894 Chiang Mai J. Sci. 2014; 41(4)

Chiang Mai J. Sci. 2014; 41(4) : 894-909http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Marine Derived Fungi of Peninsular Malaysia – aBiochemical PerspectiveAudra Shaleena Paliany [a], Yasodha Sivasothy [b], Khalijah Awang [b],Mohammed Rizman-Idid [a,c] and Siti Aisyah Alias*[a,c][a] Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia.[b] Department of Chemistry, University of Malaya, Kuala Lumpur 50603, Malaysia.[c] Institute of Ocean and Earth Sciences, C308 IGS Building, University of Malaya,

Kuala Lumpur 50603, Malaysia.*Author for correspondence; e-mail: saa@um.edu.my

Received: 12 December 2013Accepted: 15 February 2014

ABSTRACTIn an effort to tap into natural products harboured by marine derived fungi in

Malaysia, selected marine derived endophytic and manglicolous fungi from the coastlinesof Peninsular Malaysia were investigated for their antibacterial potential. Forty-onestrains were isolated from marine associated plants, comprised and comprised 12 and 19endophytic strains from Vitex rotundifolia and Ipomoea pes-caprae respectively, while10 manglicolous strains were from decaying mangrove wood collected in PeninsularMalaysia. In preliminary experiments, a plug assay was employed to study the antibacterialactivities of all 41 fungi isolates. Fifteen of the endophytic isolates and nine of themanglicolous isolates displayed antibacterial activities against at least one of the testbacteria. Based on the plug assay, the endophytic fungi from Ipomoea pes-caprae wereshown to display higher antibacterial potential in comparison to the endophytic fungifrom Vitex rotundifolia. In particular, Minimidochium sp. and Bipolaris sp. (ISB0014)displayed antibacterial activities against 5 or more test bacteria. Potential fungi isolateswith good antibacterial activities were further analysed through a broth microdilutionassay. Minimidochium sp. and Dyfrolomyces rhizophorae exhibited promising antibacterialactivities with minimum inhibitory concentrations not higher than 0.5 mg/ml. Bioactivity-guided fractionation of D. rhizophorae extracts resulted in the isolation of fatty acids,palmitic and linoleic acid. Though ubiquitous in nature, linoleic acid is known as anessential health supplement and both fatty acids are used as biodiesel replacingconventional fuels.

Keywords: natural products, antibiotics, manglicolous fungi, endophytic fungi,bioactivity

1. INTRODUCTIONMarine natural products are known to

possess a plethora of chemical variations andcompounds that when harnessed, arebelieved to potentially exhibit a wide range

Chiang Mai J. Sci. 2014; 41(4) 895

of properties,such as, antimicrobial,anticancer, antituberculosis, antiviral,antiparasitic, antihelmintic, antimalarial,antiprotozoal, anticoagulant, antiplatelet,antiinflammatory, antidiabetic, andantitumor bioactivities [1,17,19]. For thepast two decades, marine and marine-relatedresources including plants, sponges,bacteria, cyanobacteria, algae and fungihave become an important source of noveland pharmacologically active chemicalstructures for the development of new drugs,antibiotics, pesticides and antifoulingsubstances, which are made possible due tothe chemical diversity of their secondarymetabolites [6,7,22,32]. It is now widelyaccepted that marine derived fungi play aprominent role as a promising source forthe discovery of novel bioactive chemicalcompounds, in line with the growing needof new natural products [7,28,35]. Studieshave shown that marine derived fungi werea class of organisms poorly studied incomparison to its counterparts such as bacteria,plants and animals [11].

The marine ecosystem is governed byharsh biological, physical and chemicalparameters which may have driven thedevelopment and evolution of novelmetabolic pathways in living marineorganisms, thus giving rise to chemicalswith interesting structures [6,7,17]. Thefirst report of a bioactive natural productfrom a marine derived fungus dates backto the 1940s when cephalosporin C wasisolated from the fungus Acremoniumchrysogenum (Thirum. & Sukapure) W.Gams collected from a sewage outlet in theMediterranean Sea [34]. Cephalosporin Cis the precursor chemical compound of themodern cephalosporin antibiotics [34].Existing modern drugs of fungal origininclude b-lactam antibiotics, griseofulvin,cyclosporine A, taxol, ergot alkaloids, and

lovastatin [7,41]. To date, studies havereported some 1100 new chemicalstructures from marine derived fungi[6,7,11]. Forty two new compounds and 35known compounds were discovered frommarine derived fungi isolated from varioussubstrates from the South China Sea [31].Although there have been several reportsin Malaysia on the potential of secondarymetabolites of endophytic fungi, most ofthem originated from medicinal herbs andnon-marine associated plants of Malaysia[40,43]. There is a general lack of informationand few studies on the secondary metabolitechemistry of marine derived fungi fromMalaysia. One of which was a study thatinvestigated the antimicrobial properties of152 marine derived fungi from Malaysiaand successfully isolated 2,2,7-trimethyl-2H-chromen-5-ol from the marine derivedfungi Fasciatispora nypae K.D. Hyde, acompound that was never before reportedto be isolated from nature [48].

Therefore, the objective of the presentstudy is to tap into and explore thepotential for bioactive metabolitesproduced by marine derived fungi inMalaysia. The two host plants in thepresent study, Vitex rotundifolia andIpomoea pes-caprae, are commonly foundgrowing on the beaches of PeninsularMalaysia. There have been no studies onthe bioactivities of endophytic fungi fromthe plant V. rotundifolia, while there wasonly one study pertaining to the bioactivity ofendophytic fungi from I. pes-caprae. Althoughthe sudy isolated exopolysaccharidefrom the endophytic fungus F. oxysporumfrom the plant I. pes-caprae, its antibacterialbioactivity was not tested [14]. Thus overall,there has been minimal work investigatingthe diversity of endophytic fungi fromthe leaves of V. rotundifolia and I. pes-caprae.

896 Chiang Mai J. Sci. 2014; 41(4)

2. MATERIALS AND METHODS2.1 Collection of Marine AssociatedPlants, Endophytic Fungi Isolation andManglicolous Fungi Used

Vitex rotundifolia was collected fromKijal Beach, from the east coast, while I.pes-caprae was collected from Port DicksonBeach, from the west coast of PeninsularMalaysia. Only leaves of healthy plantswere taken for the isolation of endophyticfungi. The plant materials were excisedusing clean scissors and placed in sterilepolythene bags and transported back to thelaboratory and processed within two hoursof collection. Plant samples were washedwith distilled water to remove surface sandand debris. The leaves and stems were thencut into 6 mm disks and surface sterilizedusing a series of chemical treatments [2].Firstly, samples were soaked in 70% ethanolfor two minutes followed by 4% bleachfor one minute, after which they weretreated again with 70% ethanol for oneminute and finally rinsed thrice in distilledwater. The surface sterilized plant materialswere then placed on Potato Dextrose agar(PDA) without antibiotic supplement andincubated at 37oC. The agar was observeddaily under the microscope for myceliasporulation; germinating mycelia werethen transferred to fresh media using a fineneedle to obtain a pure fungi culture. Thepure fungi cultures were then transferredto agar slants to make triplicates of eachpure culture.

In this study, ten marine derivedmanglicolous fungal strains comprising ofsix genera/species that were previouslyisolated from the decaying wood found inmangroves of Peninsular Malaysia werescreened for their antibacterial activity. Allstrains used in this study were obtained fromthe Institute of Biological Sciences (ISB),University of Malaya culture collection.

2.2 Identification of Endophytic FungiUsing Sequence Analysis of InternalTranscribed Spacer (ITS) Sequences

Endophytic fungi mycelia wereharvested after an optimum incubationtime of 7 days, frozen in liquid nitrogenand macerated into fine powder with amortar and pestle. Total genomic DNAwas extracted using DNeasy Plant Mini Kit(QIAGEN, Germany). The internaltranscribed spacer regions of the 5.8SrRNA of the fungi were PCR amplifiedusing primers ITS4; 5’- TCCTCCGCTTATTGATATGC-3’ and ITS5; 5’GGAAGTAAAAGTCGTAACAAGG3’. PCRreaction mixtures of 25μl consisted of 2.5μL1 × PCR buffer, 0.2mM dNTP, 0.2mMprimer pairs, 0.5UTaq DNA polymeraseand 0.1mg template genomic DNA. PCRamplifications were performed on thermo-cycler with an initial denaturation of 96oCfor 5minutes, followed by 35 cyclescomprising of 95oC for 30 seconds, 52oCfor 30 seconds, and 72oC for 90 seconds,before ending with a final extension stepof 72oC for 10 minutes. PCR productswere visualized on 1% TBE agarose gel.DNA sequencing was outsourced andperformed by First Base Laboratories SdnBhd. Forward and reverse sequencechromatograms were checked for ambiguityand edited, before assembling the contigsequences for each fungal isolates usingChromas 2.33 (Technelysium Pt. Ltd.;Australia). The Basic Local AlignmentSearch Tool (BLAST) program at the USNational Centre for BiotechnologyInformation (NCBI), (http://www.ncbi.nlm.gov/) was employed for speciesidentification of the fungi isolate sequences.Fungi isolates with an ITS sequencesimilarity ≥ 96% were considered tobe the same species [37]. The identificationsmust be treated with some caution

Chiang Mai J. Sci. 2014; 41(4) 897

however, as many of the ITS sequence datain GenBank is wrongly named [24].

2.3 Phylogenetic AnalysisBefore constructing the phylogenetic tree,

the best evolutionary model for the ITSsequence alignment dataset was chosenbased on Akaike Information Criterion(AIC) of the jModeltest 2.1.3 software [33].The jModeltest result suggested theTransitional Evolutionary Model withinvariable and gamma distribution(TIM2+I+G; I=0.2330; G=1.8540) forthe ITS sequences. Unequal base frequencieswere also observed with 0.2045, 0.2945,0.2589 and 0.2421 for A, C, G, and T,respectively. The substitution rate matrixwere [A-C] = 1.7504; [A-G] = 2.6362;[A-T] = 1.7504; [C-G] = 1.0000; [C-T] =3.7294; [G-T] = 1.0000. MaximumLikelihood tree was constructed usingRandomized Axelerated MaximumLikelihood (RAxML) web server [38],while incorporating the parametersobtained from the jModeltest analysis.

2.4 Test BacteriaThe test bacteria used comprised of

five Gram positive and two Gram negativestrains. The Gram positive bacteria usedwere Bacillus cereus Frankland andFrankland (ATCC 11778), Bacillussubtilis (Ehrenberg) Cohn. (ATCC 6051),Enterococcus faecalis (Andrewes andHorder) Schleifer and Kilpper-Balz(ATCC 29212), Micrococcus luteus(Schroeter) Cohn (ATCC 49732) andStaphylococcus aureus Rosenbach MTCC96 (ATCC 9144). The Gram negativebacteria used were Escherichia coli (Migula)Castellani & Chalmers MTCC 443 (ATCC25922) and Pseudomonas aeruginosa(Schroeter) Migula (ATCC 27853). Thetest bacteria were provided by the

Microbiology Department, University ofMalaya.

2.5 Screening of Fungi Isolates throughPlug Assay Method

The preliminary screening forantibacterial activity of marine derivedfungi were carried out using a plug assaymethod [13,16,48]. Plates containing PDAwithout antibiotic supplement were seededwith 24 hour old test bacteria on LuriaBroth (LB) agar and a round well of 6 mmin diameter was cut out from the centre ofthe agar. Plugs of actively growing fungalmycelia (6 mm) from 30 days old PDAcultures were then transferred into thesewells, and incubated at 37oC for 24 hours.The diameters of the zones of inhibitionsurrounding the well were measured andrecorded in millimetres (mm) using a ruler.An inhibition zone of 8 mm or higherindicates a positive antibacterial response.All assays were carried out in independenttriplicates. Paper discs containingantibiotics penicillin and streptomycinserved as negative controls.

2.6 Cultivation, Extraction and Bioassayof Fungal Secondary Metabolites

Based on the preliminary resultsobtained from the plug assay (Tables 1 &2), three endophytic and three manglicolousstrains were chosen for further analysis oftheir antibacterial potential through thebroth microdilution assay following theprotocols of [12]. The fungal isolates weregrown on sterile PDA without antibioticsupplements in Petri-plates for 14-30 daysat 25oC. Active growing fungi mycelia wasthen cut into 6 mm plugs and added into250 ml conical flasks containing potatodextrose broth (PDB) and the flasks wereincubated at 25oC under shaken phase of120 rpm for 30 days. After the incubation

898 Chiang Mai J. Sci. 2014; 41(4)

period, the fungal biomass was separatedby filtration. The filtrate was thenextracted thrice with equal volumes of ethylacetate (1:1 v/v). The organic ethyl acetatelayers were then combined and evaporatedto dry the crude extract using a rotaryevaporator at 25oC. The dried crudesecondary metabolite was then storedat -4oC prior to the broth microdilutionassay. Crude extract of the fungi wasdissolved in 1% dimethyl sulfoxide(DMSO) and prepared at a two-foldconcentration ranging from 2.0mg/ml to0.0156mg/ml. The crude extract at varyingconcentrations was then added to a 96 wellplate seeded with bacterial suspension at5 × 105 cfu/ml. The plates were incubatedat 37oC for 24 hours, after whichp-Iodonitrotetrazolium violet (INT) at aconcentration of 0.4mg/ml was added intoeach well and incubated for 20 minutes.The colour change from clear to pinkindicates positive bacterial growth and anegative inhibition. The lowest concentrationable to inhibit bacterial growth was recordedas the minimum inhibitory concentration(MIC) value. Antibiotics penicillin andstreptomycin ranging from concentrations2 mg/ml to 0.0156 mg/ml were used asnegative controls.

2.7 Isolation of Active Constituents ofDyfrolomyces rhizophorae

A strain of D. rhizophorae waschosen for further chemical analysisbased on results displayed in the brothmicrodilution assay. Key proceduresleading to the isolation of activeconstituents comprised thin layerchromatography (TLC), column chro-matography, nuclear magnetic resonancespectroscopy (NMR), Ultra-violetspectroscopy and Liquid Chromato-graphy-Mass Spectrometry (LCMS).

Analytical and preparative thin layerchromatography (TLC) was carried out onMerck 60 F254 silica gel plates (absorbentthickness: 0.25). Column chromatographywas performed using silica gel (Merck230-400 mesh, ASTM). Nuclear magneticresonance (NMR) spectra were recordedin deuterated chloroform (CDCl3) (Merck,Germany) with tetramethylsilane as aninternal standard, using JEOL ECA400MHz NMR spectrometer. InfraredIR spectra were recorded using a Perkin-Elmer Spectrum 400 FT-IR Spectrometer.Ultraviolet (UV) spectra were recordedusing a Shimadzu 1650 PC UV-Vis Spectrophotometer. The Liquidchromatography-Mass spectrum-Iontrap-Time of flight (LC-MS-IT-TOF)spectra were recorded on a Ultra-fast liquidchromatography (UFLC) ShimadzuLiquid Chromatograph with a SPD-M20Adiode array detector coupled to a IT-TOFThe IT-TOF was operated in positive ionelectrospray mode.

2.8 Bioassay Guided Fractionation ofDyfrolomyces rhizophorae Extract

Dyfrolomyces rhizophorae was chosenfor further chemical analysis based onpromising antibacterial activity againstboth Gram positive and negative bacteriaat low MIC values. The crude ethyl acetateextract of D. rhizophorae (900mg) waspartitioned in sequence using solvents ofincreasing polarity starting with n-hexane(3×, 0.5L), dichloromethane (3×, 0.5L) andmethanol (3×, 0.5L) at room temperature,affording 3 primary extracts (110mg,320mg, and 235mg, respectively). Theactive extract, identified as the n-hexaneextract, was re-dissolved in n-hexane andsubjected to silica gel column chromatographyusing a gradient elution with mixtures ofn-hexane : ethyl acetate (100:0, 90:10, 85:15,

Chiang Mai J. Sci. 2014; 41(4) 899

80:20, 75:25, 70:30, 65:35, 60:40, 55:45,50:50 and 0:100 v/v), affording 7 fractions.The antibacterial activities of all 7 fractionswere evaluated, whereby fractions 2(51.1mg) and 3 (24.5mg) were found to bethe most active. Final purification offraction 2 and 3 via preparative TLC usingn-hexane: ethyl acetate (75:25 v/v), yieldedcompounds 1 (3.2mg) and 2 (2.5mg),respectively.

3. RESULTS AND DISCUSSION3.1 Identification of Marine DerivedEndophytic Fungal Strains

Maximum Likelihood tree shows theclustering of isolates with their referenceITS sequences (Figure 1). Based on thestrong support of the monophyleticgroupings, most isolates were able to betentatively identified to the species level.Only a few isolates were identified up tothe genus or order level. A total of 31 fungiisolates were isolated from V. rotundifoliaand I. pes-caprae (Table 1). Phoma sp. werecommon to both plant species while thefungi isolates Bipolaris sp., Cochliobolusgeniculatus R.R. Nelson, Curvularia sp.and Phoma sp. had more than oneoccurrence on the same host.

3.2 Antibacterial Activities of MarineDerived Fungi

Endophytic fungi have been known toproduce unique compounds with interestingbioactivities and recent years has seen agrowing interest in the exploration ofmarine derived endophytic fungi for newand novel metabolites of pharmacologicalimportance [7,20,22]. One example was theisolation of antibacterial indole alkaloidsfrom the algal endophyte Eurotiumcristatum (Raper & Fennell) Malloch &Cain [10]. In the present study, five andten of the endophytic fungi strains from

V. rotundifolia and I. pes-caprae respectivelydisplayed positive antibacterial activity(Table 1) The broadest exhibition ofantibacterial activity by the endophyticfungi from V. rotundifolia were by thefungal strains Curvularia sp. andPaecilomyces sp. which were active againstthree out of the seven test bacteria. Theendophytic fungi from I. pes-caprae,however, displayed a wide spectrum ofantibacterial potential with Bipolaris sp.(ISB0014) being active against six testbacteria. Bipolaris sp. derived from theseagrass, Halophila ovalis, has been knownto contain a dimeric chromanone and aphthalide with antibacterial activity againstS. aureus [3]. It was observed that onlyendophytic fungi from I. pes-capraeexhibited antibacterial activities againstGram negative bacteria, namely the strainsBipolaris sp. (ISB0014), Dothideomycete sp.(ISB0019), Minimidochium sp. (ISB0023) andPenicillium sp. (ISB0027).

Both the marine associated plants inthe present study have been exploited intraditional medicine and the habitats ofboth plants are constantly surrounded byextreme conditions. V. rotundifolia hasbeen reported to be used for cold remediesand headaches in Japan and is also used asraw material in traditional Chinesemedicine [21]. It has also been reported topossess anticancer properties [23]. As forI. pes-caprae, it is used as an herbal drug inMexico mainly in the treatment of kidneycomplaints, digestive disorders, hypertension,arthritis, rheumatism, skin infections andother inflammatory conditions. Nineantibacterial oligosaccharides were isolatedfrom I. pes-caprae while there has been noreports of antibacterial compounds fromV. rotundifolia which may reflect the host’sinfluence on the fungi’s antibacterialactivities [29]. This may account for the

900 Chiang Mai J. Sci. 2014; 41(4)

Figure 1. Maximum Likelihood tree based on ITS sequences of 31 endophytic fungi(isolate ID indicated in parenthesis) and 69 reference taxa obtained from GenBank (withaccession numbers) constructed using Raxml sofware. Only bootstrap values over 70%are shown above the branches.

Chiang Mai J. Sci. 2014; 41(4) 901

End

ophy

tic

fung

iT

est

bact

eria

(zon

e of

inhi

biti

on in

mm

)V

itex

rot

undi

foli

aS.

aur

eus

B. c

ereu

sB

. su

btili

sM

. lut

eus

E.

faec

alis

E.

coli

P.

aeru

gino

saC

ochl

iobo

lus

geni

cula

tus

(ISB

0001

)-

--

--

--

Coc

hlio

bolu

s ge

nicu

latu

s (I

SB00

02)

-14

.00

± 0.

0014

.83

± 0.

29-

--

-C

urvu

lari

a sp

. (I

SB00

03)

16.1

7 ±

0.29

14.8

3 ±

0.29

13.0

0 ±

0.00

--

--

Cur

vula

ria

sp.

(ISB

0004

)-

--

--

--

Fung

al s

p. (

ISB0

005)

--

--

--

-Le

tend

raea

hel

min

thic

ola

(ISB

0006

)-

--

--

--

Nem

ania

pri

mol

utea

(IS

B000

7)-

--

--

--

Paec

ilom

yces

sp.

(IS

B000

8)16

.00

± 0.

0016

.17

± 0.

2915

.00

± 0.

00-

--

-Ph

oma

sp.

(ISB

0009

)-

--

--

--

Phom

a sp

. (I

SB00

28)

--

-8.

000

± 0.

000

--

-Ph

oma

sp.

(ISB

0029

)10

.830

± 0

.290

--

10.1

70 ±

0.2

90-

--

Xyl

aria

ceae

(IS

B001

2)-

--

--

--

Ipom

oea

pes-c

apra

eS.

aur

eus

B. c

ereu

sB

. su

btili

sM

. lut

eus

E.

faec

alis

E.

coli

P.

aeru

gino

saBi

pola

ris

sp.

(ISB

0013

)-

--

--

--

Bipo

lari

s sp

. (I

SB00

14)

21.0

00 ±

0.0

0014

.830

± 0

.290

10.0

00 ±

0.0

0018

.670

± 0

.290

15.6

70 ±

0.5

808.

00 ±

0.0

00-

Bipo

lari

s sp

. (I

SB00

15)

--

10.1

70 ±

0.2

9025

.000

± 0

.000

--

-C

lado

spor

ium

sp.

(IS

B001

6)-

--

--

--

Coc

hlio

bolu

s sp

. (I

SB00

17)

--

--

--

-C

olle

totr

ichu

m s

p. (

ISB0

018)

--

--

--

-D

othi

deom

ycet

e sp

. (IS

B001

9)10

.170

± 0

.290

11.1

70 ±

0.2

90-

10.0

00 ±

0.0

00-

-8.

667

± 0.

236

Fung

al e

ndop

hyte

(IS

B002

0)-

--

--

--

Fusa

rium

sp.

(IS

B001

0)-

--

--

--

Gui

gnar

dia

man

gife

rae

(ISB

0022

)-

12.3

3 ±

0.58

14.0

0 ±

0.00

--

--

Min

imid

ochi

um s

p. (

ISB0

023)

8.33

3 ±

0.47

18.

333

± 0.

236

13.3

30 ±

0.5

8010

.830

± 0

.290

-8.

667

± 0.

236

-M

onta

gnul

acea

e sp

. (I

SB00

24)

24.6

70 ±

0.2

9027

.330

± 0

.580

19.8

30 ±

0.2

9024

.000

± 0

.000

--

-M

ycol

epto

disc

us i

ndic

us (

ISB0

025)

--

--

--

-M

yrot

heci

um s

p. (

ISB0

026)

8.33

3 ±

0.47

18.

333

± 0.

471

8.33

3 ±

0.23

6-

--

-Pe

nici

llium

sp.

(IS

B002

7)8.

000

± 0.

000

8.00

0 ±

0.00

0-

--

8.66

7 ±

0.23

6-

Phom

a sp

. (I

SB00

28)

--

-8.

000

± 0.

000

--

-Ph

oma

sp.

(ISB

0029

)10

.830

± 0

.290

--

10.1

70 ±

0.2

90-

--

Pleo

spor

ales

sp.

(IS

B003

0)-

--

--

--

Xyl

aria

sp.

(IS

B003

1)-

--

--

--

Tab

le 1

. End

ophy

tic fu

ngi a

nd t

heir

ant

ibac

teri

al a

ctiv

ities

.

902 Chiang Mai J. Sci. 2014; 41(4)

broader spectrum of antibacterial activityby the endophytic fungi from I. pes-capraein the present study. Exhibition of similarbioactivities between plant and itsendophytic fungi have been reported [45,46]. Hence, the choice of endophytic fungihosts for bio-prospecting and bioactivitystudies should consider; firstly, theuniqueness of the host’s environment orecological niche, such as harsh environmentsthat require specific survival strategies;secondly, plants that possess an ethnobotanical history and thirdly, plants beingexploited for their medicinal values[36,39,41].

The ability of mangrove fungi tosynthesize unique secondary metabolites wasconsidered as an adaptive response towardsits harsh environment with high salinity andextreme intertidal conditions [27,44,47].Table 2 shows that nine out of the tenmanglicolous strains displayed positiveantibacterial activity. The fungal strainsD. rhizophorae, Henningsomyces sp. and theISB004 strain of Dactylospora haliotrephawas active against four or more test bacteriawith D. rhizophorae being the only strainactive against the Gram negative bacteriaE. coli. The antibacterial activities of somemanglicolous fungi strains similar to thepresent study had been previouslyreported whereby a novel antibacterialagent corollosporine with phthalide wasisolated from the marine derived fungiCorollospora maritima [25]. The compoundcorollosporine exhibited activity againstS. aureus and B. cereus [30]. Metabolitesenalin A and B had been previously isolatedfrom the manglicolous fungi Verruculinaenalia (Kohlm.) Kohlm. & Volkm.-Kohlm., from a salt lake in the Bahamas[26]. The compound enalin A is acoumaranone with known antimicrobialactivities [26]. The V. enalia strain in the

present study displayed no activity againstthe Gram negative E. coli based on theplug assay, despite previous report ofantibacterial activity against S. aureus,B. subtilis and E. coli [48]. In the brothmicrodilution assay, Dactylosporahaliotrepha (ISB003) and Henningsomycessp. exhibited antibacterial activities againstthe Gram positive test bacteria (Table 3).However, D. rhizophorae was the onlymanglicolous fungi with antibacterialactivities against four Gram positive andone Gram negative test bacteria (Table 3).Since D. rhizophorae displayed antibacterialpotential against both bacterial Gramtypes, the chemistry of its metabolites wasfurther analysed. The D. rhizophoraeisolate used in the present study wasisolated from the drift wood of Rhizophoraapiculata.; a mangrove tree, which belongsto a genus previously explored for itsantibacterial secondary metabolites [18].Although the antibacterial potential of D.rhizophorae was previously unknown,another species of the same genus,Dyfrolomyces mangrovei, has beenreported to possess antibacterial activitiesagainst B. subtilis and S.aureus [48].

3.3 Active Constituents of Dyfrolomycesrhizophorae

Compound 1 was isolated from sub-fraction 2 and compound 2 was isolatedfrom sub-fraction 3, both from then-hexane extract (Table 4). The gaschromatography-mass spectrum of 1exhibited a molecular ion peak M+ atm/z 280. Comparison of its massspectrum with the reference spectra in theNIST 05 Library suggested that 1 waslinoleic acid that was confirmed from theIR spectrum, 1H NMR , 13C NMR, andDEPT experiments. The absorption bandsaround 3276, 2922, 2847 and 1715 cm-1 were

Chiang Mai J. Sci. 2014; 41(4) 903

Man

glic

olou

s fu

ngi

Tes

t ba

cter

ia (z

one

of in

hibi

tion

in m

m)

SAB

CB

SM

LE

FE

CP

AC

orol

losp

ora

mar

itim

a (I

SB00

1)-

10.3

33 ±

0.2

3610

.166

7 ±

0.23

6-

--

-D

acty

losp

ora

halio

trep

ha (I

SB00

2)10

.833

± 0

.236

-10

.333

± 0

.236

--

--

Dac

tylo

spor

a ha

liotr

epha

(ISB

003)

11.0

00 ±

0.4

08-

10.8

33 ±

0.2

36-

--

-D

acty

losp

ora

halio

trep

ha (I

SB00

4)13

.000

± 0

.408

13.8

33 ±

0.2

3614

.333

± 0

.236

10.8

33 ±

0.2

36-

--

Dac

tylo

spor

a ha

liotr

epha

(ISB

005)

13.0

00 ±

0.4

0814

.333

± 0

.236

13.8

33 ±

0.2

36-

--

-Fu

sari

um s

p. (

ISB0

06)

--

--

--

-H

enni

ngso

myc

es s

p. (

ISB0

07)

14.6

67 ±

0.2

3614

.333

± 0

.236

13.6

67 ±

0.2

3610

.333

± 0

.236

--

-D

yfro

lom

yces

rhi

zoph

orae

(IS

B008

)15

.833

± 0

.236

16.1

67 ±

0.2

3615

.000

± 0

.408

14.6

67 ±

0.2

36-

8.00

0 ±

0.00

0-

Ver

rucu

lina

enal

ia (

ISB0

09)

11.0

00 ±

0.4

0813

.833

± 0

.236

12.1

67 ±

0.2

36-

--

-V

erru

culin

a en

alia

(IS

B010

)10

.833

± 0

.236

12.1

67 ±

0.2

3611

.000

± 0

.408

--

--

Tab

le 2

. Mar

ine

deri

ved

man

glic

olou

s fu

ngi a

nd t

heir

ant

ibac

teri

al a

ctiv

ities

.

Fun

giB

acte

ria

S. a

ureu

sB

. cer

eus

B.

subt

ilis

E.

faec

alis

M. l

uteu

sP

. ae

rugi

nosa

E.

coli

Endo

phyt

ic fu

ngi

Bipo

lari

s sp

. (I

SB00

14)

0.50

0 ±

00.

250

± 0

NA

NA

1.00

0 ±

0N

AN

AM

inim

idoc

hium

sp.

(IS

B002

3)0.

125

± 0

0.06

3 ±

00.

125

± 0

0.25

0 ±

00.

250

± 0

NA

NA

Mon

tagn

ulac

eae

sp.

(ISB

0012

4)2.

000

± 0

0.50

0 ±

0N

AN

A1.

000

± 0

NA

NA

Man

glic

olou

s fu

ngi

Dac

tylo

spor

a ha

liotr

epha

(IS

B003

)0.

125

± 0

0.12

5 ±

00.

063

± 0

NA

0.50

0 ±

0N

AN

AH

enni

ngso

myc

es s

p.0.

250

± 0

1.00

0 ±

00.

417

± 0.

15N

A1.

000

± 0

NA

NA

Dyf

rolo

myc

es r

hizo

phor

ae0.

500

± 0

0.25

0 ±

00.

052

± 0.

02N

A0.

500

± 0

NA

0.50

0 ±

0

Mea

n ±

stan

dard

dev

iatio

n, n

=3;

NA

= n

o ac

tivity

Tab

le 3

. Min

imum

inhi

bito

ry c

once

ntra

tion

(MIC

) val

ues

of s

elec

ted

mar

ine

deri

ved

endo

phyt

ic a

nd m

angl

icol

ous

fung

i in

mg/

ml.

904 Chiang Mai J. Sci. 2014; 41(4)

Frac

tion

/Poo

l/C

ompo

und

Bac

teri

aS.

aur

eus

B. c

ereu

sB.

subt

ilis

E. fa

ecal

isM

. lut

eus

P. a

erug

inos

aE.

coli

Cru

de0.

500 ±

00.

250 ±

00.

052 ±

0.02

NA

0.50

0 ± 0

NA

0.50

0 ± 0

Frac

tion

n-he

xane

2.00

0 ± 0

0.25

0 ± 0

0.12

5 ± 0

1.00

0 ± 0

0.50

0 ± 0

NA

2.0

Frac

tion

dich

loro

met

hane

NA

NA

NA

NA

NA

NA

NA

Frac

tion

met

hano

lN

AN

AN

AN

AN

AN

AN

ASu

b-fr

actio

n 1

NA

NA

NA

NA

NA

NA

NA

Sub-

frac

tion

20.

500 ±

00.

250 ±

00.

250 ±

00.

250 ±

00.

500 ±

0N

AN

ASu

b-fr

actio

n 3

0.50

0 ± 0

0.25

0 ± 0

0.25

0 ± 0

NA

NA

NA

NA

Sub-

frac

tion

4N

AN

AN

AN

AN

AN

AN

ASu

b-fr

actio

n 5

NA

NA

NA

NA

NA

NA

NA

Sub-

frac

tion

6N

AN

AN

AN

AN

AN

AN

ASu

b-fr

actio

n 7

NA

NA

NA

NA

NA

NA

NA

Lino

leic a

cid1.

000 ±

0N

A0.

500 ±

0N

AN

AN

AN

APa

lmiti

c acid

0.50

0 ± 0

0.25

0 ± 0

0.25

0 ± 0

NA

0.50

0 ± 0

NA

NA

Peni

cillin

0.00

4 ± 0

0.06

3 ± 0

0.06

3 ± 0

0.50

0 ± 0

0.12

5 ± 0

0.50

0 ± 0

0.12

5 ± 0

Chl

oram

phen

icol

0.03

1 ± 0

0.01

6 ± 0

0.01

6 ± 0

0.50

0 ± 0

0.01

6 ± 0

0.12

5 ± 0

0.00

8 ± 0

Mea

n ±

stan

dard

dev

iatio

n, n

=3;

NA

= n

o ac

tivity

Tab

le 4

. Min

imum

inhi

bito

ry c

once

ntra

tion

(MIC

) of c

rude

, fra

ctio

ns a

nd p

ure

com

poun

ds fr

om D

yfro

lom

yces

rhi

zoph

orae

in m

g/m

l.

Chiang Mai J. Sci. 2014; 41(4) 905

indicative of the hydroxyl group, thealiphatic chain and the carbonyl group,respectively. The absorption band at 1686cm-1 indicates the double bonds in linoleicacid. The 13C NMR spectrum of 1 indicated18 signals of which one was methyl, fourolefinic carbons twelve methylenes and onequaternary carbon. The carbon resonancesat δC 180.7 and 14.1 were attributed toC-1 and C-18, respectively. The carbonresonances at 128.1-130.2 were indicativeof the olefinic carbons. The remainingsignals in the highfield region between δC

24.8-34.3 were indicative of the methylenecarbons of the aliphatic chain. The 1HNMR spectrum was assigned with the aidof the HSQC spectrum, and was furtherconfirmed by the COSY and HMBCexperiments. The triplets at δH 2.35, 2.78and 0.90 and the multiplet at δH 1.65 wererespectively assigned to H-2, H-11, H-18and H-3, the signals around δH 5.30-5.42were attributed to the olefinic protons,while the signals around δH 1.31-2.09 wereattributed to the methylene protons of thealiphatic chain. The complete NMRspectral results of 1 are presented inTable 5. The gas chromatography-mass

spectrum of 2 exhibited a molecular ionpeak M+ at m/z 256. Comparison of itsmass spectrum with the reference spectrain the NIST 05 Library suggested that 2was n-hexadecanoic acid (palmitic acid),which was confirmed with the IRspectrum, 1H NMR, 13C NMR, and DEPTexperiments. The absorption bands around3348, 2924, 2854 and 1713 cm-1 wereindicative of the hydroxyl group, thealiphatic chain and the carbonyl group,respectively. The 13C NMR spectrum of2 indicated 16 signals of which one wasmethyl, 14 methylenes and one quaternarycarbon. The carbon resonances at δC 180.2and 14.3 were attributed to C-1 and C-16,respectively. The remaining signals in theupfield region between δC 22.9-34.3 wereindicative of the methylene carbons of thealiphatic chain. The 1H NMR spectrumwas assigned with the aid of the HSQCspectrum, and was further confirmed bythe COSY and HMBC experiments. Thetriplets at δH 2.35 and 0.89 and themultiplet at δH 1.64 were respectivelyassigned to H-2, H-16 and H-3, while thesignals around δH 1.26 were attributed tothe methylene protons of the aliphatic

Position δδδδδHa δδδδδC1 180.72 2.35 t (7.8) 34.33 1.64 m 24.84 1.31-1.38 mb 29.2e

5 1.31-1.38 mb 29.3e

6 1.31-1.38 mb 29.3e

7 1.31-1.38 mb 29.5e

8 2.04-2.09 mb 27.4d

9 5.30-5.42 mb 130.2f

Table 5. The 1H and 13C NMR 400Hz data of linoleic acid.

Position δδδδδHa δδδδδC10 5.30-5.42 mb 128.1g

11 2.78 t (6.0) 25.812 5.30-5.42 mb 128.3g

13 5.30-5.42 mb 130.4f

14 2.04-2.09 mb 27.3d

15 1.31-1.38 mb 29.8e

16 1.31-1.38 mb 22.8c

17 1.31-1.38 mb 31.7c

18 0.90 t (7.3) 14.1

aCoupling constants (J) in Hz are indicated in parenthesesbOverlapping signalsc,d,e,f,gChemical shifts are interchangeable

906 Chiang Mai J. Sci. 2014; 41(4)

chain. The complete NMR spectral resultsof 2 are presented in Table 6.

Palmitic acid and linoleic exhibitedantibacterial activity against only Grampositive bacterial strains in this study whichcould be attributed to its hydrophobicnature [4]. Certain fatty acids, such aspalmitic acid, have been known to displayantibacterial activities against oralmicroorganisms [15]. The antibacterialactivity exhibited by linoleic acid in thepresent study is similar to previous studies[8]. Linoleic acid and palmitic acid are alsoknown to be common fatty acidconstituents in essential oils [42]. Thecomposition of 44.8% oleic acid and 33.7%of linoleic acid in argan oil has been provento possess nutritional benefits in thereduction of atherosclerosis consequentlypreventing occurrences of cardiovasculardiseases in humans [5]. Furthermore, fattyacids from microorganisms have also beenshown to be sustainable feedstock forbiodiesel production [9,49].

ACKNOWLEDGEMENTSWe thank the University of Malaya for

the Postgraduate Grant (PPP) (PV068-2012A) and the University of MalayaResearch Grant (UMRG) (RG203-12SUS).We also thank the Ministry of Science,Technology and Innovation (MOSTI) forthe eScienceFund grant (12-02-03-2092).

REFERENCES

[1] Acevedo M.S., Puentes C., CarrenoK., Leon J.G., Stupak M., Garcia M.,Perez M. and Blustein G., Antifoulingpaints based on marine naturalproducts from Colombian Carribean,International Biodeterioration &Biodegradation, 2013; 83: 97-104.

[2] Aly A.H., Ebel R.E., Wray V., MullerW.E.G., Kozytska S., Hentschel U.,

Proksch P. and Ebel R., Bioactivemetabolites from the endophyticfungus Ampelomyces sp. Isolated fromthe medicinal plant Urospermumpicroides, Phytochemistry, 2008; 69:1716-1725.

[3] Arunpanichlert J., Rukachaisirikul V.,Tadpetch K., Phongpaichit S., TowatanaN.H., Supaphon O. and Sakayaroj J., Adimeric chromanone and a phthalide:Metabolites from the seagrass-derivedfungus Bipolaris sp. PSU-ES64,Phytochemistry Letters, 2012: 5: 604-608.

[4] Bajpai V.K., Sharma A. and BaekK.H., Antibacterial mode of action ofCudrania tricuspidata fruit essentialoil, affecting membrane permeabilityand surface characteristics of food-borne pathogens, Food control., 2013;32: 582-590.

Position δδδδδHa δδδδδC1 180.22 2.35 t (7.3) 22.93 1.64 m 32.24 1.26 mb 29.3c

5 1.26 mb 29.3c

6 1.26 mb 29.5c

7 1.26 mb 29.5c

8 1.26 mb 29.6c

9 1.26 mb 29.6c

10 1.26 mb 29.7c

11 1.26 mb 29.7c

12 1.26 mb 29.8c

13 1.26 mb 29.9c

14 1.26 mb 24.9d

15 1.26 mb 34.3d

16 0.89 t (6.4) 14.3

Table 6. The 1H and 13C NMR 400Hzdata of palmitic acid.

aCoupling constants (J) in Hz are indicated inparentheses

bOverlapping signalsc,dChemical shifts are interchangeable

Chiang Mai J. Sci. 2014; 41(4) 907

[5] Cherki M., Berrougui H., Drissi A.,Adlouni A. and Khalil A., Argan oil:Which benefits on cardiovasculardiseases?, Pharmacological Research,2006; 54: 1-5.

[6] Debbab A., Aly A.H. and Proksch P.,Endophytes and associated marinederived fungi-ecological and chemicalperspectives, Fungal Diversity, 2012;57: 45-83.

[7] Debbab A., Aly A.H. and Proksch P.,Mangrove derived fungal endophytes-a chemical and biological perception,Fungal Diversity, 2013; 61: 1-27.

[8] Dilika F., Bremner P.D. and MeyerJ.J.M., Antibacterial activity oflinoleic and oleic acids isolated fromHelichrysum pedunculatum: a plantused during circumcision rites,Fitoterapia, 2000; 71: 450-452.

[9] Dominguez L.A., Urbina E.C.,Jimenez A.M., Ortiz C.M.F., LealE.R., Garcia F.J.E., Noyola M.T.P.and Varaldo H.M.P., Polyunsaturatedfatty acids in bacteria, algae and fungi-A review, Environmental engineeringand management journal, 2012; 11: 97.

[10] Du F.Y., Li X.M., Li C.S., Shang Z.and Wang B.G., Cristatumins A-D,new indole alkaloids from the marine-derived endophytic fungus Eurotiumcristatum EN-220, Bioorganic &Medicinal Chemistry Letters, 2012; 22:4650-4653.

[11] Ebel R., Natural products frommarine-derived fungi. Application ofmarine fungi; in Jones E.B.G. and PangK.L., eds., Marine Fungi and Fungal-like organisms: Marine and FreshwaterBotany, 1st ed, 2012: 411-440.

[12] Eloff J.N., A sensitive and quickmicroplate method to determine theminimal inhibitory concentration ofplant extracts for bacteria, PlantaMedica., 1998; 64: 711-713.

[13] Ezra D., Hess W.M. and Strobel G.A.,New endophytic isolates of Muscodoralbus, a volatile-antibiotic producingfungus, Microbiology, 2004; 150: 4023-4031.

[14] Guo S., Mao W., Li Y., Tian J. andXu J., Structural elucidation of theexopolysaccharide produced byfungus Fusarium oxysporum Y24-2,Carbohydrate Research, 2012; 365: 9-13.

[15] Huang C.B., Alimova Y., Myers T.M.and Ebersole J.L., Short and mediumchain fatty acids exhibit antimicrobialactivity for oral microorganisms,Archives of oral biology, 2011; 56: 650-654.

[16] Hoskisson P.A., Hobbs G. andSharples G.P., Antibiotic production,accumulation of intracellular carbonreserves, and sporulation inMicromonospora echinospora (ATCC15837), Canadian Journal ofMicrobiology, 2001; 47: 148-152.

[17] Imhoff J.F., Labes A. and Wiese J.,Bio-mining the microbial treasures ofthe ocean: New natural products,Biotechnology Advances, 2011; 29: 468-482.

[18] Joel E.L. and Bhimba V., Isolationand characterization of secondarymetabolites from the mangrove plantRhizophora mucronata, Asian PacificJournal of Tropical Medicine, 2010;602-604.

[19] Jones E.B.G., Bioactive compounds inmarine organisms, Botanica Marina,2008; 51: 161-162.

[20] Jones E.B.G., Stanley S.J. and PinruanU., Marine endophyte sources of newchemical natural products: A review,Botanica marina, 2008; 51: 163-170.

[21] Kawazoe K., Yutani A. and TakaishiY., Aryl naphthalenes norlignans fromVitex rotundifolia, Phytochemistry,

908 Chiang Mai J. Sci. 2014; 41(4)

1999; 52: 1657-1659.[22] Kjer J., Debbab A., Aly A.H. and

Proksch P.. Methods for isolation ofmarine-derived endophytic fungi andtheir bioactive secondary products,Nature protocols, 2010; 5: 3.

[23] Ko W.G., Kang T.H., Lee S.J., KimN.Y., Kim Y.C., Sohn D.H. and LeeB.H., Polymethoxyflavonoids fromVitex rotundifolia inhibit proliferationby inducing apoptosis in humanmyeloid leukemia cells, Food andChemical Toxicology, 2000; 38: 861-865.

[24] Ko T.W.K., Stephenson S.L., BahkaliaH. and Hyde K.D., From morpho-logy to molecular biology: can we usesequence data to identify fungalendophytes?, Fungal Diversity, 2011;50: 113–120.

[25] Liberra K., Jansen R. and LindequistU., Corollosporine, a new phthalidederivative from the marine fungusCorollospora maritima Werderm.1069, Pharmazie., 1998; 53: 578-581.

[26] Lin Y., Wu X., Deng Z., Wang J.,Zhou S., Vrijmoed L.L.P. and JonesE.B.G., The metabolites of themangrove fungus Verruculina enaliaNo. 2606 from a salt lake in theBahamas, Phytochemistry, 2002; 59:469-471.

[27] Loilong A., Sakaroj J., RungjindamaiN., Choeyklin R. and Jones E.B.G.,Biodiversity of fungi on the palm Nypafruticans; in Jones E.B.G. and PangK.L., eds., Marine Fungi and fungal-like organisms: Marine and freshwaterbotany, 1st ed, 2012: 273-290.

[28] Lu X., Cao X., Liu X. and Jiao B.,Marine Microbes-Derived Anti-Bacterial Agents, Mini-Reviews inMedicinal Chemistry, 2010; 10: 1077-1090.

[29] Martinez C.E., Morales S.C., SerranoM.F., Rahman M.M., Gibbons S. and

Miranda R.P., Characterization of axylose containing oligosaccharide, aninhibitor of multidrug resistance inStaphylococcus aureus, from Ipomoeapes-caprae, Phytochemistry, 2010; 71:1796-1801.

[30] Neumann H., Strubing D., Lalk M.,Klaus S., Hubner S., Spannerberg A.,Lindequist U. and Beller M., Synthesisand antimicrobial activity of N-analogous corollosporines, Organicand Biomolecular Chemistry, 2006; 4:1365-1375.

[31] Pan J.H., Jones E.B.G., She Z.G., PangJ.Y. and Lin Y.C., Review of bioactivecompounds from fungi in the SouthChina Sea, Botanica Marina, 2008; 51:179-190.

[32] Pang K.L. and Jones E.B.G., Epilogue:Importance and impact of marinemycology and fungal-like organisms:challenges for the future; in JonesE.B.G. and Pang K.L., eds., MarineFungi and Fungal-like Organisms:Marine and Freshwater Botany, 1st ed,2012: 510-517.

[33] Posada D., jModelTest: phylogeneticmodel averaging, Molecular biologyand evolution, 2008; 25(7): 1253-1256.

[34] Proksch P., Ebel R., Edrada R.A.,Riebe F., Liu H., Diesel A., Bayer M.,Li X., Lin W.H., Grebenyuk V.,Muller W.E.G., Draeger S., ZuccaroA. and Schulz B., Sponge-associatedfungi and their bioactive compounds:the Suberites case, Botanica Marina,2008; 51: 208-218.

[35] Rateb M.E. and Ebel R., Secondarymetabolites of fungi from marinehabitats, Natural Products Report,2011; 28: 290-344.

[36] Schulz B., Boyle C., Draeger S.,Rommert A.K. and Krohn K.,Endophytic fungi: a source of novelbiologically active secondary

Chiang Mai J. Sci. 2014; 41(4) 909

metabolites, Mycological Research,2002; 106(9): 996-1004.

[37] Smith M.E., Douhan G.W. and RizzoD.M., Intra-specific and intra-sporocarp ITS variation ofectomycorrhizal fungi as assessed byrDNA sequencing ofsporocarps and pooled ectomycorrhizalroots from a Quercus woodland,Mycorrhiza., 2007; 18: 15–22.

[38] Stamatakis A., Hoover P. andRougemont J. A Rapid BootstrapAlgorithm for the RAxML Web-Servers, Systematic Biology, 2008;75(5): 758-771.

[39] Strobel G. and Daisy B., Bioprospectingfor Microbial Endophytes and TheirNatural Products, Microbiology andMolecular Biology Reviews, 2003;67(4): 491-502.

[40] Sultan S., Shah S.A.A., Sun L.,Ramasami K., Cole A., Blunt J., H.G.M.M. and Weber J.F.F., Bioactivefungal metabolites of 9PR2 isolatedfrom roots of Callophyllum Ferrugineum,International Journal of Pharmacy andPharmaceutical Science, 2011; 3: 7-9.

[41] Suryanarayanan T.S., ThirunavukkarasuN., Govindarajulu M.B., Sasse F.,Jansen R. and Murali T.S., Fungalendophytes and bioprospecting, FungalBiology Reviews, 2009; 23: 9-19.

[42] Tang T.F., Liu X.M., Ling M., Lai F.,Zhang L., Zhou Y.H. and Sun R.R.,Constituents of the essential oil andfatty acid from Malania oleifera,Industrial Crops and Products, 2013; 43:1-5.

[43] Tong W.Y., Darah I. and Latiffah Z.,Antimicrobial activities of endophyticfungi isolates from the medicinal herbOrthosiphon stamineus Benth, Journalof Medicinal Plants Research, 2011;5(5): 831-836.

[44] Wu J., Xiao Q., Xu J., Li M., Pan J.

and Yang M., Natural products fromtrue mangrove flora: Source, chemistryand bioactivities, Natural ProductReports, 2008; 25: 955-981.

[45] Yin H. and Sun H., Vincamine-producing endophytic fungus isolatedfrom Vinca minor, Phytomedicine,2011; 18: 802-805.

[46] You X., Feng S., Luo S., Cong D., YuZ., Yang Z. and Zhang J., Studies ona rhein producing endophytic fungusisolated from Rheum palmatum L.,Fitoterapia, 2013; 85: 161-168.

[47] Yu H., Zhang L., Li L., Zheng C.,Guo L., Li W., Sun P. and Qin L.,Recent developments and futureprospects of antimicrobial metabolitesproduced by endophytes,Microbiological Research, 2010; 165:437-449.

[48] Zainuddin N., Alias S.A., Lee C.W.,Ebel R., Othman N.A., MukhtarM.R. and Awang K., Antimicrobialactivities of marine fungi fromMalaysia, Botanica Marina, 2010; 53:507-513.

[49] Zheng Y., Yu X., Zeng J. and ChenS., Feasibility of filamentous fungi forbiofuel production using hydrolysatefrom dilute sulphuric acid pretreatmentof wheat straw, Biotechnology forBiofuels, 2012; 5: 50.