Endophytic fungus strain 28 isolated from Houttuynia cordata possesses wide-spectrum antifungal activity

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h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

Endophytic

fungus

strain

28 isolated

from

Houttuynia

cordata

possesses

wide-spectrum

antifungal

activity

Feng

Pan

1

_,

_Zheng-Qiong

_Liu

1

_,

_Que

_Chen,

_Ying-Wen

_Xu,

_Kai

_Hou,

_Wei

_Wu

∗ AgronomyCollege,SichuanAgriculturalUniversity,Chengdu,Sichuan,PRChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31October2013

Accepted7October2015

Availableonline2March2016

AssociateEditor:EleniGomes

Keywords:

Endophyticfungi

HouttuyniacordataThunb.

Chaetomiumglobosum

Antifungalactivity

Processparameter

a

b

s

t

r

a

c

t

Theaimofthispaperistoidentifyandinvestigateanendophyticfungus(strain28)that

wasisolatedfromHouttuyniacordataThunb,afamousandwidely-usedTraditionalChinese

Medicine.BasedonmorphologicalmethodsandaphylogeneticanalysisofITSsequences,

thisstrainwasidentiﬁedasChaetomiumglobosum.Anantifungalactivitybioassay

demon-stratedthatthecrudeethylacetate(EtOAc)extractsofstrain28hada wideantifungal

spectrumandstrongantimicrobialactivity,particularlyagainstExserohilumturcicum(Pass.)

LeonardetSuggs,BotrytiscinereapersoonandBotrytiscinereaPers.exFr.Furthermore,the

fermentationconditions,extractionmethodandtheheatstabilityofantifungalsubstances

fromstrain28werealsostudied.Theresultsshowedthatoptimalantifungalactivitycan

beobtainedwiththefollowingparameters:usingpotatodextrosebroth(PDB)asthebase

culturemedium,fermentationfor4–8d(initialpH:7.5),followedbyextractionwithEtOAc.

Theextractwasstableattemperaturesupto80◦C.Thisistheﬁrstreportontheisolation

ofendophyticC.globosumfromH.cordatatoidentifypotentialalternativebiocontrolagents

thatcouldprovidenewopportunitiesforpracticalapplicationsinvolvingH.cordata.

anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Endophyticfungiareconsideredasnovelsourcesforbioactive

compoundsthathaveimportantapplicationsintheﬁeldsof

agricultureandmedicine.Thesefungimaypossessthe

poten-tial toproducebioactive compounds, and this potential to

producebioactivecompounds isequalor similartothatof

theirhost.1–3_Studies_have_shown_positive_symbiosis_between

∗ _{Corresponding}_author.

E-mail:ewuwei@gmail.com(W.Wu).

1 _The_ﬁrst_two_authors_contributed_equally_to_this_work.

endophyticfungiandtheirhosts.Forexample,thesecondary

metabolites of endophytic fungi can facilitate the growth

oftheir hostplantsdirectlyor indirectlyand improvehost

resistancetoabioticorbioticstresses.4_Substantial_renewed

attentionisnowbeingpaidtoendophyticfungi,whichhave

inhibitoryactivitytowardpathogenicfungi5_and_play_an

indis-pensableroleinfungalbiocontrol.6_Recently,_an_endophytic

fungal strainwas isolatedbySantiagoet al.7_from _the

Cin-namomummollissimumplant.Onebioactivityofametabolite

http://dx.doi.org/10.1016/j.bjm.2016.01.006

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fromthisplant,whichwasidentiﬁedasapolyketide,wasthe

efﬁcientkillingofthefungalpathogenAspergillusniger(IC50

1.56␮gmL−1).7

Conventional chemical fungicides have been shown to

beharmful tothe environment and topublichealth,8 _and

theycan cause pathogens to becomeresistant whenused

foralongtime.9_By_contrast,_extracts_from_natural_products

showed advantages withregards to environmental

protec-tionandhumanhealth.For example,Yinetal.10 _suggested

thatanethylacetate(EtOAc)extractofTrichodermaharzianum

fermentedbrothcan controlthe tomato graymold caused

by Botrytis cinerea, without fungicide resistance and in an

environmentally friendly manner. In addition, secondary

metaboliteshavebeenacontinuoussourceofnewlead

com-poundsandchemicalentitiesintheﬁeldsofagricultureand

medicine.Forinstance,multiplefungalmetabolites,suchas

12␤-hydroxy-13␣-methoxyverruculogenTR-2,fumitremorgin

B,verruculogen,andhelvolicacidandothersfroman

endo-phyticfungus(strainAspergillusfumigatusLN-4)exhibitedhigh

antifungal activities against multifarious phytopathogenic

fungi.11

HouttuyniacordataThunb. isa signiﬁcant plantresource

toscreen forendophytic fungi that could produce speciﬁc

metabolicproductsforuseasbiofungicides.Infact,agreat

numberofmedicinalplantshavebeenusedinChinatocure

variousdiseasesforalongtime,andabundantplantuse

expe-riencehasbeenaccumulated.InTraditionalChineseMedicine

(TCM),H.cordatahasbeenusedtocureinﬂammation,

bronchi-tisinfectionsoftheupperrespiratorycavity,coughs,arthritis,

conjunctivitisandinfectionsoftheurethralchannelbecause

of its antifungal, antiviral, anti-mutagenic, anti-oxidative,

andantileukemicactivitiesand foritsimmunity-increasing

effects. Nevertheless, little published work has focusedon

testingendophyticfungifromH.cordata.Consequently,our

groupisinterestedinthesefungiandinparticularinthose

withantifungalactivity.

Basedonmetabolicstudiesaboutusingendophyticfungias

biofungicides,12_we_isolated_an_endophytic_fungal_strain_from

fresh,symptomlessH.cordatatissuesthatwerecollectedfrom

Sichuanprovince,southwestChina.Weprimarilyinvestigated

theirantifungalactivity and best processingparameters to

laya foundation forthe furtheruse ofeffective biocontrol

agents.

Materials

and

methods

Isolationoftheendophyticfungus

Sourceorganism:Theendophyticfungus(strain28)was

iso-latedfromfresh,intactH.cordata,whichwascollectedfrom

YaanCity,southwestChinaand identiﬁedbyProfessorWu

Wei.Theabovesite hasa subtropicalhumidmonsoon

cli-matewithabundantrainfall.Strain28wasthenstoredina

refrigeratorat4◦C.

Theisolationprocedurewasbasedonthearticle13,14_with

minormodiﬁcations:Healthysampleswerecollectedin

plas-ticbagsandplacedinaniceboxatonce,andthentheywere

transportedtothelaboratorywithinamaximumof48h.The

samplesurfaceswerewashedwithrunningwatertoremove

soilandthensterilizedasfollows:thesampleswererinsed

withsteriledistilledwater3times;theyweresubmergedin

75%ethanolfor1minandrinsed5timeswithsterilewater,

followedby0.1%HgCl2for1min;andlastly,theywererinsed

withsteriledistilledwater5moretimes.Thesamplesurfaces

weredriedwithsterileblottingpaper.Thesurface-sterilized

tubers were cut into 0.5cm-longsegments, and the center

ofthe sterilized leaveswas cutinto 10mm×10mmpieces

with sterile blades. The tissues (4 segments or pieces per

plate) were placed on potato dextroseagar (PDA)medium

(containing morethan 30␮gmL−1 streptomycin and

ampi-cillin) and incubated at 28◦C in the dark for 5–7d. Three

replicateplates were usedforeachtest. Thecolonieswere

examinedperiodically,andtheendophyticfungalhyphaethat

emergedfromthesegmentsweretransferredontofreshPDA

medium.Transferswerenotrepeateduntilapureculturewas

obtainedaccordingtothecolonymorphology.Tovalidatethe

effectofsurfacedisinfection,uncuttissuesthatwere

steril-izedaccordingtothesamestepswereplacedonPDAmedium

andincubatedunderthesameconditionsasthecontrol.In

addition, the last drift lotion from the surface-sterilization

process (1–2 drops)wasadded tothe PDAculturemedium

andcoatedevenlyasanothercontrol.Finally,weexamined

theeffectofdisinfectionaftercheckingwhethercolonieshad

formed.

Morphologicalcharacterization

Strain28 wasincubatedonPDAmediumat28◦Cfor7d.A

morphologicalcharacterizationwasthenusedtoidentifythe

strain using acombination ofthe fungal characteristics in

parallelwiththoseintheliterature15_including_the_mycelia,

sporangium,and sporemorphologyaswell astheexternal

formofthecolony.

Molecularidentiﬁcation,ITSsequenceanalysis16

After a strain 28 subculture was grown in an Erlenmeyer

ﬂask (250mL) containing 150mL of potato dextrose broth

(PDB)(200gL−1potatoand20gL−1dextrose,initialpH6.6–7.0)

at 130 revolutions per minute (revmin−1) for 7 dat 26◦C,

the mycelia and broth were separated by vacuum

suc-tion ﬁltration. The mycelia were then scraped from the

ﬁlter and groundinto powder withthe aid ofliquid

nitro-gen. Genomic DNA was extracted by CTAB method.17 _The

quality and integrity of the DNA were estimated by

elec-trophoresisona1%(w/v)agarosegel.TheITS1-5.8SrRNA-ITS2

region, which was the target rDNA region, was ampliﬁed

usingprimersITS1(5-TCCGTAGGTGAACCTGCGG-3)andITS4

(5-TCCTCCGCTTATTGATATGC-3).18_Each₂₅_␮L_reaction

con-tained 2.5mM10×buffer (withMg2+_), _2.0_mM ₍₁₀_␮mol_L−1₎

dNTPs,2.0mM(5␮molL−1)ofeachprimer,1.0mM(3U)Taq

DNApolymerase,and1mMgenomicDNA.Polymerasechain

reaction (PCR) ampliﬁcations were performed ina thermal

cycler(ABI2720,USA)withaninitialdenaturingstepat94◦C

for 5min, followed by 35 ampliﬁcation cycles of 94◦C for

30s,60◦Cfor 30s, 72◦C for60sand a ﬁnalextensionstep

of 72◦C for 10min. Theampliﬁcation products were

sepa-ratedbyelectrophoresisona 1%(w/v) agarosegel at100V

for30minin1×TAEbufferandstainedwithethidium

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toShanghaiSangonBiologicalEngineeringTechnology&

Ser-vicesCo. Ltd.forsequencing.Thesequencewassubmitted

totheNationalCentreforBiotechnologyInformation(NCBI)

GenBankandanalyzedbyBLAST.

Cultureconditionsduringfermentation

Strain28wasstoredintherefrigerator,andthenitwas

incu-batedonPDAmediumat28◦Cfor6–7d.Afungusplug(4mm

diameter)ofmycelialinoculumwascutfromthemarginof

activelygrowing strain 28 coloniesandtransferred into an

Erlenmeyerﬂask(250mL)containing150mLoffermentation

medium(4plugsperﬂask).Underbasecultureconditions,the

strainswereincubatedinPDBat26◦Cfor7dandinitialpH

val-uesof6.6–7.0.Therotationrateoftheincubatorwasﬁxedat

130revmin−1.Thecultureconditionswerevariedonthebasis

oftheexperimentaldesign.

Metaboliteextraction

Fungal mycelia were separated from the culture broth by

vacuumﬁltrationanddiscardedafterthefermentation

proce-dures.Theﬁltratewascollectedandconcentratedinaslittleas

10%oftheoriginalvolume(v/v)inavacuumrotaryevaporator

at40–45◦C,thenextractedthreetimesattheliquid-liquid

par-titionwithEtOAcsolvent1:1(v/v).Theresultingcrudeextracts

werecollectedandconcentratedtodrynessinavacuumrotary

evaporatorat40–45◦C,thendissolvedinto 3mLofacetone

per 150mL of initialculture volume, followed byﬁltration

through0.22␮mMilliporeﬁlterstoobtaintheEtOAc-extracted

solution.

Antifungalactivitybioassay

Theantifungalactivity ofthecrudemetaboliteswas

deter-minedbymycelialgrowthmethods19_against_the_following₁₅

speciesofplantpathogens:FusariumgraminearumSehw.(B1

forshort),Alternariasolani(Ell.etMart.)Sorauer(B2),

Phytoph-thoracapsiciLeonian(B3),Fusariumoxysporumf.sp.niveum(B4),

Sclerotiniasclerotiorum(Lib.)deBary(B5),Exserohilumturcicum

(Pass.)LeonardetSuggs (B6),Aphanomycesraphani Kendrick

(B7),PucciniazoysiaeDiet.(B8),FusariummoniliformeSheld.(B9),

Alternariaalternata (Fries) Keissler (B10),Botrytis cinerea

per-soon(B11),AlternariasolaniSorauer(B12),Thielaviopsisbasicola

(Berk.et Br.)Ferraris(B13),Botrytis cinereaPers.exFr. (B14),

and Bipolarismaydis (Nisikado et Miyake)Shoeml (B15).All

thepathogensabovewereprovidedbythelaboratoryof

phy-topathology at Sichuan Agricultural University, China. The

pathogenswereactivatedfromdormantstates.Afterthat,the

fungusplugs(4mmdiameter)of15pathogenicfungi(B1–B15)

fromthemarginofactivelygrowingcolonieswereplacedin

thecenterofPDAmediumplatescontaining10%ofthe

EtOAc-extractedsolution(v/v).Inaddition,extract-freeacetone(10%

inmedium) was usedas a control. Theexperiments were

performedintriplicateforeachpathogenicfungusand

con-trol.Thoseplatesthen were incubatedinthe darkfor7d,

andthecolonydiameterwasmeasuredintwoperpendicular

directions.

Optimizationofprocessparameters

Toscreenforthebestprocessingparameters, theeffectsof

thecultureandthefermentdisposalconditionsonantifungal

crudeextractproductionwereinvestigatedusingsingle-factor

experiments.Theinhibitoryrateofthecrudestrain28extracts

against B4 served as the indicator of antifungal activity.

Thebasecultureconditionsandthecrudeextractionofthe

metaboliteswerecompletedaccordingtotheabovemethod.

Extract-freeacetonewasusedasanegativecontrol.All

exper-imentswereperformedintriplicate.

MediaOptimization:Strain28wasinoculatedintoeight

dif-ferentliquidmedia(GauseNo.1liquidmedium,Czapek–Dox

broth,Sabouraud’sdextrosebroth,BSE(0.5%succinate,0.04%

K2HPO4, 0.04% KH2PO4, 0.02% MgSO4·7H2O, 0.05% yeast

extract;pH6.5),Martin’sbroth,LBbroth,mediumA,andPDB)

toselectasuitablemedium.

InitialpHtest:Strain28wasinoculatedontodifferentPDB

basicmediaatdifferentinitialpHvalues(initialpHvaluesof

6.0,6.3,6.6,6.9,7.2and7.5).

Incubationtimetest:Tenconicalﬂasks(250mL),eachof

whichcontained150mLofthePDBbasicmedium,were

incu-batedfor3,4,5,6,7,8,9,10,11and12d.

Thermal stability of antifungal activity assay: Seven

uniform-boreglasstubeswithﬁnegradationswerenumbered

ascontrolkeeping(CK)and1–6.Threemilliliterofthe

extract-ing solution was pouredinto the ﬂasks, which were then

heated inawaterbathfor30minattemperaturesof40◦C,

50◦C,60◦C,70◦C,80◦C,90◦C,and100◦C,respectively.

Antifungalcompoundextraction:Toinvestigatetheeffect

oftheextractionsolventonthefermentationbroth,5

differ-entextractionsolvents(petroleumether,diethylether,n-butyl

alcohol,EtOAcandalcohol)werechosentoextractthe

anti-fungalcompounds.

Antifungal activity assay: We prepared enough 50-mL

Erlenmeyerﬂaskssothateachcontainedprecisely30mLof

PDB medium[including10% oftheextracted solution(v/v)]

foreachprocessparameterabove.Next,this30mLmixture

was pouredinto threePetridishesper condition.Afungus

plug(4mmdiameter)ofmycelialinoculumwascutfromthe

marginofactivelygrowingB4coloniesasanindicatorfungus,

anditwasplacedinthecenterofthePDAmediumofeach

dish.Havingbeeninoculated,thesedisheswerewrappedwith

plasticwrap(whichwaspurchasedinthesupermarket),and

incubatedinanincubatorat28◦Cfor7d.Finally,the

suppres-sionofB4byfermentedbrothextractwasmeasuredintwo

perpendiculardirectionsusingagrowthratemethod.

Statisticalanalysis

Theinhibitoryrateofthecrudestrain28extractsagainsteach

funguswascalculatedbyusingthefollowingformula:

anti-fungal rate=[(average colony diameter of control−average

colony diameter of treatment)/average colony diameter of

control]×100.20 _The_colony_diameter_was _measured_in_two

perpendiculardirectionsasdescribedabove.

All theresultswereexpressedasthe average±standard

deviationoftriplicatedeterminations.Ananalysisof

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0.20000 mm

A

B

C

D

0.05000 mm

Fig.1–Thecolonyandmicrostructureofstrain28:colonyfrontsides(A),colonybacksides(B),ascocarps(C)andspore morphology(D).ThecolonialmorphologywasobservedaftergrowingonaPDAplateat28◦Cfor6d,andthemicrostructure ofthestrainwasobservedunderanOlympusBX51ﬂuorescencemicroscope(OLYMPUSOPTICAL.CO.LTD.Tokyo,Japan) aftergrowingfor7d.

analysis of variance. Data analysis was performed with

MicrosoftOfﬁceExcel2010software.

Results

and

discussion

Thestrain 28 colonies(which grew onPDA plates at 28◦C

for6d)weresimilarlyﬂattened,black-gray,whiteandlobed

onthe edge, and theygrew relatively slowly (Fig. 1A). The

reversesidesofthecoloniesweredarkyellowtoaurantium

(Fig.1B),and theascocarpswereoliveorgreen-yellowwith

ascomalhairs(Fig.1C).Moreover,thesporeswerepalebrown

andlemon-shaped(Fig.1D).Therefore,thesecharacteristics

correspondedwellwiththoseofChaetomiumglobosum.15

The ampliﬁed ITS1-5.8S rDNA-ITS2 region of the

selected isolates (GenBank accession number: KF697163)

was sequenced and compared with the ITS sequences of

organisms represented in the NCBI GenBank database by

usingaBLASTsearchand DNAMANtogeneratea

phyloge-netictree(Fig.2)byNeighbor-joiningmethod.Thesequences

that showed E=0.0 and the highest % similarity with the

ampliﬁedsequenceswereincludedforalignmentand

boot-strappingusingCLUSTALX.Thephylogeneticrelationofthis

isolatetorelated fungiisshowninFig. 2. Thus,thisstrain

wasidentiﬁedasC.globosumofChaetomiumsp.basedonits

morphologicalcharacteristicsandtherDNAsequencingofits

region data.Chaetomium sp.is cosmopolitanindistribution

anditrepresentsaconsiderablemicroorganismalresource.21

TheresultsinTable1showthatthecrudeEtOAcextracts

ofstrain28 couldeffectively inhibit15typesofpathogenic

fungi,especiallyB6,B11andB14,theinhibitionratiosofwhich

wereall100.00%aswellasB15andB7at91.67±0.24%and

86.18±4.28%,respectively(Fig.3).Onthewhole,the

inhibi-tionratiosforallthepathogenicfungiwereabove50%,except

forB13andB12,whichhadinhibitionratiosof41.67±1.27%

and 45.93±1.74%, respectively. This result shows that the

crude extractsofstrain28 havebroad-spectrumantifungal

abilities. In 1954, Tveit22 _suggested _that _C. _globosum _could

control the wheat seedling blight caused by

Helminthospo-rium victoriae. Abundant new reports have indicated that

Chaetomiumareinterestingmicrobialantagoniststhatareused

tocontrolphytopathogens.Therehavebeenmorethan200

compoundsreportedfromthisgenustodate.11_Many_of_them

areendophyticfungifromplants.EndophyticChaetomiumwas

reportedtobeantagonistictothedevelopmentofdiseasein

wheatleavescausedbyPyrenophoratritici-repentis.23_A_gliotoxin

isolated from endophytic C. globosum that originated from

Ginkgo bilobashowed good antifungalactivity against Fusa-riumgraminearum.24_A_novel_cytotoxic_agent_that_was_derived

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0.05 Achaetomium_strumarium_I AF048788.1 I Chaetomium_globosum_I HM01 6879.1 I Chaetomium_globosum_I HM01 6896.1 I 28.seq Chaetomium_cruentum_I HM365266.1 I Chaetomium_globosum_I HQ529775.1 I Chaetomium_murorum_I GQ376100.1 I Neurospora_crassa_I JN198494.1 I Chaetomium_elatum_I JN209872.1 I Chaetomium_elatum_I JN209873.1 I Chaetomium_subglobosum_I JN209930.1 I Chaetomium_angustispirale_I JN209862.1 I Chaetomium_cruentum_I JN209871.1 I Chaetomium_spirochaete_I JN209921.1 I Chaetomium_murorum_I JF502431.1 I 66 97 100 100 100 100 100

Fig.2–Clusteranalysisofstrain28.ThesequencesthatshowedE=0.0andthehighest%similaritywiththeamplified sequenceswereincludedforalignmentandbootstrappingusingCLUSTALX.Thisfigureshowsthephylogeneticrelationsof thisisolatewithrelatedfungi.Thus,thisstrainwasidentifiedasChaetomiumglobosumofChaetomiumsp.throughtherDNA sequencingofITSregiondata.ThistreewasconstructedonthebasisoftherDNAsequence(ITS1,5.8SandITS2)by Neighbor-joiningmethod.Bootstrapvalues>50%(1000replicates)areshownatthebranches.Thebarindicatesa5% sequencedivergence.

isolatedfromtheculturesofC.globosumthatoriginatedfrom

Ginkgo biloba.25 _C._globosum _EF18_endophytic _fungus_extract

originatingfromWithaniasomniferaisreportedtobe

signiﬁ-cantlyeffectiveagainstSclerotiniasclerotiorum.26

Fromageneticstandpoint,plantendophyticfungiareable

tosynthesizebioactivecompoundsthatareequalorsimilarto

thosesynthesizedbytheirhosts,possiblyasaresultof

hori-zontalgenetransfer27_between_endophytic_fungi_and_their

cor-respondinghosts.Furthermore,C.globosumisabletodegrade

lignin,28_which_is_beneﬁcial_to_horizontal_gene_transfer._Other

workhasshownthatarecombinantplasmidcontainingthe

TUB2-resistancegeneconferred300timeshigherressistance

tocarbendazimwhentransformedintoaChaetomiumsp.

pro-toplastusingthe PEGmethod.29 _However,_H._cordata_shows

potentantifungalactivitiesasaTCMoveralongperiod.

There-fore,strain28mightdisplayeffectiveantifungalpropertiesas

aresultofhorizontalgenetransferwithitshost.

Plantpathogensareharmfultothedevelopmentofcrops,

andthustherehavebeenmanyattemptstocontrolthem.30

However,abundantnovelpathwaysorsubstanceswith

efﬁ-cient and environmentally friendly characteristics require

further research because of the high-frequency heritable

Table1–Theinhibitionof15typesofpathogensbystrain28fermentationﬁltrateinvitro.

Pathogens Averagediameterwithextract(cm)a _Control_average_diameter_(cm)a _Inhibition_ratio_(%)a

B1 3.82±0.22 8.62±0.03 59.06±2.66 B2 2.83±0.15 7.67±0.12 67.44±2.13 B3 3.37±0.23 8.67±0.06 64.90±2.76 B4 3.62±0.08 8.63±0.06 61.68±0.94 B5 3.63±0.12 8.27±0.25 59.66±1.49 B6 0.50±0.00 1.50±0.20 100.00±0.00 B7 1.63±0.35 8.70±0.00 86.18±4.28 B8 3.73±0.25 8.47±0.32 59.41±3.16 B9 3.00±0.10 8.70±0.00 69.51±1.22 B10 2.80±0.20 7.80±0.26 68.49±2.74 B11 0.50±0.00 3.20±0.20 100.00±0.00 B12 3.60±0.10 6.23±0.25 45.93±1.74 B13 5.28±0.10 8.70±0.00 41.67±1.27 B14 0.50±0.05 6.25±0.48 100.00±0.87 B15 0.91±0.01 5.38±0.08 91.67±0.24

a _Each_value_is_the_average_±_standard_deviation_of_triplicate_{determinations.}_Fusarium_graminearum_Sehw._(B1_for_short),_Alternaria_solani_(Ell._et Mart.)Sorauer(B2),PhytophthoracapsiciLeonian(B3),Fusariumoxysporumf.sp.niveum(B4),Sclerotiniasclerotiorum(Lib.)deBary(B5),Exserohilum turcicum(Pass.)LeonardetSuggs(B6),AphanomycesraphaniKendrick(B7),PucciniazoysiaeDiet.(B8),FusariummoniliformeSheld.(B9),Alternaria alternata(Fries)Keissler(B10),Botrytiscinereapersoon(B11),AlternariasolaniSorauer(B12),Thielaviopsisbasicola(Berk.etBr.)Ferraris(B13),

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B6-CK Control +Extr acts of str ain 28 B7-CK _B11-CK _B14-CK B15-CK B6 B7 _B11 _B14 B15

Fig.3–AntifungalactivityofcultureextractsproducedwithChaetomiumglobosumstrain28.Fungusplugs(4mmdiameter) ofpathogenicfungi(B6,B7,B11,B14andB15)fromthemarginsofactivelygrowingcolonieswereplacedinthecenterof PDAmediumcontainingthe10%EtOAcextractingsolutionofstrain28(v/v).Inaddition,theextract-freeacetone(10%in medium)wasusedasacontrol(B6-CK,B7-CK,B11-CK,B14-CKandB15-CK).

variationofpathogenswithbiotopechangesanddemandsin

agriculturaldevelopment.Furthermore,endophyticfungiare

becominginterestingsourcesofbioactivecompounds

includ-ingantifungals. Ourworkhas shownthatthe crudeEtOAc

extracts ofstrain 28 were broad-spectrum and efﬁcient in

termsoftheirantifungalability.Therefore,itispromisingand

interestingtostudythisfungusfurtheranddevelopitsutility

valueasabiocontrolagent.

Wealsodiscussthe optimizationofprocessparameters

using single-factor experiments. A large amount of

endo-phyticfungalmetabolitesareobtainedprincipallybymeansof

liquidfermentation,inwhichcultureconditionsplayacrucial

role.Threevariables(culturemedium,initialpHand

fermen-tationtime)that signiﬁcantlyinﬂuencefungal growthwere

selectedasthekeycultureconditionfactorspriorto

optimiza-tion.

Culture medium directly affects the biosynthesis and

accumulationofsecondarymetabolites because itsupplies

nutrientsandservesasaspatialcarrierduringthe

fermen-tationprocess.Table2indicates thatthereweresigniﬁcant

differences (p<0.05) amongseveral culturemedia. Martin’s

broth displayed the most obvious ability to contribute to

theaccumulationofantifungalsubstancesduringstrain28

growths, and it possessed the maximum inhibition ratio

(82.53%±1.95%)(p<0.05).

The initial pH is widely recognized as a critical factor

infermentationbecause it can affectindividual

physiolog-ical phenomena, such as fungal growth, sporulation, and

the production of enzymes, and it can inﬂuence enzyme

stability,31 _the _surface _charge _of _protoplasts _and _nutrient

utilizationefﬁciency.32_Therefore,_research_is_needed_to

deter-minetheoptimalinitialpH.Therangeofinitialexperimental

pHvalues givenhere was basedon preliminary tests. The

resultsshowobvioussigniﬁcantdifferences(p<0.05)frompH

6.0–pH 7.5, and pH value of 6.9 is the most beneﬁcial for

theaccumulationofantifungalsubstancesandresultsinthe

highestinhibitionratioof81.69%±1.81%(Table3)inspiteof

showingno signiﬁcant difference(p<0.05)from that ofpH

6.0–6.9.

Theinhibitionratioincreasedsubstantially(p<0.05)from

3dto4doffermentationtime,andthentherewasno

sig-niﬁcant difference from 4 d to 8 d, immediately followed

by a signiﬁcant decrease (p<0.05) with an increase in the

fermentationtime(Table4).Secondarymetabolites(suchas

antibiotics)begintobeproducedneartheonsetofthe

sta-tionarygrowthphaseofamicrobialgrowthcurve,wherethe

speciﬁc growth rateis zero.33,34 _Therefore, _the _species _and

content ofantifungal substances increased withincreased

fermentationtime.Eventually,however,moreandmore

fun-galcelldeathoccurred,usuallybecausethecellsburstopen,

which is also known as cell lysis, and this lysis resulted

Table2–TheinhibitionofB4bystrain28when

cultivatedindifferentliquidmediuminvitro.

Media B4

Averagediameter (cm)a

Inhibitionratio (%)a GauseNo.1liquid

medium – – Czapek–Doxbroth 8.80±0.00a* _0.00_±_0.00_f* Sabouraud’sdextrose broth 2.53±0.08e 75.50±0.92b BSE 5.52±0.03b 39.56±0.35e Martin’sbroth 1.95±0.23f 82.53±1.95a LBbroth 4.02±0.03c 57.63±0.35d MediumA 3.90±0.17cd 59.04±2.09cd PDB 3.60±0.23d 62.65±2.76c Control 8.80±0.05a 0.00±0.60f

a _Each _value _is _the _average_±_standard _deviation _of _triplicate determinations.

∗ _Values_with_the_same_letter_are_not_{signiﬁcantly}_different_(p_<_0.05) accordingtotheone-wayanalysisofvariance(ANOVA).

(7)

Table3–TheinhibitionofFusariumoxysporumf.sp.

niveumbystrain28whencultivatedinliquidmedium

invitroatdifferentpHvalues.

InitialpH Fusariumoxysporumf.sp.niveum

Averagediameter(cm)a _Inhibition_ratio_(%)a

6.0 2.25±0.28cd* _78.92_±_3.35_ab* 6.3 2.40±0.20cd 77.11±2.41ab 6.6 2.33±0.13cd 77.95±1.52ab 6.9 2.02±0.16d 81.69±1.81c 7.2 2.53±0.06c 75.54±0.70b 7.5 3.05±0.15b 69.28±1.94a Control 8.80±0.05a 0.00±0.60d

∗ _Values _with _the _same _letters _are _not _{signiﬁcantly} _different (p<0.05)accordingtotheone-wayANOVA.

in reduced inhibition ratios after more than 8 d. To save

timewithoutcompromisingonthecontentofantifungal

sub-stance,4doffermentationwaschosenasasuitabletime.

Temperature treatments of the crude extracts over the

courseofconcentrationandsolventextractionisalsocritical

apartfromthecultureconditionsbecausesomecompounds

aretemperature-sensitiveorhavedifferentsolubilitiesat

dif-ferent temperatures. Therefore, the conditions of ferment

disposalwereoptimized.

Thethermalstabilityofextractsfromfermentationbroth

shouldbeinvestigatedasmuchaspossibletoprevent

ther-mal degradation.35 _We _tested _the _thermal _stability _of _the

extractsin awaterbathat differenttemperatures. Table 5

liststhechangesinantifungalactivityagainsttheindicator

pathogen(B4)aftertheEtOAcextractedsolutionwasheated

for30minattemperaturesof40◦C,50◦C,60◦C,70◦C,80◦C,

90◦C, and 100◦C. The antimicrobial activity of the EtOAc

extractingsolutionvariedfrom0.00±0.00%to55.12±0.35%.

Thereweresigniﬁcantdifferences(p<0.05)above70◦C,and

Table4–Theantifungalabilityofstrain28when

cultivatedfordifferenttimesinvitro.

Days Fusariumoxysporumf.sp.niveum

3 8.60±0.17a* _2.41_±_2.09_d* 4 3.82±0.08d 60.04±0.92a 5 3.63±0.12d 62.25±1.39a 6 3.60±0.10d 62.65±1.20a 7 3.77±0.06d 60.64±0.70a 8 3.92±0.18d 58.84±2.12a 9 4.65±0.09c 50.00±1.04b 10 4.75±0.05c 48.80±0.60b 11 5.35±0.13b 41.57±1.59c 12 5.58±0.16b 38.76±1.94c Control 8.80±0.05a 0.00±0.60d

∗ _Values_with_the_same_letter_are_not_{signiﬁcantly}_different_(p_<_0.05) accordingtotheone-wayANOVA.

Table5–Inhibitionratiooftheantimicrobialsubstances

instrain28whentreatedatdifferenttemperatures

invitro.

Temperature (◦C)

Fusariumoxysporumf.sp.niveum

40 4.20±0.26de* _54.88_±_3.23_ab* 50 4.18±0.03e 55.12±0.35a 60 4.38±0.18de 52.68±2.14ab 70 4.58±0.08cd 50.24±0.93bc 80 4.73±0.08c 48.41±0.93c 90 7.97±0.15b 8.90±1.86d 100 8.70±0.00a 0.00±0.00e Control 8.70±0.09a 0.00±1.06e

∗ _Values _with _the _same _letters _are _not _{signiﬁcantly} _different (p<0.05)accordingtotheone-wayANOVA.

theinhibitionratiodecreasedrapidlyto0at100◦C.Clearly,

this resultindicatesthattheantimicrobialsubstancesfrom

strain28wereheat-stableafterthermaltreatmentupto80◦C.

Theantimicrobialactivitiesoftheextractsthatweretreated

attemperatureshigherthan80◦Cdeclinedsharply,possibly

becauseofthermaldegradation.ItisnotablethatZhaoetal.36

drewasimilarconclusionwhentheantibacterialmetabolites

ofamarinefungus(NJ0104)werethermallystableupto80◦C.

Hence,thereisajustiﬁcationinthatpartoftheantibacterial

metabolitescanmaintainbiologicalactivityatleastatmarine

temperatures.

Bioactive antifungalcompounds areextracted efﬁciently

onlybysuitableextractionsolventsbecause oftheir

differ-entpolarityandsolubilityproperties.Thereweresigniﬁcant

differences(p<0.05)amongﬁveextractionsolvents(Table6).

EtOAc extractexhibitedthestrongestantifungalability

fol-lowedbydiethyletherandn-butylalcoholextracts.

Onamoregeneralnote, thereisalsoaneedforfurther

study on howto usesecondary metabolitesfrom strain 28

as biocontrol agents. For example,there is alack of

stud-iesondirectantifungaleffectsincrops,andphytopathogenic

fungusisoneofthevitallinkstodevelopingbio-antifungal

Table6–Inhibitionratioofthefermentationﬁltrates

fromstrain28whenextractedindifferentsolvents

invitro.

Extraction solvents

Fusariumoxysporumf.sp.niveum

Averagediameter(cm)a _Inhibition_ratio_(%)a Petroleumether 4.22±0.26b* _55.18_±_3.09_d* Diethylether 2.12±0.06d 80.48±0.70b N-butylalcohol 2.20±0.26cd 79.52±3.19bc Ethylacetate 1.91±0.02e 83.01±0.28a Alcohol 2.27±0.06c 78.67±0.70c Control 8.80±0.05a 0.00±0.60e

∗ _Values_with_the_same_letter_are_not_{signiﬁcantly}_different_(p_<_0.05) accordingtotheone-wayANOVA.

(8)

agents.Previousstudiesonlyrevealedtheantifungalactivity

ofthecrudeEtOAcextractsinspiteoftheactivityofthe

sin-glecomponents.However,thisinvestigativedirectionwould

leadtofascinatingscientiﬁcresearchandpractical

applica-tionvaluebecause oftheextract’s obviousbroad-spectrum

antifungalability.Thisarticleisalsotheﬁrstreportonthe

isolationofendophyticC.globosumfromH.cordataasa

biocon-trolagent.Moreover,preliminarydatafromprocessparameter

optimizationassays will provide good guiding signiﬁcance

forharvesting more secondary metabolites from strain 28.

Inaddition,numeroussecondarymetabolitesfrommultiple

C.globosum strainshave beenreported andshowed a

vari-etyofbiologicalactivitiesincludingcytotoxicity,antibacterial

activity,phytotoxicity,elicitoractivityandothers.37_Therefore,

studyingthisorganismanditsantifungalcompoundsfurther

todeveloppracticalapplicationswillbeapromisinglineof

researchforthefuture.

Author

contributions

FP, ZQL and WWconceived and designed the experiment.

ZQL, QC, and YWX participated in sample collection and

the isolation of the strain. FP, ZQL and KH analyzed and

interpretedthe sequence data and identiﬁedthe strain. In

addition, the antifungal activity bioassay and optimization

ofprocessparametersincludingstatisticalanalysiswere

per-formedbyFPand ZQL.Moreover,FP, ZQLand WWdrafted

andrevisedthemanuscript.Allauthors readand approved

theﬁnalmanuscript.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

This work was ﬁnancial supported by the “211” Project

Double-SupportPlanofSichuanAgriculturalUniversity(No.

00170705).Wearegratefultotheassistanceofthelaboratory

ofphytopathology ofSichuan agriculturalUniversity,China

forprovidingplantpathogens.

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