgent need to find effective and safe non-fungicide sheets were from Merck Limited, Mumbai, India. Various
meansofcontrollingpostharvestpathogensmainlybecauseofthe microbiological media used in the study were from Hi-Media
toxicity of the synthetic fungicide residues to humans and the LaboratoriesLimited,Mumbai,India.Chemicalusedforprepar-
environment [11]. In recent years, some antagonist microorgan- ing the fermentation medium was purchased from SRL Labora-
isms have been applied in biocontrol of postharvest diseases of toriesLtd.,Mumbai,India.Chemicalsusedforantimicrobialand
agricultural products. Saccharomyces cerevisiae and lactic acid cytotoxicityassayswerepurchasedfromSigma-Aldrich,USA.All
bacteria(LAB)representuniquegroups,whicharewidelyusedin other reagents were of analytical grade and the other chemicals
foodpreservation [12]. used in this study were of highest purity. The software used for
Inthecourseofstudiesonentomopathogenicnematode(EPN), drawing chemical structure was Chemsketch Ultra, Toranto,
a new EPN belonging to the genus Rhabditis and subgenus Canada.
Oscheius was isolated from sweet potato weevil grubs collected
from Central Tuber Crops Research Institute (CTCRI) farm, Test fungal strains
Thiruvananthapuram,Kerala,India[13].AspecificB.cereuswas Five Aspergillus species used in the present study were
associatedwithEPN,whichwaspathogenictovariousagricultur- Aspergillus flavus MTCC 277, Aspergillus niger MTCC 282,
ally important insects [13]. Based on molecular characteristics, Aspergillus tubingensis MTCC 2425, Aspergillus fumigatus
Rhabditis(oscheius)sp.resemblesRhabditisisolateatD2andD3 MTCC 3376 and Aspergillus parasiticus MTCC 2796. Other
(nucleotide sequence region) expansion segments of 28S rDNA testfungiusedwereCryptococcusgastricusMTCC1715,Candida
[13].ThecellfreeculturefiltrateofB.cereuswasfoundtoinhibit albicansMTCC3017,CandidatropicalisMTCC184,Trichophy-
severalpathogenicbacteria,fungiandaplantparasiticnematode ton rubrum MTCC 296, Fusarium oxysporum MTCC 284,
(Meloidogyne incognita) [14], suggesting that it could be a rich Rhizoctonia solani MTCC 4634, and Penicillium expansum
sourceofbiologicallyactivecompounds.Recentlywereportedthe MTCC 2006. All the fungal strains were purchased from
antifungal activity of the crude extract obtained from a modified Microbial Type Culture collection Centre, IMTECH (Institute
liquid medium against Penicillium expansum and Candida ofMicrobialTechnology),Chandigarh,Indiaandweregrownon
albicans [15]. This modified media is superior to TSB for the potatodextroseagar(PDA)at30uCfor3daysandstoredat4uC
production of secondarymetabolites by B.cereus. forfurther studies.
ThepotentialofBacillusspeciestoproduceantibioticshasbeen
recognizedforpast50years.Bacillusspeciesproducestructurally Peanut kernels
diverse classes of secondary metabolites, such as lipopeptides,
Peanut kernels with a commercial level of maturity were used
polypeptides,cyclicdipeptides,macrolactones,fattyacids,polyke-
immediatelyafterharvest,orstoredat4uCfornolongerthan48h
tides, lipoamides, and isocoumarins [16–17]. These structurally
before using. Before treatments, the peanut kernels were washed
versatile compounds exhibit a wide range of biological activities,
with tap water, then surfaced-disinfected with 0.1% sodium
includingantimicrobialandanticancereffects[16–17].AsBacillus
hypochlorite for 1 min, cleaned with sterile water, and air dried
strainsrapidlygrowinliquidmediaevenunderstressfulconditions
prior towounding.
andreadilyformsresistantspores,itmightbeusefulasaneffective
biocontrol agentagainstvariousphytopathogens [18].Structures,
B. cereus: Antifungal compounds producing bacteria
synthesis,andspecificfunctionsofdiverseantibioticsproducedby
The antifungal compound producing B. cereus was isolated
B. subtilis haveelaborately beenreviewed [19].
from3rdstageinfectivejuvenilesofthenematodesamplecollected
ThepresentmanuscriptdealswiththebiocontrolpropertyofB.
from sweet potato weevil grubs. The strain was identified as B.
cereus in controlling Aspergillus species, in peanut food model.
cereus (Accession No. HQ200404) based on 16S rDNA and
Moreover the manuscript also deals with the purification and
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BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure1.EffectofB.cereusoncontrolofAspergillusspecies.(A)autoclavedmodifiedliquidmedium;(B)cellfreeculturefiltrateofmodified
liquidmediumalone;(C)16108CFU/mlB.cereuscellsuspension;and(D)steriledistilledwaterasacontrol.Valuesfollowedbydifferentletterswere
significantlydifferentaccordingtoDuncan’smultiplerangetestp=0.05.
doi:10.1371/journal.pone.0106041.g001
BLASTanalysis.ThestrainwascurrentlydepositedinIMTECH Efficacy of B. cereus on control of Aspergillus species
and theaccessionnumber isMTCC 5234. in vivo
Peanut kernels were wounded (5mm diameter and approxi-
In vitro antifungal assay of B. cereus against Aspergillus mately3 mmdeep)usingasterileborer.Thenthepeanutkernels
species were treated with 25ml of one of the following treatments: (A)
To evaluate the interactions between the B. cereus and the autoclaved modified liquid medium alone; (B) bacterial cell free
Aspergillusspeciesinculture,6-mmdiameterplugswerecutfrom culturefiltrateofmodifiedliquidmedium;(C)16108CFU/mlB.
4-day-oldPDAculturesofA.flavusandA.nigerandthenplaced cereuscellsuspension;and(D)control(steriledistilledwater).Two
on another PDA plate (15ml/plate) seeded with 1.0 ml of hours later, 10ml of A. flavus and A. niger spores at 56104
different treatments. The treatments used for the study were as spores/ml was inoculated into each peanut kernels. The peanut
follows:(A)autoclavedmodifiedliquidmediumalone;(B)bacterial kernels were placed in BOD incubator and incubated at 28uC.
cell free culture filtrate of modified liquid medium; (C) 16108 ThenumberofpeanutkernelsthatwereinfectedwithAspergillus
CFU/mlB.cereuscellsuspension;and(D)control(steriledistilled species was recorded after 7 days inoculation. There were three
water). Autoclaved cultures were prepared by autoclaving replicates of20peanutkernels pertreatment andtheexperiment
modified liquid culture broth for 20min at 121uC. Bacterial cell was conducted twice.
free culturefiltrates wereprepared byfilteringthesupernatant of
centrifugedcultureoftheB.cereusthrougha0.22mmmembrane EffectofdifferentincubationtimesofB.cereusoncontrol
filter after 48h fermentation [20]. Bacterial cells from 72h of Aspergillus species in vivo
cultures were adjusted to 16108 CFU/ml by adding additional
Thepeanutkernelswerewoundedasdescribedabove.20 mlof
culture filtrate. After inoculation the plates were incubated for 7 16108CFU/mlcellsuspensionofB.cereusthathadbeengrown
days at 37uC. Fungal growth was recorded after 8 days. Growth innutrientbrothfor12,24,36,48,60and72hwasrespectively
inhibition was calculated as the percentage of inhibition of radial
inoculated into the wounds of the peanut kernels, and sterile
growthrelativetothecontrol[21].Threereplicateswereusedper distilled water was used as control. After 2 h, 10ml of 56104
treatment and theexperiments were repeatedtwice.
spores/mlsuspensionofA.flavusandA.nigerwasinoculatedinto
eachwound.ThepeanutkernelswereplacedinaBODincubator
as describedabove.
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BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure2.EffectofdifferentincubationtimesofB.cereusoncontrolofAspergillusspecies.Thecontrolistreatedwithsteriledistilledwater.
ValuesfollowedbydifferentlettersweresignificantlydifferentaccordingtoDuncan’smultiplerangetestp=0.05.
doi:10.1371/journal.pone.0106041.g002
Effect of different concentrations of B. cereus on control a0.22 mmfilter,toobtaincellfreeculturefiltrate.Twentylitresof
of Aspergillus species in vivo cell free culture filtrate were neutralized with concentrated
hydrochloric acid and extracted with an equal volume of ethyl
Thepeanutkernelswere woundedasdescribedabove.TheB.
cereus cell suspensions were adjusted toconcentrations of 16106, acetatethrice.Theethylacetatelayerswerecombined,driedover
16107, 16108, 16109 and 16101uCFU/ml with sterile distilled anhydrous sodium sulphate, and concentrated at 30uC using a
water, respectively. 20ml of 16106, 16107, 16108, 16109 and rotary flash evaporator (Buchi, New Castle, USA). The initial
16101uCFU/ml cell suspension respectively was inoculated into antifungalactivityofthecrudeextractwastestedagainstA.flavus
by disc diffusionmethod.
each wound, and sterile distilled water was used as control. After
2 h10mlof56104spores/mlsuspensionofA.flavusandA.niger
Purification of antifungal compounds
was inoculated to each wound. Treated peanut kernels were
The browny yellow residue (6.38 g) obtained after drying was
incubated ina BODincubatoras describedabove.
loadedonasilicagelcolumn(306600mm)previouslyequilibrat-
ed with hexane and eluted successively with 200ml of 100%
Isolation, purification and characterization of antifungal
hexane,200mloflineargradient hexane:dichloromethane (95:5
compounds produced by B. cereus
to 5:95 v/v), 200ml of 100% dichloromethane, 200ml of linear
Preparationofcrudeantifungalsubstancebyfermenting
gradientdichloromethane:ethylacetate(95:5to5:95 v/v),200ml
B. cereus. The bacterial fermentation was carried out using
of100%ethylacetateandfinallywith200mlof100%methanol.
modifiedliquidmedium.Theliquidmediumwascomposedof(g/
About 88fractionsmeasuring 100mleach were collected.
l):fructose,10.0;beefextract,10.0;K HPO ,1.0;KH PO ,1.0;
2 4 2 4 Purity of the compounds was tested by TLC (20620cm,
MgSO , 1.0; NaCl, 2.0; Na SO , 1.0. The medium pH was
4 2 4 precoated silica gel 60 GF254 plates) and HPLC. The HPLC
adjusted to 7.0 before autoclaving using NaOH or HCl solution.
analysis was performed using a Shimadzu LC-10AT liquid
Afterpreparingtheliquidmedium,B.cereuswasinoculatedinto chromatography (LC; Shimadzu, Singapore) equipped with a
the flasks containing 100ml sterile liquid media. The flasks were quaternary pump, a degasser, an autosampler, a thermostated
incubatedinanorbitalshaker(Remi,Mumbai,India)(120 rpm)at column compartment, and a variable wavelength detector.
30uCindarkfor24h.Whentheopticaldensityofthecultureat Separations were carried out with isocratic elution on a C18
600nm was approx 1.7, the bacterial cultures were transferred reversed-phase column (5 mm, 4.66250mm, Shimadzu, Singa-
asepticallyinto400mlsterilemediumandincubatedintheorbital pore) connected with a C18 security guard column (3 mm
shaker at 30uC in dark for 96h. The culture media were then ID64 mm, Phenomenex, Torrance, USA). The mobile phase
centrifuged(10,000g,20min,4uC)followedbyfiltrationthrough consistedof100%HPLCgradeMeOH.Theinjectionvolumewas
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BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure3.EffectofdifferentconcentrationsofB.cereusoncontrolofAspergillusspecies.Thecontrolistreatedwithsteriledistilledwater.
ValuesfollowedbydifferentlettersweresignificantlydifferentaccordingtoDuncan’smultiplerangetestp=0.05.
doi:10.1371/journal.pone.0106041.g003
15ml and detection wavelength was 240nm. The flow rate was resuspendedinH O(100 ml),and1%Marfey’sreagentinacetone
2
1 ml/min, andthecolumntemperature was set to25uC andrun (200 ml)and1 MNaHCO (40 ml)wereadded.Themixturewas
3
time was10minforeach sample. heatedat40uCfor1 hinashakingwaterbath,afterwhichitwas
removed and cooled. The mixture was quenched with 2M HCl
Structure determination of antifungal compounds (20 ml),dried,anddissolvedinMeOHforHPLCanalysis.Amino
OpticalrotationofthecompoundswasmeasuredonaRudolph acidstandards(1mgeach)werederivatizedwithMarfey’sreagent
Research Autopol III polarimeter (Hackettstown, NJ, USA). UV in same manner similar as described above. The HPLC analysis
spectra were measured on a Shimadzu UV-VIS spectrophotom- was performed using two solvent systems: (Shimadzu LC-20AD,
eter UV-2450 (Shimadzu, Japan). Nuclear magnetic resonance C18column;5 mm,4.66250mm;1.0 ml/min)at30uCusingthe
(NMR)spectroscopy(BrukerDRX500NMRinstrument,Bruker, followinggradientprogram:solventA,water+0.2%TFA;solvent
Rheinstetten, Germany) equipped with a 2.5-mm microprobe. B, MeCN; linear gradient 0 min25% B, 40min 60%B, 45min
NMRSpectrometerusingDMSO-d6wasdeployedtomeasure1H 100%B; UV detection at340nm [22].
and 13C. All spectra were recorded at 23uC. Chemical shifts are
reported relative to the solvent peaks. (DMSO-d6:1H d 2.50 and Antifungal assay
13Cd39.51).High-resolutionmassspectrophotomer(HRMS)was Minimuminhibitoryconcentration(MIC). TheMICwas
performed on a Thermo Scientific Exactive Mass Spectrometer performedbybrothmicrodilutionmethodsaspertheguidelinesof
(Thermo Fisher Scientific, Bremen, Germany) with an electro- Clinical and Laboratory Standard Institute (CLSI) (formerly, the
sprayionizationmode.Themeltingpointofthepurecompounds National Committee for Clinical Laboratory Standards) [23–24],
wasmeasuredwithadifferentialscanningcalorimeterinaMettler with RPMI 1640 medium containing L-glutamine, without
Toledo DSC 822e instrument (Mettler-Toledo, Schcoerfenbach, sodium bicarbonate and buffered to pH 7.0. Twofold serial
Switzerland). Temperature ranges from 30uC to 300uC were dilutions of the test compounds were prepared in media in
employed. amountsof100mlperwellin96-wellU-bottommicrotiterplates
(Tarson,Mumbai,India).Thetestfungalsuspensionswerefurther
Absolute configuration determination of compounds by dilutedinmedia,anda100mlvolumeofthesedilutedinoculums
the HPLC analysis of Marfey’s derivatives wasaddedtoeachwelloftheplate,resultinginafinalinoculumof
Asolutionoffourcompounds(1.5 mg)in6MHCl(1 ml)was 0.56104to2.56104CFU/mlforCandidaspeciesand0.46104to
heated to 125uC for 24h. The dried aqueous solution was 56104 CFU/ml for other fungi. The final concentration of test
PLOSONE | www.plosone.org 5 August2014 | Volume 9 | Issue 8 | e106041
BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
compounds ranged from 1 to 1000mg/ml. The medium without
the test compounds was used as a growth control and the blank
H)
O control used contained only the medium. Amphotericin B served
e
M asthestandarddrugcontrol.Themicrotiterplateswereincubated
2, at35uCfor48hforCandidaspeciesand30uCfor72hforother
0
0. fungi. After incubation OD600 nm was measured using a
(c, microplate reader, and the MIC was defined as the lowest
n
o concentration of the antifungal agents that prevented fungal
otati growth.
Opticalr 2a]118D 145a]+D 2167a]D 2a]065D AnCtiofmunpgouanldassswaeyrebsycrdeeisnceddiffofrutshioeinr atentcihfunnigqauleactivity against
[ [ [ [
test fungi by disc diffusion method [25–26]. The fungal cultures
n) weregrownonpotatodextrosebroth.Themyceliamatoffungiof
mi 7-dayoldculturewassuspendedinnormalsalinesolutionandtest
(
e inoculum was adjusted to 16105 CFU/ml. Inocula (0.1ml) were
m
ti applied on the surface of the PDA plate and spread by using a
n
o cotton swab. Subsequently, filter paper discs (6 mm in diameter)
enti containingMICconcentrationoftestcompoundswereplacedon
Ret the agar plates and incubated at 35uC for 24–48h. Afterwards,
PLC 534 654 121 823 thediameter oftheinhibition zonewas measured.
H 1. 4. 6. 7.
The antifungal compound from B. cereus as
C) biopreservative agent in peanut food model system
(u
point 05.55 7.34 65.58 65.67 50Emiglhotfpdeisatnilluetdswkaertenrelfsor(a5php,roaxnidmtahteelnya8u.t1ocgl)avwederaets1o2a1kueCdfoinr
elting 2.51–2 5.1–26 2.23–2 1.56–1 2ac0etmatien.eTxthraecatuotfomclaovdeidfiepdeamneudtiukemrnaenlsdwpeurreescooamkepdouinndcrfuodre8ehthaytl
M 20 26 26 16 room temperature and then transferred to Petri dishes. Cooked
peanuts soaked in methanol and modified medium alone were
m) usedascontrols.AsporesuspensionofA.flavusandA.nigerwas
n
n( prepared (107 spores/100 ml distilled water); and approximately
o
pti 10mlofthesporesuspensionofA.flavusandA.nigerwasspread
absor 4 276 278.4 ofonrp2edaanyustakte3r0nueCls.,aTnhdetihneocfuunlagtaeldgproewanthutwkaesrnexealsmwinereedienvceurbyadteady
UV 212 278. 238, 268, for10days [27].
g)
m Cytotoxicity test
(
eld The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazo-
Yi 32 44 26 33 lium bromide) assay was used to determine the cytotoxicity of
diketopiperazine.FSnormalfibroblastandVEROcellswereused
fortesting.ThecelllineswerepurchasedfromNationalCentrefor
M M
C C CellScience,Pune,IndiaandmaintainedinDulbecco’sModified
D D
e e n n Eagle Medium (DMEM) supplemented with 10% FBS with
n n i i
vent hexa hexa etate etate caenltlisb(i3o6tic1s0a3/nwdealln)twimeyrecosteiecsdeadt3in7u0C.2inmaloCfOth2eimnceudbiuatmor(.DBMrieEflMy,
unds. Columnsol 30%DCMin 75%DCMin 15%ethylac 35%ethylac warefitmtehro1v0iinn%gcuPbthBaetSio)dnirn,ug9c6yctwoonetoltlaxpiinclaiitntyegs,wmtraeesdaitaem,de2wa5siuthmrelddro.ufgFMsofrTorTt7h2issohlu.atafitnoendr
o (5 mg/ml in PBS) and 75ml of complete medium were added to
p
m wells(untreatedandtreated)andincubatedfor2 h.Attheendof
Detailsofthepureco2. Compounds Cyclo-(L-Pro-Gly) Cyclo(L-Tyr-L-Tyr) Cyclo-(L-Phe-Gly) Cyclo(4-hydroxy-L-Pro-L-Trp) 71/journal.pone.0106041.t002 5St(viaBnh7inta0ceiaAdobutoo-libiilrlfpsniaastttuctydtiiiuacncoatibtaEnnirlsaleLtdtMpiaaecIetedSnaneTrlAdsacfTiaoeltysninrrealseatysamaialssnyagdipsstoeeelt5erbshw7u6we6a0fr8fes1en04r0rcem,0ahwpl)Ccweaa[uars2tellfa8iroa3fet]ord7e.mrmdudnCeeei(dda.Aa,sAtwuo5Utir7tet0tShhhdAoeeSuf)P.westiSnrTeneSdlghlas(oetaVe(f0dprei.enlr1aslscaatimuoetmibnvlrp/aee1lwtaei7cdo/e.een0lAllr;,l)
Table S.No 1 2 3 4 doi:10.13 SastsPapnS,dSa,0rI.dn0c5d..,eDvCiaahttiaicoafnogsor.,dIiLsc,dUifSfuAs)i.oSntaatsissatyicawlassigpnriefisceanntecdewasamsdeeafninse6d
PLOSONE | www.plosone.org 6 August2014 | Volume 9 | Issue 8 | e106041
BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure4.Thestructureofdiketopiperazines.(A)Cyclo-(L-Pro-Gly),(B)Cyclo(D-Tyr-D-Tyr),(C)Cyclo-(L-Phe-Gly)and(D)Cyclo(4-hydroxy-L-Pro-
L-Trp).
doi:10.1371/journal.pone.0106041.g004
Results of the water control and autoclaved culture were above 95% for
bothfungi,whereasthetreatmentwiththebacteriacellsuspension
Efficacy of B. cereus on the growth of Aspergillus species at 16108 CFU/ml was 18% for A. flavus and 9% for A. niger,
in vitro which was significantly lower than the treatments with the
In in vitro tests, different treatments exhibited different autoclaved culture, and water control. Meanwhile, the average
inhibitory effects on Aspergillus species. The percentage of percentage ofthe diseaseincidence in thetreatment with thecell
inhibition of culture filtrate and cell suspension (16108 CFU/ml) free culture filtrate was 33 and 25%, which also recorded
against A. flavus was 26 and 20, respectively and whereas for A. remarkable decaycontrol comparedwiththetreatmentsofwater
niger it was 29 and 22, respectively (Table1). Sterile distilled control, autoclaved mediaalone.
water and autoclaved modified medium alone did not inhibit the
growth of Aspergillus species, because the fungal growth reached EffectofdifferentincubationtimesofB.cereusoncontrol
theedge ofthe Petriplates. of rot caused by Aspergillus species in vivo
Different incubation times of B. cereus significantly influenced
Efficacy of B. cereus on control of decay caused by the effectiveness of the diseasecontrol in peanut kernels at 28uC.
Aspergillus species in vivo The results recorded that better biocontrol was obtained when
AsshowninFig.1,thecellsuspensionofB.cereuswasthemost longerincubationtimeofB.cereuswasapplied(Fig.2).Whenthe
effectivetreatmentoncontroloftherotsinpeanutkernelscaused incubationtimeofB.cereuswas72h,therateofdecaydeclinedto
byAspergillusspecies.After7daysat28uC,thediseaseincidence 15%and5%forA.flavusandA.niger,respectively.Sothebest
PLOSONE | www.plosone.org 7 August2014 | Volume 9 | Issue 8 | e106041
BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure5.HPLCchromatogramofdiketopiperazinesonareversed-phaseC18column(LC-20AD).Samplesof15mlwereinjectedtoa
column(250mm64.6mm65mm),elutedwith100%methanol.Retentiontimeis2.778min.Thecalculatedpurityis96%basedonthepeakarea.
(A)Cyclo-(L-Pro-Gly),(B)Cyclo(D-Tyr-D-Tyr),(C)Cyclo-(L-Phe-Gly)and(D)Cyclo(4-hydroxy-L-Pro-L-Trp).
doi:10.1371/journal.pone.0106041.g005
control effect was obtained by the cell suspension of B. cereus which recorded significant antifungal activity and these fractions
whentheincubation timewas 60–72h. werefurtherpurifiedbycrystallisationusingbenzeneandhexane
to yield four crystal compounds. The column solvent, yield and
Effect of different concentrations of B. cereus on control UVabsorptionofthepurecompoundswereshownintheTable 2.
of rot caused by Aspergillus species in vivo Initialbioactivityofthepurecompoundswasconfirmedbytesting
ThediseaseincidenceonB.cereustreatedkernels(treatedwith against A.flavus.
20ml of 16105, 16106, 16107 and 16108 CFU/ml cell
suspension, respectively) was significantly lower than the control. Identification of bioactive compound
The concentrations of B. cereus significantly influenced disease The pure compounds were subjected to various spectroscopic
incidence on peanut kernels (Fig.3). The results showed that the analyses,i.e.UV,NMRandHRMS.Thestructureofcompounds
lower disease incidence was recorded when the concentrations of corresponded to a four different diketopiperazines (DKPs). The
B. cereus increases. When the concentration of B. cereus was DKPs identified were cyclo(L-Pro-Gly), cyclo(D-Tyr-D-Tyr),
16108 CFU/ml and spore suspension of A. flavus and A. niger cyclo(L-Phe-Gly) and cyclo(4-hydroxy-L-Pro-L-Trp) (Fig.4). The
wasat56104cells/ml,therateofdecayinpeanutkernelsdeclined melting point, HPLC retention time and optical rotation of the
to20%and 12%,respectively. compoundsareshowninTable 2.InHPLCanalysisthepurityof
the compounds reached greater than 96% according to the peak
Isolation and purification of antifungal compounds area (Fig.5).
Thecrudeethylacetateextractofthecellfreeculturefiltrateof DKP 1: Cyclo-(L-Pro-Gly); hexahydropyrrolo[1,2-
the B. cereus recorded significant antifungal activity against A. a]pyrazine-1,4-dione was obtained as white crystals. The
flavus,theinitialtestfungus.Silicagelcolumnchromatographyof spectral data of DKP 1 (see File S1). The spectral data were in
the crude ethyl acetate extract yielded four bioactive fractions, agreement with thosereported earlier inliterature [29].
PLOSONE | www.plosone.org 8 August2014 | Volume 9 | Issue 8 | e106041
BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
n
o
biti
him)
inm
neofa.in 61.52 60 61.52 61.15 61.52 61.73 61.15 61.2 61.15 61.15 61.73
B Zo(Di 24 31 16 19 24 24 24 21 25 30 23 36
n
ci
eri
hot ml)
Amp MIC(g/m 8 4 16 32 16 2 16 32 16 8 2 4
n
o
biti
him)
inm
neofa.in 60.57 60 60.57 60.57 60 60.57 60.57 60 61.2 61.2 61 61
Zo(Di 32 29 27 24 30 22 29 24 19 21 25 30
4 ml)
DKP MIC(g/m 2 4 8 16 16 8 16 16 16 32 16 4
n
o
biti
him)
inm
neofa.in 60 61 61.15 61.15 60.57 61 61.73 61.73 61.52 60
Zo(Di 25 27 26 30 20 24 – – 30 33 25 23
3 ml)
DKP MIC(g/m 64 64 125 250 250 500 – – 32 125 500 64
n
o
biti
him)
inm
neofa.in 60 60.57 61.15 61.15 61.15 61 61.73 60 61.73 61 61.73 61
Zo(Di 22 17 19 21 23 15 22 21 11 22 27 15
2 ml)
DKP MIC(g/m 16 32 16 8 64 64 16 64 250 125 125 250
es. on
zin biti
pera inhimm)
diketopi Zoneof(Dia.in – – – – – – – – – – – 6111
of 3
0
y 0
ngalactivit DKP1 MIC(g/ml)m – – – – – – – – – – – 500 00g/ml.mone.0106041.t
AntifuTable3. Testfungi A.flavus A.niger A.tubingensis A.fumigatus A.parasiticus C.albicans F.oxysporum R.solani P.expansum C.gastricus C.tropicalis T.rubrum –noactivityupto10doi:10.1371/journal.p
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BiocontrolofAspergillusspp.onPeanutbyBacilluscereus
Figure6.Microscopicimages(CarlZeissstereomicroscope,Stemi200C)ofthegrowthofA.flavusincontrolandpeanutkernels
treatedwithcrudeethylacetateextractandcyclo(4-hydroxy-L-Pro-L-Trp)after2days.(A)Controlplates:solventcontrol-treatedwith
methanol,mediumalone-treatedwithautoclavedmodifiedmediumanduntreated-peanutkernelswithoutmethanolandmodifiedmedium(B)
crudeextract(C)cyclo(4-hydroxy-L-Pro-L-Trp).16,26,and36arethe1-fold,2-fold,and3-foldMICconcentrationofcyclo(4-hydroxy-L-Pro-L-Trp)or
crudeethylacetateextract.
doi:10.1371/journal.pone.0106041.g006
DKP 2: Cyclo(L-Tyr-L-Tyr); 3,6-bis(4- AbsoluteConfigurationDeterminationofcompoundsby
hydroxybenzyl)piperazine-2,5-dione was obtained as the HPLC analysis of Marfey’s derivatives
colorless solid. The spectral data of DKP 2 (see File S1).
The modified Marfey’s method which successfully applied for
Thesedata were similar tothosepreviously reported [30].
determining the absolute stereochemistry of the four compounds.
DKP 3: Cyclo-(L-Phe-Gly); 3-benzylpiperazine-2,5-dione
Regarding the absolute stereochemistry, all the compounds
was obtained as white crystal. The spectral data of DKP 3
contain L amino acids except glycine with is achiral (Figure S1–
(seeFileS1).Thesedataweresimilartothosepreviouslyreported
S4 inFileS1).
[31].
DKP 4: Cyclo(4-hydroxy-L-Pro-L-Trp); 7-hydroxy-3-(1H-
Antifungal activity
indol-2-ylmethyl)hexahydropyrrolo[1,2- a]pyrazine-1,4-
MICvaluesofcompoundsagainsttwelvefungiareprovidedin
dione wasobtained ascolorless needles. The spectral data
Table 3. Compounds recorded antifungal activity against all the
of DKP 4 (see File S1). The spectra were identical to those
tested fungi except DKP 1. DKP 4 recorded significant activity
recorded inliterature [32].
against all test fungi especially against Aspergillus species
(Table 3).TheresultofdiscdiffusionassaybyMICconcentration
of thecompounds isalsopresented intheTable 3.
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