Research Article Antagonistic Evaluation of Bioactive...

Research ArticleAntagonistic Evaluation of Bioactive Metabolite fromEndophytic Fungus Aspergillus flavipes KF671231

Ankita Verma12 B N Johri1 and Anil Prakash2

1 Department of Biotechnology Barkatullah University Bhopal 462026 India2Department of Microbiology Barkatullah University Bhopal 462026 India

Correspondence should be addressed to Ankita Verma ankitaverma1234gmailcom

Received 25 May 2014 Revised 14 July 2014 Accepted 15 August 2014 Published 27 August 2014

Academic Editor Zia U Khan

Copyright copy 2014 Ankita Verma et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Of the total 40 endophytic fungi isolated from foliar tissues ofmedicinal plant Stevia rebaudianaBertoni a fungal isolateAspergillusflavipes was subjected to bioassay guided fractionation The fractionation was found active against medicinal plant pathogenSclerotinia sclerotiorum with an inhibition zone of 29mm in size Further the metabolite was extracted which shows 20 growthinhibition in 24 h and 46 after 48 h respectively Bioassay guided chemical compound was identified as 12-benzenedicarboxylicacid mono(2-ethylhexyl) ester On the basis of morphological characters and rDNA sequencing of ITS region the endophyte wasidentified as Aspergillus flavipes which showed promising plant growth promotory properties

1 Introduction

Bioprospecting refers to the search for novel products of eco-nomic importance from animal plant and microbial sources[1] Traditionally we always relied on natural remedies fortreating andhealing our ailmentsNatural products have beenexploited for human use for thousands of years and plantshave been a major source of compounds of medicinal usePlants are found to be associated withmicroorganisms whichare capable of producing molecules possessing remarkablebiological activities Microorganisms that live in the intercel-lular spaces of different parts of plants showed no evidentexpression of their presence and are called endophytes [2]The relationship between plant and endophyte is consideredto be mutualistic the former being protector and feeder forthe latter which in return produces bioactive substances thatprovide protection to plant and also enhance the growth andcompetitiveness of the host in nature [3]

Sclerotinia sclerotiorum a phytopathogenic fungus occursworldwide and infects a wide array of plants resulting inconsiderable losses Although a significant number of naturalbioactive compounds have been reported to antagonize thisfungus [4] Stevia (Stevia rebaudiana Bertoni Asteraceae)

an exotic annual plant originating from Paraguay containsglucosides of a diterpenoid nature which are used as a low-caloric sweetener in some South American and South EastAsian countries [5] The main active ingredient of Stevia is astevioside which is 100 to 300 times sweeter than sucrose

Traditionally farmers use agrochemicals to protect plantsfrom diseases and to optimise the crop yields Prolonged andpersistence use of pesticides including fungicides and her-bicides made several organisms resistant to such chemicalsbesides causing the environmental pollution This presentstudy represents a step towards understanding the endo-phytes as a means to develop effective biocontrol agents forStevia rebaudiana Bertoni with broader implications for useagainst other crops

2 Materials and Methods

21 Sample Collection Leaves of Stevia rebaudiana Bertoni(Family Asteraceae) were randomly collected from MisrodAgricultural field near Bhopal India (Lg N 23090211015840 andLt E 077∘271015840043) Immediately after collection leaves werewashed with tap water and processed for isolation of endo-phytic fungi

Hindawi Publishing CorporationJournal of MycologyVolume 2014 Article ID 371218 5 pageshttpdxdoiorg1011552014371218

2 Journal of Mycology

22 Media Preparation Potato dextrose agar (PDA Hi-media) was used to isolate endophytic fungi Chlorampheni-col (02 gLminus1) was added to the medium to avoid bacterialcontamination

23 Isolation of Endophytic Fungi Endophytic fungi wereisolated from healthy leaves employing surface sterilizationprocedure [6] Briefly the tissues were surface sterilized with70 ethanol for 2min followed by treatment with 4 sodiumhypochlorite for 2min Finally tissues were washed withsterile distilled water for 2 sec Sterile leaves were placedon blotting sheet and cut into 5mm pieces which weretransferred to PDA plates Plates were incubated at 28∘C for3ndash6 days Hyphal tips of the developing fungal colonies weretransferred to fresh PDA plates to get pure cultures

24 Confrontation Bioassay Primary screening for antago-nism was done by a confrontation assay [7] The endophyteand the pathogenic fungus (Sclerotinia sclerotiorum) werecocultured on 90mm petri dish containing PDA and incu-bated at 28∘C for 3ndash5 days to check the interaction betweenthem A culture of the pathogen Sclerotinia sclerotiorum wasobtained from the Directorate of Soybean Research IndorePlates were observed regularly and antagonismwas expressedby the presence of inhibition zone at the point of interaction

25 Molecular Identification of Antagonistic Endophyte Mor-phological identification of the organism was carried outat National Fungal Culture Collection of India (NFCCI)Agharkar Research Institute Pune For molecular identifi-cation total genomic DNA of the endophytic fungus wasisolated directly from actively growing mycelium growingin potato dextrose broth (PDB) using CTAB method [8]DNA amplification was performed by PCR using primer pairITS1 TCCGTAGGTGAACCTGCGG and ITS4 TCCTCC-GCTTGATATGC [9] PCR was carried out according tothe following protocol initial denaturation at 95∘C for 5mindenaturation at 95∘C for 1min annealing at 55∘C for 45 secextension at 72∘C for 10min steps 2ndash4were repeated 35 timesSequencing of PCR product was carried at Xcelris Labs LtdAhmedabad The sequenced data was subjected to BLASTalgorithm and submitted to Genebank for accession number

26 Plant Growth Promoting Attributes Plant growth pro-moting attributes of the antagonistic endophyte such asproduction of IAA [10] and siderophore [11] and phosphatesolubilization activity [12] was assayed both qualitatively andquantitatively

27 Evaluation of Bioactivity Bioactivity of the culture filtrateagainst Sclerotinia sclerotiorum was evaluated over growthand temperature

28 Influence of Growth Period A 48 h old fungal culturegrown on PDA was inoculated in Erlenmeyer flasks contain-ing 100mLPDBThe flasks were incubated at 28∘C at 120 rpmfor 10 d Culture broth was recovered at 2 4 6 8 and 10 d

after incubation by centrifugation at 12000 rpm for 20min at4∘C A disc diffusion assay was carried out to determine thebioactivity of the culture filtrate obtained after 6 d of fungalgrowth

29 Influence of Temperature TheErlenmeyer flasks contain-ing 100mLPDB were incubated at 22 24 26 28 30 and32∘C at 120 rpm for 6 d to assess the influence of temperatureon bioactive metabolite The culture filtrate was harvestedand antagonistic activity of the filtrate was checked by diskdiffusion assay against Sclerotinia sclerotiorum

210 Extraction of Antifungal Metabolite For extraction ofantifungal metabolite the culture filtrate was centrifuged at12000 rpm for 20min at 4∘C and the culture supernatant(pH 58) was acidified (1NHCl) to a final pH 20 [13] Theacidified filtrate was subjected to organic extraction by ethylacetate (1 1 vv) Organic extract was dried under vacuumin a rotary evaporator The dried crude was dissolved in2mLofmethanol For antagonistic assay 40120583L ofmethanolicextract was impregnated on sterile paper discs MeOH coateddiscs were used as control Plates were incubated at 28∘Cand inhibition of fungal growth was recorded after 24 h and48 h Growth inhibition was calculated as per the followingformula

GI = (119860 minus 119861)119860times 100 (1)

where119860 = radial diameter of test fungus 119861 = radial diameterof test fungus against culture

211 Characterization of Bioactive Compound

2111 Chromatographic Detection and Partial Purification ofBioactive Metabolite Thin layer chromatography (TLC) wasperformed on crude extracted from the culture broth of theendophyte (TLC silica gel 60 20 times 0 05mm Merk CoInc) For this the crude fraction was spotted (30120583L) on theTLC plate and chromatography was performed by employ-ing solvent system dichloromethane methanol (95 5 vv)Spots were visualized by spraying with ceric sulphate silicaresidue was extracted and centrifuged and the supernatantwas transferred to a microcentrifuge tube The silica-freesupernatant was checked for antifungal activity PreparativeTLC was carried out to obtain the partial purified samplewhich showed antifungal activity

2112 GC-MS Analysis GC-MS analysis was carried outat Central Instrumentation Facility JNU (New Delhi) Gas-chromatography mass spectrometry (GC-MS) analysis of thecrude was performed on a Shimadzu GCMS-QP-2010 plussystem RTx-5 SilMS column (30m times 025mm id times025 filmthickness) was used for the analysisThe operating conditionsof the column were as follows oven temperature programfrom 80∘C to 210∘C at 4∘Cmin with holding time of 2minand from 210∘C to 300∘C at 15∘Cmin with holding timeof 5min and the final temperature was kept for 20minThe injector temperature was maintained at 270∘C and

Journal of Mycology 3

the volume of injected sample was 03120583L pressure 854 kPatotal flow 768mLmin column flow 121mLmin linearvelocity 405 cmsec purge flow 30mLmin split ratio 600ion source temperature 230∘C scan mass range119898119911 40ndash600and interface line temperature 280∘C The identification ofcompounds was performed by comparing the mass spectrawith data from NIST05 (National Institute of Standards andTechnology US) WILEY 8 and FFNSC13 (Flavour andFragrance Natural and Synthetic Compounds) libraries

3 Results

31 Confrontation Assay and Identification of Potential Antag-onistic Endophytic Fungi The confrontation assay showedstrong inhibition of Sclerotinia scleretiorum and produced aninhibition zone of 29mm after 48 h The antagonistic endo-phytic fungus was identified by rDNA sequencing of ITSregion wherein it showed 99 similarity with Aspergillusflavipes (accession number KF671231)

32 Plant Growth Promoting Attributes Aspergillus flavipeswas positive for siderophore production as evident by anorange halo on CAS medium It solubilized inorganic phos-phorus as determined by a clear zone around the cultureon Pikovskayarsquos agar IAA production was confirmed bythe appearance of stable pink colour when reacted withSalkowsky reagent This fungal strain produced 73120583gmLminus1of IAA whereas siderophore level after 2 d was estimated at2272mgmLminus1 endophyte was found as a promising phos-phate solubilizer (125 120583gmlminus1)

33 Extraction and Evaluation of Bioactivity of MetaboliteThe culture filtrate from 48 h old culture inoculated withPDB showed no bioactivity on 2 d whereas an inhibition zoneof 10mm was observed on 4 d maximum bioactivity wasobtained on 6 d with zone size of 17mm There was reducedbioactivity with further incubation and no antagonism wasobtained with 10 d old culture filtrate The culture filtraterecovered after 6 d of incubationwas acidified in the pH range(1ndash6)The filtrate showedmaximum activity at pH 20 that is19mm inhibition zone zone size was reduced to 9mm at pH40

The inhibitory activity of the culture filtrate was maxi-mum at 28∘C that is 15mm inhibition zone a 10mm zonewas obtained at 24∘C whereas there was further reductionat 30ndash32∘C During the temperature dependent incubationbiomass was constantly increased up to 28∘C (05ndash102 g drywt100mL) followed by a decline (Figure 1) The metabolitewas extracted through ethyl acetate The dried extract wasdissolved in methanol and subjected to disk diffusion assayPercent growth inhibition against Sclerotinia sclerotiorumwas2045 after 24 h and 462 after 48 h (Figure 2)

34 Characterization of Bioactive Compound Thin layerchromatographic analysis and partial purification of bioac-tive compound the crude extract prepared from the cell-freeculture filtrates showed strong antagonistic activity againstthe pathogenic fungus S sclerotiorum No inhibitory activity

0

02

04

06

08

1

12

0

2

4

6

8

10

12

14

16

18

22 24 26 28 30 32

Biom

ass (

gm1

00 m

L)

Inhi

bitio

n zo

ne (m

m)

Inhibition zone (mm)Biomass (dry wt gm100 mL)

Temperature (∘C)

Figure 1 Effect of temperature on biomass and inhibition zone(mm) The inhibitory activity of the culture filtrate was maximumat 28∘C whereas there was further reduction at 30ndash32∘C Duringthe temperature dependent incubation biomass was constantlyincreased up to 28∘C followed by a decline as the temperatureincreases

was observed in the supernatant The crude extract wassubjected to TLC analysis for the separation of the antifungalmolecule Two fractions designated as 1st and 2nd wereobserved when developed in dichloromethane methanol(95 5) on silica gel TLC plates and sprayed with ceric sul-phateThesewere eluted out and checked for antifungal activ-ity only 2nd fraction having119877

119891= 082 exhibited the antifun-

gal activity The spot showed purplish colour when sprayedwith ceric sulphate Preparative TLCwas carried out to obtainsufficient crude material for further analysis

35 GC-MSAnalysis Thecrude extract was partially purifiedby TLC analysisThe partially purified crude was subjected toGC-MS analysis which showed one sharp peak at RT 14648which covers maximum area (Table 1) This matched at98 level in the standard library (NIST) with 12-benzenedicarboxylic acid mono(2-ethylhexyl) ester The antifungalproperty is likely to be due to this compound (Figure 3)

4 Discussion

Anitha et al [14] recently reported occurrence of an endo-phyte from the stem of endemicmedicinal plants of Tirumalahills while others have earlier reported A flavipes from innerbark of Acanthus ilicifolius [15] Endophytic presence of Aflavipes has also been recorded in flowers of Calotropis gigan-tea [16] However to the best of our knowledge this is the firstreport on A flavipes from the foliar tissues of Stevia rebau-diana Bertoni a medicinal plant Sclerotina sclerotiorum isthe worldrsquos most successful and omnivorous fungal pathogen


Table 1 Percentage area covered by the antifungal compounds

Peak R time Area Area Name1 6348 119244 109 1-Pentanamine N-nitroso-N-pentyl2 8057 331055 3043 8594 395652 3634 9144 317567 292 1012-Tricosadiynoic acid methylester5 9558 276323 254 16-Cyclodecadiene1-methyl-5-Met6 10217 156155 143 Hexadecanoic acid methyl ester7 10549 974536 895 Dibutyl phthalate8 11337 326456 300 9-Octadecenoic acid (Z)-methyl ester9 14648 7995418 7340 12-Benzenedicarboxylic acid mono(2-ethylhexyl) ester10 10892406 100

(a) (b) (c)

Figure 2Disc diffusion assay (a) control plate (b) inhibition after 24 hrs and (c) inhibition after 48 hrs (a)Control plate having discs impreg-nated with methanol (b) Zone of inhibition observed after 24 h on plate having discs impregnated with methanolic extract (c) Zone ofinhibition observed after 48 h on plate having discs impregnated with methanolic extract

40 60 80 100 120 140 160 180 200 220 240 260 280000

025

050

075

100149

167577143 113104 27983 132121

O

OOHO

(times10000)

Figure 3Mass spectrum of 12-benzene dicarboxylic acid mono(2-ethylhexyl) ester compound from NIST Library

with a host range of more than 400 plant species Despitedecades of dedicated efforts resistant germplasm is still lack-ing in economically important crops This was an importantreason for screening antagonistic endophytes against thisphytopathogen for current studyMatroudi et al [17] screenedthree species of Trichoderma spp which showed 85 growthreduction of Sclerotina sclerotiorum in dual culture assayRocha et al [18] observed a reduction in the growth rate of Ssclerotiorum ranging from 467 to 500 by different strainsof endophytic fungi isolated from Comfrey (Symphytumofficinale L) Powthong et al [19] studied the fungal extracts

of endophytic fungi for antimicrobial activity against Bacillussubtilis and Candida albicans Evaluation of the antifungalactivity of Aspergillus flavipes from Stevia rebaudiana wasperformed wherein it was maximum on 6th day at pH 20Ethyl acetate worked as the best solvent system for extractionof metabolite GC-MS analysis of the bioactive metaboliteshowed that the compound is 12-benzene dicarboxylic acidmono(2-ethylhexyl) ester which showed antifungal activityagainst S sclerotiorum a similar compound along with1-tetradecamineNN-dimethyl squalene and phytol werereported from Muscodor tigerii as novel endophyte fromCinnamomum camphora [20] This showed 100 inhibitionof Alternaria alternata and 7167 inhibition of Rhizoctoniasolani the compounds were also active against Candidaalbicans and Staphylococcus aureus Present investigationwas carried out against S sclerotiorum which showed 20growth inhibition in 24 h and 46 growth inhibition in 48 hWe also recovered Alternaria alternata as endophyte fromhealthy leaves of Stevia rebaudiana However productionof 12-benzene dicarboxylic acid mono(2-ethylhexyl) esterproduced by Aspergillus flavipes might prevent Alternariaalternata to cause infection in Stevia rebaudiana although leafspot disease caused by A alternata in Stevia rebaudiana was


reported [21] A marine Burkholderia cepacia also produced12-benzene dicarboxylic acid (2-ethylhexyl) ester whichshowed potential antibacterial activity against Aeromonashydrophila Edwardsiella tarda and Vibrio ordalii [22] Thesestudies showed that the compound 12-benzene dicarboxylicacid (2-ethylhexyl) ester is a potential antifungal and antibac-terial in endophytic fungi

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The first author is supported as Junior Research Fellowshipfrom MP Biotechnology Council Bhopal The authors aregrateful to Dr SK Singh Coordinator National Facility(NFCCI) Pune for identification of fungi They are thankfulto Analytical Instrumentation Facility JNU (New Delhi) forGC-MS analysis and result interpretation The first author isextremely thankful to Dr Ankit Kumar Research AssociateIARI New Delhi for his valuable suggestions in the prepara-tion of the paperThe authors are also thankful toMr SandeepSaini and Mrs Nidhi Gujar for their help in the conduct ofthis research

References

[1] M Stinson D Ezra W M Hess J Sears and G Strobel ldquoAnendophytic Gliocladium sp of Eucryphia cordifolia producingselective volatile antimicrobial compoundsrdquo Journal of PlantScience vol 165 pp 913ndash922 2003

[2] H Lu W X Zou J C Meng J Hu and R X Tan ldquoNew bioac-tive metabolites produced by Colletotrichum sp an endophyticfungus inArtemisia annuardquo Journal of Plant Science vol 151 pp67ndash73 2000

[3] G A Strobel ldquoRainforest endophytes and bioactive productsrdquoCritical Reviews in Biotechnology vol 22 no 4 pp 315ndash3332002

[4] H Garg H Li K Sivasithamparam andM J Barbetti ldquoDiffer-entially expressed proteins and associated histological and dis-ease progression changes in cotyledon tissue of a resistant andsusceptible genotype of Brassica napus infected with Sclerotiniasclerotiorumrdquo PLoS ONE vol 8 no 6 Article ID e65205 2013

[5] S K Goyal and R K Goyal ldquoStevia (Stevia rebaudiana) a bio-sweetener a reviewrdquo International Journal of Food Sciences andNutrition vol 61 no 1 pp 1ndash10 2010

[6] O Petrini ldquoTaxonomy of endophytic fungi of aerial planttissuesrdquo inMicrobiology of the Phylosphere N J Fokkenna and JVanDenHeuvel Eds pp 175ndash187 Cambridge University PressCambridge UK 1986

[7] S Gopalakrishnan P Humayun B K Kiran et al ldquoEvaluationof bacteria isolated from rice rhizosphere for biological controlof charcoal rot of sorghum caused byMacrophomina phaseolina(Tassi) GoidrdquoWorld Journal of Microbiology and Biotechnologyvol 27 no 6 pp 1313ndash1321 2011

[8] J Sambrook and D W Rusell ldquoRapid cloning of yeast DNArdquoin Molecular Cloning A Laboratory Manual J Sambrook and

DW Rusell Eds pp 631ndash632 Cold Spring Harbor LaboratoryNew York NY USA 2001

[9] T J White T Bruns S Lee and JW Taylor ldquoAmplification anddirect sequencing of fungal ribosomal RNA genes for phyloge-neticsrdquo in PCR Protocols A Guide to Methods and ApplicationsM A Innis D H Gelfand J J Sninsky and T J White Edspp 315ndash322 Academic Press New York NY USA 1990

[10] J M Bric R M Bostock and S E Silverstone ldquoRapid in situassay for indoleacetic acid production by bacteria immobilizedon a nitrocellulose membranerdquo Applied and EnvironmentalMicrobiology vol 57 no 2 pp 535ndash538 1991

[11] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[12] R I Pikovskaya ldquoMobilization of phosphorus in soil in connec-tion with the vital activity of some microbial speciesrdquoMicrobi-ology vol 17 pp 362ndash370 1948

[13] M Grover L Nain S B Singh and A K Saxena ldquoMolecularand biochemical approaches for characterization of antifungaltrait of a potent biocontrol agent bacillus subtilis RP24rdquoCurrentMicrobiology vol 60 no 2 pp 99ndash106 2010

[14] D Anitha T Vijaya D Pragathi et al ldquoIsolation and character-ization of endophytic fungi from endemic medicinal plants ofTirumala hillsrdquo International Journal of Life Science amp PharmaResearch vol 2 no 3 pp 364ndash373 2013

[15] Z Lin G Zhang T Zhu R Liu H Wei and Q Gu ldquoBioactivecytochalasins from Aspergillus flavipes an endophytic fungusassociated with the mangrove plant Acanthus ilicifoliusrdquo Hel-vetica Chimica Acta vol 92 no 8 pp 1538ndash1544 2009

[16] S Selvanathan I Indrakumar and M Johnpaul ldquoBiodiversityof the endophytic fungi isolated from Calotropis gigantea (L)Rbrrdquo Recent Research of Science and Techechnology vol 3 no4 pp 94ndash100 2011

[17] S Matroudi M R Zamani and M Motallebi ldquoAntagonisticeffects of three species of Trichoderma sp on Sclerotinia scle-rotiorum the causal agent of canola stem rotrdquo Egyptian Journalof Biology vol 2 pp 37ndash44 2009

[18] R Rocha D E da Luz C Engels et al ldquoSelection of endophyticfungi from comfrey (Symphytum officinale l) for in vitro biolog-ical control of the phytopathogen Sclerotinia sclerotiorum (lib)rdquoBrazilian Journal of Microbiology vol 40 no 1 pp 73ndash78 2009

[19] P Powthong B Jantrapanukorn AThongmee and P Suntorn-thiticharoen ldquoEvaluation of endophytic fungi extract for theirantimicrobial activity from Sesbania grandiflora (L) Persrdquo Inter-national Journal of Biomedical amp Pharmaceutical Research vol3 no 2 pp 132ndash136 2012

[20] S Saxena V Meshram and N Kapoor ldquoMuscodor tigeriinov-Volatile antibiotic producing endophytic fungus from theNortheastern Himalayasrdquo Annals of Microbiology 2014

[21] C K Maiti S Sen R Acharya and K Acharya ldquoFirst reportof Alternaria alternata causing leaf spot on Stevia rebaudianardquoPlant Pathology vol 56 no 4 p 723 2007

[22] Y M Gohar ldquoCharacterization of marine Burkholderia cepaciaantibacterial agentsrdquo Journal of Natural Product vol 3 pp 86ndash94 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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Signal TransductionJournal of


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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


22 Media Preparation Potato dextrose agar (PDA Hi-media) was used to isolate endophytic fungi Chlorampheni-col (02 gLminus1) was added to the medium to avoid bacterialcontamination

23 Isolation of Endophytic Fungi Endophytic fungi wereisolated from healthy leaves employing surface sterilizationprocedure [6] Briefly the tissues were surface sterilized with70 ethanol for 2min followed by treatment with 4 sodiumhypochlorite for 2min Finally tissues were washed withsterile distilled water for 2 sec Sterile leaves were placedon blotting sheet and cut into 5mm pieces which weretransferred to PDA plates Plates were incubated at 28∘C for3ndash6 days Hyphal tips of the developing fungal colonies weretransferred to fresh PDA plates to get pure cultures

24 Confrontation Bioassay Primary screening for antago-nism was done by a confrontation assay [7] The endophyteand the pathogenic fungus (Sclerotinia sclerotiorum) werecocultured on 90mm petri dish containing PDA and incu-bated at 28∘C for 3ndash5 days to check the interaction betweenthem A culture of the pathogen Sclerotinia sclerotiorum wasobtained from the Directorate of Soybean Research IndorePlates were observed regularly and antagonismwas expressedby the presence of inhibition zone at the point of interaction

25 Molecular Identification of Antagonistic Endophyte Mor-phological identification of the organism was carried outat National Fungal Culture Collection of India (NFCCI)Agharkar Research Institute Pune For molecular identifi-cation total genomic DNA of the endophytic fungus wasisolated directly from actively growing mycelium growingin potato dextrose broth (PDB) using CTAB method [8]DNA amplification was performed by PCR using primer pairITS1 TCCGTAGGTGAACCTGCGG and ITS4 TCCTCC-GCTTGATATGC [9] PCR was carried out according tothe following protocol initial denaturation at 95∘C for 5mindenaturation at 95∘C for 1min annealing at 55∘C for 45 secextension at 72∘C for 10min steps 2ndash4were repeated 35 timesSequencing of PCR product was carried at Xcelris Labs LtdAhmedabad The sequenced data was subjected to BLASTalgorithm and submitted to Genebank for accession number

26 Plant Growth Promoting Attributes Plant growth pro-moting attributes of the antagonistic endophyte such asproduction of IAA [10] and siderophore [11] and phosphatesolubilization activity [12] was assayed both qualitatively andquantitatively

27 Evaluation of Bioactivity Bioactivity of the culture filtrateagainst Sclerotinia sclerotiorum was evaluated over growthand temperature

28 Influence of Growth Period A 48 h old fungal culturegrown on PDA was inoculated in Erlenmeyer flasks contain-ing 100mLPDBThe flasks were incubated at 28∘C at 120 rpmfor 10 d Culture broth was recovered at 2 4 6 8 and 10 d

after incubation by centrifugation at 12000 rpm for 20min at4∘C A disc diffusion assay was carried out to determine thebioactivity of the culture filtrate obtained after 6 d of fungalgrowth

29 Influence of Temperature TheErlenmeyer flasks contain-ing 100mLPDB were incubated at 22 24 26 28 30 and32∘C at 120 rpm for 6 d to assess the influence of temperatureon bioactive metabolite The culture filtrate was harvestedand antagonistic activity of the filtrate was checked by diskdiffusion assay against Sclerotinia sclerotiorum

210 Extraction of Antifungal Metabolite For extraction ofantifungal metabolite the culture filtrate was centrifuged at12000 rpm for 20min at 4∘C and the culture supernatant(pH 58) was acidified (1NHCl) to a final pH 20 [13] Theacidified filtrate was subjected to organic extraction by ethylacetate (1 1 vv) Organic extract was dried under vacuumin a rotary evaporator The dried crude was dissolved in2mLofmethanol For antagonistic assay 40120583L ofmethanolicextract was impregnated on sterile paper discs MeOH coateddiscs were used as control Plates were incubated at 28∘Cand inhibition of fungal growth was recorded after 24 h and48 h Growth inhibition was calculated as per the followingformula

GI = (119860 minus 119861)119860times 100 (1)

where119860 = radial diameter of test fungus 119861 = radial diameterof test fungus against culture

211 Characterization of Bioactive Compound

2111 Chromatographic Detection and Partial Purification ofBioactive Metabolite Thin layer chromatography (TLC) wasperformed on crude extracted from the culture broth of theendophyte (TLC silica gel 60 20 times 0 05mm Merk CoInc) For this the crude fraction was spotted (30120583L) on theTLC plate and chromatography was performed by employ-ing solvent system dichloromethane methanol (95 5 vv)Spots were visualized by spraying with ceric sulphate silicaresidue was extracted and centrifuged and the supernatantwas transferred to a microcentrifuge tube The silica-freesupernatant was checked for antifungal activity PreparativeTLC was carried out to obtain the partial purified samplewhich showed antifungal activity

2112 GC-MS Analysis GC-MS analysis was carried outat Central Instrumentation Facility JNU (New Delhi) Gas-chromatography mass spectrometry (GC-MS) analysis of thecrude was performed on a Shimadzu GCMS-QP-2010 plussystem RTx-5 SilMS column (30m times 025mm id times025 filmthickness) was used for the analysisThe operating conditionsof the column were as follows oven temperature programfrom 80∘C to 210∘C at 4∘Cmin with holding time of 2minand from 210∘C to 300∘C at 15∘Cmin with holding timeof 5min and the final temperature was kept for 20minThe injector temperature was maintained at 270∘C and



3 Results






0

02

04

06

08

1

12

0

2

4

6

8

10

12

14

16

18

22 24 26 28 30 32

Biom

ass (

gm1

00 m

L)

Inhi

bitio

n zo

ne (m

m)


Temperature (∘C)






4 Discussion





(a) (b) (c)


40 60 80 100 120 140 160 180 200 220 240 260 280000

025

050

075

100149

167577143 113104 27983 132121

O

OOHO

(times10000)








Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results






0

02

04

06

08

1

12

0

2

4

6

8

10

12

14

16

18

22 24 26 28 30 32

Biom

ass (

gm1

00 m

L)

Inhi

bitio

n zo

ne (m

m)


Temperature (∘C)






4 Discussion





(a) (b) (c)


40 60 80 100 120 140 160 180 200 220 240 260 280000

025

050

075

100149

167577143 113104 27983 132121

O

OOHO

(times10000)








Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(a) (b) (c)


40 60 80 100 120 140 160 180 200 220 240 260 280000

025

050

075

100149

167577143 113104 27983 132121

O

OOHO

(times10000)








Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Antagonistic Evaluation of Bioactive...

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