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
P a g e | 1
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.
P a g e | 2
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)
P a g e | 3
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).
P a g e | 3
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).
P a g e | 3
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).
P a g e | 4
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).
P a g e | 5
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).
P a g e | 5
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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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
P a g e | 9
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
P a g e | 10
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
P a g e | 11
(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).
P a g e | 13
(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).
P a g e | 14
(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).
P a g e | 17
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
P a g e | 20
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
P a g e | 22
(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
P a g e | 23
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
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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).
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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
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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
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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 -
Isolate 10 Penicillium dravuni[AY494856]
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)
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 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
27 unknown compounds
-
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
13 unknown compounds
3 known compounds
microsphaerone B paxilline manzamin JN-Oxid
21 unknown compounds
-
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)
This compound was isolated from methanol extract of fungal strain, Isolate 2 (closely
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
This compound was isolated from methanol extract of fungal strain, Isolate 2 (closely
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
This compound was isolated from methanol extract of fungal strain, Isolate 2 (closely
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
This compound was isolated from methanol extract of fungal strain, Isolate 2 (closely
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).
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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.
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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.
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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).
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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.
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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
Identification of Endophytic Fungi
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
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(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
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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
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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
Isolate 1 Penicillium dravuni[AY494856]
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
Isolate 10 Penicillium dravuni[AY494856]
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
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 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.
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(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
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(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.
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(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.
101
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
102
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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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 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
Identification of Endophytic Fungi
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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