Screening of mangrove endophytic fungi for bioactive compounds · 2016-12-05 · Screening of...

Screening of Mangrove Endophytic Fungi forBioactive Compounds

by

ONN MAY LING

A thesis

presented in fulfillment of the requirements

for the degree of

Masters of Science (by Research)

at Swinburne University of Technology

2013

Abstract

Endophytic fungi are an underexplored group of microorganisms as only a few plants have

been studied with regards to this community. They live inside the tissues of other

organisms, such as mangrove plants that provide protection to them and in return

endophytic fungi support their hosts by fighting off pathogens through the production of

antimicrobial compounds. These bioactive compounds are secondary metabolites which are

often produced as waste- or by-products. Besides, endophytic fungi also help the host plant

in adapting to (extreme) environments, for example by removing harmful heavy metals. In

Malaysia, mangrove forests continue to be threatened by heavy metal pollution, resulting

from industrial waste water pollution and urbanization.The presence of heavy metals can

lead to severe damage as they are bioaccumulative and toxic. In the present study,

endophytic fungi isolated frommangrove plants were characterized and assessed for their

antimicrobial, cytotoxicity activity and heavy metal biosorption potential. Twelve

endophytic fungi were isolated and identified (using molecular methods) to belong to 7

families: Penicillium, Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya and

Eupenicillium. Antimicrobial activities of these 12 fungal endophytes were tested against

gram positive bacteria (Bacillus subtilis and Staphylococcus aureus among others), gram

negative bacteria (Escherichia coli among others), yeast (Saccharomyces cerevisiae) and

fungi (Candida albicans and Aspergillus niger). Two strains; Isolate 7 and Isolate 13

(related to Guignardia sp. and Neosartoya sp., respectively) showed strong antimicrobial

(and antifungal) activity which was indicated by the formation of clear zone of inhibition,

whereas the rest showed no activity. Compounds were isolated from the extracts of both

isolates and screened using HPLC. Whereas for cytotoxicity assay, two strains; Isolate 3

and Isolate 9 (related to Diaporthe sp. and Eupenicillium sp., respectively) displayed

toxicity against the matured brine shrimps at concentrations of 500 ppm after 24 hours

incubation. For heavy metal biosorption, Isolate 2, which is closely related to Curvularia

sp., is the most efficient in removing Cu, up to 25mg Cu/g biomass (using dead biomass).

On the other hand, Isolate 8 and Isolate 13 (both related to Neosartorya sp.) are the most

efficient in removing zinc (also using dead biomass), with a removal of up to 24 mg Zn/g

biomass.The findings clearly indicate the potential of mangrove endophytic fungi to be

used for drug development and also in wastewater bioremediation.

ACKNOWLEDGEMENT

I would like to express the deepest appreciation to my supervisor, Dr. Moritz Mueller, who

has the attitude and substance of a genius: he continually and convincingly conveyed a

spirit of adventure in regard to research, and an excitement in regard to teaching his

students. Without his guidance and persistent help, this research as well as dissertation

would not have been possible.

I would also like to thank my co-supervisors, Dr. Lim Po Teen and Dr. Aazani Mujahid,

whose work demonstrated that science and technology should always transcend academia.

In addition, a thank you to my senior lab mate, Noreha Mahidi, who gave the permission to

use her required equipment and the necessary materials to complete the benchwork.

Besides, her stimulating suggestions and encouragement definitely has helped me to

coordinate my project.

I would also like to thank my fellow colleagues of the lab, Felicity Kuek, Chua Jia Ni,

Nurul and Fika for the guidance and help throughout my benchwork period. Deepest

appreciation for the time spent helping me out with some equipments as well as other tasks.

Lastly, I would like to thank Professor Peter Proksch from the Institut für Pharmazeutische

Biologie und Biotechnologie, University of Düsseldorf,Germany for the HPLC analysis

conducted on my research samples.

DECLARATION

I, Miss Onn May Ling, Masters of Science (By Research), Faculty of Engineering,

Computing and Science, hereby declare that my project work titled “Screening of

Mangrove Endophytic Fungi For Bioactive Compounds” is original and contains no

material which has been accepted for the award to the candidate of any other degree or

diploma, except where due reference is made in the text of the examinable outcome; to the

best of candidate’s knowledge contains no material previously published or written by

another person except where due reference is made in the text of the examinable outcome;

and where the work is based on joint research or publications, discloses the relative

contributions of the respective workers or authors. All the given information is true to best

of my knowledge.

……………………………

(ONN MAY LING)

DATE: 1.7.2013

I

Table of ContentsList of Figures ...................................................................................................................... VI

List of Tables......................................................................................................................VIII

1. Introduction ................................................................................................................ 1

1.1 Infectious diseases, drug resistance, and bioactive compounds................................... 1

1.2 Sources of bioactive compounds.................................................................................. 4

1.3 Fungi ............................................................................................................................ 6

1.3.1 Fungi as sources of bioactive compounds............................................................. 7

1.4Endophytic fungi ........................................................................................................... 7

1.4.1 Endophytic fungi as sources of bioactive compounds .......................................... 9

1.5 Mangroves.................................................................................................................. 17

1.5.1 Mangrove endophytic fungi ................................................................................ 19

1.5.2Threats to mangroves ........................................................................................... 21

1.5.3 Heavy metal pollution ......................................................................................... 21

1.5.4 Heavy metal uptake and removal ........................................................................ 23

1.5.5Biosorption by Marine Fungi ............................................................................... 24

1.6Aim of the project and scope of study ........................................................................ 25

2. Materials and methods ............................................................................................. 26

2.1 Sampling .................................................................................................................... 26

2.1.1 Field site sampling .............................................................................................. 26

2.2 Isolation of mangrove endophytic fungi .................................................................... 27

2.2.1 Plant samples....................................................................................................... 27

2.2.2 Soil samples ........................................................................................................ 28

2.3 Fungal Cultivation...................................................................................................... 31

2.3.1 Fungal Culture for Short Term Storage .............................................................. 31

2.3.2 Fungal Culture for Long Term Storage............................................................... 31

2.3.3 Fungal Culture for Extraction of Bioactive Compounds .................................... 31

2.4 Endophytic fungi identification.................................................................................. 33

2.5 Extraction of bioactive compounds............................................................................ 36

2.5.1 Solvent-solvent extraction................................................................................... 36

II

2.6 Biological Assays....................................................................................................... 40

2.6.1 Primary Screening of antimicrobial activity ....................................................... 40

2.6.2 Secondary screening of antimicrobial activity .................................................... 41

2.6.3 General Cytotoxicity assay ................................................................................. 41

2.7 Heavy Metal Analysis ................................................................................................ 45

2.7.1 Determination of heavy metal-resistant fungi..................................................... 45

2.7.2 Heavy metal biosorption by dead fungal cells .................................................... 45

3. Results and Discussion............................................................................................. 48

3.1 Fungi identification .................................................................................................... 48

3.2 Biological assays ........................................................................................................ 52

3.2.1 Primary screening of antimicrobial activity ........................................................ 52

3.2.2 Secondary screening of antimicrobial activity .................................................... 54

3.2.3 Cytotoxic activity ................................................................................................ 56

3.3 Bioactive compounds isolated from endophytic fungi............................................... 58

3.3.1 Citreonigrin F ...................................................................................................... 62

3.3.2 Gancidin(cycloleucylprolyl) ............................................................................... 62

3.3.3 Citreodrimene B .................................................................................................. 62

3.3.4 2-Hydroxy-3-methylbenzoic acid ....................................................................... 62

3.3.5 Altechromone A .................................................................................................. 63

3.3.6 Fatty Acid............................................................................................................ 63

3.3.7 Cerebroside ......................................................................................................... 64

3.3.8 Cyclo(prolylvalyl) ............................................................................................... 64

3.3.9 Kahalalide B........................................................................................................ 65

3.3.10 Cyclo(tyrosylprolyl) .......................................................................................... 65

3.3.11 Citreoisocoumarin and Diachlordiaportin......................................................... 65

3.3.12 Sumiki’s acid..................................................................................................... 66

3.3.13 Cyclochalasin H ................................................................................................ 66

3.3.14 Naamine A ........................................................................................................ 66

3.3.15 Citrinin hydrate ................................................................................................. 66

3.3.16 Quinolactacin .................................................................................................... 67

3.3.17 Altenusin ........................................................................................................... 67

III

3.3.18 Citrinin .............................................................................................................. 67

3.3.19 Sclerotigenin ..................................................................................................... 68

3.3.20 Cladosporin ....................................................................................................... 68

3.3.21 Trihydroxy tetralone.......................................................................................... 68

3.3.22 Cyclopenin ........................................................................................................ 68

3.3.23 Graphislactone derivative.................................................................................. 69

3.3.24 Phenylacetic acid............................................................................................... 69

3.3.25 Isofistularin-1 .................................................................................................... 69

3.3.26 8E-6-3-3 Aurantiamine ..................................................................................... 70

3.3.27 Aureonitol ......................................................................................................... 70

3.3.28A new gamma-pyrone ........................................................................................ 70

3.3.294,5-dibromopyrrole-2-carboxylic acid ............................................................... 71

3.3.30 Adenosine.......................................................................................................... 71

3.3.31Dienone dimethoxyketal .................................................................................... 71

3.3.3211, 19-dideoxyfistularin ..................................................................................... 72

3.3.33Triterpene acetate ............................................................................................... 72

3.3.34 Microsphaerone B ............................................................................................. 72

3.3.35 3,4-Dihydromanzamine..................................................................................... 72

3.3.36 Paxilline ............................................................................................................ 73

3.3.37 Manzamine J N-Oxide ...................................................................................... 73

3.3.38 Pavetannin A1 Ac ............................................................................................. 73

3.3.39 Epicatechin........................................................................................................ 74

3.3.40 9alpha-OH-Pinoresinol ..................................................................................... 74

3.3.41 Rocaglamide A.................................................................................................. 74

3.3.42 Procyanidin B3 o. B6 ........................................................................................ 75

3.3.43 Trimeric Catechin.............................................................................................. 75

3.3.44 Helenalin ........................................................................................................... 76

3.3.45 Catechin............................................................................................................. 76

3.3.46 Triandrin............................................................................................................ 76

3.4 Heavy metal analysis.................................................................................................. 76

3.4.1 Determination of heavy metal resistance fungi................................................... 76

IV

3.4.2 Heavy metal biosorption by dead fungal cells .................................................... 79

4. PRELIMINARY RESULTS OF SCREENING OF MANGROVE ENDOPHYTICFUNGI FOR BIOACTIVE COMPOUNDS ........................................................................ 83

ABSTRACT..................................................................................................................... 83

INTRODUCTION ........................................................................................................... 84

MATERIALS AND METHODS .................................................................................... 85

Isolation of Endophytic Fungi...................................................................................... 85

Identification of Endophytic Fungi .............................................................................. 85

Antimicrobial Assay ..................................................................................................... 86

Cytotoxic assay............................................................................................................. 86

Extraction of Bioactive Compounds............................................................................. 86

High-Performance Liquid Chromatography (HPLC).................................................. 87

RESULTS AND DISCUSSION ...................................................................................... 87

Identification of Endophytic Fungi .............................................................................. 87

Antimicrobial Assay ..................................................................................................... 87

Cytotoxic assay............................................................................................................. 88

Extraction of Bioactive Compounds............................................................................. 90

CONCLUSION................................................................................................................ 92

ACKNOWLEDGEMENT ............................................................................................... 92

TABLES........................................................................................................................... 93

FIGURES ......................................................................................................................... 95

5. BIOSORPTION OF COPPER (CU) AND ZINC (ZN) BY MANGROVEENDOPHYTIC FUNGI..................................................................................................... 100

ABSTRACT................................................................................................................... 100

INTRODUCTION ......................................................................................................... 101

MATERIALS & METHODS ........................................................................................ 102

Isolationof Endophytic Fungi..................................................................................... 102

Identification of Endophytic Fungi ............................................................................ 103

Preparation of reagents and materials ...................................................................... 103

Determination of heavy metal-resistant fungi............................................................ 103

Biosorption studies by dead fungal cells.................................................................... 104

V

RESULTSAND DISCUSSION ..................................................................................... 104

Identification of Endophytic Fungi ............................................................................ 104

Heavy metal-resistant fungi ....................................................................................... 104

Heavy metal biosorption by dead fungal cells ........................................................... 107

CONCLUSION.............................................................................................................. 109

ACKNOWLEDGEMENT ............................................................................................. 109

TABLES......................................................................................................................... 110

FIGURES ....................................................................................................................... 111

6. CONCLUSION...................................................................................................... 113

REFERENCES................................................................................................................... 115

VI

List of Figures

Figure 1: Penicillin(source: websters-online-dictionary.org) ................................................ 2Figure 2: Cephalosporin (source: websters-online-dictionary.org) ....................................... 2Figure 3: Vancomycin (source: websters-online-dictionary.org) .......................................... 3Figure 4: Doxorubicin (source: websters-online-dictionary.org)........................................... 3Figure 5: Staurosporine (source: websters-online-dictionary.org)......................................... 3Figure 6: Dolastatin (source: sigmaldrich.com)..................................................................... 5Figure 7: Marinomycin (source: molecular-networks.com) .................................................. 5Figure 8: Taxol..................................................................................................................... 10Figure 9: Compounds (a) and (b) were extracted from the endophytic fungus Gliomastixmurorum. The fungus is isolated from the Chinese medicinal plant (c) Paris polyphylla var.yunnanensis, which is widely used in China as medicinal herb due to its anti-tumor,analgesia, anti-inflammatory, and antifungal properties (Liu & Ji 2012)............................ 13Figure 10: Camptothecin (a), a modified monoterpene indole alkaloid, was first isolatedfrom the stems of Camptotheca accuminata (b) in 1966. This compound (a) was found toexhibit clinical anti-tumor activity by inhibiting DNA topoisomerase I, an enzyme involvedin DNA recombination, repair, replication, and transcription (Sun et al. 2011). It was laterfound to be produced by Entrophospora infrequens, an arbuscularmycrorrhiza(Meenakshisundaram & Santhagur 2010), isolated from Nothapodytes foetida, which is theonly native species isolated from the Orchid Island, commonly used for hedges or firewoodand cultured in Taiwan (Wu et al. 2008).............................................................................. 14Figure 11: 5-Hydroxyramulosin (a), a polyketide compound extracted from an endophyticfungus morphologically similar to Phoma sp. (b)................................................................ 15Figure 12: Cytochalasin H2 (a), a new compound was extracted from the endophyticfungus, Xylaria sp. (b) which was isolated from Annona squamosa (c).............................. 15Figure 13: Palmarumycin CP 2 (a), palmarumycin CP 17 (b), and preusommerin EG (c),were isolated from Edenia sp. and cercosporin (d), a fungal toxin was isolated fromMycosphaerella sp.These compounds were found to possess antiparasitic activity againstthe parasite, Leishmania donovani (e), a protozoan parasite known to cause Leishmaniasis,a worldwide disease known to cause serious disfigurement and which may be fatal(Martinez-Luis et al. 2011). ................................................................................................. 16Figure 14: Kampung Pasir Pandak (Sampling site) situated near Kampong Batu, indicatedby the Blue Point (Source: Google Map) ............................................................................. 26Figure 15: Schematic overview of isolation of mangrove endophytic samples from plantsamples................................................................................................................................. 29Figure 16: Schematic overview of isolation of mangrove endophytic samples from soilsamples................................................................................................................................. 30Figure 17: Fungal Cultivation for short term storage, long term storage, and extraction ofbioactive compounds............................................................................................................ 32Figure 18:Endophytic fungi identification using molecular tools........................................ 35

VII

Figure 19: Extraction of bioactive compounds using ethyl acetate ..................................... 38Figure 20:Extraction of bioactive compounds using solvent-solvent (methanol and n-hexane) extraction ................................................................................................................ 39Figure 21: Primary and Secondary screening of antimicrobial activity............................... 43Figure 22:Cytotoxicity assay................................................................................................ 44Figure 23:Determination of heavy metal resistant fungi using minimum inhibitoryconcentration (MIC) and heavy metal biosorption .............................................................. 47Figure 24: 18S gene-based phylogenetic tree representing the twelve endophytic fungalisolates. The phylogenetic tree was generated with distance methods, and sequence

distances were estimated with the neighbor-joining method. Bootstrap values ≥50 areshown and accession numbers for the reference sequences are indicated. .......................... 49Figure 25: Zone of inhibition (ZOI) for Isolate 7 and Isolate 13. (a) Isolate 7 againstBacillus cereus; (b) Isolate 13 against Candida albicans. ................................................... 54Figure 26: Zone of inhibition (ZOI) for Isolate 7 extract and Isolate 13 extract. (a) Isolate 7extract against Bacillus cereus; (b) Isolate 13 extract against Candida albicans. Scale isindicated at the bottom. ........................................................................................................ 56Figure 27: 18S gene-based phylogenetic tree representing the twelve endophytic fungalisolates. The phylogenetic tree was generatedwith distance methods, and sequence

distances were estimated with the neighbor-joining method. Bootstrap values ≥50 areshown and accession numbers for the reference sequences are indicated. .......................... 95Figure 28: Zone of inhibition (ZOI) for strains Isolate 7 and Isolate 13. (a) Strain Isolate7against Bacillus cereus; (b) Strain Isolate 13 against Candida albicans. Scale is indicated atthe bottom. ........................................................................................................................... 96Figure 29: HPLC chromatograms of Ethyl Acetate extracts of (a) Isolate 7 and (b) Isolate13 recorded at 235 nm.......................................................................................................... 97Figure 30: HPLC chromatograms of compounds from Isolate 7 that had similar structuresto (a) Pavetannin A1 Ac, (b) Epicatechin, and (c) 9alpha-OH-Pinoresinol. Chromatogramswere recorded at 235 nm and library hits are indicated at the top right of the picture. ....... 98Figure 31: HPLC chromatograms of compounds from Isolate13 that had similar structuresto (a) Trimeric Catechin, (b) Epicatechin, and (c) Helenalin. Chromatograms were recordedat 235 nm and library hits are indicated at the top right of the picture. ............................... 99Figure 32: 18S gene-based phylogenetic tree representing the twelve endophytic fungalisolates. The phylogenetic tree was generated with distance methods, and sequence

distances were estimated with the neighbor-joining method. Bootstrap values ≥50 are

shown and accession numbers for the reference sequences are indicated. ........................ 111Figure 33: Two fungal strains: (a) Isolate 1 and (b) Isolate10 closely related to Penicilliumdravuni but having different morphological characteristics and growth patterns whereIsolate 10 grows at a faster rate within a week compared to Isolate 1, as seen from thepictures of both plates taken during 1 week incubation. .................................................... 112

VIII

List of TablesTable 1: Characteristics of the soil conditions of the three different sampling sites ........... 27Table 2: Overview of the closest relatives found for each endophytic isolate, their querycoverage in base pairs and %, as well as the source of the sample from which the isolateoriginates. ............................................................................................................................. 48Table 3: Antimicrobial activity of endophytic fungi strains (Primary screening) ............... 53Table 4: Antimicrobial activity of endophytic fungi strains (Secondary Screening) .......... 55Table 5: Mortality of brine shrimps observed at different concentrations (0.5, 5, 50 and 500ppm) of crude extracts of fungal strains............................................................................... 57Table 6: Overview of the amounts (in mg) obtained for each fraction ................................ 58Table 7: Overview of HPLC results obtained for the three fractions (ethyl acetate, methanoland n-hexane). Number of compounds related to known structures/compounds is indicatedand details listed below, as well as number of compounds showing no similarityto knowncompounds (unknown compounds). Note: Number of known compounds is based onlibrary hits available. ............................................................................................................ 59Table 8: Overview of HPLC results obtained for the three fractions of Isolate7 andIsolate13 (ethyl acetate, methanol and n-hexane). Number of compounds related to knownstructures/compounds is indicated and details listed below, as well as number ofcompounds showing no similarity to known compounds (unknown compounds). Note:Number of known compounds is based on library hits available. ....................................... 61Table 9: Minimum inhibitory concentration (MIC) of heavy metal Copper (Cu) and Zinc(Zn) in living biomass of fungi ............................................................................................ 77Table 10: Copper (Cu) Biosorption capacity by dead fungal cells ...................................... 80Table 11: 18S rRNA phylogenetic results for endophytic fungi ......................................... 93Table 12: Antimicrobial activity of endophytic fungi strains .............................................. 94Table 13: Mortality of the brine shrimps at different concentration of crude extract .......... 94Table 14: Minimum inhibitory concentration (MIC) of heavy metal Copper (Cu) and Zinc(Zn) in living biomass of isolated endophytic fungi (in µg/ml). The most and the leastresistant species are highlighted in bold, as are their respective MIC values. ................... 110Table 15: Copper (Cu) and Zinc (Zn) Biosorption capacity, Q, by dead fungal cells(calculated as amount of metal ions (mg) bioabsorbed per gm (dry mass)). The mostefficient species is highlighted in bold, as is their respective Q value............................... 110

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1. Introduction

1.1 Infectious diseases, drug resistance, and bioactive compounds

The emergence of new infectious diseases such as H1N1, influenza, and SARS has

become a major challenge towards human health. Many of these new diseases are related to

microorganisms that are becoming more and more drug resistant; hence the search for new

bioactive compounds has emerged as an important approach to combat these diseases

(Bhatia & Narain 2010). For instance, in South East Asia, signs of infections with

Plasmodium falciparum (protozoan parasite known to cause malaria) disappear later after

the beginning of treatment with the malaria drug, indicating that the parasite is becoming

more resistant to the commonly used medicine, for instance artemisinin, in Thailand

(Science Daily 2012).The resistance to antibiotics is a phenomenon by which a

microorganism is no longer affected by the antimicrobial compound to which it was

previously sensitive (WHO 2012).

These so called bioactive compounds have been gaining attention due to their ability

to reduce the incidenceof diseases such as cancer and diabetes. They have been profoundly

used as antibiotics such as penicillin (Figure 1), cephalosporin (Figure 2), and vancomycin

(Figure 3) which are effective against infectious diseases.

Drugs commonly used against carcinoma are for example doxorubicin (Figure 4)

and staurosporine (Figure 5) (Kim & Bhatnagar 2010). Their ability has been associated

with their various degrees of bioactivity such as anti-cancer, anti-diabetic, and many other

properties which are useful in biomedical research and drug development (Strobel& Daisy

2003). In the following, we describe some of the sources for these compounds.

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Figure 1: Penicillin(source: websters-online-dictionary.org)

Penicillin was first discovered by Alexander

Fleming, in 1928, produced by a rare mold,

Penicillium notatum (Derderian 2007).

It was found to be especially active against

Gram-positive bacteria but some semi-

synthetic penicillin, such as ampicillin, are

also effective against Gram-negative

bacteria (Behal 2000). It was widely used

for the treatment of infections such as

syphilis, pneumonia, diphtheria, bacterial

meningitis, and septicemia (Muniz et al.

2007).

Cephalosporin was discovered by Giuseppe

Brotzu and was extracted from

Cephalosporium acremonium, and found to

show antibiotic activity against

Staphylococcus aureus, Salmonella typhi,

and Escherichia coli (Muniz et al. 2007). Figure 2: Cephalosporin (source: websters-online-dictionary.org)

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Figure 3: Vancomycin (source: websters-online-dictionary.org)

Vancomycin was isolated from

Streptomyces orientalis and found active

against most gram positive organisms,

including penicillin-resistant staphylococci

(Levine 2006). However, in 1997,

Staphylococcus aureus was found resistant

towards vancomycin, despite that

compound being the only defense

available then (Nicolaou et al. 1999).

Doxorubicin is an anthracycline antineoplastic

antibiotic that is potent and widely used in

clinical oncology (Yu et al. 2012; Yurekli et

al. 2005).

Figure 4: Doxorubicin (source: websters-online-dictionary.org)

Figure 5: Staurosporine (source: websters-online-dictionary.org)

Staurosporine was discovered in 1977

from the bacterium Streptomyces

staurosporeus. It has been shown to

possess an array of important biological

properties such as anti-fungal, anti-

hypertensive and platelet aggregation

inhibition (Hewavitharana et al. 2009).

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al. 2005).










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al. 2005).










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1.2 Sources of bioactive compounds

Bioactive compounds are naturally derived metabolites and/or by-products from

microorganisms, plants, or animals that are also referred to as secondary metabolites as

they are not used for basic primary cell survival but instead often produced as waste

products (Behal 2000). Plants have historically been the main source of compounds used

for medicine; however, many research studies are now focusing on the role of the

microorganisms living inside the plants and the plants themselves in producing bioactive

compounds (Refer to section 1.3 Endophytic Fungi for a more detailed discussion).

Microbial secondary metabolites include antibiotics (as mentioned), pigments (astaxanthin),

toxins (Conus toxin), enzymes (clavulanic acid) and many more which have been of great

use to humans, animals and even plants (Demain 1998; Martins et al. 2011; Kim &

Bhatnagar 2010).

Bioactive compounds have been isolated from microorganisms originating from

various terrestrial and marine environments (Strobel & Daisy 2003; Ortholand & Ganesan

2004). Although organisms from the terrestrial environment have been the main source of

antibiotics for decades, the marine environment is proving to be the new area of interest

with several studies showing marine organisms to be producers of anti-cancer compounds

and also compounds which act against infectious diseases and inflammation. Well known

examples are dolastatin (Figure 6), a compound produced by marine cyanobacteria (Tan

2007) and marinomycin (Figure 7), a compound isolated from the marine actinomycete,

Marinispora sp.; both showing antitumor activity (Kwon et al. 2006). With marine

organisms being able to survive in extreme conditions due to their metabolic and

physiological capabilities; they provide an enormous potential for the production of unique

bioactive compounds that are not present in terrestrial organisms. This is not surprising as

the marine environment constitutes a large (mainly unexplored) reservoir with a long

evolutionary history and has “produced” organisms with unique biological properties

compared to terrestrial ones (Aneiros & Gateirax 2004; Belarbi et al. 2003). However,

despite the many successful applications of bioactive compounds from marine organisms,

the marine microorganisms are still under-explored with regards to their exploration as

sources of bioactive compounds (Kim &Bhatnagar 2010).

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The discovery of new bioactive compounds requires analysis of previous diversity

studies, because by knowing the types of microorganisms that reside in a certain

environment, we will be able to design cultivation techniques adapted to capture all the

microbial communities present in a certain environment (Mercado et al. 2012). Major

sources of bioactive compounds are fungi which will be introduced and discussed in the

following.

Figure 6: Dolastatin (source: sigmaldrich.com)

Dolastatin is one of the important

marine cyanobacterial molecules

that were discovered in

preclinical testing as anticancer

agents. This compound was

initially isolated from the sea

hare (Tan 2006).

Figure 7: Marinomycin (source: molecular-networks.com)

Marinomycin is a polyketide

with antibacterial and antitumor

properties produced by marine

actinomycete, Marinspora sp.

(Olano, Mendez & Salas 2009;

Lam 2006).

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following.








hare (Tan 2006).







Lam 2006).

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following.








hare (Tan 2006).







Lam 2006).

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1.3 Fungi

Fungi as important agents of plant and human diseases, producers of industrial and

pharmacological products and even as decomposers have spurred the attention of scientists

worldwide to study their nature. They are heterotrophic, eukaryotic organisms that are

unicellular in nature although they appear as multicellular during the vegetative phase

(Ireland & Bugni 2004; Sag & Kutsal, 2001). This means that they lack chlorophyll and

thus do not have the ability to photosynthesize their own food. Hence, they obtain nutrients

from substrates by absorption through their tiny thread-like filaments called hyphae that

branch in all directions (Ellis, Boehm & Mitchell 2008).

Fungi is referred to as the monophyletic true fungi although mycologists use the

term ‘‘fungi’’ to define all organisms traditionally studied (i.e. true fungi, slime molds,

water molds). The kingdom of fungi is organized into groups or better known as phyla. The

major phyla that have been identified within the true fungi are the Chytridiomycota,

Zygomycota, Ascomycota, and Basidiomycota (Lutzoni et al. 2004). The three main fungal

phyla, Zygomycota, Ascomycota, and Basidiomycota, were said to have diverged from the

Chytridiomycota approximately 550 million years ago (Guarro, Gene & Stchigel 1999).

Chytridiomycota are a phylum of fungi that reproduce through the production of

motile spores known as zoospores, typically propelled by a single directed flagellum. They

include unicellular or filamentous forms that produce flagellated cells at some point in their

life cycle and which occur in aquatic and terrestrial habitats. On the other hand, the

Zygomycota comprise a diverse assemblage of taxa that include soil saprobes (Mucorales),

symbionts of arthropod guts (Trichomycetes and Harpellales), the widespread arbuscular

mycorrhizae of plants (Endogonales) and pathogens of animals, plants, amoebae and

especially other fungi (Lutzoni et al. 2004; Abdel-Azeem 2010).

Many Ascomycota and Basidiomycota produce complex macroscopic fruiting

bodies, such as gilled mushrooms, cup fungi, coral fungi, and other forms. Ascomycota

constitute by far the largest group of fungi so far known. A large number of this species are

economically important, for instance, Fusarium sp., Colletotrichum sp., and

Mycosphaerella sp. The basic characteristic which differentiates Ascomycota from other

fungi is the presence of asci inside the ascomata. Many are free-living saprobes including

species which may be cellulose decomposers, chitinolytic, keratinolytic, or coprophilous,

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others are parasitic forms including species which cause serious plant diseases. Others that

are considered symbiotic forms contain species which live in association with insects or

algae (lichens) or roots of plants (mycorrhizas) (Abdel-Azeem 2010; Guarro et al. 1999).

The phylum Basidiomycota consists of 3 subphyla: Agaricomycotina, Pucciniomycotina

and Ustilaginomycotina (Wang et al. 2009). The most characteristic feature of

basidiomycetes is the formation of basidia (Guarro et al. 1999).

1.3.1 Fungi as sources of bioactive compounds

Fungi are prominent producers of bioactive compounds and have shown

antibacterial, antifungal, larvicidal, molluscicidal, antioxidant and free-radical scavenging

activities (Doss et al. 2010). All these activities have been associated with specific

bioactive compounds produced by fungi and exploration of fungal bioactive secondary

metabolites was initiated by the discovery of penicillin in 1928 by Alexander Fleming

which led to an expansion in the field of drug development using microorganisms (Fleming

1929). A prolific group of fungi producing bioactive compounds are the endophytic fungi.

The following provides an introduction to endophytic fungi (section 1.4) as well as various

classes of compounds produced by them (sections 1.4.1.1 to 1.4.1.3).

1.4Endophytic fungi

Endophytes are referred to as a group of fungi that reside in living tissues of plants

without causing any adverse effects towards the host plant itself. Several studies have

suggested that most fungal communities have become endophytes through invasion of

plants via wounds made by insects and plant host’s stomata (Kaul et al. 2008; Tran et al.

2010). The route of entry for these fungal endophytes transmission can be classified as

horizontal and vertical transmission. Systemic endophytes are said to transmit vertically via

the seeds, while non-systematic endophytes transmit horizontally with host colonization

arising from the surrounding environment. Endophytic fungal vertical transmission is

described as seed reproduction, which is the same as the reproduction of most plants.

However, reports on mechanism of endophytic fungal horizontal transmission are still rare

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(Dai et al. 2010; Lemons, Clay & Rudgers 2005). Fungal endophytes can be classified into

three basic ecological groups which are:

Mycorrhizal fungi

Balansiaceous or “grass endophytes”

Non-balansiaceous

Mycorrhizal fungi are a major functional group of soil organisms that forms a

symbiotic relationship with the root cells of higher green plants. The most common

mycorrhizal types form with arbuscular mycorrhizal fungi, which penetrate the host cells,

but do not modify the external appearance of the root (Amaranthus 1998). The mycorrhizal

fungi occur in most vegetation types and have been found to be one of the major

constituents of the tropical soil microflora with increased resistance towards pathogens, and

even heavy metal stress. Some of the mycorrhizal fungal species reported are Acaulospora

sp., Glomus sp., and Sclerocystis sp. (Albert & Sathianesan 2009). On the other hand, the

grass endophytes create a unique group of closely related species whose ecological

requirements and adaptations are significantly different from those of other endophytes.

They grow systemically and intercellularly within all above ground grasses, resulting in

vertical transmission of the endophytes through the seeds. For instance, the Neotyphodium

sp.and Epichloe sp. are some of the grass endophytes (Eaton, Cox & Scott 2011). Lastly,

non-balansiaceous refers to endophytes that mostly belong to the Ascomycota of various

genera such as Acremonium, Alternaria, Cladosporium, Coniothyrium, Epicoccum,

Fusarium, Geniculosporium, Phoma, and Pleospora (Devaraju & Satish 2010).

Identification of endophytic fungi can be done using microscopic and

morphological characters, and molecular sequencing analysis (Ravindran et al. 2012).

Fungal taxonomy has been traditionally based on comparative morphological features, such

as ascospore and peridium morphology, glebacolour, odour, and other organoleptic

characteristics (Lu et al. 2011). However, special caution should be taken when identifying

closely related or morphologically similar endophytes as their morphological characteristics

might be medium dependent and hence, culturing conditions can substantially affect

vegetative and sexual compatibility. On the other hand, molecular techniques exhibit higher

sensitivity and specificity for microorganism’s identification, thus, can be used for

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classifying microbial strains at diverse hierarchical taxonomic levels. Several studies have

shown that genetic methods can be successfully used in the studies of endophytic fungi.

Most of the endophytic fungi were detected and identified by comparative analyses of the

ribosomal DNA sequences, especially the internal transcribed spacer (ITS) region (ITS 1

and ITS 2) (Huang et al. 2011).

Endophytic fungi are an under-explored group of microorganisms as only a few

plants have been studied with regards tothis endophytic community. However, they

arecurrently gaining attention as they were found to be responsible for a variety of

functional benefits to their hosts. Understanding the relationship between the fungi and

their host plants will help to understand productivity in ecosystems better; in terrestrial as

well as in marine environments (Arnold & Lutztoni 2007). The endophytes play their role

in protecting their host plants from diseases or pathogens, promoting plant growth and also

enhancing their host resistance to morphological, biochemical changes and unfavorable

environmental conditions (Prabavathy & Nachiyar 2011; Dai et al. 2010). In return, host

plants are responsible forproviding shelter, protection, and even nutrients to the endophytes

(Faeth & Fagan 2002). This symbiotic relationship where both sides benefit from the

interaction, explains why plants that are infected with a broad diversity of endophytes

exhibit a lower susceptibility to insects and pathogens. Some of the bioactive compounds

produced were found to be antifungal and antibacterial and so strongly inhibit the growth of

other pathogenic microorganisms invading the host plants (Gao, Dai & Liu 2010).

1.4.1 Endophytic fungi as sources of bioactive compounds

Taxol (Figure 8) was isolated from the endophytic fungus Taxomyces andreanae

(Stierle, Strobel & Stierle 1993) and is probably the most famous compound produced by

endophytic fungi. Since that study, the search for other endophytic fungi that produce

taxolstill continues to this day and in a recent study, the endophytic fungus Phoma betae

was isolated from leaves of Ginkgo biloba and found to be a potential source of taxol

(Kumaran et al. 2012). It was shown to display high cytotoxic activity against human

cancer cells in an apoptotic assay. Taxol or better known as paclitaxel, a natural source of

the anti-cancer drug, was actually first extracted from the Pacific Yew tree, Taxus

brevifolia (Schiff &Horwitz 1980). However, overuse of plants for this purpose will not

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only affect the biodiversity but has also been found to be time consuming and results in low

yields (Zhou et al. 2010). Hence, the discovery of endophytic fungi as producers of taxol

provides a suitable approach to solve the problem especially with the possibilities of

endophytic fungi producing metabolites similar to their host plant (Redko et al. 2006).

Adding to that, only a few studies have been undertaken on the fungal endophytes diversity

among Malaysian plant species (Hazalin et al. 2009). In the following, we discuss chosen

studies that display the ability of endophytic fungi as producers of bioactive compounds

with various pharmaceutical properties. It is noteworthy that an individual endophyte may

be able to produce not only one but several bioactive compounds.

Figure 8: Taxol

Taxol or also known as

paclitaxel was first isolated

from the bark of the yew tree

(Taxus brevifolia). This tree is

a slow-growing evergreen

shrub or small tree. In 1993,

Stierle and colleagues (1993)

reported the first finding of

taxol from endophytic fungus

Taxomyces andreanae (Guo et

al. 2006).

1.4.1.1 Antimicrobial compounds

Antimicrobial compounds can be used not only as drugs but also as food

preservatives to control the occurrence of food spoilage and also food-borne diseases

during the food production. For instance, biopreservation, a biological method for food

preservation where the extension of shelf life and food safety is by the use of natural or

controlled microbiota and/or their antimicrobial compounds (Ananou et al. 2007).

Most of the endophytic antimicrobial compounds belong to several structural

classes such as alkaloids, peptides, steroids, terpenoids, phenols, quinines, and flavonoids

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(Premjanu& Jayanthy 2012). The following are some examples of antimicrobial

compounds recently isolated.

Besides, two antimicrobial compounds were also extracted from the fungus

Gliomastix murorum which was isolated from the Chinese medicinal plant, Paris

polyphylla var. yunnanensis. These two compounds were identified as ergosta-5,7,22-trien-

3-ol (Figure 9a) and 2,3-dihydro-5-hydroxy-α,α-dimethyl-2-benzofuranmethanol (Figure

9b) and found to be active against various test organisms such as Agrobacterium

tumefaciens, Escherichia coli, Pseudomonas lachrymans, Ralstonia solanacearum,

Xanthomas vesicatoria, Bacillus subtilis and Staphylococcus haemolyticus (Zhao et al.

2012).

1.4.1.2 Cytotoxic compounds

Cancer is one of the major causes of the worldwide high mortality rate (WHO

2012). As mentioned earlier, taxol, the first billion dollar anticancer drug, was the first

major anticancer product (Schiff & Horwitz 1980). The alkaloid camptothecin (Figure

10a), an antineoplastic agent isolated from the stems of Camptotheca acuminate (Figure

10b) in China, is another famous anticancer compound which is efficient against lung,

ovarian and uterian cancer. It was then later found to be produced by Entrophospora

infrequens (Figure 10c), an endophyte isolated from the medicinal plant Nothapodytes

foetida (Figure 10d; Premjanu & Jayanthy 2012), proving once more evidence of the

importance of endophytic fungi in the production of bioactive compounds.

A local study was also undertaken on cytotoxic activity of endophytic fungus. A

fungus found to be related to Phoma sp. (Figure 11b) was isolated from Cinnamom

mollissimum, a medicinal plant collected at the Universiti Kebangsaan Malaysia Forest

Reserve, Selangor, Malaysia. The bioactive compound extracted from this fungus showed

maximum cytotoxic activity against murine leukemia cells and was found to be a

polyketide termed as 5-Hydroxyramulosin (Figure 11a, Santiago et al. 2011).

A new cytochalasin, cytochalasin H2 (Figure 12a), was extracted from the

endophytic fungus Xylaria sp. (Figure 12b), which was isolated from leaves of the

medicinal plant Annona squamosa (Figure 12c). Although it shows weak cytotoxic activity

towards HeLa cell lines, cytochalasins are a group of fungal secondary metabolites which

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have cytotoxic activities that include disruption of actin microfilaments in both non-tumor

and tumor cells (Li et al. 2012).

1.4.1.3 Antiparasitic compounds

Edenia sp. and Mycosphaerella sp. strains, endophytic fungi isolated from plants

collected from Panama’s protected areas Coiba, Barro Colorado Islands, and Altos De

Campana National Park, showed strong antiparasitic activity against the pathogenic parasite

Leishmania donovani. This parasite is known worldwide for causing serious disfigurement

and death (Dey & Singh 2006). Hence, several antiparasitic metabolites isolated from an

Edenia sp. strain are shown in Figure 16, for example palmarumycin CP2 (Figure 13a),

palmarumycin CP 17 (Figure 13b), and preusommerin EG (Figure 13c), whereas

cercosporin (Figure 13d) was isolated from Mycosphaerella sp.(Martinez-Luis et al. 2011).

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(a) ergosta-5,7,22-trien-3-ol (Zhao et al. 2012)

(b) 2,3-dihydro-5-hydroxy-α,α-dimethyl-2-

benzofuranmethanol (Zhao et al. 2012)

(c) Paris polyphylla var. yunnanensis (Source: EOL)

Figure 9: Compounds (a) and (b) were extracted from the endophytic fungus Gliomastix murorum. The fungus is isolated

from the Chinese medicinal plant (c) Paris polyphylla var. yunnanensis, which is widely used in China as medicinal herb

due to its anti-tumor, analgesia, anti-inflammatory, and antifungal properties (Liu & Ji 2012).

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(a) Camptothecin(Source: Gbioscience)

(b) Camptotheca acuminate (Source: atreeaday)

(c) Entrophospora infrequens (Source: Invam) (d) Nothapodytes foetida (Source: Flickr)

Figure 10: Camptothecin (a), a modified monoterpene indole alkaloid, was first isolated from the stems of Camptotheca

accuminata (b) in 1966. This compound (a) was found to exhibit clinical anti-tumor activity by inhibiting DNA

topoisomerase I, an enzyme involved in DNA recombination, repair, replication, and transcription (Sun et al. 2011). It was

later found to be produced by Entrophospora infrequens, an arbuscularmycrorrhiza (Meenakshisundaram & Santhagur

2010), isolated from Nothapodytes foetida, which is the only native species isolated from the Orchid Island, commonly

used for hedges or firewood and cultured in Taiwan (Wu et al. 2008).

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(a) 5-Hydroxyramulosin (Santiago et al. 2012) (b) Phoma sp. (Source: mold-insp)

Figure 11: 5-Hydroxyramulosin (a), a polyketide compound extracted from an endophytic fungus morphologically similar

to Phoma sp. (b).

This fungus was isolated from Cinnamom mollissimum, a species popularly used in herbal medicines. Essential oil extract

of their leaf parts showed antifungal activity (Santiago et al. 2012).

(a) Cytochalasin H2 (Source: Li et al. 2012)(b) Xylaria sp. (Source: SpringerImages)

(c) Annona squamosa (Source: Africamuseum)

Figure 12: Cytochalasin H2 (a), a new compound was extracted from the endophytic fungus, Xylaria sp. (b) which was

isolated from Annona squamosa (c).

This tree (c) which bears edible fruits, originates from the West Indies and South America, and has been found associated

with antibacterial, antidiabetic, antioxidant and antitumor activity (Pandey & Barve 2011).

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(a) R = H(b) R = OH

(c) (d)

(e) Leishmaniadonovani (Source: medicine.cmu)

Figure 13: Palmarumycin CP 2 (a), palmarumycin CP 17 (b), and preusommerin EG (c), were isolated from Edenia sp.

and cercosporin (d), a fungal toxin was isolated from Mycosphaerella sp.These compounds were found to possess

antiparasitic activity against the parasite, Leishmania donovani (e), a protozoan parasite known to cause Leishmaniasis, a

worldwide disease known to cause serious disfigurement and which may be fatal (Martinez-Luis et al. 2011).

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Strobel and Daisy proposed in 2003 that endemic plants are good potential sources

of novel endophytes and bioactive compounds as they have a long history of growing in

areas of great biodiversity. It was also reported that out of the nearly 3,000,000 plant

species that exist on the earth, each individual plant is the host to one or more endophytes.

Besides, medicinal plants used by indigenous people are also recognized as a great source

of fungal endophytes as studies reported that these medicinal properties might be mediated

by their endophytes (Huang et al. 2008; Dai et al. 2010). Strobel and Daisy (2003) also

indicated that plants living under unique and extreme environmental conditions, for

instance mangrove forests, show great promise as well. In this study, we focused on

endophytic fungi from mangroves and in the following we introduce mangroves and their

endophytic fungi.

1.5 MangrovesMangroves are intertidal forest wetlands established at the interface between land

and sea in tropical and sub-tropical latitudes (Kathiresan & Bingham 2001). They are

unique for their well known adaptation towards their extreme environmental conditions of

high salinity, changes in sea level, high temperatures and anaerobic soils (Shearer et al.

2007).

Most of the mangrove genera and families are not closely related to each other, but

what they do have in common is their highly developed morphological, biological,

physiological, and ecological adaptability to extreme environmental conditions. The most

important characteristics to achieve this kind of adaptability are (a) pneumatophoric roots,

(b) stilt roots, (c) salt-excreting leaves, and (d) viviparous water-dispersed propagules. The

species composition and structure depend on their physiological tolerances and competitive

interactions (Kuenzer et al. 2011). The differential ability in adapting to high-salinity

seawater distinguishes the mangrove species. With that, mangrove species usually have

differentiated salt resistance-associated anatomic structures.

The pneumatophores (a) arise vertically from cable roots and have evolved

independently in at least five mangrove families and genera: Laguncularia (Combretaceae),

Avicennia (Avicenniaceae), Bruguiera (Rhizophoraceae), Xylocarpus (Meliaceae), and

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Sonneratia (Sonneratiaceae) (Yanez-Espinosa and Flores 2011). These specialized roots

contain spongy tissue connected to the exterior of the root via small pores called lenticels

which allows transportation of oxygen from the atmosphere to the root system. During low

tide, when lenticels are exposed to theatmosphere, oxygen is absorbed from the air and

transported to and even diffused outof the roots below ground (Shearer et al. 2007). This

diffusion of oxygen maintains an oxygenated microlayer around the roots that enhances

nutrient uptake. The microlayer also avoids toxicity of compounds such as hydrogen sulfide

that otherwise accumulate under such conditions (NOAA 2010).

The stilt roots (b) are alternately inundated and exposed by tidal fluctuations, easily

entrapping floating debris. Besides, they become hosts for various algae, sponges, and other

small plantlife, and when fully developed the roots and underlying mud become the habitat

ofa number of semi-aquatic organisms, such as various mollusks and crustaceans that

furnish food for both man and other animals (West 1976).

For (c) salt-excreting leaves, there are special organs or glands found in the leaves

which remove salts from the plant tissues. Avicennia and Laguncularia are those mangrove

species that have special, salt-secreting glands leading to formation of salt crystals on the

leaf surfaces. These crystals would be removed when blown or washed away by the rain.

Besides, leaf fall also allows eliminating excess salt in mangroves (NOAA 2010).

Lastly, the viviparous water-dispersed propagules (d) are an adaptation towards the

extreme environment that can be observed in most mangroves. Vivipary is a condition

where germination takes place while the offspring is still attached to the parent tree. The

offspring has no dormant stage, but grows out of the seed coat and the fruit before

detaching from the plant. Because of this, mangrove propagules are actually seedlings, and

not seeds. Hence, vivipary helps mangroves cope with the varying salinities and frequent

flooding of their intertidal environments, and increases the likelihood of survival.

Sincemost non-viviparous plants disperse their offspring in the dormant seed stage;

vivipary presents a potential problem for dispersal. However, these species would solve this

problem by producing propagules containing substantial nutrient reserves that can float for

an extended period. In this way, the propagule can survive for a relatively long time before

establishing itself in a suitable location (NOAA 2010; Sun, Wong & Lee 1998; Shi et al.

2005).

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Mangroves are found in 112 countries and dominate one fourth of the world’s

coastline, covering a total area of about 181,000 km2 (Maria & Sridhar 2004). According to

a study in 2004 led by the Food and Agriculture Organization of the United Nations (FAO),

South East Asia has the largest mangrove coverage on earth with 4.9 million hectare,

representing almost 35 percent of the world’s total. Developed mangroves grow along

humid sheltered tropical coasts for example in the delta systems of major rivers (Ganges,

Mekong and Amazon), and coastlines protected by large land masses (Madagascar, the

Indonesian Archipelago and Papua New Guinea). Mangroves extend into temperate regions

but are largely confined to the regions between 30o north and 30o south of the equator. They

also occur naturally along arid coastlines (Saudi Arabia, Yemenand northern Africa), and

along the west coast of Australia and north-eastern coast of Brazil (Macintosh & Ashton

2002).

Mangroves play an important role in the environment by providing a wide range of

ecological services such as protection against floods and hurricanes, reduction of shoreline

and riverbank erosion, and most importantly maintenance of biodiversity (Ronnback 1999).

Mangrove stands and associated waterways are important sites for gathering and small-

scale cultivation of shellfish, finfish and crustaceans (Alongi 2002). Besides, it remains as

an ecosystem of great importance for the ecological balance, being responsible for the

supply of nutrients to the marine environment and forms forests of salt tolerance

plantspecies with harbor a great number of marine microorganisms, with fungi being one of

them (Silva et al. 2011). Fungi are -among others- also aiding with recycling the detritus of

mangrove trees, thereby re-generating nutrients and making them available for other

organisms again. This aids in promoting an ecological balance in the mangrove

environment (Bharathidasan & Panneerselvam 2011).

1.5.1 Mangrove endophytic fungi

The unique mangrove ecosystem adjacent to the coastal waters provides a wide

variety of organic substrates and a significant salinity gradient caused by daily changes in

the sea level (Shearer et al. 2007). This constitutes an ideal environment for the bases of

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trunks and submerged aerating roots of mangrove plants, making mangrove forests an

important source for unique endophytic fungi (Xing et al. 2011). Mangrove fungi were

reported as the second largest group among the marine fungi (Hyde 1990).

Several studies have been conducted on the endophyte communities of mangrove

plants found along the coastlines of the Indian, Pacific and Atlantic Ocean (Xing et al.

2011), however not along the Sarawak coast. Current studies on mangrove fungi have been

focusing more on South East Asia because the unique mangrove-associated fungi are more

frequently found in that area (Sarma & Hyde 2001; Schmidt & Shearer 2003).

As mangrove endophytic fungi were found to be partly responsible for the

mangrove’s ability in adapting to the extreme environment (Silva et al. 2011), their

bioactive compounds are of interest. These bioactive compounds are found to be widely

distributed in the mangrove environment, making mangroves a potential source for the

discovery of new bioactive compounds-producing endophytes (Nag, Bhattacharya & Das

2012). For instance, their increasing recognition as sources of bioactive compoundswas

shown in a recent study by Joel and Bhimba (2012) on bioactive compounds produced by

Hypocrea lixii, a fungal endophyte isolated from the leaves of mangrove plants found to

possess antioxidant, anticancer and antimicrobial activity. The fungal extract showed

maximum antibacterial activity against Pseudomonas aeruginosa, a pathogen known for

respiratory infections among cystic fibrosis patients (Sadikot et al. 2005; Morosini et al.

2005). In addition to that, another genus found in the mangrove fungal community is the

Diaporthe sp. This genus has also been reported to have potential use in biological control,

development of antibiotics and growth promotion, due to its ability in producing enzymes

and bioactive compounds (Sebastianes et al. 2011). Another recent study by Ebrahim et al.

(2012) reported on two new compounds, Pullularins E and F, extracted from the endophytic

fungus Bionectriao chroleuca which was isolated from the leaves of the mangrove plant

Sonneratia caseolaris. These compounds were found to show moderate cytotoxic activity

against mouse lymphoma cells (Ebrahim et al. 2012).

Mangroves are –as mentioned above- at the interface between land and sea and are

therefore directly affected by disturbances to both land and sea regions. In the following we

discuss some of the threats faced by mangroves.

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1.5.2Threats to mangroves

A study by Polidoro and colleagues (2010) on the mangrove extinction risk and the

geographic areas of global concern showed that 11 out of 70 species (16%) of true

mangrove species studied qualified for one of the three International Union for the

Conservation of Nature (IUCN) Red List of Threatened Species categories; Critically

Endangered, Endangered, or Vulnerable. Climate change is one of the components that

affects mangroves in terms of changes in sea-level, high water events, storminess,

precipitation, temperature, atmospheric CO2 concentration, ocean circulation patterns,

health of functionally linked neighboring ecosystems, as well as human responses to

climate change (Gilman et al. 2008).

The primary threats to all mangrove species are long known and have always been

associated with human-caused pollution; for instance, habitat destruction and removal of

mangrove areas for conversion to aquaculture, agriculture, urban and coastal development,

and waste pollution. Conversion of mangrove area for agricultural fields not only involves

habitat destruction but also runoff from agricultural fields which contains organic

chemicals that become contaminants to the mangrove ecosystems (NOAA 2010). Of these,

clear-felling, aquaculture and over-exploitation of fisheries in mangroves are expected to be

the greatest threats to mangrove species in the next coming years (Alongi 2002).

Studies of oil spills in the Caribbean have shown that mangroves exhibit increased

mutation rates and long recovery times after repeated exposure. Contamination by

petroleum hydrocarbons from oil spills and oil refineries is a major threat to mangroves

throughout the tropics. The presence of hydrocarbons reduced the diversity and numbers of

saprotrophic fungi on intertidal mangrove wood. The presence of hydrocarbons on the

substratum surface and mangrove mud reduces aeration and slows down the activity of

micro-organisms such as fungi (Tsui et al. 1998).

Another most prominent human-caused pollution resulting from land conversion

and development is heavy metal pollution which will be discussed in the following.

1.5.3 Heavy metal pollutionThe definition of a heavy metal refers to elements with a specific gravity

above five (density more than 5 g/cm3) and is frequently used for a vast range of metals and

metalloids such as copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), nickel (Ni), cobalt

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(Co), cadmium (Cd), and arsenic (As). At certain or low concentrations, metals such as Cu,

Zn, Co, or Ni are considered essential micronutrients involved in functional activities that

sustain growth and development of living organisms. As they are natural constituents of the

earth crust, and have been long persistent, they cannot be degraded or destroyed, and can

enter the human body through food, air, and water and bio-accumulate over a period of

time (Duruibe et al. 2007). However, when at excess concentrations, even highly reputable

trace elements such as Zn and especially Cu metal ions can become detrimental to living

organisms, including plants (Hossain et al. 2012). Copper easily interacts with radicals

(oxygen molecule) making copper potentially very toxic; resulting in many organisms

being very sensitive to copper. The toxicity is based on the production of hydroperoxide

radicals and on interaction with the cell membrane (Nies 1999; Sharma et al. 2012). On the

other hand, zinc is less toxic than copper and serves as a co-factor for dehydrogenating

enzymes and in carbonic anhydrase. However, Zn has also been reported to cause the same

signs of illness as lead and symptoms of zinc poisoning can easily be mistaken for lead

poisoning. When taken in excess, zinc can cause system dysfunctions resulting in

impairment of growth and the reproduction system (Nies 1999; Duruibe et al. 2007).

Environmental pollution by heavy metals is very notable in areas of mining and old

mine sites, where these metals are leached out by weathering processes or due to the

chemicals used and are then carried downstream as acidic and often highly toxic run-off.

This process is called Acid Mine Drainage (AMD) (Mallo 2011; Manaka et al. 2007) and

the toxic fluids are ultimately transported to the sea making water bodies along the way

highly polluted with heavy metals. The metals are transported through rivers and streams,

in the form of dissolved species or an integral part of suspended sediments which then is

later stored in river bed sediments or seep into the underground water thereby

contaminating water from underground sources. Groundwater obtained from particularly

wells could then be contaminated depending on the proximity of the well to the mining site.

Wells located near mining sites have been reported to contain heavy metals at levels

exceeding drinking water criteria (Duruibe et al. 2007; Li et al. 2012). In addition to that,

heavy metals are bioaccumulative which leads to a transfer of toxic elements to the human

food chain (Tumin et al. 2008). Their toxicity to humans has been associated with many

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acute and chronic diseases, hormonal imbalances, nutritional deficiencies, autoimmune and

neurological disorders (Patcharee et al. 2009).

Mangrove forests in Malaysia continue to be threatened by heavy metal pollution,

resulting from industrial waste water pollution and urbanization since the 1990’s (Ayub et

al. 1998; Tsui et al. 1998) and endophytic fungi from mangrove plants should in theory

possess the ability to deal with high levels of heavy metal contamination. The use of

biological means (most in the form of bacteria or fungi) to remove these metals is termed

bioremediation and is one of the most promising technique and research areas for the future

(Hiraishi et al. 2001; Gadd 2010). In the following we introduce some of the mechanisms

how organisms, in particular fungi, deal with heavy metals.

1.5.4 Heavy metal uptake and removal

The involvement of microbes in biogeochemical cycling of elements, mineral

formation and deterioration (which includes bioweathering and processes leading to soil

and sediment formation), and chemical transformations of metals, metalloids and

radionuclides are major areas of geomicrobiology and most of these processes involve

metal and mineral transformations (see for example Ehrlich 1996, Macalady & Banfield

2003; Bottjer 2005; Choroveretal 2007; Gleeson et al. 2007; Gadd 2008).

Many approaches have been made to eliminate heavy metals from wastewater,

sludge and other heavy metal contaminated areas. Some of these elimination methods are

by means of chemical precipitation, ion exchange, solvent extraction, electrochemical

treatment, reverse osmosis, membrane technologies, evaporation recovery and chemical

oxidation-reduction which are complex and expensive methods, and frequently resulting in

the production of toxic products instead. Hence, these toxic products become another

source of environmental pollution (Kannan, Hemambika & Rani 2011; Leitao 2009). With

that in mind, many researchers have looked at developing new cost-effective methods to

address this heavy metal contamination, and microorganisms (bacteria and fungi) have been

found to be one of the alternatives (White & Gadd 1995; Wang & Chen 2009). Fungi are

always present in the aerial and subsoil environments where they maintain the soil structure

through their filamentous branching growth and by exopolymer production. They were

found to be excellent biogeochemical cycling agents of elements such as carbon, nitrogen,

P a g e | 24

phosphorus and even metals in the soil. Besides, they are good bioaccumulators of soluble

and particulate forms of metals which makes them very adaptive to extreme environments

with Penicillium sp. reported as one of the most prominent ones (Leitao 2009; Gadd 2007).

Biosorption is one of these above mentioned alternatives. The mechanism has been

known for a few decades, however has emerged as a promising low-cost technology in the

last decade (Das 2005). Biosorption can be divided into (a) metabolism dependent (living

cells biomass) and (b) non-metabolism dependent (dead cells biomass). Metabolism

dependent refers to the uptake of metals across the cell membrane, defined as intracellular

uptake, active uptake or bioaccumulation. On the other hand, non-metabolism dependent

refers to the surface binding of metal ions to cell walls, or in other words known as

biosorption or passive uptake (Sag & Kutsal 2001; Bishnoi, Pant & Garima 2004). The

difference between live and treated biosorbents is that live biosorbents are organisms that

carry out the sorption process actively, whereas in dead or treated biomass, sorption mostly

occurs via intracellular binding. For this biosorption system to take place, many chemical

processes are involved; adsorption, ion exchange and covalent bonding with the biosorptive

sites of the microorganisms, extra and intracellular precipitation and active uptake. All

these can be summarized as categories of (i) biosorption of metal ions on the surface, (ii)

intracellular uptake of metal ions and (iii) chemical transformation of metal ions (Iskandar

et al. 2011; Leitao 2009). Besides removing heavy metals, biosorption systems can also be

used to recover precious metals such as gold (Volesky 1990; Gadd 2009; Wang & Chen

2009).

1.5.5Biosorption by Marine Fungi

One of the main reasons why fungi are able to survive in high metal concentrations

is that they possess a high surface to volume ratio which makes them more tolerant to

heavy metals compared to bacteria or actinomycetes (Gadd 2007). Therefore fungi’s unique

physiology is one of the main reasons behind the uptake of heavy metals by the cell. The

uptake of heavy metals by the fungal biomass has been associated with their cell wall

which consists mainly of polysaccharides. The phosphate and glucouronic acid and chitin-

chitosan complex found in these cell walls are the major contributors to the binding of

heavy metals through ion exchange and coordination (Sag & Kutsal 2001).

P a g e | 25

Some examples of marine fungi being used as a biosorbent for heavy metals are the

very common Aspergillus flavus and Rhizopus spp. which shown tolerance towards arsenic

(Vala & Sutariya 2012). Besides, Aspergillus cristatus was isolated from the heavy metals

polluted areas in the Mediterranean Sea, Egypt and has been found to be a potential

biosorbent and bioaccumulator of cadmium (II). Fungal cells both living and dead, such as

Penicillium, Rhizopus, and Saccharomyces have also been applied in metal removal from

aqueous streams using either batch or continuous modes (Hassan & Kassas 2012). It was

also reported by Gomathi and colleagues (2012) that mangrove-derived fungi, the

Aplanochytrium sp. was found to be efficient for the removal of chromium in waste water

treatment.

1.6Aim of the project and scope of studyThe overall aim of this thesis is to assess the potential of endophytic fungi from a

mangrove plant for their use in medicine and bioremediation. Objectives are to:

(a) Isolate and identify (using molecular methods) endophytic fungi associated with

the mangrove plant Avicennia sp.

(b) Evaluate their antimicrobial activity and cytotoxicity

(c) Assess the heavy metal biosorption potential of these endophytes isolated.

The approach to test for both their production of antimicrobial compounds (see

Section 2.6.1 and 2.6.2 for primary and secondary screening of antimicrobial activity)

as well as their biosorption capacity (see Section 2.7.2 heavy metal biosorption) helped to

gain new insights into the role that these fungi might play for their host plant.

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2. Materials and methods

2.1 Sampling

2.1.1 Field site sampling

Plant and soil samples were collected from the mangrove forests in Kampung Pasir

Pandak, Sarawak on 26 November 2010. Figure 14 shows Kampung Pasir Pandak located

near Kampong Batu, situated north of Kuching town.

Figure 14: Kampung Pasir Pandak (Sampling site) situated near Kampong Batu, indicated by the Blue Point (Source:Google Map)

P a g e | 27

Plant and soil samples were collected from three different sites (island, freshwater stream,

and village). At each of the sites, samples were collected in triplicate (see Table 1 for an

overview of the GPS coordinates).

All samples were collected during low tide at 12 noon. Plant materials were

collected and placed on ice in aluminium bags. Soil samples were collected in sterile

centrifuge tubes and placed in a cooling box to be transported back to the laboratory within

4 hours where they were kept at 4oC until further analysis.

Table 1: Characteristics of the soil conditions of the three different sampling sites

Location Coordinates of GPS

Island Station A N01o 42’11.2” E110o 18’ 20.5”

Station B N01o 42’11.5” E110 o 18’ 19.1”

Station C N01o 42’11.5” E110o 18’ 19.6”

Freshwater Stream Station A N01o42’11.0” E110o 18’ 18.5”

Station B N01o 42’11.2” E110o 18’ 18.8”

Station C N01o 42’11.6” E110o 18’ 18.9”

Village Station A N01o 42’03.8” E110o 18’ 44.6”

Station B N01o 42’01.4” E110o 18’ 43.3”

Station C N01o 42’02.8” E110o 18’ 44.1”

2.2 Isolation of mangrove endophytic fungi

2.2.1 Plant samples

Surface sterilization is the first and an obligatory step for endophyte isolation in

order to kill all the surface microbes. It is usually accomplished by treatment of plant

tissues with oxidant or general sterilant for a given period, followed by a sterile rinse. In

general, the sterilization procedure should be optimized for each plant tissue, especially the

sterilization time since the sensitivity varies with plant species, age and organs (Qin et al.

2011). Surface sterilization involving the use of a variety of solutions is important to kill

the unwanted phylloplane fungal propagules adhering to the surface of the cuticle of the

leaves (Gangadevi et al. 2008).

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The collected plant material (related to Avicennia sp.) was rinsed under running tap

water for 10 minutes, and then air-dried. The surface treatment usually initializes with plant

material being washed in running tap water, by means of detergent or not, to remove

extraneous matter (Seena & Sridhar 2004). The plant material was then cut into 1 cm long

fragments using sterile surgical blades and the fragments were surface sterilized by

immersing them sequentially in 70% ethanol solution for 3 minutes and 0.5% sodium

hypochlorite for 1 min.

Thereafter, the fragments were rinsed thoroughly with sterile distilled water and

surface-dried on sterile filter paper before being placed onto Petri dishes containing Potato

Dextrose Agar (PDA) (Difco). The plates were incubated at 28oC for 1 week. After

incubation, hyphal tips of the fungi could be seen growing out from the plant fragments and

they were then transferred to a new PDA plate using a sterile straw (see Figure 15 for a

schematic overview of the procedure; Kumaresan & Suryanarayanan 2002; Bharathidasan

& Panneerselvam 2011).

2.2.2 Soil samples

The soil samples were analysed for endophytic fungi using a modified method

based on Nopparat et al. (2007), in which the Pikovskaya agar is substituted with PDA.

Each sample was added to 9ml of sterile distilled water with 10-fold dilution series. 0.1ml

dilution was then plated onto PDA agar and incubated for one week at 28oC. After a few

days of incubation, fungal colonies that were seen growing were selected and re-inoculated

on PDA agar for purification of fungi cultures (see Figure 16 for a schematic overview of

the procedure).

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Figure 15: Schematic overview of isolation of mangrove endophytic samples from plant samples

Plant samples

Rinse with running tapwater

Cut into 1cm x 1cmfragments

Air Dried

Surface Sterilization

70% ethanolsolution

3 mins

0.5% sodiumhypochlorite

1 min

Sterile distilledwater

1 min

Surface Dried

Placedonto PDA

Incubation at28oC

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Figure 16: Schematic overview of isolation of mangrove endophytic samples from soil samples

Soil samples

Added into 9ml of 0.85%w/v saline with 10-fold

dilution series

0.1ml of each dilution plated ontoPDA agar

Incubation at 28oC

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2.3 Fungal Cultivation

In any case, the preservation of fungal strains, as type material or reference stocks,

becomes a strategic approach to acquire reproductive outcome. However, the choice of

preservation method depends on the service asked for, the laboratory availabilities and

other factors (Gallo et al. 2008).

2.3.1 Fungal Culture for Short Term Storage

Cylindrical pieces were cut using sterilized straw from pure fungal cultures and grown on

PDA media at 25oC for several days. Once the fungal hyphae covered ¾ of the whole

surface of the PDA medium, the cultures were then kept in 4oC till further use. The short

term storage can be used for maximum 6 months before re-inoculating onto new PDA

plates (see Figure 17 for an overview of the different storage procedures; Nakasone,

Peterson & Jong 2004).

2.3.2 Fungal Culture for Long Term Storage

Cylindrical pieces were cut using sterilized straw from fungi grown plates of one week old

and placed onto sterilized barley media in universal bottles. Each universal bottle was filled

with sterilized barley up to half of the bottles. The fungal culture was then incubated at

25oC for one week before being kept in 4oC for further usage (Figure 17; Nath,

Raghunatha & Joshi 2012).

2.3.3 Fungal Culture for Extraction of Bioactive Compounds

Cylindrical pieces were cut using sterilized straw from fungi grown plates of one week old

and inoculated into 20 ml of potato dextrose broth (PDB) (Difco, USA). The fungal broth

culture was then incubated at 25oC for one week before being used for solvent extraction of

bioactive compounds (Figure 17; Kjer et al. 2010). Two fungi strains (Isolate 7 and Isolate

13) were further cultivated in large scale volume for extraction of increased amount of

bioactive compounds.

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Figure 17: Fungal Cultivation for short term storage, long term storage, and extraction of bioactive compounds

SHORT TERM STORAGE

Cylindrical pieces of pure fungalcultures were grown on PDA at

25oC

Fungal hyphae covered ¾ of thesurface of PDA

Plates kept at 4oC until further use

LONG TERM STORAGE

Barley media are placed inuniversal bottles and sterilized

Cylindrical pieces of pure fungalcultures were placed in the sterilized

barley media

Incubated at 25oC for one week

EXTRACTION OF BIOACTIVECOMPOUNDS

Cylindrical pieces of pure fungalcultures were grown in 20 ml

PDB

Incubated at 25oC for one week

Ready to be used with solvent

Kept in 4oC for further usage

P a g e | 33

2.4 Endophytic fungi identification

Molecular identification has made it possible to study theecology of fungi in their

dominant but in conspicuous mycelial stage and not only by means of fruiting bodies

(Bellemain et al. 2010). The internal transcribed spacer (ITS) region of the nuclear

ribosomal repeat unit has become the primary genetic marker for molecular identification

of many groups of fungi (Nilsson et al. 2011). The entire ITS region has commonly been

targeted with traditional Sanger sequencing approaches and typically ranges between 450

and 700 bp (Bellemain et al. 2010).

The endophytic fungi were identified using molecular tools. Genomic DNA was

extracted from 5-day old fungi cultures grown on plates using a modified thermolysis

method (Zhang et al. 2010). The edge of the mycelium colony with the size of a sesame

seed was picked using a sterilized toothpick and placed into a 1.5 ml microcentrifuge

containing 100µl pure distilled water. The mixture was vortexed for 1 minute and then

centrifuged at the speed of 8,000 g for 1 minute. The supernatant was discarded and 100 µl

of Tris-EDTA (TE) (First Base, Malaysia) buffer was added into the tube. The tube was

then immersed in water bath at 93oC for 20 minutes and stored at -20oC until use.

Fungal DNA was amplified using universal primers of fungal DNA ITS1 (5’–TCC

GTA GGT GAA CCT GCG G-3’) and ITS4 (5’–TCC TCC GCT TAT TGA TAT GC-3’;

1st Base, Malaysia). Each sample ready for amplification contained 2 µl of 10X PCR buffer

(Fermentas, Germany), 1.2 µl of dNTP mixture (2.5mmol l-1 each), 0.8 µl of deioned

formamide, 0.4 µl of MgCl2 (25mmol l-1), 0.8 µl of each primer (10µmol l-1), 0.2 µl of Taq

DNA polymerase (5 U µl-1) and 1 µl of genomic DNA in a total volume of 20 µl. PCR was

carried out as follows:

Step 1 Initial denaturation 94oC 5 mins

Step 2 Denaturation 94oC 50 s

Step 3 Annealing 54oC 50 s

Step 4 Elongation 72oC 50 s

Step 5 Final Elongation 72oC 10 mins

Step 6 Storage 4oC until use

Steps 2 to 4 were repeated 35 times before proceeding to step 5.

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DNA fragments were purified using PureLink PCR purification kit (Invitrogen,

U.S.) following the protocol provided by the supplier and then sent for sequencing to the

Beijing Genomic Institute, BGI, China.Nucleotide sequences were determined using the

dideoxynucleotide method by cycle sequencing of the purified PCR products and

sequences were analyzed against the NCBI database. Sequences were aligned and

phylogenetic trees were created with MEGA5 using the neighbor-joining method (see

Figure 18 for an overview; Manikprabhu & Lingappa, 2012).

P a g e | 35

Figure 18:Endophytic fungi identification using molecular tools

1)EXTRACTION OF GENOMIC DNA

Edge of the mycelium colony wasplaced into microcentrifuge tube

containing 100µl pure distilled water

Mixture was vortexed andcentrifuged

Supernatant was discarded and100µl TE buffer was added

Supernatant was discarded and 100µl TEbuffer was added

Tube was immersed in water bath at93oC for 2 minutes before stored at 4oC

2)DNA AMPLIFICATION

PCR mixture was prepared:

- 2 µl of 10X PCR buffer- 1.2 µl of dNTP mixture- 0.8 µl of deionedformamide- 0.4 µl of MgCl2

- 0.8 µl of each primer- 0.2 µl of Taq DNA polymerase- 1 µl of genomic DNA

PCR cycle was run:

a) Initial denaturation (94oC - 5 mins)b) Denaturation (94oC - 50 s)

c) Annealing (54oC - 50 s)d) Elongation (72oC - 50 s)

e) Final Elongation (72oC - 10 mins)f) Storage (4oC - until use)

3)SEQUENCING

PCR mixture were purified usingPureLink PCR purification kit

Sent for sequencing to the SarawakBiodiversity Centre

Sequences obtained were analyzedagainst the NCBI database

Sequences were aligned andphylogenetic tree was constructed

P a g e | 36

2.5 Extraction of bioactive compounds

Crude extract from each fungal isolate was extracted using ethyl acetate solvent.

This extraction method is particularly useful for extraction of extracellular (excreted by

fungi into the medium) and intracellular bioactive compounds.

20 ml of ethyl acetate was added into the fungal broth that was cultivated as

described in 2.3.3 and left standing for two hours. The mixture was then filtered with the

mycelium residues being discarded and the filtrate collected in 50ml centrifuge tubes. The

filtrate containing ethyl acetate phase and the medium were collected. The ethyl acetate

phase was then separated from the broth medium with centrifugation at 8,000 rpm for 10

minutes and also separation funnel. The top layer which consists of the ethyl acetate phase

was removed and transferred to new tubes. Another 20 ml of ethyl acetate were added into

the remaining broth and the extraction was repeated three times. The ethyl acetate extract

was then dried in the fumehood to give a solid and oily residue. The dried extract was then

kept in -20oC until further use (see Figure 19 for an overview).

2.5.1 Solvent-solvent extraction

Solvent-solvent partitioning of the ethyl acetate extracts was performed using n-

hexane and 90% (vol/vol) aqueous methanol in a ratio of 1:1 (vol/vol) with a total volume

of 20 ml being added into the fungal ethyl acetate dried extracts (see Figure 19). The

mixture was again left standing for two hours. The mixture was filtered and the filtrate was

collected in 50ml centrifuge tubes. The filtrate containing the n-hexane and 90% methanol

was then separated through centrifugation at 8,000 rpm for 10 minutes and separation

funnel. The top layer which consists of the n-hexane phase was removed and transferred to

new tubes. Another 20 ml of solvent mixture (containing n-hexane and 90% aqueous

methanol in a ratio of 1:1) were added into the remaining extract and the extraction was

repeated three times. The n-hexane extract was collected and dried in the fumehood to give

a solid and oily residue and the dried extract was stored in the freezer (-20oC) until further

use. On the other hand, the remaining aqueous methanol extract was also dried in the

fumehood to give a solid and oily residue and the dried extract was kept in -20oC until

further use. All fractions of dried extract were submitted for High Performance Liquid

Chromatography (HPLC) analysis to the laboratory of Professor Peter Proksch from the

P a g e | 37

Institut für Pharmazeutische Biologie und Biotechnologie, University of Düsseldorf,

Germany (see Figure 20 for an overview).

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Figure 19: Extraction of bioactive compounds using ethyl acetate

Fungal Cultures in 20ml PDB

Mixture Broth

Mycelium Filtrate

Discard

Ethyl Acetate phase Medium

MixtureDried EthylAcetate extract

Additionsof ethyl

acetate 3X

Centrifuged,Separating Funnel

MediumEthyl Acetate phase

Evaporated

Filtered

Addition of 20ml Ethyl Acetate, stand for 2 hours

P a g e | 39

Figure 20: Extraction of bioactive compounds using solvent-solvent (methanol and n-hexane) extraction

Residue Solvent phase (90% aqueousmethanol: n-hexane

Dried Ethyl Acetateextract

Mixture Broth

n-hexane 90% aqueousmethanol

Dried n-hexaneextract

Dried methanolextract

Addition of mixture (90%aqueous methanol: n-hexane)

Filtered

Centrifuged,Separating Funnel

EvaporatedEvaporated

P a g e | 40

2.6 Biological Assays

2.6.1 Primary Screening of antimicrobial activityAll fungal isolates were screened for their antimicrobial and cytotoxic activities and

the approaches used are described in the following. The antimicrobial assay includes the

testing of fungal isolates for their antibacterial and antifungal activity using a modified

preliminary screening method (Alias et al. 2010; Ding et al. 2010).

For antibacterial activity, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus,

and Micrococcus luteus were selected as examples of Gram positive bacteria, whereas

Escherichia coli, Pseudomonas aeruginosa, and Vibrio anguillarum were chosen as

representatives of Gram negative bacteria. Saccharomyces cerevisiae was used as an

example of yeast. Although the strains used in this thesis were not pathogenic, all species

chosen represent common human pathogens.

Gram positive bacteria have long been known to cause many infectious diseases.

For instance, Bacillus cereus is an uncommon but potentially serious bacterial pathogen

causing infections of the bloodstream, lungs, and central nervous system of preterm

neonates (Hilliard et al. 2003). Besides, Staphylococcus aureus is known to infect and

destroy normal healthy tissue, causing skin and wound infections, bloodstream infection

(BSI), pneumonia, osteomyelitis, endocarditis, lung abscess, and pyomyositis (Rivera and

Boucher 2011; Woodford & Livermore 2009). Micrococcus luteus has been implicated as

the causative agent in cases of intracranial abscesses, meningitis, pneumonia and septic

arthritis in immune-suppressed or immune-competent hosts (Altuntas et al. 2004).

Pathogenicity or virulence of Gram-negative bacteria is strictly dependent on the

presence of a secretion system in their cells, through which they secrete proteins or

nucleoproteins involved in their virulence in the apoplast or inject in the host cell

(Buonaurio 2008). For instance, Escherichia coli were first known to be associated with

diarrhea and now with outbreaks of foodborne diseases (Doyle et al. 2006).

The test organisms were prepared in nutrient broth (Difco) and incubated at 30oC

for 24 hours. After incubation, the test pathogens were then streaked evenly onto nutrient

agar (Difco) and left for five minutes to dry before being used for the screening of

antibacterial assay. Cylindrical pieces of 1 x 1 cm size agar plugs were cut from one week

old fungi grown plates and placed on the agar previously streaked with test organisms.

P a g e | 41

Each plate was placed with six cylindrical pieces of different fungi isolate at a regular

distance (in triplicates). The plates were incubated for 24 hours and observed for clear

inhibition zones (Alias et al. 2010).

For antifungal activity, Candida albicans and Aspergillus niger were chosen as

representatives for fungi. Candida albicans is commonly known to colonize the human

gastrointestinal, respiratory, reproductive tracts and the skin whereas Aspergillus niger is

one of the most common Aspergillus infecting species along with Aspergillus flavus and

Aspergillus fumigatus (Shoham and Levitz, 2005).

Cylindrical pieces of 1 x 1 cm size agar plugs were cut from one week old fungi

grown plates and placed opposite of the fungi test pathogen and incubated for one week at

25oC. Each plate contains one fungi isolate and one test pathogen. All tests were prepared

in triplicate. The clear inhibition zones were measured after the incubation period (see

Figure 21 for an overview of the primary and secondary screening; Ding et al. 2010).

2.6.2 Secondary screening of antimicrobial activitySecondary screenings were undertaken after primary screening using the agar well

diffusion method. For the secondary antimicrobial assay only Bacillus cereus, Bacillus

subtilis, Vibrio anguillarum, Micrococcus luteus, and Candida albicans were selected as

they were inhibited by the isolates during the primary screening. The test organisms were

grown as described above and antimicrobial activity was determined using the agar well

diffusion method. Ethyl acetate extracts were obtained from the isolates (see Section 2.5

Extraction of Bioactive Compounds for details of the extraction procedure and also

Figure 19) and dissolved in 1 ml of dimethylsulfoxide (DMSO). Small wells (5mm in

diameter) were made in the agar plates using sterilized straws. 20 µl of the extract of each

isolate were added to each well and the plates incubated overnight at 37oC under static

conditions. After 24 hours, the zones of inhibition around the wells were measured and

recorded in cm. All tests were perfomed in triplicate and a control using DMSO alone was

prepared (see Figure 21 for an overview).

2.6.3 General Cytotoxicity assay

Bioactive compounds are almost always toxic in high doses. Thus, in vivo lethality

in a simple zoologic organism can be used as a convenient monitor for screening and

P a g e | 42

fractionation in the monitoring of bioactive natural products (McLaughlin, Rogers&

Anderson 1998). Brine shrimps lethality assay is a rapid and useful method as a

preliminary screening for cytotoxic activity as it has been used in detection of fungal

toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and

cytotoxicity testing (Harwig & Scott 1971; Carballo et al. 2002; Manilal et al. 2009). For

this study, the assay was used to determine the toxicity of a compound hence; it was

applied to the fungal ethyl acetate extracts.

The eggs of the brine shrimp, Artemia salina, were hatched in artificial seawater (38

g/L) for 48 hours. Each fungal ethyl acetate extracts was mixed with 10% DMSO and

diluted with artificial seawater to obtain concentrations of 0.5, 5, 50 and 500ppm. The

compounds were prepared by dissolving in DMSO in the suggested maximum volume to

prevent possible false effects coming from DMSO’s toxicity to the experimental results

(Arslanyolu & Erdemgil 2006).

A 96-well microtitre plate was used for this analysis and 10 matured shrimps were

applied to each well containing 50µl of each fungal extract of different concentrations. The

number of brine shrimps that died after 24 hours were counted using a stereomicroscope

and the lethal concentration at which 50% of the brine shrimps died (LC50) was determined

by looking at the percent of mortality of the brine shrimp calculated for every

concentration. Experiments were performed in triplicates and a negative control using

DMSO alone was prepared (see Figure 22 for an overview; Milon et al. 2012; Manilal et

al. 2009).

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Figure 21: Primary and Secondary screening of antimicrobial activity

PRIMARY SCREENING OFANTIMICROBIAL ACTIVITY

ANTIBACTERIALASSAY

Test pathogens grownin nutrient broth

Cylindrical pieces (1cm x 1cm) of fungi isolatesplaced on the agar (nutrient agar and PDA)

Antibacterial assay – Incubation 24 hrs

Antifungal assay – Incubation 1 week

ANTIFUNGALASSAY

Test pathogens grownon PDA

Incubated at 30oCfor 24 hours

Incubated at 25oCfor one week

Test pathogen streakedonto nutrient agar

Test pathogen (cylindrical piece)placed on the other half of the

PDA plates

SECONDARY SCREENING OFANTIMICROBIAL ACTIVITY

Small wells (5mm diameter) weremade in the nutrient agar and PDA

20 µl of the ethyl acetate extract offungi isolates added to each well of

nutrient agar and PDA plates

Ethyl acetate extract of fungi isolates(Figure 35) dissolved in 1 ml of DMSO

Test pathogens streakedonto nutrient agar

Test pathogens placed on theother half of the PDA plates

Antibacterial assay – Incubation 24 hrs

Antifungal assay – Incubation 1 week

P a g e | 44

Figure 22: Cytotoxicity assay

PREPARATION

Ethyl acetate extract of fungi isolates(Figure 35) dissolved with 10ml of DMSO

Eggs of the brine shrimp hatched inartificial seawaterfor 48 hours

DMSO Extract diluted with artificialseawater to obtain different concentrations

(0.5, 5, 50 and 500ppm)

CYTOTOXICITY ASSAY

50µl of each extracts of differentconcentrations were placed into the

wells of the plates

A 96-microtitreplate was used

10 matured shrimpswere added to each of

those wells

Number of brine shrimps that diedafter 24 hours were counted using

stereomicroscope

LC50 wasdetermined

P a g e | 45

2.7 Heavy Metal Analysis

2.7.1 Determination of heavy metal-resistant fungiTolerance of the fungal isolates towards the heavy metals, Copper (Cu) and Zinc

(Zn), wasdetermined as the minimum inhibitory concentration (MIC). MIC is defined as

the lowest concentration of metal which inhibits visible growth of the isolate. For this

study, the MIC was determined based on the percentage (%) of biomass dry weight

measured. The dry weight of the fungi biomass suggests that the growth pattern is relative

to the tolerance development or adaptation of the fungi to the presence of heavy metals, at

which as the metal concentration increases, a reduction in growth would be observed from

the measured dry weight (Lairini et al. 2009).

Cu2+ and Zn+ ions were added separately to PDB at concentrations of 50 to 200

µg/ml. For the preparation of Cu2+ and Zn+ ions, Copper (II) sulphate and Zinc sulphate

were used. The broth was inoculated with 1 cm2 agar plugs from young fungal colonies that

were pre-grown on PDA plates for 5 days. Three replicates of each concentration and

controls without metal were prepared. The inoculated broth was then incubated at 25oC for

one week under static conditions. The broth was filtered using sterile filter paper (Whatman

filters No.1, USA) and the biomass obtained was dried in the oven at 60oC. The dried

biomass was then weighed and its dry weight obtained (see Figure 23 for an overview;

Iskandar et al. 2011).

2.7.2 Heavy metal biosorption by dead fungal cells

Dead biomass is more preferred to living cells in industrial applications as systems

using living cells were found to be more sensitive to metal ion concentration (toxicity

effects) and adverse operating conditions (pH and temperature). Also, constant nutrient

supply is needed for systems using living cells, and recovery of metals and regeneration of

biosorbent is more complicated. For preparation of dead biomass, cells can be killed

through physical treatment methods, for instance heat treatment, autoclaving and vacuum

drying or chemicals like acids, alkalies and detergents, or other chemicals like

formaldehyde or by mechanical disruption (Bishnoi, Pant & Garima 2004).

For adsorption by dead fungal cells, biomass was prepared by grinding dried fungal

biomass using mortar and pestle and then passed through a 0.45 µm sieve to standardize the

P a g e | 46

particle size. Working standards of 50 µg/ml copper and zinc ion solutions in 150mM NaCl

solution (added to prevent cell damage caused by osmotic pressure) were prepared. 0.1 g of

the powdered biomass was then inoculated in the Cu2+ and Zn+ solutions and the cell

suspension incubated at 150 rpm and 30oC for 72 hours in the dark. Samples were filtered

using sterile filter paper (Whatman filters No.1, USA) and cell-free filtrates obtained were

analyzed for the remaining Cu2+ (µg/ml) using atomic absorption spectrometry (AAS;

Kannan, Hemambika & Rani 2011).

The detection of trace metals can be done by various methods but in this study the

AAS technique was used, which is relatively simple, versatile, accurate and free from

interferences (Raghav et al. 2003). The calibration curve of well prepared standards and an

accurate Atomic Absorption Spectrophotometer should present as a linear curve and our

standards did so as can be seen in Figure XY.

Bioadsorption capacity was measured based on the amount of metal ions (mg)

bioadsorbed per gm (dry mass) of biomass calculated using the following equation:

Q = [(Ci – Cf)/m)] V

Q = mg of metal ion bioadsorbed per gm of biomass, Ci = initial metal ion concentration, mg/L, m = mass of

biomass in the reaction mixture gm, V = volume of the reaction mixture (L)

See Figure 23 for an overview of the approach used to determine MIC and heavy metal

biosorption (Cruz et al. 2009).

47

Figure 23: Determination of heavy metal resistant fungi using minimum inhibitory concentration (MIC) and heavy metal biosorption

DETERMINATION OF HEAVYMETAL RESISTANT FUNGI

Cu and Zn ions were added separately toPDB at 50 to 200 µg/ml concentration

Cylindrical pieces (1cm x 1cm) of fungiisolates were added into the broth mixture

HEAVY METAL BIOSORPTION

Cylindrical pieces (1cm x 1cm) of fungiisolates were added into the PDB

Incubated at 25oC for oneweek under static condition

Incubated at 25oC for one weekunder static condition

The broth was filtered

The biomass obtainedwas dried at 60oC

The percentage (%) of biomass dryweight was measured

The broth was filtered and the biomassobtained was dried at 60oC

Dried biomass grind and passed through a0.45 µm sieve

0.1g of the powdered biomass was inoculated intothe 50 µg/ml Cu and Zn ion solutions

Incubated at 150 rpm and 30oC for 72 hours

Solution filtered and measured with AAS

48

3. Results and Discussion

In this chapter we discuss the results obtained during the various experiments conducted.

Selected data was submitted for publication and the detailed discussions of the relevant

results are presented in the form of submitted manuscripts in chapters 4 (bioactive

compounds) and 5 (biosorption potential). The data that was not part of these submissions

is presented and discussed in the following.

3.1 Fungi identification

Table 2: Overview of the closest relatives found for each endophytic isolate, their query coverage in base pairs and %, aswell as the source of the sample from which the isolate originates.

FUNGALSTRAINS

CLOSEST RELATIVE[accession number]

IDENTITIESLOCATION / SOURCE

ISLAND FRESHWATER VILLAGE

Isolate 1 Penicillium dravuni[AY494856]

399 / 409(98%)

- Root -

Isolate 2 Curvularia affinis isolate S255[HM770741]

469 / 469(100%)

- - Soil

Isolate 3 Diaporthe sp. SAB-2009astrain Q1160 [FJ799940]

454 / 459(99%)

- - Leaves

Isolate 4 Diaporthe sp. 138SD/T[GU066697]

471 / 473(99%)

- - Leaves

Isolate 5 Penicillium citrinum strainSGE29 [JX232276]

408 / 408(100%)

- Root -

Isolate 6 Aspergillus sp. Da91[HM991178]

501 / 501(100%)

- Root -

Isolate 7 Guignardia mangiferae strainSCIW10 [HM150733]

426 / 439(97%)

Leaves - Leaves

Isolate 8 Neosartorya stramenia isolateNRRL 4652 [EF669984]

349 / 357(98%)

Root Root Root

Isolate 9 Eupenicillium sp. 5 JH-2010culture-collection

CBS:118134 [GU981610]

447 / 449(99%)

- Root -


399 / 409(98%)

Leaves - -

Isolate 12 Cladosporiumsphaerospermum strain

SCSGAF0054[JN851005]

478 / 479(99%)

- - Root

Isolate 13 Neosartorya hiratsukae strainKACC 41127 [JN943580]

460 / 464(99%)

- - Root

49

Figure 24: 18S gene-based phylogenetic tree representing the twelve endophytic fungal isolates. The phylogenetic tree

was generated with distance methods, and sequence distances were estimated with the neighbor-joining method.

Bootstrap values ≥50 are shown and accession numbers for the reference sequences are indicated.

From the isolation of plant and soil samples, a total of 222 strains were isolated and

subcultured. A total of twelve endophytic fungi isolated from the plant samples (Avicennia

sp.) were selected for further studies; molecular identification, antimicrobial screening,

bioactive compounds isolation, cytotoxic activity and heavy metal analysis. The twelve

isolates were identified using molecular methods and found belonging to 7 families;

Penicillium, Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya, Cladosporium

and Eupenicillium (see Table 2 for an overview of the closest matches as well as Figure 24

for phylogenetic tree generated based on ITS sequences of the fungal isolates).

Species of Penicillium are ubiquitous saprobes, whose numerous conidia are easily

distributed through the atmosphere. This species has been found with the potential for

50

increasing plant growth, especially in the Chinese radish. Some species of Penicillium are

well known for their activities to produce antibiotics (for instance Penicillin, as mentioned

above in section 1.1 Infectious diseases, drugs resistance and bioactive compounds)

(Phuwiwat & Soytong 2001).

Curvularia sp. is one of the marine-derived fungi, which have been known as rich source of

biologically active secondary metabolites for instance lunatin, curvularin and others

(Geetha et al. 2011). It has also been reported by Madavasamy and Panneerselvam (2012)

as one of the endophytic fungi out of twenty two species isolated from the leaves of

Avicennia marina. For this study, strain Isolate2 was identified as Curvularia affinis based

on the similarity comparison of ITS sequences. For our study, strain Isolate 2 was isolated

from the soils of mangrove forests in Kampung Pasir Pandak, Sarawak. Endophytic fungi

are not only those fungi that live entirely within plant tissues but also may grow within

roots (Singh, Gill & Tuteja 2011); hence, there might be transmission of endophytes from

the roots to the soil that lead to occurrence of Isolate 2 found in soil. Studies have also

reported on Curvularia sp. being isolated from mangrove soils (Thatoi et al. 2012; Zakaria

et al. 2011). One study reported on Curvularia being isolated from the peat soils of

Sarawak, where the sampling sites were Pelitanah, Maludam National Park and Cermat

Ceria (Omar, Ismael & Ali 2012). Besides, Curvularia sp. was reported with the potential

of degrading polycyclic aromatic hydrocarbons (PAH), a group of environmental pollutants

that can be found as contaminants at industrial sites, especially those associated with

petroleum or gas production and wood preserving processes (Juckpech, Pinyakong &

Rerngsamran 2012). In this study, strain Isolate 2 was found to possess biosorption

potential as it was able to remove heavy metal copper (Cu), an environmental pollutant

(refer to section 3.4.2 Heavy metal biosorption by dead fungal cells).

Isolates 3 and 4 were linked to Diaporthe sp. (Figure 24) which is a marine lignicolous

fungus. They are an important group that is able to degrade fiber, and commonly derived

from marine algae, mangrove plants, seawoods and rotten wood. Their metabolites were

associated with their ability to retain their predominance on fibered material (Lin et al.

51

2005). This genus is commonly found in mangrove fungal communities and has been

described as an antibiotic producer (Sebastianes et al. 2011).

Naikwade and colleagues (2012) reported on a total of 17 species of fungi being isolated

from leaves of the mangrove plant Ceriops tagal, out of which 9 fungal species belonged to

Aspergillus, making it the dominant genus. Besides, Aspergillus species were also isolated

from mangrove forests in Borneo Island, Sarawak. The locations reported were Sematan,

Lundu, Kampung Bako and Bako (Seelan, Ali & Muid 2009) whereas our study was

undertaken in Kampung Pasir Pandak, Sarawak. Aspergillus flavus, isolated from

mangrove plant Avicennia officinalis, was associated with antioxidant potency which might

be responsible for the mutualistic association of plant and endophyte against various biotic

and abiotic stresses (Ravindran et al. 2012). Isolate 6 was grouped with Aspergillus sp.

Da91 (Figure 24) however it was the only isolate among twelve belonging to the genus

Aspergillus. Avicennia species therefore seem to harbor distinctively different endophytic

fungal communities.

The strain Guignardia sp. was isolated for the first time from Undaria pinnatifida, a type of

seaweed in Changdao Sea (Wang 2012). The genus Guignardia is also one of the

endophytic fungi commonly isolated from mangrove forests and known for their cytotoxic

activities (Bhimba et al. 2011). Isolate 7 was grouped with Guignardia species (Figure 24),

however a detailed discussion of Isolate 7 can be found in the following chapter.

The genus Neosartorya (family Trichocomaceae) was first established by Malloch and

Cain in 1972 to allow telemorphs of species belonging to the Aspergillus fischeri series of

the Aspergillus fumigatus species group (Varga et al. 2000). This genus was reported with a

higher frequency of occurrence (%) in rhizome (11.1%) compared to in stems (3.7%) of

mature plants Cyperus malaccensis that dominates about one-third of the areas of estuaries

and mangroves (Karamchand, Sridhar & Bhat 2009). It is also one of the several

endophytic fungi of rhizome found in other tissues as endophytes. In this study, both strains

Isolate 8 and Isolate 13 (found closely related to Neosartorya sp.) were found highly

52

occurring in roots of mangrove plants collected at the island, freshwater and also near the

village in Kampung Pasir Pandak.

The genus Cladosporium is one of the largest genera of dematiaceous hyphomycetes where

most of the species belonging to this genus are characterized by a coronate scar structure

(Bensch et al. 2010). Cladosporium cladosporioides was reported as one of the endophytes

isolated from leaves of the mangrove plant Rhizophora apiculata (Kumaresan &

Suryanarayanan 2002). Besides, as reported earlier on Curvularia sp., Cladosporium sp.

has also been reported by Madavasamy and Panneerselvam (2012) as one of the endophytic

fungi out of twenty two species isolated from the leaves of Avicennia marina. Isolate 12

was related to Cladosporium sphaerospermum strain SCSGAF0054 confirming previous

findings and indicating a common distribution of Cladosporium in Avicennia.

The genus Eupenicillium was introduced by Ludwig in 1892 for an ascomycete species

(Houbraken & Samson 2011). It also belongs to the family, Trichocomaceae (Aly et al.

2010), similar to the genus Neosartorya sp. Trichocomaceae comprise of a relatively large

family of fungi, with the most well-known species belonging to the genera Aspergillus,

Penicillium and Paecilomyces. They are well-known for their secretion of secondary

metabolites that are known as mycotoxins while others are used as pharmaceuticals,

including antibiotics such as penicillin (Houbraken & Samson 2011). Isolate 9 was related

to Eupenicillium sp. 5 JH-2010 (Table 2 and Figure 24); however, it did not show

antimicrobial activity in our tests.

3.2 Biological assays

In the following, the main results of the various assays are presented as well as their

discussion.

3.2.1 Primary screening of antimicrobial activity

Antimicrobial activity was determined using the agar plug method (see section 2.6.1

Primary Screening of Antimicrobial Activity for description of the method). Cylindrical

pieces, or agar plugs, cut from one week old PDA (Potato dextrose agar) plate cultures of

53

12 strains were screened for their antimicrobial activity against ten (10) test organisms. A

positive result of antimicrobial activity was based on the presence of a clear zone (or also

known as zone of inhibition) (see Figure 25(a) and (b) for exemplary plates).

The results obtained showed that only two strains, Isolate 7 (related to Guignardia sp.) and

Isolate 13 (related to Neusartorya sp.) displayed significant antimicrobial activity (> 6mm

inhibition zone, see Table 3) against two or more test organisms (detailed discussionin the

following chapter). Activity was observed against Gram positive bacteria (Bacillus cereus,

Bacillus subtilis and Micrococcus luteus), Gram negative bacteria (Vibrio anguilarum), and

fungus (Candida albicans) (see Table 3).

Table 3: Antimicrobial activity of endophytic fungi strains (Primary screening)

Zone of inhibition is measured in mm and the Mean + Standard Deviation are displayed. Only the strains thatshowed activity are displayed, the others have been omitted for reasons of clarity. BC: Bacillus cereus; BS:Bacillus subtilis; SA: Staphylococcus aureus; ML: Micrococcus luteus; EC: Escherichia coli; PA:Pseudomonas aeruginosa; VA: Vibrio anguilarum; CA: Candida albicans; SC: Saccharomyces cerevisiae;AN: Aspergillus niger

Zone of inhibition (mm) (Mean + SD)

BC BS SA ML EC PA VA CA SC AN

Isolate 1 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 2 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 3 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 4 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 5 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 6 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 7 7.33

+ 0.58

7.00

+ 1.00

0 + 0 0 + 0 0 + 0 0 + 0 7.67

+ 0.58

0 + 0 0 + 0 0 + 0

Isolate 8 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 9 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 10 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate 12 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

54

Isolate 13 0 + 0 0 + 0 0 + 0 9.67 + 1.53 0 + 0 0 + 0 0 + 0 10.67

+ 0.58

0 + 0 0 + 0

(a) (b)

Figure 25: Zone of inhibition (ZOI) for Isolate 7 and Isolate 13. (a) Isolate 7 against Bacillus cereus; (b) Isolate 13 against

Candida albicans.

Scale is indicated at the bottom.

3.2.2 Secondary screening of antimicrobial activity

To confirm and determine the ability of the two fungal strains as potential producers of

antimicrobial compounds, the strains which displayed relatively broad antimicrobial

activity in the primary assay, were selected for secondary assay, Isolate 7 and Isolate 13,

respectively.

Antimicrobial activity was determined using the agar well diffusion method. The ethyl

acetate extracts obtained after 1 week incubation and extraction were dissolved in 1 ml of

dimethyl sulfoxide (DMSO). As shown in Table 3, the two strains were indeed able to

produce antimicrobial compounds, and an even higher antimicrobial activity as compared

to the primary screening as can be seen from a larger zone of inhibition (Figure 26 (a) and

(b)).

5mm 5mm

55

Table 4: Antimicrobial activity of endophytic fungi strains (Secondary Screening)




Isolate. 1 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 2 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 3 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 4 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 5 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 6 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 7 14.00 +

1.00

13.00

+ 2.65

0 + 0 0 + 0 0 + 0 0 + 0 12.33

+ 1.15

0 + 0 0 + 0 0 + 0

Isolate. 8 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 9 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 10 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 12 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0

Isolate. 13 0 + 0 0 + 0 0 + 0 8.00 +

0.00

0 + 0 0 + 0 0 + 0 11.67 +

1.15

0 + 0 0 + 0

56

(a) (b)

Figure 26: Zone of inhibition (ZOI) for Isolate 7 extract and Isolate 13 extract. (a) Isolate 7 extract against Bacillus

cereus; (b) Isolate 13 extract against Candida albicans. Scale is indicated at the bottom.

3.2.3 Cytotoxic activity

The cytotoxic assay was undertakenby testing the ethyl acetate extracts obtained for each

isolate (after 1 week incubation) against matured shrimps at different concentrations (0.5,

5, 50, and 500 ppm). Table 5 shows that Diaporthe sp. strain Isolate 3 and Eupenicillium

sp. strain Isolate 9 displayed toxicity against the matured brine shrimps at concentrations of

500 ppm after 24 hours incubation. Isolate 3 showed a significantly stronger cytotoxicity

and was able to kill 100% of brine shrimps, whereas Isolate 9 only killed 10% (Table 5).

The percentage (%) refers to the number of brine shrimps that were killed over the total of

brine shrimps; which is 10 in total.

5mm 5mm

57

Table 5: Mortality of brine shrimps observed at different concentrations (0.5, 5, 50 and 500 ppm) of crude extracts of

fungal strains

Mortality at different Concentration (%)

500 ppm 50 ppm 5 ppm 0.5 ppm

Isolate. 1 100% 100% 100% 100%

Isolate. 2 100% 100% 100% 100%

Isolate 3 0% 100% 100% 100%

Isolate 4 100% 100% 100% 100%

Isolate 5 100% 100% 100% 100%

Isolate 6 100% 100% 100% 100%

Isolate 7 100% 100% 100% 100%

Isolate 8 100% 100% 100% 100%

Isolate 9 90% 100% 100% 100%

Isolate 10 100% 100% 100% 100%

Isolate 12 100% 100% 100% 100%

Isolate 13 100% 100% 100% 100%

58

3.3 Bioactive compounds isolated from endophytic fungi

In this part, we discuss the compounds obtained from the various isolates after subjecting

them to solvent-solvent extraction. Table 6 shows an overview of the amounts (in mg)

obtained for each fraction (ethyl acetate, methanol and n-hexane) for each isolate. Initially

all isolates were incubated in 20 ml PDB for 1 week and the extracts obtained were sent to

Professor Peter Proksch (Institut für Pharmazeutische Biologie und Biotechnologie,

University Düsseldorf, Germany) for High Performance Liquid Chromatography (HPLC)

analyses and identification of the active compounds. Unfortunately, the amounts were too

low for many fractions and results could only be obtained for some of the fractions (Table

7). The isolates that showed antimicrobial activity (Isolate7 and Isolate13) were

subsequently incubated in 250 ml for 5 weeks to obtain sufficient extracts for HPLC

analysis. Table 8 shows the compounds that were analysed for Isolate7 and Isolate13.

Table 6: Overview of the amounts (in mg) obtained for each fraction

StrainsAmounts (in mg) for each fractions

Ethyl Acetate (mg) Methanol (mg) n-Hexane (mg)

Isolate 1 5.0 1.5 17.6

Isolate 2 0.9 5.2 2.4

Isolate 3 2.4 12.0 2.2

Isolate 4 4.7 12.9 1.7

Isolate 5 9.3 22.9 11.0

Isolate 6 2.7 1.0 4.9

Isolate 7 1.4 14.6 1.8

Isolate 8 2.0 0.6 2.8

Isolate 9 12.7 10.3 3.0

Isolate 10 1.3 10.5 2.0

Isolate 12 1.0 0.5 2.1

Isolate 13 1.2 22.6 1.8

59

Table 7: Overview of HPLC results obtained for the three fractions (ethyl acetate, methanol and n-hexane). Number

of compounds related to known structures/compounds is indicated and details listed below, as well as number of

compounds showing no similarityto known compounds (unknown compounds). Note: Number of known

compounds is based on library hits available.

Strains

Compounds Analysis based on fractions

Ethyl Acetate (mg) Methanol (mg) n-Hexane

(mg)

Isolate 1 - - -

Isolate 2 - 13 known compounds

Isox-brom-derivat citreonigrin F meta-Chloro-para-hydroxy-

phenyl-essigsaureamid MA-Medium D gancidin(cycloleucylprolyl) citreodrimene B 2-Hydroxy-3-methylbenzoic

acid altechromone A Fatty Acid amin.-Chlor.-Phe.-Essigsr. cerebroside brom. Dipheter 7 hydroxydienoic acid methyl

ester

8 unknown compounds

-

Isolate 3 14 known compounds

cyclo(prolylvalyl) kahalalide B kahalalide D cyclo(tyrosylprolyl) 4-hydroxyscytalon Desoxyfunicon Desmethyldichlorodiaportin Diaportinsaure Citreoisocoumarin Diachlordiaportin kealjinine A sumiki’s acid gancidin(cycloleucylprolyl) methoxy-methyl Agistatin D

9 unknown compounds

- -

Isolate 4 - Seven known compounds

cyclochalasin H kahalalide D

60

Fatty Acid cyclo(prolylvalyl) new emericellin derivative naamine A Sumiki’s acid

Sixteen unknown compounds

Isolate 5 21 known compounds

isox-brom-derivat benzyl-pyridin A meta-Chloro-para-hydroxy-

phenyl-essigsaureamid citrinin hydrate quinolactacin 8-hydroxy-4-Quinolone new emericellin derivative altenusin citrinin 8E-6-3-2 bastadin 11 benzyl-pyridin B 3,4,5-Tribromo-3-(2,4-dibromo-

phenoxy)-phenol bastadin 3 sclerotigenin Sumiki’s acid Cladosporin sarasinside H2 8E-2-5-1 22-Dehydrocampesterol

16 unknown compounds

23 known compounds

Sumiki’s acid benzyl-pyridin A trihydroxy tetralone Amin.-Chlor.-Phe.-Essigsr. citrinin hydrate quinolactacin butyl 2-(4-hydroxyphenyl)

acetate N-ethylene-renieron citrinin hydroxyanthranilic acid benzyl-pyridin B stevensin cyclopenin bastadin 3 graphislactone derivative cyclopenol Fatty Acid naamine F renieron altenusin cladosporin 8E-2-5-1 S16


-

Isolate 6 Seventeen known compounds

phenylacetic acid hydroxysydonic acid PC 3.3.21.E Isofistularin-1 8E-6-3-3 Aurantiamine cyclopenol aureonitol benzyl-pyridin A A new gamma-pyrone di-iso-Octylphtalat

(Weichmacher) 4,5Dibr.pyrrol2carba sarasiniside A sarasiniside K sarasinside 12 adenosine benzyl-pyridin B

9 known compounds

dienone dimethoxyketal phenylacetic acid citrinin hydrate 8-hydroxy-4-Quinolone 11,19-deoxyfistularin 2-Hydroxy-3-methylbenzoic

acid PC 3.3.6.6.3.A sarasiniside K triterpene acetate

4 unknown compounds

-

61

22-dehydrocampesterol

Five unknown compounds

Isolate 7 - - -

Isolate 8 - - -

Isolate 9 Four known compounds

microsphaerone B sclerotigenin 3,4-Dihydromanzamine naamine F


3 known compounds

microsphaerone B paxilline manzamin JN-Oxid


-

Isolate 10 - - -

Isolate 12 - - -

Isolate 13 - - -

Table 8: Overview of HPLC results obtained for the three fractions of Isolate7 and Isolate13 (ethyl acetate,

methanol and n-hexane). Number of compounds related to known structures/compounds is indicated and details

listed below, as well as number of compounds showing no similarity to known compounds (unknown compounds).

Note: Number of known compounds is based on library hits available.

StrainsCompounds Analysis based on fractions

Ethyl Acetate (mg) Methanol (mg) n-Hexane (mg)

Isolate 7 Three knowncompounds

Pavetannin A1Ac Epicatechin 9alpha-OH-

Pinoresinol

23 unknowncompounds

Four knowncompounds

Pavetannin A1Ac Rocaglamid A Salicifoliol Procyanidin B3 o.

B6

45 unknowncompounds

Five knowncompounds

Pavetannin A1Ac Trimeric Catechin Helenalin Catechin Rocaglamid A

4 unknowncompounds

Isolate 13 Three knowncompounds

Trimeric Catechin Epicatechin Helenalin

14 unknowncompounds

Five knowncompounds

CS-H2O-2 9-OH-Pinoresinol Helenalin Triandrin Trimeric Catechin

45 unknowncompounds

Five knowncompounds

Pavetannin A1Ac Catechin Rocaglamid A Helenalin

1 unknowncompound

62

In the following, information about some of the compounds listed in Tables 6 and 7 will be

provided and related to our isolates where possible. These compounds discussed were

based on other findings whereas some of the compounds listed in Tables 6 and 7 might not

be discussed as they were no available literature reported on it.

3.3.1 Citreonigrin F

This compound was isolated from methanol extract of fungal strain, Isolate 2 (closely

related to Curvularia). Citreonigrin A, was reported in a conference abstract (Ebel et al.

2006) as one of the bioactive metabolites isolated from marine derived fungi, Penicillium

citreonigrum obtained from the Indonesian sponge Pseudoceratina purpurea. Other

additional citreonigrins (inclusive of Citreonigrin F) were reported in a doctoral thesis

(Rusman 2006).

3.3.2 Gancidin(cycloleucylprolyl)


related to Curvularia) and ethyl acetate extract of Isolate 3 (closely related to Diaporthe).

A similar compound was reported by Rhee 2002, as an antibiotic, cyclo (L-leucyl-L-prolyl)

isolated from the Streptomyces sp., an actinomycete strain was reported active against

vancomycin-resistant enterococci strains and leukemia cell lines.

3.3.3 Citreodrimene B


related to Curvularia). This was also reported in a doctoral thesis (Rusman 2006) just like

the compound Citreonigrin F (section 3.3.1).

3.3.4 2-Hydroxy-3-methylbenzoic acid


related to Curvularia) and isolated from methanol extract of Isolate6 (closely related to

Aspergillus). The 2-Hydroxy-3-methylbenzoic acid compound, reported as a new benzoic

acid derivative, was first isolated by Ali and colleagues (1998), from the Stocksia brahuica

plant.

63

3.3.5 Altechromone A


related to Curvularia). This compound, a chromone derivative, was first reported isolated

from Alternaria sp., an endophytic fungus. Chromones are known common entities in

natural products, drug development as well as technical applications (Konigs et al. 2010).

Besides, Altechromone A was reported by Gu (2009) as one of the seven compounds

isolated from ethyl acetate extracts of Alternaria brassicicola, an endophytic fungi isolated

from the leaves of Malus halliana. It was reported very active against Bacillus subtilis,

Escherichia coli, Pseudomonas fluorescens and Candida albicans. However, in this study,

Isolate 2 did not exhibit antimicrobial activity towards any of the test pathogens. The

difference would be that in this study, only primary antimicrobial screening was conducted

using agar plugs of fungal strains whereas Gu (2009) performed antimicrobial assay using

crude extracts of Alternaria where the compounds of interest were already isolated. Hence,

the extracts of Isolate 2 could be further studied for their bioactive potential.

3.3.6 Fatty Acid

This compound was isolated from methanol extracts of fungal strains, Isolate 2 (closely

related to Curvularia), Isolate 4 (closely related to Diaporthe) and Isolate 5 (closely related

to Penicillium). In this study, fatty acid was isolated from three fungal strains with all of

them from methanol extracts.

Fatty acid was commonly reported isolated from fungi for instance from Glomerella

cingulata (plant pathogenic fungus) and Epichloe festucae (fescues pathogenic fungus)

(Richardson et al. 1997; Tenguria, Khan & Quereshi 2011). For the endophyte infecting

fine fescues (Epichloe festucae), the major fatty acids isolated were C18 and C16

compounds, which were found similar to other ascomycetes fungi. Quite a number of fatty

acid methyl esters were also reported isolated from all the fungal isolates of Thai medicinal

plants, Hiptage benghalensis, Betula alnoides, and Houttuynia cordata with antioxidant

properties (Theantana et al. 2012).

64

3.3.7 Cerebroside

Cerebrosides are neutral glycosphingolipids that contain a monosaccharide, a glucose or

galactose, in 1-ortho-beta-glycosidic linkage with the primary alcohol of an N-acyl

sphingoid (ceramide). They are also known as ceramide monohexosides (CMHs) as they

contain one sugar unit, which differs from gangliosides in that the latter contain at least one

sialic acid residue. Barreto-Bergter and colleagues (2004) also reported that cerebrosides

seem to be present in almost all fungal species studied so far (for instance, Aspergillus sp.,

Penicillium sp., Fusarium sp., etc).

In this study, this compound, cerebroside was isolated from methanol extracts of fungal

strain Isolate 2 (closely related to Curvularia sp.), which showed similarity towards the

findings by Wang and colleagues (2009) in which the fungal endophytes responsible for

this compound were both from the sediment samples of mangroves. For the study reported

by Wang and colleagues (2009), three new cerebrosides compounds were isolated from the

ethyl acetate extract of the halotolerant fungal strain, identified as Alternaria raphani (from

sediment in the Hongdao sea salt field, China). The cerebrosides belonging to the

halotolerant fungal strain showed weak antibacterial activity against Escherichia coli,

Bacillus subtilis, and Candida albicans. However, in this study, the ethyl acetate extract for

the Isolate2 strain was not tested against these test pathogens as the secondary screening

assay done was only to confirm the activity of the two selected fungal strains (Isolate 7 and

Isolate 13) which displayed antimicrobial activity in the preliminary assay.

3.3.8 Cyclo(prolylvalyl)

Cyclo(prolylvalyl) is classified as a diketopiperazine according to Smelcerovic and

colleagues (2002), who isolated cyclo(prolylvalyl) from a marine actinomycete using high

speed countercurrent chromatography (HCCC), which is a tool for separating natural

products. This compound was isolated from ethyl acetate extract of fungal strain Isolate 3

(closely related to Diaporthe) and methanol extract of fungal strain Isolate 4 (closely

related to Diaporthe). This compound was also reported by Kim and colleagues (2005) as

one of the structures determined isolated from the methanol extract of the mushroom

Sarcodon aspratus through ethyl acetate extraction where the compound showed

65

antioxidant activity by scavenging DPPH radical and superoxide radical which could be

tested in the future for above mentioned isolates.

3.3.9 Kahalalide B

Kahalalide B is a cyclic depsipeptide formed by seven different amino acids (Gly, thr, Pro,

D-Leu, Phe, D-Ser, Tyr), and the fatty acid 5-methylhexanoic (5-MeHex), an aliphatic

isoacid which is also present in the structure of other members of the series (Lopez-Macia

et al. 2000). The finding of a compound with a similar structure from fungal strain Isolate 3

(closely related to Diaporthe sp.), which also showed cytotoxic activity against mature

brine shrimps, is therefore highly promising and warrants further studies to isolate the

compound and enumerate its structure.

3.3.10 Cyclo(tyrosylprolyl)

Cyclo(L-tyrosyl-L-prolyl), known as a cyclic dipeptide, hasbeen reported in many studies

with potential biological activity. Killian and colleagues (2011) reported that this particular

compound possess antibacterial activity in vitro. Besides, this compound was also reported

by Milne and colleagues (1998), with a potential to be used in muscle relaxants, anti-

tumour compounds and antibiotics. This compound was isolated from fungal strain, Isolate

3 (which is closely related to Diaporthe).

3.3.11 Citreoisocoumarin and Diachlordiaportin

Citreoisocoumarin, along with diachlordiaportin, [3-(3,3-dichloro-2-hydroxy-propyl)-8-

hydroxy-6-methoxyisochromen-1-one] have been reported to be produced by has been

reported to be produced by Penicillia related to Eupenicillium and other filamentous fungi

(Frisvad et al. 2004; Brien et al. 2006). This compound was also reported to be the first

isolated from a Phoma species by Sorensen and colleagues (2010). Both compounds were

isolated from fungal strain Isolate 3 (which is closely related to Diaporthe) in this study

lending support to previous findings.

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3.3.12 Sumiki’s acid

According to Jadulco and colleagues (2001), Sumiki’s acid, also known as furan carboxylic

acid, together with its new derivative, acetyl Sumiki’s acid showed antimicrobial activity

against Bacillus subtilis and Staphylococcus aureus. However, in this study, fungal strains

Isolate 3 and Isolate 4 (which are closely related to Diaporthe sp.) and Isolate 5 (which is

closely related to Penicillium) were found to possess only Sumiki’s acid, hence this might

be the possibility for these fungal strains to not exhibit any inhibition towards Bacillus

subtilis and Staphylococcus aureus, when tested against these pathogens.

3.3.13 Cyclochalasin H

The cytochalasins are a class of fungus-derived metabolites with diverse effects on cellular

functions (Udagawa et al. 2000). Cytochalasin H, metabolite of the endophytic fungi

Endothia gyrosa was reported by Xu and colleagues (2009) with cytotoxic activity against

human leukaemia cell lines, comparable to the positive reference 5-fluorouracil. In this

study, this compound was isolated from fungal strain Isolate 4 (which is closely related to

Diaporthe).

3.3.14 Naamine A

Naamine A, an alkaloid, isolated from two marine sponges, Leucetta chagosensis and

Leucetta cf chagocensis, was collected from the Great Barrier Reef and the Fiji Islands

(Gross et al. 2002). The same compound was also reported in another study by Dunbarand

colleagues (2000) in the isolation from Red Sea sponge Leucetta cf chagocensis and it was

found to possess antifungal properties. This compound was found in the methanol extract

of Isolate 4, which is closely related to Diaporthe.

3.3.15 Citrinin hydrate

Citrinin hydrate, isolated from the Penicillium sp. was found to exhibit strong inhibitory

activity against arylalkylamine N-acetyltransferase (AA-NAT). AA-NAT plays key roles in

several disorders, such as depression and delayed sleep-phase syndrome. Hence, with the

strong inhibitory activity towards AA-NAT, this could possibly lead to the discovery of

67

useful antidepressive drugs (Kim et al. 2001). Citrinin hydrate was also reported to have

been isolated from the Penicillium sp. by Kadam and colleagues (1994) and in this current

study itself from the ethyl acetate extract of Isolate 5, which is found also closely related to

Penicillium sp. Besides Penicillium sp., this compound is also isolated from fungal strain,

Isolate6 (which is closely related to Aspergillus).

3.3.16 Quinolactacin

Quinolactacin, known as an alkaloid was reported to be isolated also from Penicillium sp.

with inhibitory activity against tumor necrosis factor (TNF) production (Sasaki et al. 2006).

Besides, quinolactacins A, B and C were also reported to be isolated from Penicillium sp.

as novel quinolone antibiotics (Kakinuma et al. 2000). Similarly, in this study, this

compound was isolated from fungal strain, Isolate 5 (which is closely related to

Penicillium).

3.3.17 Altenusin

Altenusin, a biphenyl derivative was reported to be isolated from endophytic fungus of

Alternaria sp. and was found to exhibit strong antifungal activity against pathogenic fungus

Paraccoccidioides brasiliensis and nonpathogenic yeast Schizosaccharomyces pombe

(Johann et al. 2012). A similar compound was produced by Isolate 5 (which is closely

related to Penicillium).

3.3.18 Citrinin

Citrinin, a common mycotoxin that was first isolated from Penicillium citrinum, was

reported by Iwahashi and colleagues (2007) indicating citrinin’s strong inhibitory action

against yeast cells. Mycotoxins are known as fungal secondary metabolites regarded as

hazardous contaminants. Similarly, in this study, this compound was also isolated from the

fungal strain, Isolate 5 which is closely related to the Penicillium species. Besides, citrinin

was also reported as a fungal secondary metabolite of fermented products of the fungus

Monascus (Hajjaj et al. 1999).

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3.3.19 Sclerotigenin

Sclerotigenin, a benzodiazepine was first isolated from dichloromethane extracts of the

sclerotia of Penicillium sclerotigenum and was found to possess antiinsectan activity (Gloer

et al. 1999). A compound with similar structure was also isolated from fungal strains that

were closely related to Penicillium species; Isolate 5 and Isolate 9 (which is closely related

to Eupenicillium), however no tests for antiinsectan activity were undertaken.

3.3.20 Cladosporin

Cladosporin, a fungal isocoumarin derivative was first reported by Scott and colleagues

(1971) as a new antifungal metabolite isolated from Cladosporium cladosporioides. This

compound was isolated from fungal strain, Isolate 5 (which is closely related to

Penicillium).

3.3.21 Trihydroxy tetralone

There have been literature reports on the discovery of a new α-tetralone derivative, (3S)-

3,6,7-trihydroxy-α-tetralone, that was isolated from the ethyl acetate extract of a culture

broth of the endophytic fungus Phoma, which showed growth inhibition against Fusarium

oxysporium and Rhizoctonia solani (Wang et al. 2012). In this study, trihydroxy tetralone

was isolated from fungal strain Isolate5 (which is closely related to Penicillium), however

the fungal strain was not tested against Fusarium oxysporium and Rhizoctonia solani,

which would then require further testing to further support the bioactiv potential of this

compound. Besides, tetralone derivative was also reported to be a potential anti-diabetes

agent when found showing moderate bioactivity against protein tyrosine phosphatase 1B

(PTP1B), a compound playing a major role in the reaction of Type-2 diabetes and obesity

(An et al. 2003).

3.3.22 Cyclopenin

This compound was isolated alongside with cyclopenol from methanol extract of Isolate 5

(which is closely related to Penicillium). Cyclopenin was also isolated from the Penicillium

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of seventeen subgenuses and found to possess potential herbicidal and anti-HIV activity

(Frisvad et al. 2004).

3.3.23 Graphislactone derivative

Graphislactones A-H and the structurally related ulocladol are highly oxygenated resorcylic

lactones produced by lichens and fungi (Altemoller et al. 2009). Cudaj and Podlech (2010)

werethe first to report on the synthesis of Graphislactone G by Cephalosporium

acremonium. Graphislactone A was characterized as the most bioactive secondary

metabolite of endophytic Cephalosporium sp. with free radical-scavenging and antioxidant

activities (Song et al. 2005). These studies show the potential bioactivities possessed by

these Graphislactones. In this study, Graphislactones derivatives compound was isolated

from fungal strain Isolate 5 (which is closely related to Penicillium), however further

studies would be required to identify the type of Graphislactone and its potential activity.

3.3.24 Phenylacetic acid

Phenylacetic acid is classified under phenolics (C6-C2), which is a compound needed by the

plants for pigmentation, growth, reproduction, resistance to pathogens and for many other

functions (Lattanzio, Lattanzio & Cardinali 2006). This compound is also known as an

antifungal metabolite produced by endophytic bacteria, Burkholderia species (Mendes et al.

2007). A compound with similar structure was isolated from fungal strain Isolate 6 (which

is closely related to Aspergillus) which did however not show antifungal activity.

3.3.25 Isofistularin-1

To date, there has been no literature citing the discovery of Isofistularin-1, but Isofistularin-

3 has been reported in several studies. Isofistularin-3 was reported as one of the brominated

isoxazoline alkaloids found accumulated in Mediterranean marine sponge Aplysina

aerophobaas part of a defensive mechanism against the polyphagous Mediterranean fish

Blennius sphinx and also possibly as a protection from invasion of bacterial pathogens

(Thoms et al. 2004). Acompound with similar structure to Isofistularin-1 was isolated from

fungal strain Isolate 6 (which is closely related to Aspergillus) in this study but did not

show any antimicrobial activity.

70

It is noteworthy that many of the compounds isolated in this study were similar in structure

to compounds reported from marine sponges (Eg 3.3.65 4,5-dibromopyrrole-2-carboxylic

acid and 3.3.69 Dienone dimethoxyketal) which shows the potential of marine life as a

source of natural products for medicinal development purposes.

3.3.26 8E-6-3-3 Aurantiamine

Aurantiamine was reported by Larsen and colleagues (1992) as a new substituted

diketopiperazine, isolated from Penicillium aurantiogriseum. This compound was isolated

from fungal strain Isolate 6 (which is closely related to Aspergillus).

3.3.27 Aureonitol

This compound was first known as a fungal metabolite isolated from Chaetomium species

and later found produced also by another endophytic fungus, Helichrysum aureo-nitens

(Aly, Debbab & Kjer 2010). Aureonitol is now being isolated from fungal strain, Isolate 6

(which is closely related to Aspergillus).

3.3.28A new gamma-pyrone

Gamme-pyrone compounds have been reported by Liou and colleagues in 1993 as potential

anti-cancer drugs, showing inhibition towards cancer cell lines.

A new gamma-pyrone was reported to be isolated from dichloromethane extract of stems

and roots of Hypericum brasiliense plant. This new gamma-pyrone compound was termed

hyperbrasilone and found to possess antifungal properties (Rocha et al. 1994). Besides,

many new gamma-pyrones have been reported, for instance, Carbonarones A and B

obtained from the culture of the marine derived fungus, Aspergillus carbonarius, to which

both compounds showed moderate cytotoxicity against KF62 cells. For this study, another

new gamma-pyrone was also reported for a fungal strain that is also closely related to

Aspergillus, Isolate 6. With that, this compound would require further structure elucidation

to identify the new compound of interest.

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3.3.294,5-dibromopyrrole-2-carboxylic acid

The 4,5-dibromopyrrole-2-carboxylic acid is one of the long-known marine alkaloids, and

was reported as a compound commonly isolated from marine sponges for instance in (a)

Astrosclera wiedenmayeri, marine sponge which inhabits the Florida coast (North Dry

Rocks) (Dembitsky 2002) and also in (b) Agelas Oroides, Maltese marine sponge, reported

by Konig and colleagues in 1998 that the 4,5-dibromopyrrole-2-carboxylic acid was found

to exhibit moderate cytotoxic activity towards cancer cell lines. In this study, this

compound was isolated from fungal strain Isolate 6 (which is closely related to

Aspergillus), but did not show any cytotoxicity towards the matured brine shrimps.

3.3.30 Adenosine

Adenosine was reported as one of the compounds isolated from cultures of Paecilomyces

sp., an endophytic fungus present in leaves of Enantia chlorantha Oliv (Annonaceae)

(Talontsi et al. 2012). Besides, this compound was also reported as natural products

isolated from medicinal plants for instance; in the fruiting bodies of the caterpillar-shaped

Chinese medicinal mushroom, DongCongXiaCao (Hong et al. 2007) and medicinal plant,

Selaginella tamariscina (Setyawan 2011). This compound was found isolated from fungal

strain Isolate 6 (which is closely related to Aspergillus).

3.3.31Dienone dimethoxyketal

Dienone was reported by Aydogmus and colleagues (1999) together with dienonediethoxy

ketal that was isolated for the first time from the ethanol extract of sponge samples

collected from the Aegean Sea. Later in 2009, a study showed the isolation of dienone

dimethoxyketal from the sponge, Pseudoceratina purpurea collected from Banyuwangi,

Indonesia. According to the study, dienone dimethoxyketal was suspected to be artefacts

formed during the extraction and purification process (Hertiani et al. 2009). This compound

was isolated from the fungal strain Isolate 6 (which is closely related to Aspergillus).

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3.3.3211, 19-dideoxyfistularin

This compound was reported by Mancini and colleagues in 1993 as one of the compound

isolated from extracts of the sponge (belonging to the order Verongida), which was

collected from two spots in the Coral Sea. 11, 19-dideoxyfistularin-3 is also known as a

bromotyrosine metabolite isolated from the ethanolic extract of Pseudoceratina sp., marine

sponge collected in Vanuatu (Lebouvier et al. 2009). In this study, this compound was

isolated from fungal strain, Isolate 6 (which is closely related to Aspergillus).

3.3.33Triterpene acetate

Triterpenes were reported with bioactivities of antioxidation, hepatoprotection, cholesterol

stasis, anti-hypertension, and inhibition of platelet aggregation. Triterpene isolated from hot

water extracts from mycelia of medicinal mushrooms, Ganoderma lucidum extracts were

reported by Lin and colleagues (2003) with anticancer activity which inhibits growth of

cancer cells, Huh-7. In this study, this compound was isolated from fungal strain, Isolate 6

(which is closely related to Aspergillus).

3.3.34 Microsphaerone B

Microsphaerone B was first isolated from the ethyl acetate extract of the culture of an

undescribed fungus of the genus Microsphaeropsis, isolated from the Mediterranean

sponge Aplysina aerophoba. This compound represents the gamma-pyrone derivatice of the

fungal genus Microsphaerosis (Wang et al. 2002) and was isolated from the ethyl acetate

and methanol extracts of fungal strain, Isolate 9 (closely related to Eupenicillium). To date,

only one literature (Wang et al. 2002) have cited on their findings of microsphaerone B.

3.3.35 3,4-Dihydromanzamine

3,4-DihydromanzA is classified as a β-carboline alkaloids, which is termed as a group of

natural and synthetic indole alkaloids. In this study, 3,4-Dihydromanzamine was isolated

from ethyl acetate extracts of fungal strain Isolate 9 (closely related to Eupenicillium),

which exhibited cytotoxic activity towards the matured brine shrimps.

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3.3.36 Paxilline

This compound was isolated from the methanol extract of the fungal strain, Isolate 9

(closely related to Eupenicillium). Paxilline is a toxic indole-isoprenoid tremorgen which

was first discovered produced by Penicillium paxilli and later found synthesized by the

endophytic fungus, Acremonium loliae (Ibba et al. 1997). It is known as a potassium

channel blocker where it inhibits the alpha-subunit of the high-conductance calcium-

activated potassium channel however; this is not within our scope of study (Sanchez &

McManus 1996).

3.3.37 Manzamine J N-Oxide

The manzamines are the most promising antimalarial compound (Sipkema et al. 2005) and

are well known for their unique class of polycyclic alkaloids identified from marine

sponges in the late 1980s. They have been reported with a number of significant biological

activities including cytotoxicity, insecticidal, antibacterial, antiflammatory, antiinfective

and antiparasitic. Manzamine J N-Oxide was first reported isolated from the Philippine

sponge Xestospongia ashmorica with a few compounds of N-oxides of Manzamine J

exhibiting strong cytotoxicity activity against mouse lymphoma cells (Edrada et al. 1996).

In this study, this compound was isolated from fungal strain, Isolate 9 (closely related to

Eupenicillium sp.) which also showed toxicity to matured brine shrimps (as can be seen in

Table 5).

3.3.38 Pavetannin A1 Ac

Pavetannin A1 is usually found in plant and not fungi. Pavetannin A1 has previously been

reported from studies on the antiviral properties of Pavettao wariensis and showed activity

against Herpes simplex (Arnasan, Mata & Romeo 1995). Antiviral tests were however not

scope of the present study but the finding of a compound with a similar structure in

endophytic fungi is interesting nonetheless and warrants further studies. This compound is

isolated from fungal strain Isolate 7 (which is closely related to Guignardia) and Isolate 13

(which is closely related to Neosartorya).

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3.3.39 Epicatechin

Epicatechin is a flavanoid that has been reported to be responsible for antibacterial activity

against Gram-positive and Gram-negative bacteria. This compound was isolated by Masika

and colleagues (2004) from Schotia latifolia, a plant commonly used in folkloric medicine.

The fungal metabolite with a similar structure to Epicatechin most likely possesses a

similar chromophore and could be responsible for the observed antibacterial activity of

Isolate 7 (which is closely related to Guignardia) against Gram-positive (Bacillus cereus

and Bacillus subtilis) and Gram-negative (Vibrio anguillarum) bacteria. As seen in this

study, this compound was isolated from ethyl acetate extracts of both fungal strains Isolate

7 (which is closely related to Guignardia) and Isolate 13 (which is closely related to

Neosartorya). However, in this case, Isolate 13 (which is closely related to Neosartorya)

only exhibited antibacterial activity against Gram-positive bacteria (Micrococcus luteus)

and not Gram-negative. This might be attributed to the other different compounds produced

by both strainsin compliment with Epicatechin to allow the reaction to take place, as the

gram-positive bacteria that were inhibited by both isolates were also different; Bacillus

cereus and Bacillus subtilis (Isolate 7) and Micrococcus luteus (Isolate13).

3.3.40 9alpha-OH-Pinoresinol

9alpha-OH-Pinoresinol was reported as a lignin with anticancer activity (Chunsriimyatav et

al. 2009); however, Isolate 7 (which is closely related to Guignardia) did not show any

cytotoxic activity in our study and the fungal metabolite with a similar structure might

therefore not be cytotoxic. Same goes to Isolate 13 (which is closely related to

Neosartorya), which was found producing this compound and not exhibiting any

cytotoxicity activity towards matured brine shrimps.

3.3.41 Rocaglamide A

Rocaglamide was reported by Janprasert and colleagues (1992) as a highly substituted

benzofuran isolated and identified as the active insecticidal constituent in the twigs of the

Chinese rice flower bush, Aglaia odorata. Besides, rocaglamide was also reported as a

novel antileukemic 1H-cyclopenta[b]benzofuran isolated from Aglaia elliptifolia by King

and colleagues (1982). However, there have been no literature citing on Rocaglamide A,

75

the compound which was isolated from the methanol and n-hexane extracts of Isolate 7

(which is closely related to Guignardia sp.) and n-hexane extracts of Isolate 13 (which is

closely related to Neosartorya sp.).

3.3.42 Procyanidin B3 o. B6

Procyanidins are a subclass of flavanoids, which are a subclass of polyphenols, a group of

compounds known ubiquitous in the plant kingdom. Oligomeric procyanidins represent one

class of polyphenols and have attracted increasing attention in the fields of medicine due to

their potential health benefits where they have shown to have potent antioxidant activity

(Hammerstone, Lazarus & Schmitz 2000). Procyanidin B3 o. B6 was found in the methanol

extract of fungal strain, Isolate 7 (closely related to Guignardia sp.). Procyanidin B3 along

with Catechin and Epicatechin were reported isolated from extracts of the guarana seeds,

showed no activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and

Pseudomonas aeruginosa (Antonelli Ushirobira et al. 2007). Hence, the antibacterial

activity of Isolate 7 (closely related to Guignardia sp.) against Bacillus subtilis might not

be related to procyanidin and epicatechin.

3.3.43 Trimeric Catechin

Trimeric Catechin is catechin in its trimeric form (also known as oligomeric form).

Catechins are polyphenols and components of condensed tannins which display

antibacterial activity by precipitating proteins of pathogenic bacteria through direct binding

(Shimamura, Zhao & Hu 2007). Besides, catechin was also reported to possess antifungal

activity against Candida albicans (Hirasawa& Takada 2004). These findings are in

agreement with our results as this compound was found in the ethyl acetate and methanol

extract of fungal strain, Isolate 13 (which is closely related to Neosartorya), which also

displayed antifungal activity against Candida albicans. However, this compound was also

found in n-hexane extracts of fungal strain, Isolate 7 (which is closely related to

Guignardia), which did not exhibit any antifungal activity against Candida albicans.

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3.3.44 Helenalin

In this study, a compound isolated from extracts of Isolate 7 (which is closely related to

Guignardia) and Isolate 13 (which is closely related to Neosartorya), displayed structure-

similarity to Helenalin, a sesquiterpene lactone commonly isolated from plant families such

as Acanthaceae, Anacardiaceae, Apiaceae, Euphorbiaceae, Lauraceae, Magnoliaceaa and

others (Chaturvedi 2011) with anti-inflammatory and antineoplastic activity. Anti-

inflammatory tests were not scope of the present study but the finding of a compound with

a similar structure in endophytic fungi is again interesting and also warrants further studies.

3.3.45 Catechin

As mentioned above, this compound might be responsible for the antifungal activity,

however, antimicrobial testing were not performed using n-hexane extracts of both fungal

strains, Isolate 7 (which is closely related to Guignardia) and Isolate 13 (which is closely

related to Neosartorya),. This would require further studies which might lead to greater

findings.

3.3.46 Triandrin

Triandrin also known as 1-O-β-D-glucopyranoside of p-coumaryl alcohol, is one of the

phenolic compounds isolated from the bark extracts of basket-willow, Salix viminalisL.

Phenolic compounds are usually extracted from plant raw materials using methanol,

ethanol or aqueous alcohol (Minakhmetov et al. 2002) and indeed, for this study, this

compound was found in the methanol extracts of Isolate 7 (closely related to Guiganardia

sp.).

3.4 Heavy metal analysis

3.4.1 Determination of heavy metal resistance fungi

The ability of the endophytic fungi to resist the heavy metal (or also known as minimum

inhibition concentration (MIC) was determined from the dry weight of the biomass present.

The MIC varied for all the endophytic fungi tested which shows the different abilities of

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withstanding the heavy metal (Table9). Isolate 3 showed the highest resistance to Cu2+ and

was able to grow in concentrations up to 600 µg/ml (Table 9). Isolate 12 and Isolate 8

showed the lowest resistance towards Cu2+ in which both were only able to grow up till the

concentration of 50 µg/ml. On the other hand, Isolate 5 and Isolate 9 showed the highest

resistance towards Zn+ with an MIC of 20,000 µg/ml. Isolate 10 showed the lowest

resistance towards Zn+ in which it was only able to grow up till the concentration of 100

µg/ml (Table 9).

From both the Table 9, it can be seen that these fungal isolates were all more resistant

towards heavy metal Zn+ that the MIC level is much higher in average compared to MIC

level towards Cu2+.

Table 9: Minimum inhibitory concentration (MIC) of heavy metal Copper (Cu) and Zinc (Zn) in living biomass of fungi

Species

Isolate1

Isolate2

Isolate3

Isolate4

Isolate5

Isolate6

Isolate7

Isolate8

Isolate9

Isolate10

Isolate12

Isolate13

MICµg/ml

Copper(Cu) 100 200 600 200 200 150 100 50 300 200 50 100

MICµg/ml(Zinc)

200 10,000 400 1,000 20,000 10,000 200 2,000 20,000 100 200 200

The MIC values suggest that the resistance level against individual metals was dependent

on the type of fungal species. Fungal strain, Isolate 3 was identified using molecular tools,

in which the purified genomic fragments were sent for sequencing and found closely

related to Diaporthe sp. So far there are no other reports of Cu-resistant Diaporthe sp. in

the literature. However, as seen from the phylogenetic tree (Figure 24), this species is

found closely related to Phomopsis sp., where a study published by European Food Safety

Authority, EFSA (2012), showed that phomopsins (a family of mycotoxins) produced by

the fungus Diaporthe toxica (formerly referred to as Phomopsis leptostromiformis) might

be responsible for the accumulation of copper in the liver. Genera Diaporthe is a sexual

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stage (telemorph), and mostly found in asexual stage (anamorphic stage) that belongs to

genera of Phomopsis. Diaporthe is also very difficult to be identified correctly

morphogically since this genera is seldom to form perithecia grown in a synthetic medium

(Ilyas et al. 2009). Besides, Phomopsis sp. is ascomycetes filamentous fungus, to which

was reported by Saiano and colleagues (2005) that this genus was able to complex various

metal species from aqueous media, mainly due to the presence of chitosan content of its

cell wall.

Fungal Isolate 12 was found closely related to Cladosporium sp. whereas Isolate8 fungal

isolate was found closely related to Neosartorya sp. Based on the study by Xinjiao 2006 on

the biosorption of Cu2+ by pretreated Cladosporium sp., the findings showed that the

pretreated Cladosporium with sodium hydroxide has a better biosorption capacity than the

non-pretreated one. However, it was still able to biosorp Cu2+ at a capacity of 4.14mg/g,

which means it was still resistant towards Cu2+ but at a very low level. However, another

study showed that Cladosporium sphaerospermum was still able to grow at the maximum

concentration of Cu tested (10mM), but still was reported as a weak growth, with an

approximation close to 0% growth in diameter as showed in their graph study

(Bridžiuvienė & Levinskaitė, 2007). Neosartorya sp. (Isolate 8), on the other hand, was

another endophytic fungus that was found to be least resistant towards Cu. Studies on

Neosartorya sp. were mostly on its ability to degrade petroleum oil (Kathi & Khan 2011;

Jain et al. 2011; Das & Chandran 2011). Another fungal isolate of the same genus, Isolate

13 also closely related to Neosartorya sp. showed a moderate tolerance towards Cu,

slightly higher with an MIC of 100 µg/ml compared to Isolate 8 (which is closely related to

Neosartorya) (50 µg/ml). Up till now, there have not been any findings on living biomass

Neosartorya’s level of resistant towards heavy metal reported yet, although there have been

findings on dried biomass of Neosartorya sp. in heavy metal removal.

For the tolerance towards Zn, Isolate 5 and Isolate 9 showed the highest resistance to Zn

with the former being closely related to Penicillium sp. and the latter being closely related

with Eupenicillium sp. According to Iram and colleagues, 2009, Penicillium sp. was among

the fungal strains tested for their degree of tolerance towards heavy metals, Zinc (Zn), Lead

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(Pb), Nickel (Ni), and Cadmium (Cd) through the measurement of the minimum inhibitory

concentration (MIC). Penicillium sp. was among the three fungal strains that showed strong

growth despite the high concentrations of Zn tested. Besides, findings by Lairini and

colleagues (2009) also supported the results for this study to which Penicillium sp. was

found to be the most tolerant to heavy metals and Zn is one of them being tested. However,

for the heavy metals tested, Cu was also one of them. For this study, besides Isolate 5 being

closely related to Penicillium sp., Isolate 1 and Isolate 10 fungal isolates are also closely

related to Penicillium sp., to which they showed a moderate tolerance level to Cu at 100-

200 µg/ml, with Isolate10 showing the least resistance towards Zn. Isolate 9 isolate which

is closely related to Eupenicillium sp. also showed high resistance towards Zn. To which, it

was reported that Eupenicillium sp. and Talaromyces sp. are telemorphic states of the

Penicillium genus (Visagie & Jacobs, 2009).

The results obtained in this study could also be supported further by Lairini and colleagues

(2009) when reporting that the isolates of the same genus could present a marked difference

in the levels of metal resistance, which may be due to the presence of different tolerance or

resistance mechanisms exhibited by different fungal isolates, especially when these fungal

isolates being tested are using the living fungal cells. Living fungal biomass biosorption

process is more complicated as bioaccumulation of heavy metal is also driven by growth,

metabolic energy and transport needs (Leitao 2009).

In addition, as reported, the results of Zn for this study was found to be at MIC in average,

as Zn is considered essential metal for all organisms, although at high concentrations, it can

be toxic, therefore, this might explain the reason for the high MIC of Zn in average as

compared to Cu (Lairini et al. 2009).

3.4.2 Heavy metal biosorption by dead fungal cells

Based on the Table 10, three isolates were observed with maximum biosorption capacity,

with Isolate 2 fungal strain in removing 25 mg Cu/g biomass and two other fungal strains,

Isolate 8 and Isolate 13 strains in removing 24 mg Zn/g biomass.

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Isolate 2 which was found to be the most efficient in removing Cu/g biomass is closely

related to Curvularia sp. (Figure 24) and –to our knowledge- this is the first reported study

on the ability of Curvularia sp. in removing heavy metal using dead biomass. Even though

no further experiments were performed to identify the mechanism by which the isolate

biosorps Cu and Zn, our results seem to indicate that Isolate 2 is adsorbing Cu on the

surface (as indicated by high Q and low MIC values) but actively adsorbs Zn (as indicated

by low Q and high MIC values).

Table 10: Copper (Cu) Biosorption capacity by dead fungal cells

Species

Isolate

1

Isolate

2

Isolate

3

Isolate

4

Isolate

5

Isolate

6

Isolate

7

Isolate

8

Isolate

9

Isolate

10

Isolate

12

Isolate

13

Q valueCopper

(Cu)18 25 11 5 8 1 8 15 4 0 15 7

Q valueZinc(Zn)

3 6 8 15 16 21 16 24 14 16 14 24

Isolate 8 and Isolate 13 showed highest efficiency in Zn/g biomass removal, with both

species being closely related to the same genus, Neosartorya. Heavy metal removal using

non-living biomass is less complicated than using living biomass, due to the absence of

metabolic activity, hence this might explain for the close proximity of heavy metal removal

capabilities for isolates of the same genus. However, findings by Simonovicova (2008)

reported results on non-living biomass of Neosartorya fisheri having the highest efficiency

of removing Cu and the lowest efficiency in removing Zn. But to take note, Isolate8 and

Isolate 13 are related to different types of species but of the same genus, to which the

former is closely related to Neosartorya stramenia and the latter being closely related to

Neosartorya hiratsukae. As mentioned earlier, to our knowledge, so far only one study has

been published regards tolerance of Neosartorya sp. towards heavy metals, and it involved

live biomass.

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Intriguingly, Isolate1 which displayed moderate tolerance towards Cu (MIC value of

100µg/ml, Table 10), showed the second-highest Cu biosorption capacity (Q of 18, Table

8) and lowest Zn biosorption capacity (Q of 3, Table 10) when used as dead biomass.

Isolate1 is closely related to Penicillium sp. and we see the opposite in the results for

Isolate10 which is also closely related to Penicillium sp. Live biomass of Isolate 10

similarly had moderate tolerance towards Cu (MIC values of 200µg/ml), but, when used as

dead biomass, it displayed the lowest Cu biosorption capacity (Q values of 0mg/g, Table

10) and third-highest Zn biosorption capacity (Q values of 16mg/g, Table 10).

Besides Isolate 10 having the lowest Cu biosorption capacity, Isolate 6 (closely related to

Aspergillus sp., Figure 1) showed similar results of moderate tolerance towards Cu (MIC

value of 150µg/ml, Table 9) but lowest Cu biosorption capacity (Q value of 1mg Cu/g,

Table 10) when used as dead biomass. This result could be further supported with the

findings of Kannan and colleagues (2011), where Aspergillus sp. was found to be an

efficient strain resistant to Cu when in the form of live biomass. It is when tested for

biosorption of Cu using dead biomass; Aspergillus sp. had the ability to adsorb maximum

level of Cu after the cell fraction was treated with sodium hydroxide (NaOH). This was due

to the dead biomass comprising of small particles with lower density, poor mechanical

strength and little rigidity (Volesky and May-Philips 1995). Again, this approach has not

been tested in this study but could potentially lead to higher biosorption capacities for

Isolate 6 and Isolate 10.

Penicillium is commonly known as a halotolerant genus isolated from mangroves and

salterns with high resistance towards metals such as copper (Leitao 2009).For this case,

although both strains were found closely related to Penicillium sp., both fungal strains were

different. Identification of Penicillium to species level requires multidisciplinary

approaches (Leitao 2009) which were beyond the scope of this study, however they should

be carried out in future on both isolates to help explain the observed different patterns.

To summarise, the results of this study show that the biosorption capacity depends on the

type of species and their cell wall’s mechanism towards tolerating heavy metals.

Biosorption of metals involves several mechanisms that differ qualitatively and

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quantitatively, according to the species used, the origin of the biomass, and its processing

procedure (Raize et al. 2004).

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4. PRELIMINARY RESULTS OF SCREENING OF MANGROVE

ENDOPHYTIC FUNGI FOR BIOACTIVE COMPOUNDS

May Ling ONN*, Po-Teen LIM2, AAZANI MUJAHID2, PETER PROKSCH3, ANDMORITZ MÜLLER1

1Biotechnology, School of Engineering, Computing and Science, Swinburne University ofTechnology, Sarawak Campus, 93350 Kuching, Malaysia

2Department of Aquatic Science, Faculty of Resource Science and Technology, UniversitiMalaysia Sarawak, 93400 Kota Samarahan, Malaysia.

3Institut fürPharmazeutischeBiologie undBiotechnologie, Universität Düsseldorf, Germany.

*Email:[email protected]

Submitted to Journal of Basic Microbiology (Manuscript ID: jobm.201200752)

ABSTRACT

Endophytic fungi are fungi that live inside the tissues of other organisms, such as mangrove

plants. The plants provide protection to the fungi and the fungi often produce antimicrobial

compounds to aid the host fighting off pathogens. These bioactive compounds are

secondary metabolites which are often produced as waste- or by-products. In the present

study, endophytic fungi isolated from mangrove plants and soils were characterized and

their antimicrobial potential assessed. Twelve endophytic fungi were isolated and identified

to belong to 7 families: Penicillium, Curvularia, Diaporthe, Aspergillus, Guignardia,

Neosartorya and Eupenicillium. Antimicrobial activities of these 12 fungal endophytes

were tested againstgram positive bacteria (Bacillus subtilis and Staphylococcus aureus

among others), gram negative bacteria (Escherichia coli among others), yeast

(Saccharomyces cerevisiae) and fungi (Candida albicans and Aspergillus niger). Two

strains; Isolate 7 and Isolate 13 (related to Guignardia sp. and Neosartoya sp., respectively)

showed strong antimicrobial (and antifungal) activity whereas the rest showed no activity

based on the formation of a clear zone of inhibition indicative of a positive activity.

84

Compounds were isolated from the extracts of both isolates and screened using HPLC.

Both isolates displayed chemically very interesting chromatograms as they possess a high

diversity of basic chemical structures and peaks over a wide range of polarities. In the ethyl

acetate extract of Isolate 7, compounds with structures similar to Pavetannin A1Ac,

Epicatechin, and 9alpha-OH-Pinoresinol were identified. In the ethyl acetate extract of

Isolate 13, compounds with structures similar to Trimeric Catechin, Epicatechin, and

Helenalin were identified.

Keywords: Mangroves; endophytic fungi; bioactive compounds; antimicrobial

INTRODUCTION

Natural products have been gaining attention in the search for novel drugs. They are

naturally derived bioactive compounds and by-products from microorganisms, plants or

animals which pose no toxicity or harm in the prevention of diseases (Tenguria, Khan &

Quereshi 2011).

Endophytic fungi reside within living tissues of plants without causing any adverse effects

towards the host plant itself (Kaul et al. 2008; Tran et al. 2010). Mangrove endophytic

fungi are increasingly recognized for their ability to produce bioactive compounds with

anti-cancer, anti-diabetic, and antimicrobial properties which are of pharmacological

importance (Strobel & Daisy 2003; Lu et al. 2010).

The genus Avicennia contains about 15 species and grows in the intertidal zone of coastal

mangrove forests distributed widely throughout tropical and warm temperate regions of the

world (Duke et al. 1998). Plants of this genus such as Avicennia marina were reported to

display antimicrobial and cytotoxic activities against carcinoma cell lines, and were

reported to be associated with endophytic fungi (Xu et al. 2005). Their adaptation to the

unique mangrove environment and the production of bioactive compounds has been linked

to their symbiotic relationship with the endophytes (Elavarasi & Kalaiselvam 2011).

85

With that, research on the bioactive compounds of endophytes can reveal the association

between the endophytes and their host plant which would promise new discoveries of

potentially life saving drugs. Thus the present study focuses on isolating, identifying and

screening endophytic fungi for antimicrobial activity as they have been displaying great

potential for discovery of new pharmacologically active metabolites. The endophytic fungi

with activity were then selected to further evaluate theirbiological activity and to identify

the bioactive compound which gives rise to the observed activity.

MATERIALS AND METHODS

Isolation of Endophytic Fungi

Endophytic fungi were isolated from plant materials (Avicennia sp.) which were collected

from Kampung Pasir Pandak, Sarawak. The protocol was adapted from Ebada and

colleagues (2008). Plant and leaf samples were surface sterilized and cultured onto potato

dextrose agar and incubated at 28oC for 1 week. After incubation period, hyphal tips of

fungi growing out from the plant fragments were transferred to new PDA plates.

Identification of Endophytic Fungi

The endophytic fungi were identified using molecular tools. Genomic DNA was extracted

from 5-day old fungi cultures grown on plates using a modified thermolysis method (Zhang

et al. 2010). Fungal DNA was amplified using universal primers of fungal DNA ITS1 (5’–

TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’–TCC TCC GCT TAT TGA TAT

GC-3’; 1st Base, Malaysia). Each sample ready for amplification contained 2 µl of 10X

PCR buffer (Fermentas), 1.2 µl of dNTP mixture (2.5mmol l-1 each), 0.8 µl of

formamidede ion, 0.4 µl of MgCl2 (25mmol l-1), 0.8 µl of each primer (10µmol l-1), 0.2 µl

of Taq DNA polymerase (5 U µl-1) and 1 µl of genomic DNA in a total volume of 20 µl.

PCR was carried out with denaturation at 94oC for 50 sec, annealing at 54oC for 50 sec and

elongation at 72oC for 50 sec. This was conducted in 35 cycles and the final elongation

reaction was set at 72oC for 10 min. PCR products were purified using PureLink PCR

purification kit (Invitrogen, U.S.) and sent for sequencing to the Beijing Genomic Institute,

BGI, China.

86

Antimicrobial Assay

Test microorganisms included four Gram-positive (Bacillus cereus, Bacillus subtilis,

Staphylococcus aureus, and Micrococcus luteus) and three Gram-negative (Escherichia

coli, Pseudomonas aeruginosa, and Vibrio anguillarum) bacteria, one yeast

(Saccharomyces cerevisiae) and two fungi (Candida albicans and Aspergillus niger). The

bacteria and yeast were grown in nutrient broth and incubated at 30oC for 24 hours whereas

the fungi were grown in potato dextrose broth and incubated at 25oC for 1 week.

The agar plug assay (Alias et al. 2010) was used to evaluate the antimicrobial activity

where cylindrical pieces of 1 x 1 cm size (agar plugs), cut from well grown and sporulated

cultures of one week old fungi strains were used. These pieces were placed on the agar

previously streaked with test organisms. For the antibacterial activity, plates were

incubated for 24 hours. For antifungal activity, agar plugs of the investigated fungi strains

were placed opposite of the fungi test pathogen and incubated for one week at 25oC.

Inhibition zones were measured after the incubation period. All tests were done in

triplicates.

Cytotoxic assayThe eggs of the brine shrimp, Artemia salina, were hatched in artificial seawater (38 g/L)

for 48 hours. Ethyl acetate extracts (in 10% DMSO) were diluted with artificial seawater to

obtain concentrations of 0.5, 5, 50 and 500ppm. A 96-well microtitre plate was used for

this analysis and 10 matured shrimps were applied to each well containing 50µl of the

extracts. The number of nauplii that died after 24 hours were counted and the lethal

concentration at which 50% of the nauplii die (LC50) was determined.

Extraction of Bioactive Compounds

A single cylindrical block (agar plug) from well grown and sporulated fungal cultures was

inoculated into 20 ml of potato dextrose broth (PDB) and incubated for one week at 25oC.

After the incubation period, 20 ml of ethyl acetate were added into the broth and left

standing for two hours. Then the mixture was filtered. The filtrate was then centrifuged at

8,000 rpm for 10 minutes and the top layer (Ethyl acetate phase) was removed and

87

transferred to new tubes. The extraction was repeated three times. The ethyl acetate extract

was then dried to give a solid and oily residue and the dried extract was stored at -20oC

until further use. Two fungal strains Isolate 7 and Isolate 13 who showed the strongest

activities were further cultivated in large volume for the extraction of bioactive compounds.

High-Performance Liquid Chromatography (HPLC)

Bioactive compounds in the ethyl acetate, methanol and n-hexane fractions were analysed

using UV-VIS High Performance Liquid Chromatography (HPLC; Dionex). 20 µl were

injected and runs performed over 60 minutes at 235, 254, 280 and 340nm. Structures of the

compounds were compared to library hits of similar structures. Future work will involve

isolation and identification of the individual compounds; however this was not in the scope

of this study.

RESULTS AND DISCUSSION


A total of twelve endophytic fungi were isolated from the plant samples (Avicennia sp.).

The twelve isolates were identified and found belonging to 7 families; Penicillium,

Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya, Cladosporium and

Eupenicillium (see Figure 27 for phylogenetic tree generated based on ITS sequences of

the fungal isolates and Table 11 for the phylogenetic results based after BLAST). Indeed,

the fungi population isolated from the species Avicennia sp. commonly consists of

Penicillium, Curvularia, and Aspergillus as reported by Madavasamy and Pannerselvam

(2012).

Antimicrobial Assay

Table 12 presents the fungal isolates that showed antimicrobial activity against the test

pathogens. Isolate 7 (related to Guignardia sp.) showed antibacterial activity against Gram

positive bacteria (Bacillus subtilisand Bacillus cereus,see Table 12 and Figure 28a) and

Gram negative bacteria (Vibrio anguilarum) with the presence of clear inhibition zones of

7-7.67 mm. Guignardia species are endophytes commonly isolated from mangrove plants

88

(Bhimba et al. 2011; Xia et al. 2009; Silva et al. 2011). This species was reported to be

isolated for the first time from the plant Undaria pinnatifida, having antibacterial and

antifungal activity (Wang 2012). Hence, the findings showing that Guignardia with

antibacterial activity supported the results of this study showing Isolate 7 having

antibacterial activity against Gram positive bacteria.

Isolate 13 (related to Neosartorya sp.) showed comparatively stronger antibacterial activity

against Gram positive bacteria (Micrococcus luteus, inhibition zone of 9.67 mm) and

antifungal activity against fungi (Candida albicans, inhibition zone of 10.67 mm; see

Table 12 and Figure 28b). The genus Neosartorya belongs to the family Trichocomaceae

(Varga et al. 2000) and Galgoczy and colleagues (2011) reported on a novel antifungal

peptide isolated from the Neosartorya fischeri and this antifungal peptide exhibited high

antifungal activity against filamentous fungi within broad pH and temperature ranges. This

finding by Galgoczy and colleagues again supports the results of this study where Isolate

13 (related to Neosartorya sp.) was found to show strong antifungal activity against

Candida albicans.

Besides, the antimicrobial results of this study showed that the antibacterial activity of the

isolates was more common towards Gram positive bacteria compared to Gram negative

bacteria. The higher resistance level of the Gram negative bacteria compared to Gram

positivecan be attributed to the differences in cell wall structure of Gram-positive bacteria

which are less complex compared to the outer membrane present in Gram-negative bacteria

thought to act as an additional barrier against antibiotics as also reported by Alias and

colleagues (2010).

Cytotoxic assay

The cytotoxic assay was done by testing the ethyl acetate extracts for each isolate obtained

after 1 week incubation and extraction against matured shrimps at different concentrations.

Table 13 shows that two fungal strains, Isolate 3 (related to Diaporthe sp.) and Isolate 9

(related to Eupenicillium sp.) displayed toxicity against the matured brine shrimps at

concentrations of 500 ppm after 24 hours incubation. Isolate 3 showed a significantly

89

stronger cytotoxicity and was able to kill 100% of brine shrimps, whereas Isolate 9 only

killed 10% (at 500ppm). The lethal concentration at which 50% of the nauplii die (LC50)

could not be determined.

Brine shrimps lethality assay is said to be a rapid and useful method for preliminary

screening of cytotoxic activity as it has been used in detection of fungal toxins, plant

extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing

(Carballo et al. 2002; Manilal et al. 2009). This was supported by Lin et al. (2005) who

reported in their study on Diaporthe sp.-an endophytic fungus isolated from leaves of

Kandelia candel plant of the mangroves in China-cytotoxic activity against lymphoma cell

lines. Eupenicillium sp. was reported by Davis et al. (2008) to exhibit strong cytotoxic

activity against human colorectal carcinoma and human lung carcinoma cells through the

production of a bioactive compound known as trichodermamide C.

Besides, extraction of bioactive compounds was performed for extracts of all fungal strains

(from Isolate 1 till Isolate 13). Particularly, for these two fungal strains, Isolate 3 (related to

Diaporthe sp.) and Isolate 9 (related to Eupenicillium sp.) which displayed toxicity against

the matured brine shrimps, the compounds extracted showed great interest. In this study, a

compound of a similar structure with Kahalalide B was extracted from fungal strain Isolate

3 (closely related to Diaporthe sp.). This compound has been reported in marine molluscs

but not yet been reported to be isolated from fungal endophytes. So far, only Kahalalide F,

a new marine-derived compound, was reported as a novel antitumor drug which showed

potent cytotoxicity activity against a panel of human prostate and breast cancer cell lines

(Suarez et al. 2003) and not Kahalalide B. Hence, itis therefore highly promising and

warrants further studies to isolate the compound and enumerate its structure, as this

compound might be responsible for the cytotoxicity activity of the fungal strain Isolate 3

(closely related to Diaporthe sp.).

Besides, fungal strain Isolate 9 (closely related to Eupenicillium) also displayed toxicity

and a compound with a structure similar to 3,4-dihydromanzamine was isolated. This

compound might be responsible for the cytotoxicity activity of the fungal strain as further

90

supported with a finding by Kobayashi and colleagues (1994) who reported on the

compound, 3,4-dihydromanzamine being isolated from the marine sponge, Amphimedon

sp collected from the Kerama Islands, Okinawa, Japan, and showed cytotoxic activity

against L1210 and KB cell lines. Yet, this is another one of the literatures that cited on

compounds isolated from marine sponges.

Extraction of Bioactive Compounds

Bioactive compounds from the extracts of both fungal isolates were screened using HPLC.

Both isolates displayed chemically very interesting chromatograms as they possess a high

diversity of basic chemical structures and peaks over a wide range of polarities (see Figure

29). In the ethyl acetate extract of Isolate 7, three compounds with structures similar to

Pavetannin A1 Ac (with a retention time of 2.56 min, Figure 30a), Epicatechin (with a

retention time of 38.77 min, Figure 30b), and 9alpha-OH-Pinoresinol (with a retention

time of 37.50 min, Figure 30c) were identified. The other 23 compounds found in the

spectrum were not identifiable and require further analyses by nuclear magnetic resonance

spectroscopy. It is noteworthy that the spectrum contained not only one major compound

but a few and over a wide range of polarity. This general picture might help explain why

Isolate 7 shows activity towards a wide range of organisms (Gram positive and Gram

negative bacteria).

A similar spectrogram was observed for Isolate 13 with several major compounds over a

wide range of polarity and the majority of compounds of an unknown nature. Again, this

might explain why Isolate 13 was able to inhibit the growth of Gram positive bacteria as

well as fungi (see Table 12).

Epicatechin is a flavanoid that has been reported to be responsible for antibacterial activity

against Gram-positive and Gram-negative bacteria. This compound was isolated by Masika

and colleagues (2004) from Schotia latifolia, a plant commonly used in folkloric medicine.

The fungal metabolite with a similar structure to Epicatechin most likely possesses a

similar chromophore and could be responsible for the observed antibacterial activity

against Gram-positive (Bacillus cereus and Bacillus subtilis) and Gram-negative (Vibrio

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anguillarum) bacteria. 9alpha-OH-Pinoresinol was reported as a lignin with anticancer

activity (Chunsriimyatav et al. 2009); however, Isolate 7 did not show any cytotoxic

activity in our study and the fungal metabolite with a similar structure might therefore not

be cytotoxic.

Similar to Epicatechin, Pavetannin A1 is usually found in plant and not fungi. Pavetannin

A1, has previously been reported from studies on the antiviral properties of Pavettao

wariensis and showed activity against Herpes simplex (Arnasan, Mata & Romeo 1995).

Antiviral tests were however not scope of the present study but the finding of a compound

with a similar structure in endophytic fungi is interesting nonetheless and warrants further

studies.

The ethyl acetate extract of Isolate13 also containedthree compounds that displayed

structures similar to known ones; Trimeric Catechin with a retention time of 37.53 min

(Figure 31a), Epicatechin with a retention time of 38.76 min (Figure 31b), and Helenalin

with a retention time of 40.88 min (Figure 31c).

Isolate 13 was found to display antibacterial activity against Gram-positive bacteria

(Micrococcus luteus) which might again be attributed to the compound with a similar

structure as epicatechin, as discussed above. Furthermore, a compound with a structure

similar to trimeric catechin was found in Isolate 13 extracts. This is catechin in its trimeric

form (also known as oligomeric form). Catechins are polyphenols and components of

condensed tannins which display antibacterial activity by precipitating proteins of

pathogenic bacteria through direct binding (Shimamura, Zhao & Hu 2007). Besides,

catechin was also reported to possess antifungal activity against Candida albicans

(Hirasawa & Takada 2004). These findings are in agreement with our results as Isolate 13

displayed activity against Gram positive bacteria as well as fungi (Table 12).

Another compound isolated displayed structure-similarity to Helenalin, a sesquiterpene

lactone commonly isolated from plant families such as Acanthaceae, Anacardiaceae,

Apiaceae, Euphorbiaceae, Lauraceae, Magnoliaceaa and others (Chaturvedi 2011) with

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anti-inflammatory and antineoplastic activity. Anti-inflammatory tests were not scope of

the present study but the finding of a compound with a similar structure in endophytic fungi

is again interesting and also warrants further studies.

The main difference between the activities observed by Isolate 7 and Isolate 13 is that

Isolate 13 displayed antifungal activity which might be explained by the existence of

compounds similar to catechin in its trimeric form. There is however no conclusive answer

possible based on the data available and future studies will aim to isolate the individual

compounds and identify and test them.

CONCLUSION

Our results indicate the potential of mangrove endophytic fungi in producing bioactive

compounds and further studies will be necessary to identify the unknown compounds found

in our isolates.

ACKNOWLEDGEMENT

The study was supported by a MOHE MyBrain15 scholarship.

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TABLESTable 11: 18S rRNA phylogenetic results for endophytic fungi

FUNGAL STRAINS CLOSEST RELATIVE IDENTITIES PHYLOGENETICDIVISION


399 / 409 (98%) Dikarya

Isolate 2 Curvularia affinis isolateS255 [HM770741]

469 / 469 (100%) Dikarya

Isolate 3 Diaporthe sp. SAB-2009astrain Q1160 [FJ799940]

454 / 459 (99%) Dikarya

Isolate 4 Diaporthe sp. 138SD/T[GU066697]

471 / 473 (99%) Dikarya

Isolate 5 Penicillium citrinumstrain SGE29 [JX232276]

408 / 408 (100%) Dikarya

Isolate 6 Aspergillus sp. Da91[HM991178]

501 / 501 (100%) Dikarya

Isolate 7 Guignardia mangiferaestrain SCIW10[HM150733]

426 / 439 (97%) Dikarya

Isolate 8 Neosartorya strameniaisolate NRRL 4652

[EF669984]

349 / 357 (98%) Dikarya

Isolate 9 Eupenicillium sp. 5 JH-2010 culture-collection

CBS:118134[GU981610]

447 / 449 (99%) Dikarya


399 / 409 (98%) Dikarya

Isolate 12 Cladosporiumsphaerospermum strain

SCSGAF0054[JN851005]

478 / 479 (99%) Dikarya

Isolate 13 Neosartorya hiratsukaestrain KACC 41127

[JN943580]

460 / 464 (99%) Dikarya

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Table 12: Antimicrobial activity of endophytic fungi strains




Isolate 7 7.33+ 0.58

7.00

+ 1.00

0 + 0 0 + 0 0 + 0 0 + 0 7.67

+ 0.58

0 + 0 0 + 0 0 + 0

Isolate 13 0 + 0 0 + 0 0 + 0 9.67 +1.53

0 + 0 0 + 0 0 + 0 10.67+ 0.58

0 + 0 0 + 0

Table 13: Mortality of the brine shrimps at different concentration of crude extract

Strains Mortality at different Concentration (%)

500 ppm 50 ppm 5 ppm 0.5 ppm

Isolate 3 0% 100% 100% 100%

Isolate 9 90% 100% 100% 100%

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FIGURES

Figure 27: 18S gene-based phylogenetic tree representing the twelve endophytic fungal isolates. The phylogenetic treewas generatedwith distance methods, and sequence distances were estimated with the neighbor-joining method.Bootstrap values ≥50 are shown and accession numbers for the reference sequences are indicated.

96

(a) (b)

Figure 28: Zone of inhibition (ZOI) for strains Isolate 7 and Isolate 13. (a) Strain Isolate7 against Bacillus cereus; (b)Strain Isolate 13 against Candida albicans. Scale is indicated at the bottom.

5mm 5mm

97

(a)

(b)

Figure 29: HPLC chromatograms of Ethyl Acetate extracts of (a) Isolate 7 and (b) Isolate 13 recorded at 235 nm.

98

(a)

(b)

(c)

Figure 30: HPLC chromatograms of compounds from Isolate 7 that had similar structures to (a) Pavetannin A1 Ac, (b)Epicatechin, and (c) 9alpha-OH-Pinoresinol. Chromatograms were recorded at 235 nm and library hits are indicated atthe top right of the picture.

99

(a)

(b)

(c)

Figure 31: HPLC chromatograms of compounds from Isolate13 that had similar structures to (a) Trimeric Catechin, (b)Epicatechin, and (c) Helenalin. Chromatograms were recorded at 235 nm and library hits are indicated at the top rightof the picture.

100

5. BIOSORPTION OF COPPER (CU) AND ZINC (ZN) BY

MANGROVE ENDOPHYTIC FUNGI

May-Ling ONN1*, Po-Teen LIM2, AAZANI MUJAHID2, and MORITZ MÜLLER1

1Biotechnology, School of Engineering, Computing and Science, Swinburne University of

Technology, Sarawak Campus, 93350 Kuching, Malaysia2Department of Aquatic Science, Faculty of Resource Science and Technology, Universiti

Malaysia Sarawak, 93400 Kota Samarahan, Malaysia.

Email:[email protected]

Keywords: Mangroves; endophytic fungi; heavy metals; biosorption; Copper (Cu); Zinc

(Zn)

Submitted to Marine & Freshwater Research (Manuscript ID: MF12341)

ABSTRACT

Endophytic fungi are fungi that live inside the tissues of other organisms such as

mangrove plants. These endophytic fungi support their hosts in adapting to (extreme)

environments, for example by removing harmful heavy metals. The presence of heavy

metals can lead to severe damage as they are bioaccumulative and toxic. Many

approaches were made towards removing heavy metals from the environment and

biosorption has been found to be a cost-effective and simple method. Biosorption

involves the use of microbial cells (live or dead biomass) to absorb and accumulate heavy

metals. In this study, we evaluated the potential of twelve endophytic fungi that were

isolated from a mangrove plant (related to Avicennia sp.) as biosorption material (both

live and dead biomass) for the heavy metals copper (Cu) and Zinc (Zn). Isolate Sp. 2,

which is closely related to Curvularia sp., is the most efficient in removing Cu, up to

25mg Cu/g biomass (using dead biomass). On the other hand, Isolate 8 and Isolate 13

(both related to Neosartorya sp.) are the most efficient in removing zinc (also using dead

biomass), with a removal of up to 24 mg Zn/g biomass. The findings clearly indicate the

potential of mangrove endophytic fungi for biosorption purposes.

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INTRODUCTION

Trace metals such as copper (Cu) and zinc (Zn) play a biochemical role in the life

processes of all aquatic plants and animal, hence should be present in the environment in

trace amounts (Saeed and Shaker 2008). However, in high enough concentrations, both

metals become detrimental to human health and unfortunately, they have been

continuously released into the environment as a result of the industrial activities and

technological development (Iskandar et al. 2011). Intensive mining and processing

activities have resulted in heavy metal pollution which poses a significant threat to the

environment, public and soil health (Petrisor et al. 2002; Iskandar et al. 2011). Copper in

excess has been associated with liver diseases and acute gastrointestinal infections (Stern

et al. 2007). This toxic heavy metal is widely used for microbial control especially in the

agriculture sector and high concentrations remain especially in soils. On the other hand,

excess zinc can be associated with system dysfunctions resulting in impairment of growth

and the reproduction system (Nies 1999; Duruibe et al. 2007).

Many conventional methods were developed to remove heavy metal ions such as

filtration, chemical precipitation, electrochemical treatment, ion exchange, oxidation or

reduction, reverse osmosis, and evaporation recovery (El-Gendy et al. 2011). Some of

these methods are complex and expensive, and frequently resulting in the production of

toxic products which would then become another source of environmental pollution

(Kannan, Hemambika & Rani 2011; Leitao 2009).

Biosorption is a physiochemical process that occurs naturally in certain biomass which

allows immobilization of metals through binding of the contaminants onto cellular

structures (Sameera et al. 2011). The advantages of biosorption over conventional

methods are the low cost, high efficiency in removing metal from dilute solution,

minimization of chemical use, no additional requirement of additives or nutrients,

regeneration of biosorbent and the possibility of metal recovery (Kumar et al. 2009).

Fungi are considered as the most promising adsorbant, whose cell walls and their

components have a major role in biosorption. It has been reported that fungal biomass

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can take up considerable quantities of organic pollutants from aqueous solution by

adsorption, even in the absence of physiological activity. Many fungal species have been

studied for their heavy metal biosorption ability, for instance, Rhizopus arrhizus,

Aspergillus niger and others (Sameera et al. 2011; Kannan, Hemambika & Rani 2011).

Microorganisms can take up metal either actively (live biomass) through bioaccumulation

and/or passively (dead biomass) through biosorption (Kannan, Hemambika & Rani

2011).

Fungi under stress develop several mechanisms to tolerate the mangrove adverse

conditions which unfold a potential source for biotechnological applications, including

the search for new endophytic species of environmental importance, for instance, with

potential for bioremediation application for polluted environments. This study aims to

isolate and identify endophytic fungi associated with the mangrove plant Avicennia sp.,

and assess their potential to biosorb the heavy metals copper (Cu) and zinc (Zn) as well

as their minimum inhibitory concentration (MIC) based on dry biomass weight.

MATERIALS & METHODS

Isolation of Endophytic Fungi

Endophytic fungi were isolated from Avicennia sp. collected at Kampung Pasir Pandak,

Sarawak. The plant and leaf samples were surface sterilized using a modified method by

Kumaresan & Suryanarayanan (2002) and cultured onto potato dextrose agar and

incubated at 28oC for one week. After the incubation period, hyphal tips of fungi growing

out from the plant fragments were transferred to new PDA plates for purification of the

strains.

The soil samples were analysed for endophytic fungi using a modified method based on

Nopparat et al. (2007), in which the Pikovskaya agar is substituted with PDA. After a few

days of incubation, fungal colonies that were seen growing were selected and re-

inoculated on PDA agar for purification of fungi cultures.

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The endophytic fungi were identified using molecular tools. Genomic DNA was

extracted from 5-day old fungi cultures grown on plates using a modified thermolysis

method (Zhang etal.2010). Fungal DNA was amplified using universal primers of fungal

DNA ITS1 (5’–TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’–TCC TCC GCT

TAT TGA TAT GC-3’; 1st Base, Malaysia). Each sample ready for amplification

contained 2 µl of 10X PCR buffer (Fermentas), 1.2 µl of dNTP mixture (2.5mmol l-1

each), 0.8 µl of deioned formamide, 0.4 µl of MgCl2 (25mmol l-1), 0.8 µl of each primer

(10µmol l-1), 0.2 µl of Taq DNA polymerase (5 U µl-1) and 1 µl of genomic DNA in a

total volume of 20 µl. PCR was carried out with denaturation at 94oC for 50 seconds,

annealing at 54oC for 50 seconds and elongation at 72oC for 50 seconds. This was

conducted in 35 cycles and the final elongation reaction was set at 72oC for 10 minutes.

PCR products were purified using PureLink PCR purification kit (Invitrogen, U.S.) and

sent for sequencing to the Beijing Genomic Institute, BGI, China.

Preparation of reagents and materials

For the determination of heavy metal-resistant fungi, heavy metal Copper Nitrate and

Zinc Nitrate solution were prepared, filtered and added separately to Potato Dextrose

Broth (PDB) to achieve final Cu or Zn concentrations of 50 to 200 µg/ml.

For adsorption by dead fungal cells, working standards of 50 µg/ml copper and zinc ion

solutions in 150mM NaCl solution (added to prevent cell damage caused by osmotic

pressure) were prepared. To obtain the dried biomass, the dead fungal cells were dried

and then ground using mortar and pestle to obtain 0.1g and subsequently passed through

a 0.45 µm sieve to standardize the particle size.

Determination of heavy metal-resistant fungi

The prepared (heavy metal solution and broth) mixture of varying concentration was

inoculated with 1 cm2 agar plugs from young fungal colonies that were pre-grown on

PDA plates for 5 days. Three replicates of each concentration and controls without metal

were prepared. The inoculated mixture was then incubated at 25oC for one week under

104

static conditions. The mixture solution was filtered using sterile filter paper (Whatman

filters No.1) and the biomass obtained was dried in the oven at 60oC. The dried biomass

was then weighed and its dry weight obtained. The minimum inhibitory concentration

(MIC) was determined based on the percentage (%) of biomass dry weight.

Biosorption studies by dead fungal cells

For adsorption by dead fungal cells, 0.1 g of the prepared dried biomass was added to the

working standards (heavy metal ion solution) and incubated at 150 rpm and 30oC for 72

hours in the dark. Samples were filtered using sterile filter paper (Whatman filters No.1)

and cell-free filtrates obtained were analysed for the remaining Cu (µg/ml) using atomic

absorption spectrometry (AAS) (Kannan, Hemambika & Rani 2011). Bioadsorption

capacity was measured based on the amount of metal ions (mg) bioadsorbed per gm (dry

mass) of biomass calculated using the following equation:

Q = [(Ci – Cf)/m)] V

where Q = mg of metal ion bioadsorbed per gm of biomass, Ci = initial metal ion concentration, mg/L, m =

mass of biomass in the reaction mixture gm, V = volume of the reaction mixture (L)

RESULTSAND DISCUSSION


A total of twelve endophytic fungi were isolated from the plant samples (Avicennia sp.).

The twelve isolates were identified and found belonging to 7 genus; Penicillium,

Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya, Cladosporium and

Eupenicillium (see Figure 32 for phylogenetic tree). Indeed, the fungi population isolated

from the species Avicennia sp. commonly consists of Penicillium, Curvularia, and

Aspergillus as reported by Madavasamy and Pannerselvam (2012). The phylogenetic tree

(Figure 32) was generated based on ITS sequences of the fungal isolates.

Heavy metal-resistant fungi

The ability of the endophytic fungi to resist the heavy metal (also known as minimum

inhibition concentration (MIC)) was determined from the dry weight of the biomass

present. The MIC varied for all the endophytic fungi tested with Isolate 3 (which is

105

closely related to Diaporthe) showing the highest resistance to Cu (See Table 14 for Cu-

MIC and Zn-MIC). It was able to grow in concentrations up to 600 µg/ml (Table 14).

Isolate 12 (which is closely related to Cladosporium) and Isolate 8 (which is closely

related to Neosartorya) showed the lowest resistance towards Cu and both were only able

to grow up to a concentration of 50 µg/ml (Table 14).

Intriguingly, the isolate with the highest MIC towards Cu (Isolate 3) is closely related to

Diaporthe sp. and there are no other reports of Cu-resistant Diaporthe sp. in the

literature. However, as seen from the phylogenetic tree (Figure 32), this species is found

closely related to Phomopsis sp.,and a study published by the European Food Safety

Authority, EFSA (2012), showed that phomopsins (a family of mycotoxins) produced by

the fungus Diaporthtoxica (formerly referred to as Phomopsis leptostromiformis) might

be responsible for the accumulation of copper in the liver. Besides, Phomopsis sp. is an

ascomycetes filamentous fungus which were reported by Saiano et al. (2005) to be able to

complex various metal species from aqueous media, mainly due to the presence of

chitosan in its cell wall.

On the other hand, Isolate 5 and Isolate 9 showed the highest resistance towards Zn with

an MIC of 20,000 µg/ml and Isolate10 showed the lowest resistance towards Zn and was

only able to grow up to a concentration of 100 µg/ml (Table14).

From both the Table14, it can be seen that the endophytic fungal isolates were all more

resistant towards Zn than Cu. The MIC is much higher in Zn (20,000) compared to the

MIC of Cu (600). Zn is considered an essential metal for all organisms which might help

to explain the higher MIC of Zn as compared to Cu (Lairini et al. 2009). Besides, this

finding is further supported by Hartikainen and colleagues (2012) whose study on the

impact of copper and zinc showed that Cu was more toxic than Zn to the ascomycetous

(Fusarium sp. and Alternaria sp. were among those tested) and basidiomycetous fungi

tested. They concluded that Cu might have a greater impact than Zn on the competition

between fungal species and therefore on the structure of fungal communities in

contaminated soil. However, the lack of another obvious trend in the MIC values

106

suggests that the resistance level against the individual metals very much depends on the

individual fungal isolate.

The two isolates showing the least resistance towards Cu (Isolate 12 and Isolate 8) were

closely related to Cladosporium sp. and Neosartorya sp., respectively. Dong (2006)

reported on the benefits of increasing the Cu adsorption ability of Cladosporium sp.

through chemical pretreatment where the biomass pretreated with 0.2M NaOH solution

for 40 min resulted in a significant improvement of Cu2+ removal in comparison with the

native biomass. This approach was not tested in this study but could potentially lead to a

higher removal capacity and therefore higher MIC for Isolate 12.

For Neosartorya, only one study so far that showed this families’ bioaccumulation

capacity using live biomass where the species Neosartorya fischeri was found more

efficient in removing Cu compared to Zn (Simonovicova 2008). However, Isolate 8 was

found to be closely related to Neosartorya stramenia (Figure 32) and despite belonging

to the same genus, this species seems to differ in its mechanism in tolerating Cu and Zn.

Isolate 9 isolate which is closely related to Eupenicillium sp. also showed relatively high

resistance towards Cu (MIC of 300µg/ml, see Table 14). It was reported that

Eupenicillium sp. and Talaromyces sp. are telemorphic states of the Penicillium genus

(Visagie and Jacobs, 2009) and this might explain why Isolate 9 showed a MIC to Cu

similar to those of Isolate 5 and Isolate 10 (200µg/ml) which all belong to the Penicillium

genus.

Isolate 5 also showed the highest tolerance towards Zn (together with Isolate 9, see Table

14). Findings by Lairini et al. (2009) support our results as their study indicated

Penicillium sp. tolerance towards zinc with MICs in the range of 7.5mM-25mM

(1420.2µg/ml – 4734.0µg/ml). For this study, the MIC of Isolate 5 and Isolate 9 were

20,000µg/ml.

107

However, isolates that show high resistance towards one of the heavy metals tested do

not necessarily show a high resistance towards the other one. Isolate 10 which is closely

related to Penicillium sp. for example, showed a moderate tolerance level towards Cu of

200 µg/ml, but displayed the least resistance towards Zn (100µg/ml, Table 1.2). This is in

agreement with a study by Lairini et al. 2009 who reported that isolates of the same genus

can display a marked difference in the levels of metal resistance. They attributed this to

the presence of different tolerance or resistance mechanisms exhibited by different fungal

isolates, especially when alive (Lairini et al. 2009). Living fungal biomass biosorption

processes are complicated to control and understand as bioaccumulation of heavy metals

isalso driven and influenced by changes and differences in growth, metabolic energy and

transport needs (Leitao 2009).

Another approach of using fungi for biosorption purposes is to use their dead biomass

and we discuss results for this approach in the following.

Heavy metal biosorption by dead fungal cells

Heavy metal removal using non-living biomass is less complicated, due to the absence of

metabolic activity and based on Table 15, three isolates were observed with maximum

biosorption capacity. Isolate 2 was the most efficient with regards to Cu and was able to

remove up to 25mg Cu/g biomass (see Table 15) while Isolate 8 and Isolate 13 were able

to remove up to 24 mg Zn/g biomass (Table 15).

Isolate 2 which was found to be the most efficient in removing Cu/g biomass is closely

related to Curvularia sp. (Figure 32) and –to our knowledge- this is the first reported

study on the ability of Curvularia sp. in removing heavy metal using dead biomass. Even

though no further experiments were performed to identify the mechanism by which the

isolate biosorps Cu and Zn, our results seem to indicate that the dead biomass of Isolate 2

is capable in adsorbing Cu (as indicated by high Q value).

108

On the other hand, for Zn biosorption capacity, Isolate 8 and Isolate 13 (both closely

related to Neosartorya sp.) were found to be the most efficient in removing Zn/g biomass

(Q value of 24mg Zn/g; Table 15). As mentioned earlier, to our knowledge, so far only

one study has been published regards tolerance of Neosartorya sp. towards heavy metals,

and it involved live biomass.

Intriguingly, Isolate 1 which displayed moderate tolerance towards Cu (MIC value of

100µg/ml, Table 14), showed the second-highest Cu biosorption capacity (Q of 18,

Table 15) and lowest Zn biosorption capacity (Q of 3, Table 15) when used as dead

biomass. Isolate 1 is closely related to Penicillium sp. and we see the opposite in the

results for Isolate 10 which is also closely related to Penicillium sp. Live biomass of

Isolate 10 similarly had moderate tolerance towards Cu (MIC values of 200µg/ml), but,

when used as dead biomass, it displayed the lowest Cu biosorption capacity (Q values of

0mg/g, Table 15) and third-highest Zn biosorption capacity (Q values of 16mg/g, Table

15). Penicillium sp. is commonly known as a halotolerant genus isolated from mangroves

and salterns with high resistance towards metals such as copper (Leitao 2009). For this

case, although both strains were found closely related to Penicillium sp., the

morphological characteristics of both fungal strains were different (Figure 33).

Identification of Penicillium to species level requires multidisciplinary approaches

(Leitao 2009) which were beyond the scope of this study, however they should be carried

out in future on both isolates.

Besides Isolate 10 having the lowest Cu biosorption capacity, Isolate 6 (closely related to

Aspergillus sp., Figure 32) showed similar results of moderate tolerance towards Cu

(MIC value of 150µg/ml, Table 14) but lowest Cu biosorption capacity (Q value of 1mg

Cu/g, Table 15) when used as dead biomass. This result could be further supported with

the findings of Kannan and colleagues (2011), where Aspergillus sp. was found to be an

efficient strain resistant to Cu when in the form of live biomass. It is when tested for

biosorption of Cu using dead biomass, Aspergillus sp. had the ability to adsorb maximum

level of Cu after the cell fraction was treated with sodium hydroxide (NaOH). This was

due to the dead biomass comprising of small particles with lower density, poor

mechanical strength and little rigidity (Volesky and May-Philips 1995). Again, this

109

approach has not been tested in this study but could potentially lead to higher biosorption

capacities for Isolate 6 and Isolate 10.

In conclusion, the results of this study show that the biosorption capacity depends on the

type of species and their cell wall’s mechanism towards tolerating heavy metals.

Biosorption of metals involves several mechanisms that differ qualitatively and

quantitatively, according to the species used, the origin of the biomass, and its processing

procedure (Raize et al. 2004).

CONCLUSION

Our results show the high potential of mangrove endophytic fungi for the removal of

heavy metals, especiallyby using dried fungal biomass. These endophytic fungi with

heavy metal biosoption potential should be studied further to determine the active sites on

the cell surfaces as well as to assess their potential to absorb other heavy metals that are

known for their high levels of toxicity such as mercury, lead and even radioactive

substances.

ACKNOWLEDGEMENT

The study was supported by MOHE MyBrain15 scholarship.

110

TABLESTable 14: Minimum inhibitory concentration (MIC) of heavy metal Copper (Cu) and Zinc (Zn) in living biomass ofisolated endophytic fungi (in µg/ml). The most and the least resistant species are highlighted in bold, as are theirrespective MIC values.

Species

Isolate

1

Isolate

2

Isolate

3

Isolate

4

Isolate

5

Isolate

6

Isolate

7

Isolate

8

Isolate

9

Isolate

10

Isolate

12

Isolate

13

MIC

µg/ml

(Copper) 100 200 600 200 200 150 100 50 300 200 50 100

MIC

µg/ml

(Zinc) 200 10,000 400 1,000 20,000 10,000 200 2,000 20,000 100 200 200

Table 15: Copper (Cu) and Zinc (Zn) Biosorption capacity, Q, by dead fungal cells (calculated as amount of metal ions(mg) bioabsorbed per gm (dry mass)). The most efficient species is highlighted in bold, as is their respective Q value.

Species

Isolate

1

Isolate

2

Isolate

3

Isolate

4

Isolate

5

Isolate

6

Isolate

7

Isolate

8

Isolate

9

Isolate

10

Isolate

12

Isolate

13

Q value

(Cupper)

18 25 11 5 8 1 8 15 4 0 15 7

Q value

(Zinc)3 6 8 15 16 21 16 24 14 16 14 24

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FIGURES

Figure 32: 18S gene-based phylogenetic tree representing the twelve endophytic fungal isolates. The phylogenetic treewas generated with distance methods, and sequence distances were estimated with the neighbor-joining method.Bootstrap values ≥50 are shown and accession numbers for the reference sequences are indicated.

112

(a) Isolate1 (b) Isolate10

Figure 33: Two fungal strains: (a) Isolate 1 and (b) Isolate10 closely related to Penicillium dravuni but having differentmorphological characteristics and growth patterns where Isolate 10 grows at a faster rate within a week compared toIsolate 1, as seen from the pictures of both plates taken during 1 week incubation.

113

6. CONCLUSION

Plants have been known as potential sourcesfor novel drug compounds and many plant

extracts have been used as alternative forms of medical treatments since the late 1990s

(Vadlapudi & Naidu 2009). Mangroves are widespread in tropical and subtropical

regions, especially in Asia, and they are unique for their saline environment which

promises discoveries of biologically active compounds, for instance antiviral,

antibacterial and antifungal. Avicennia, a mangrove plant species, has been known to be a

source of many bioactive compounds which could be found within the bark, leaves, fruit

and also roots of the plants.

The present study shows that the bioactive properties of Avicennia collected from the

mangroves in Kampung Pasir Pandak, Kuching, Sarawak, might be due to the activity of

fungal endophytes foundwithin the leaves and roots of the mangrove plant. Two fungal

strains, Isolate 7 (closely related to Guignardia sp.) and Isolate 13 (closely related to

Eupenicillium sp.) were found to posess antimicrobial activity against gram positive and

gram negative bacteria as well as fungi. The antimicrobial activity was studied further by

isolating the bioactive compounds from the extracts of both the fungal strains.

A compound similar to epicatechin was isolated from the ethyl acetate extracts of both

fungal strains. Epicatechin was reported by Masika and colleagues (2004) as a compound

that might be responsible for the antibacterial activity and might be responsible for the

the antibacterial activity observed for both Isolate 7 and Isolate 13. Besides epicatechin,

another interesting finding was a compound with a similar structure to trimeric catechin,

isolated from fungal strain Isolate13, which has been shown to displayantifungal activity

against Candida albicans (Hirasawa and Takada, 2004) and might be responsible for the

antifungal activity of Isolate 13.

Furthermore, the fungal isolates were also tested for their cytotoxicity using brine

shrimps. Two fungal strains, Isolate 3 (related to Diaporthe sp.) and Isolate 9 (related to

Eupenicillium sp.) displayed toxicity against the matured brine shrimps at concentrations

of 500 ppm after 24 hours incubation. The bioactive compounds isolated from the

114

extracts of these two strains also showed interesting results. For instance, a compound of

a similar structure to Kahalalide B was extracted from fungal strain Isolate 3 which might

be responsible for the displayed cytotoxic activity. As Kahalalide F was reported as a

novel antitumor drug showing potent cytotoxicity activity against a panel of human

prostate and breast cancer cell lines, hence, it is highly promising and would require

further studies to isolate the compound and enumerate its structure. Besides, a compound

with a structure similar to 3,4-dihydromanzamine was isolated from fungal strain Isolate

9 which might be responsible for the cytotoxicity activity.

Our heavy metal biosorption experiments, which involved the use of microbial cells (live

and dead biomass) to absorb and accumulate heavy metals, showed highly promising

results. For instance, Isolate 2 (which is closely related to Curvularia sp.), ishighly

efficient in removing Cu, up to 25mg Cu/g biomass (using dead biomass) and fungal

strains Isolate 8 and Isolate 13 (both related to Neosartorya sp.) are the most efficient in

removing zinc (also using dead biomass), with a removal of up to 24 mg Zn/g biomass.

Further studies are required to understand the mechanism of the heavy metal uptake by

the fungal strains. By understanding the mechanism of uptake in more detail, we could

then improve the existing uptake mechanism and apply the process in larger scales for

application in wastewater remediation.

To conclude, we were able to show that different fungal endophytes fulfil different

important functions in Avicennia sp. and help the host with defence against microbes and

heavy metal stress.

115

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