Alternariol 9-methyl ether from the endophytic fungus Alternaria sp. Samif01 and its bioactivities

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

Industrial

Microbiology

Alternariol

9-methyl

ether

from

the

endophytic

fungus

Alternaria

sp.

Samif01

and

its

bioactivities

Jingfeng

Lou

a

_,

_Ruiting

_Yu

a

_,

_Xiaohan

_Wang

a

_,

_Ziling

_Mao

a

_,

_Linyun

_Fu

a

_,

_Yang

_Liu

b

_,

Ligang

Zhou

a,∗

a_{MOAKeyLaboratoryofPlantPathology,DepartmentofPlantPathology,CollegeofAgronomyandBiotechnology,ChinaAgricultural}

University,Beijing100193,China

b_{InstituteofAgro-productsProcessingScienceandTechnology,ChineseAcademyofAgriculturalSciences,Beijing100193,China}

a

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o

Articlehistory:

Received7August2013 Accepted16November2014

AssociateEditor:EleniGomes

Keywords:

SalviamiltiorrhizaBunge Endophyticfungus

Alternariasp.,Alternariol9-methyl ether

Bioactivity

a

b

s

t

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t

Onebioactivecompound,identifiedasalternariol9-methylether,wasisolatedfromthe crudeextractoftheendophyticfungusAlternariasp.Samif01residingintherootsofSalvia miltiorrhizaBunge.Alternariol9-methyletherwasactiveagainstbacteriawithminimum inhibitoryconcentrationvaluesrangingfrom25to75␮g/mLandmedianinhibitory concen-tration(IC50)valuesrangingfrom16.00to38.27␮g/mL.TheIC50valueofalternariol9-methyl

etheragainstsporegerminationofMagnaportheoryzaewas87.18␮g/mL.Alternariol9-methyl etheralsoshowedantinematodalactivityagainstBursaphelenchusxylophilusand Caenorhab-ditiseleganswithIC50valuesof98.17␮g/mLand74.62␮g/mL,respectively.Thisworkisthe

firstreportonalternariol9-methyletheranditsbiologicalactivitiesfromtheendophytic fungusAlternariasp.Samif01derivedfromS.miltiorrhizaBunge.Theresultsindicatethe potentialofAlternariasp.Samif01asasourceofalternariol9-methyletherandalsosupport thatalternariol9-methyletherisanaturalcompoundwithhighpotentialbioactivityagainst microorganisms.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

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

Introduction

Plantendophyticfungiaredefinedasfungithatlive asymp-tomatically within plant tissues.1 _{They are rich potential}

resourcesforproducingbioactivemetabolitessuchas antimi-crobial, insecticidal, anti-viral, anti-tumor and antioxidant compounds.2,3_{Someendophyticfungihavetheabilityto}

pro-ducethesameorsimilarbioactivecompoundsastheonesthat

∗ _{Correspondingauthorat:DepartmentofPlantPathology,CollegeofAgronomyandBiotechnology,ChinaAgriculturalUniversity,Beijing}

100193,China.

E-mail:lgzhou@cau.edu.cn(L.Zhou).

originatefromtheirhostplants.4_{Isolationoftheendophytic}

fungithatproducebioactivesubstanceshasalsobecomean efficientmethodtoscreenbroad-spectrum,stableagentswith lowphytotoxicity.5,6

SalviamiltiorrhizaBunge(Lamiaceae)hasbeenwidelyused intraditionalorientalmedicineforimprovingbodyfunctions, treatment of cardiovascular diseases, and liver diseases.7,8

Some endophytic fungi isolated from S. miltiorrhiza were

http://dx.doi.org/10.1016/j.bjm.2015.11.004

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

determinedtocontaintanshinonesbyTLC,HPLC,andLC-MS analyses,9,10_{thoughthesetanshinone-producingendophytic}

fungishouldbefurtherverified.

Inthiswork,wereporttheendophyticAME-producing fun-gusAlternariasp.Samif01derivedfromS.miltiorrhiza.Boththe fungusandtheAMEstructurewereidentified.The antimicro-bialandantinematodalactivitiesofAMEwerealsoevaluated toprovidedatasupportingthedevelopmentandutilizationof

Alternariasp.Samif01.

Materials

and

methods

General

Themelting point(m.p.)ofthecompound wasdetermined onanXT4-100Bmicroscopicmelting-pointapparatus (Tian-jin Tianguang Optical Instruments Company, China) and uncorrected.NMRspectrawererecordedonaBrukerAvance DRX-400(1_Hat400MHzand13_{Cat100MHz)NMR}

spectrome-terusingtetramethylsilane(TMS)asaninternalstandard,and chemicalshiftswererecordedasıvalues.Ahigh-resolution electrospraymass spectrometry (HR-ESI-MS) spectrumwas measured using a Bruker Apex IV FTMS mass spectrome-ter.Amicroplatespectrophotometer(PowerWaveHT,BioTek Instruments,USA)was appliedtomeasurelightabsorption valuesofthesamples.Thesilicagel(200–300mesh)for col-umnchromatography(CC)andsilicagelGF254forthinlayer

chromatography(TLC)werepurchasedfromQingdaoMarine Chemical Company, China. Sephadex LH-20 and silica gel RP-18 were obtained from Pharmacia Biotech, Sweden. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)waspurchasedfromAmresco(USA).Streptomycin sul-fateand carbendazim were purchased from Sigma–Aldrich (USA).Avemectinwithapurityof97.2%waskindlyprovidedby Dr.ShankuiYuanattheInstitutefortheControlof Agrochemi-cals,ChineseMinistryofAgriculture.Allotherchemicalsused inthestudywereofanalyticalgrade.

Plantmaterialsandisolationofendophyticfungi

Thethree-yearoldhealthyrootsofS.miltiorrhizaBungewere collectedfromtheInstituteofMedicinalPlantDevelopment (116◦1627E, 40◦159N), Chinese Academyof Medical Sci-ences,Beijing, China,inJuly2011.Theplantwasidentified accordingtoits morphologicalfeaturesbyProf.YuhaiGuo, a botanist from the College of Agronomy and Biotechnol-ogyatChinaAgriculturalUniversity.Thevoucherspecimen (BSMPMI-201107001)ofthisplantwasdepositedinthe Herbar-ium ofthe Institute ofChinese MedicinalMaterials, China Agricultural University. The plant samples were stored in sealedplasticbags at4◦Cforprocessingwithin24hof col-lection. The isolation of endophytic fungi was performed accordingtopreviousreports11,12_{withsomemodifications.}

Therootsampleswererinsedthoroughlywithtapwater toremovesoilresiduesanddust,sterilizedwith75%ethanol for2minandimmersedin0.2%mercuricchloridefor20min, thenrinsed4timeswithsteriledistilledwater.Aftersurface sterilization,bothrootepidermisandremnanttissueswere cut into small pieces of 0.5cm×0.5cm and placed under

aseptic conditions on potato dextrose agar (PDA) plates containing500␮g/mLofstreptomycinsulfateandincubated at25◦CuntilthemyceliawereapparentonPDAplates.Pure cultureswerefinallyisolatedbyhyphaltipisolationonPDA plateswithoutantibioticsandstoredat4◦C.

Morphologicalcharacterization

The morphological characteristics of the fungal isolate Samif01wereobservedanddescribedaccordingtothe meth-ods of Ainsworth et al.,13 _{Photita et al.}14 _{and Li et al.,}11

includingcolonymorphologyandmicroscopicobservationof myceliaandasexual/sexualspores.

DNAextraction,ITS-rDNAamplificationandsequence analysis

Total genomic DNA of the fungal isolate Samif01 was prepared according to the protocols described by Wang et al.15 _{and Jasalavich et al.}16 _{The ITS region was}

ampli-fied by polymerase chain reaction (PCR) with the primer pair ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3)asdescribedpreviously.11,12,17

For identification, the PCR product was purified using the QIA quick Gel Purification Kit (Qiagen, Hilden, Germany) asdescribedbythemanufacturer’sprotocolandsequenced usingtheprimerpairITS1andITS4ontheABIPRISM3730 sequencer (Applied Biosystem, USA). Then, the sequence was run by the BLASTN program against the database (National Center for Biotechnology Information website:

http://www.ncbi.nlm.nih.gov) and submitted to GenBank,

wheretheaccessionnumberwasobtained.

Myceliasuspensioncultureandcrudeextractpreparation

After growing on a PDA plate at 25◦C for 5 days, two or threemyceliaplugsfromtheactivelygrowingcolonyedgeof theendophyticfungusSamif01wereinoculatedinto1000mL Erlenmeyer flaskscontaining 300mLpotato dextrosebroth (PDB).Allflaskswereincubatedinarotaryshakerat150rpm and25◦Cfor15days.Afterwards,atotalof30Lfermentation brothwasharvested.Thefermentedbrothwasfilteredunder vacuumtoseparatethemyceliafromthefiltrate.Themycelia weredriedandpowdered(40.2g),thenextractedinmethanol withultrasound.Themethanolsolutionwasconcentratedin vacuumat50◦Ctoobtainacrudemethanolextract(12.6g), whichwasfurtherthoroughlymixedwithwaterand fraction-atedwithethylacetatetoobtainaconcentratedethylacetate fraction(4.6g).Thefiltratewasfractionatedwithethylacetate toaffordaconcentratedethylacetatefraction(3.0g).Asthe TLCprofilesofthemyceliaandfiltratefractionswere simi-lar,theywerecombinedtoobtainacrudetotalethylacetate extract(7.6g).

Separationandidentificationofalternariol9-methylether

The crude ethyl acetate extract (7.0g) was first subjected tochromatographyoverasilicagelcolumn(200–300mesh) elutedwithpetroleumether-ethylacetate(3:2,v/v)toobtain eightfractions(fractionsA–H)usingTLCdetection.According

totheTLC-bioautographyassayoftheseeightfractions, frac-tionHpresentedastrongantimicrobialactivity.Itwasselected forfurtherseparation andpurification ofthe antimicrobial metabolites.Thus,fractionH(2.0g)wasfurtherfractionated on a silica gel column eluted with petroleum ether-ethyl acetate(from100:0–0:100,v/v)togivefivesubfractions (frac-tions I–V). Fraction III (0.16g) obtained by elution with petroleumether-ethylacetate(100:3,v/v)wasfurtherpurified by Sephadex LH-20 (CHCl3–MeOH=1:1, v/v) and

recrystal-lizedtoobtainonecompound(30mg),whichwasidentified usingphysicochemicalandspectrometricmethods(i.e., crys-talshape,m.p.,HR-ESI-MS,andNMR)aswellasthedatain theliterature.

Biologicalactivityassay

Amodifiedbrothdilution-colorimetricassayusingthe chro-mogenic reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, purchased from Amresco, USA) wasusedtodetecttheantibacterialactivityofthesamples according to our previous reports.18,19 _{The antimicrobial}

activityofthesampleswasevaluated bybothantibacterial andantifungalactivityassays.Sixbacterialstrainsincluding twoGram-positive (Bacillus subtilis ATCC 11562 and Staphy-lococcus haemolyticus ATCC 29970) and four Gram-negative (Agrobacterium tumefaciens ATCC 11158, Pseudomonas lachry-mans ATCC 11921, Ralstonia solanacearum ATCC 11696, and

Xanthomonas vesicatoriaATCC 11633) bacteriawere selected todetecttheantibacterialactivityofthesamples.The min-imum inhibitoryconcentration(MIC)value forthebacteria wasdefinedasthelowestsampleconcentrationthatinhibits visiblegrowth,asindicatedbyMTTstaining.Inaddition,the determinationofAMEantifungalactivitywasperformedby

the spore germinationassay.18 _{Spores were prepared from}

7-day-old cultures of Magnaporthe oryzae according to our previousreport.19_{Theantinematodalactivityofthesamples}

was determined according to the method ofWang et al.20

BothBursaphelenchusxylophilusandCaenorhabditiseleganswere usedastestnematodes.Themedianinhibitoryconcentration (IC50)valueofeach samplewascalculatedusing thelinear

relationbetweentheinhibitoryprobabilityandthe concen-trationlogarithmaccordingtothemethodofSakuma.21_Allof

thesebacterial,fungal,andnematodestrainswereprovided bytheDepartmentofPlant PathologyofChinaAgricultural University.

Results

and

discussion

IdentificationoftheendophyticfungusSamif01

Atotalof57endophyticfungalisolateswereseparatedfrom the root epidermis and remnant tissues of S. miltiorrhiza.

Accordingtotheirmorphologicalcharacters(e.g.,theshapeof conidia,typeofconidiophores,growthrate,colonycolorand texture), 14representative fungal isolateswereselectedfor furtherbioactivityscreening.22_{Amongthesefungi,theextract}

isolatedfromtheSamif01strainbyTLC-bioautographyassay showedthegreatestantimicrobialactivity.23

The colony of isolate Samif01 grew rapidly on a PDA plate at 25◦C,reaching 60–70mm indiameter after5days ofincubation.Thecoloraroundthe colonyvariedfrom ini-tially white to darkgreen (Fig. 1A), whereas the back side ofthecolonydisplayedayellowishcolor.Theconidia were obclavate,obpyriform withthreetoseven transversesepta (commonlyfour), zero tothreelongitudinal septa,smooth,

C

A

B

Alternaria alternata KC134318 Alternaria sp. KC147580 Alternaria sp. KC139492 Samif01 KC878695

100

20 μm

Fig.1–Thecolonyfrontview(A),conidiospores(B)andphylogenetictree(C)oftheendophyticfungusAlternariasp. Samif01fromS.miltiorrhiza.

Intens. x106 4 3 2 1 0 230

A

11.8244 10.3563 7.2327 7.2291 6.7326 6.7268 6.6531 6.6471 6.6261 6.6216 3.9114 3.3196 2.7384 2.5029 2.4992 1.2285 166.17164.69 164.13 158.58 152.64 138.46 137.80 117.61 108.81 103.42 101.63 99.19 98.48 55.8540.15 39.94 39.73 39.52 39.3139.10 38.90 25.02 3.39 12.50 9.40 9.78 3.04 3.03 3.26 3.29 2.84 2.61 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 ppm 200 180 160 140 120 100 80 60 40 20 0 ppm 46.86 240 250 260 270 280 290 m/z 273.07605 261.13094 253.06277 240.06657 [M+H]+

B

C

Fig.2–HR-ESI-MSspectrum(A),1_{HNMRspectrum(B)at400MHzand}13_{CNMRspectrum(C)at100MHzofalternariol}

9-methyletherinDMSO-d6.ThechemicalshiftsareshowninppmwithTMSasaninternalstandard.

brown to dark brown (Fig. 1B). These morphological char-acteristicsenabledtheidentificationofisolateSamif01asa speciesinthegenusAlternaria.TheITS1-5.8S-ITS4(ITS)partial sequenceofisolateSamif01wassubmittedtoNCBIGenBank toobtain itsaccessionnumber,KC878695.Thesequence of theisolateSamif01showed100%similaritytotheAlternaria

sp.(KC139492),Alternariasp.(KC147580)andAlternaria alter-nata(KC134318)fungiinGenBank.Thephylogenetictreeof theendophyticfungusSamif01wasshowninFig.1C.Onthe basisoftheITSsequenceandmorphologicalcharacteristics, thefungusSamif01wasconsideredamemberofthegenus

AlternariaandidentifiedasAlternariasp.Samif01.11,13,14 Structuralidentificationofalternariol9-methylether

A very effective antimicrobial compound was successfully obtainedfromtheethylacetatefractionofthecrudemethanol

extractoftheendophyticfungusSamif01basedon bioassay-guided fractionation. Its characterization data were given as follows: yellowish needle-like crystals (acetone); m.p. 267–270◦C.ThemolecularformulawasassignedasC15H12O5

byHR-ESI-MS(m/z273.0761[M+H]+_{;calculatedforC} 15H13O5

as273.0758)showninFig.2A,whichjustmatchedtheresults for alternariol 9-methyl ether previously reported in the literature.241_{HNMR(400MHz,DMSO-d} 6,Fig.2B)ı(ppm),2.74 (3H,s,CH3-1),6.73(1H,d,J=2.3Hz,H-2),10.36(1H,s,OH-3),6.65 (1H,d,J=2.4Hz,H-4),11.82(1H,s,OH-7),6.62(1H,d,J=1.8Hz, H-8),3.91(3H,s,CH3O-9),7.23(1H,d,J=1.6Hz,H-10);13CNMR (100MHz,DMSO-d6,Fig.2C)ı(ppm),138.46(C-1),25.02(CH3 -1),117.61(C-2),158.58(C-3),101.63(C-4),152.64(C-4a),164.69 (C-6),98.48(C-6a),164.13(C-7),99.19(C-8),166.17(C-9),55.85 (CH3O-9), 103.42(C-10),137.80 (C-10a),108.81(C-10b). After

comparingthedatawithreportsintheliterature,19,24_the

Table1–Biologicalactivitiesofalternariol9-methylether.

Testorganism Alternariol9-methylether(␮g/mL) CK+_(␮g/mL)

MIC IC50 MIC IC50 A.tumefaciens 75.0 38.07±2.78 25.0 11.96±1.25 B.subtilis 50.0 22.83±1.06 37.5 20.58±0.88 P.lachrymans 25.0 16.00±0.30 25.0 4.69±0.79 R.solanacearum 75.0 38.27±1.80 100.0 41.26±2.41 S.haemolyticus 37.5 19.90±1.62 12.5 4.24±0.94 X.vesicatoria 75.0 31.99±1.34 50.0 22.83±1.37 M.oryzae nd 87.18±1.39 nd 6.25±0.19 B.xylophilus nd 74.62±0.62 nd 12.90±0.27 C.elegans nd 98.17±0.92 nd 14.00±0.19

Note:Thepositivecontrols(CK+_{)forbacteria,fungusandnematodeswerestreptomycinsulfate,carbendazimandavermectin,respectively.The} ‘nd’meansnotdetected.

O 1 2 3 4 4a 6 5 7 6a 8 9 10 10a 10b O OH OCH₃ HO CH₃

Fig.3–Chemicalstructureofalternariol9-methylether.

AME,djalonensone,and 3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one),andthestructureisshownin

Fig.3.

Alternariol 9-methyl ether (AME) is a dibenzo-␣-pyrone analog. It was reported previously as a mycotoxin pro-ducedbythephytopathogenicfungiinthegenusAlternaria.25

Recently, it has been isolated from a series of endophytic fungi, mainly including Alternaria fungi, such as Alternaria

sp.fromPolygonumsenegalense,24_{AlternariabrassicicolaML-P08}

fromMalushalliana,26_{Alternariasp.fromDaturastramonium,}27

and Alternaria sp. N.SBA10 from Scutellaria baicalensis.28

Other AME-producing endophyticfungi include Cephalospo-riumacremoniumIFB-E007fromTrachelospermumjasminoides,29 Hyalodendriellasp.Ponipodef12fromthehybrid‘Neva”of Popu-lusdeltoidesMarshxP.nigraL.,19_{Nigrosporasphaericafromthe}

lichenParmelinellawallichiana,30 _{andPhialophorasp.fromthe}

lichenCetreliabraunsiana.30

Bioactivityofalternariol9-methylether

The antimicrobial activity of alternariol 9-methyl ether (AME) was evaluated by broth-dilution-colorimetric and sporegerminationassays,withtheresultsshowninTable1. AMEwas foundto displayantibacterial activity toward six differentbacteriastrainswithIC50valuesvaryingfrom16.00

to38.27␮g/mL. AME showed stronger antibacterial activity against R. solanacearum than the conventional antibiotic

streptomycin sulfate. This result indicates that the R. solanacearumstrainmightbeastreptomycin-resistantstrain. The IC50 value of AME against spore germination of M.

oryzae was 87.18␮g/mL. AME also showed antinematodal activityagainstC.elegansandB.xylophiluswithIC50valuesas

74.62␮g/mLand98.17␮g/mL,respectively.

PreviousreportsshowedthatAMEexhibitedstrong anti-fungal activity against Microbotryum violacerum,31 _induced

mitochondrial apoptosisinhumancoloncarcinomacells,32

and exhibited DNA strand breaks, micronuclei, and gene mutationsinvariousculturedmammaliancells.33_Theresults

inthis workfurthersupportedtheantimicrobialactivityof AME.However,themechanismsofactionofAMEneedtobe furtherstudied.Thesefindingshaveattractedtheattentionof manyresearchersallovertheworldregardingthesynthesis andapplicationsofAME.25

Conclusion

ThisworkisthefirstreportoftheAME-producingendophytic fungusAlternariasp.Samif01fromthemedicinalplantS. mil-tiorrhiza. Inthis study,AMEwasfoundtobeactiveagainst bacteria, fungus and nematodes. The results indicate the potentialofAlternariasp.Samif01asasourceofAMEandalso supportthatAMEisanaturalcompoundwithstrong antimi-crobialactivity.

Theremarkableantimicrobialandantinematodalactivity ofAME suggests thatendophytic fungussuchasAlternaira

sp. Samif01 could protect S. miltiorrhiza plants by produc-ing bioactive metabolites that are toxic or even lethal to phytopathogens.FromTLC-bioautographyexamination, addi-tional antimicrobialcompoundshavenotyetbeen isolated fromAlternariasp.Samif01.Theseparationandpurification of these compounds are still in progress. In addition, the phylogeneticidentificationoftheSamif01strainrequires com-plementaryinvestigation.

Conflicts

of

interest

Acknowledgements

ThisworkwassupportedbygrantsfromtheNationalBasic ResearchProgramofChina(2013CB127805)andtheHi-Tech R&DProgramofChina(2011AA10A202).

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