Screening of Mangrove Endophytic Fungi for Bioactive 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 Contents List 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 ENDOPHYTIC FUNGI 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 MANGROVE ENDOPHYTIC 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) ...... 2 Figure 2: Cephalosporin (source: websters-online-dictionary.org) ...... 2 Figure 3: Vancomycin (source: websters-online-dictionary.org) ...... 3 Figure 4: Doxorubicin (source: websters-online-dictionary.org)...... 3 Figure 5: Staurosporine (source: websters-online-dictionary.org)...... 3 Figure 6: Dolastatin (source: sigmaldrich.com)...... 5 Figure 7: Marinomycin (source: molecular-networks.com) ...... 5 Figure 8: Taxol...... 10 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)...... 13 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)...... 14 Figure 11: 5-Hydroxyramulosin (a), a polyketide compound extracted from an endophytic fungus morphologically similar to Phoma sp. (b)...... 15 Figure 12: Cytochalasin H2 (a), a new compound was extracted from the endophytic fungus, Xylaria sp. (b) which was isolated from Annona squamosa (c)...... 15 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)...... 16 Figure 14: Kampung Pasir Pandak (Sampling site) situated near Kampong Batu, indicated by the Blue Point (Source: Google Map)...... 26 Figure 15: Schematic overview of isolation of mangrove endophytic samples from plant samples...... 29 Figure 16: Schematic overview of isolation of mangrove endophytic samples from soil samples...... 30 Figure 17: Fungal Cultivation for short term storage, long term storage, and extraction of bioactive compounds...... 32 Figure 18:Endophytic fungi identification using molecular tools...... 35 VII

Figure 19: Extraction of bioactive compounds using ethyl acetate ...... 38 Figure 20:Extraction of bioactive compounds using solvent-solvent (methanol and n- hexane) extraction ...... 39 Figure 21: Primary and Secondary screening of antimicrobial activity...... 43 Figure 22:Cytotoxicity assay...... 44 Figure 23:Determination of heavy metal resistant fungi using minimum inhibitory concentration (MIC) and heavy metal biosorption ...... 47 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...... 49 Figure 25: Zone of inhibition (ZOI) for Isolate 7 and Isolate 13. (a) Isolate 7 against Bacillus cereus; (b) Isolate 13 against Candida albicans...... 54 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...... 56 Figure 27: 18S gene-based phylogenetic tree representing the twelve endophytic fungal isolates. The phylogenetic tree was 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...... 95 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...... 96 Figure 29: HPLC chromatograms of Ethyl Acetate extracts of (a) Isolate 7 and (b) Isolate 13 recorded at 235 nm...... 97 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 at the top right of the picture...... 98 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 right of the picture...... 99 Figure 32: 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...... 111 Figure 33: Two fungal strains: (a) Isolate 1 and (b) Isolate10 closely related to Penicillium dravuni but having different morphological characteristics and growth patterns where Isolate 10 grows at a faster rate within a week compared to Isolate 1, as seen from the pictures of both plates taken during 1 week incubation...... 112 VIII

List of Tables Table 1: Characteristics of the soil conditions of the three different sampling sites ...... 27 Table 2: Overview of the closest relatives found for each endophytic isolate, their query coverage in base pairs and %, as well as the source of the sample from which the isolate originates...... 48 Table 3: Antimicrobial activity of endophytic fungi strains (Primary screening) ...... 53 Table 4: Antimicrobial activity of endophytic fungi strains (Secondary Screening) ...... 55 Table 5: Mortality of brine shrimps observed at different concentrations (0.5, 5, 50 and 500 ppm) of crude extracts of fungal strains...... 57 Table 6: Overview of the amounts (in mg) obtained for each fraction...... 58 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...... 59 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...... 61 Table 9: Minimum inhibitory concentration (MIC) of heavy metal Copper (Cu) and Zinc (Zn) in living biomass of fungi ...... 77 Table 10: Copper (Cu) Biosorption capacity by dead fungal cells ...... 80 Table 11: 18S rRNA phylogenetic results for endophytic fungi ...... 93 Table 12: Antimicrobial activity of endophytic fungi strains ...... 94 Table 13: Mortality of the brine shrimps at different concentration of crude extract...... 94 Table 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 least resistant species are highlighted in bold, as are their respective MIC values...... 110 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...... 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

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 Figure 1: Penicillin(source: websters-online-dictionary.org) 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, Figure 2: Cephalosporin (source: websters-online- and Escherichia coli (Muniz et al. 2007). dictionary.org) P a g e | 3

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).

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

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)

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).

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

Dolastatin is one of the important marine cyanobacterial molecules that were discovered in preclinical testing as anticancer Figure 6: Dolastatin (source: sigmaldrich.com) agents. This compound was initially isolated from the sea hare (Tan 2006). Marinomycin is a polyketide with antibacterial and antitumor properties produced by marine actinomycete, Marinspora sp. (Olano, Mendez & Salas 2009; Lam 2006).

Figure 7: Marinomycin (source: molecular-networks.com) P a g e | 6

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, P a g e | 7

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 P a g e | 8

(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.

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)

Figure 8: Taxol 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 P a g e | 12

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

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

(a) ergosta-5,7,22-trien-3-ol (Zhao et al. 2012) 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). P a g e | 15

(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). P a g e | 16

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

(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 Mangroves Mangroves 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 P a g e | 18

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). P a g e | 19

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. P a g e | 21

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 pollution The 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 study The 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. P a g e | 26

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). P a g e | 28

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). P a g e | 29

Plant samples

Rinse with running tap water Air Dried

Cut into 1cm x 1cm fragments

Surface Sterilization

Surface Dried

70% ethanol 0.5% sodium Sterile distilled Placed Incubation at solution hypochlorite water onto PDA 28oC

3 mins 1 min 1 min

Figure 15: Schematic overview of isolation of mangrove endophytic samples from plant samples P a g e | 30

Soil samples

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

0.1ml of each dilution plated onto PDA agar

Incubation at 28oC

Figure 16: Schematic overview of isolation of mangrove endophytic samples from soil samples P a g e | 31

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. P a g e | 32

SHORT TERM STORAGE LONG TERM STORAGE EXTRACTION OF BIOACTIVE COMPOUNDS

Cylindrical pieces of pure fungal Barley media are placed in Cylindrical pieces of pure fungal cultures were grown on PDA at universal bottles and sterilized cultures were grown in 20 ml 25oC PDB

Cylindrical pieces of pure fungal Fungal hyphae covered ¾ of the cultures were placed in the sterilized Incubated at 25oC for one week surface of PDA barley media

Ready to be used with solvent Plates kept at 4oC until further use Incubated at 25oC for one week

Kept in 4oC for further usage

Figure 17: Fungal Cultivation for short term storage, long term storage, and extraction of bioactive compounds P a g e | 33

2.4 Endophytic fungi identification Molecular identification has made it possible to study theecology of fungi in their dominant but in conspicuous mycelial stage and not only by means of fruiting bodies (Bellemain et al. 2010). The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit has become the primary genetic marker for molecular identification of many groups of fungi (Nilsson et al. 2011). The entire ITS region has commonly been targeted with traditional Sanger sequencing approaches and typically ranges between 450 and 700 bp (Bellemain et al. 2010). The endophytic fungi were identified using molecular tools. Genomic DNA was extracted from 5-day old fungi cultures grown on plates using a modified thermolysis method (Zhang et al. 2010). The edge of the mycelium colony with the size of a sesame seed was picked using a sterilized toothpick and placed into a 1.5 ml microcentrifuge containing 100µl pure distilled water. The mixture was vortexed for 1 minute and then centrifuged at the speed of 8,000 g for 1 minute. The supernatant was discarded and 100 µl of Tris-EDTA (TE) (First Base, Malaysia) buffer was added into the tube. The tube was then immersed in water bath at 93oC for 20 minutes and stored at -20oC until use. Fungal DNA was amplified using universal primers of fungal DNA ITS1 (5’–TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’–TCC TCC GCT TAT TGA TAT GC-3’; 1st Base, Malaysia). Each sample ready for amplification contained 2 µl of 10X PCR buffer (Fermentas, Germany), 1.2 µl of dNTP mixture (2.5mmol l-1 each), 0.8 µl of deioned -1 -1 formamide, 0.4 µl of MgCl2 (25mmol l ), 0.8 µl of each primer (10µmol l ), 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. P a g e | 34

DNA fragments were purified using PureLink PCR purification kit (Invitrogen, U.S.) following the protocol provided by the supplier and then sent for sequencing to the Beijing Genomic Institute, BGI, China.Nucleotide sequences were determined using the dideoxynucleotide method by cycle sequencing of the purified PCR products and sequences were analyzed against the NCBI database. Sequences were aligned and phylogenetic trees were created with MEGA5 using the neighbor-joining method (see Figure 18 for an overview; Manikprabhu & Lingappa, 2012). P a g e | 35

1)EXTRACTION OF GENOMIC DNA 2)DNA AMPLIFICATION 3)SEQUENCING

Edge of the mycelium colony was PCR mixture was prepared: PCR mixture were purified using placed into microcentrifuge tube PureLink PCR purification kit containing 100µl pure distilled water - 2 µl of 10X PCR buffer - 1.2 µl of dNTP mixture - 0.8 µl of deionedformamide Sent for sequencing to the Sarawak Biodiversity Centre Mixture was vortexed and - 0.4 µl of MgCl2 centrifuged - 0.8 µl of each primer - 0.2 µl of Taq DNA polymerase Sequences obtained were analyzed Supernatant was discarded and - 1 µl of genomic DNA against the NCBI database 100µl TE buffer was added PCR cycle was run: Sequences were aligned and Supernatant was discarded and 100µl TE o phylogenetic tree was constructed buffer was added a) Initial denaturation (94 C - 5 mins) b) Denaturation (94oC - 50 s) c) Annealing (54oC - 50 s) Tube was immersed in water bath at o o o d) Elongation (72 C - 50 s) 93 C for 2 minutes before stored at 4 C o e) Final Elongation (72 C - 10 mins) f) Storage (4oC - until use)

Figure 18:Endophytic fungi identification using molecular tools P a g e | 36

2.5 Extraction of bioactive compounds Crude extract from each fungal isolate was extracted using ethyl acetate solvent. This extraction method is particularly useful for extraction of extracellular (excreted by fungi into the medium) and intracellular bioactive compounds. 20 ml of ethyl acetate was added into the fungal broth that was cultivated as described in 2.3.3 and left standing for two hours. The mixture was then filtered with the mycelium residues being discarded and the filtrate collected in 50ml centrifuge tubes. The filtrate containing ethyl acetate phase and the medium were collected. The ethyl acetate phase was then separated from the broth medium with centrifugation at 8,000 rpm for 10 minutes and also separation funnel. The top layer which consists of the ethyl acetate phase was removed and transferred to new tubes. Another 20 ml of ethyl acetate were added into the remaining broth and the extraction was repeated three times. The ethyl acetate extract was then dried in the fumehood to give a solid and oily residue. The dried extract was then kept in -20oC until further use (see Figure 19 for an overview).

2.5.1 Solvent-solvent extraction Solvent-solvent partitioning of the ethyl acetate extracts was performed using n- hexane and 90% (vol/vol) aqueous methanol in a ratio of 1:1 (vol/vol) with a total volume of 20 ml being added into the fungal ethyl acetate dried extracts (see Figure 19). The mixture was again left standing for two hours. The mixture was filtered and the filtrate was collected in 50ml centrifuge tubes. The filtrate containing the n-hexane and 90% methanol was then separated through centrifugation at 8,000 rpm for 10 minutes and separation funnel. The top layer which consists of the n-hexane phase was removed and transferred to new tubes. Another 20 ml of solvent mixture (containing n-hexane and 90% aqueous methanol in a ratio of 1:1) were added into the remaining extract and the extraction was repeated three times. The n-hexane extract was collected and dried in the fumehood to give a solid and oily residue and the dried extract was stored in the freezer (-20oC) until further use. On the other hand, the remaining aqueous methanol extract was also dried in the fumehood to give a solid and oily residue and the dried extract was kept in -20oC until further use. All fractions of dried extract were submitted for High Performance Liquid Chromatography (HPLC) analysis to the laboratory of Professor Peter Proksch from the P a g e | 37

Institut für Pharmazeutische Biologie und Biotechnologie, University of Düsseldorf, Germany (see Figure 20 for an overview). P a g e | 38

Fungal Cultures in 20ml PDB

Addition of 20ml Ethyl Acetate, stand for 2 hours Mixture Broth Filtered

Mycelium Filtrate Centrifuged, Separating Funnel Discard

Ethyl Acetate phase Medium Evaporated Additions of ethyl Dried Ethyl Mixture acetate 3X Acetate extract

Ethyl Acetate phase Medium

Figure 19: Extraction of bioactive compounds using ethyl acetate P a g e | 39

Dried Ethyl Acetate extract Addition of mixture (90% aqueous methanol: n-hexane)

Mixture Broth Filtered

Residue Solvent phase (90% aqueous methanol: n-hexane

Centrifuged, Separating Funnel

n-hexane 90% aqueous methanol Evaporated Evaporated Dried n-hexane Dried methanol extract extract

Figure 20: Extraction of bioactive compounds using solvent-solvent (methanol and n-hexane) extraction P a g e | 40

2.6 Biological Assays

2.6.1 Primary Screening of antimicrobial activity All 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 activity Secondary 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). P a g e | 43

PRIMARY SCREENING OF SECONDARY SCREENING OF ANTIMICROBIAL ACTIVITY ANTIMICROBIAL ACTIVITY

Ethyl acetate extract of fungi isolates ANTIBACTERIAL ANTIFUNGAL (Figure 35) dissolved in 1 ml of DMSO ASSAY ASSAY

Small wells (5mm diameter) were Test pathogens grown Test pathogens grown made in the nutrient agar and PDA in nutrient broth on PDA

Test pathogens streaked Test pathogens placed on the Incubated at 30oC Incubated at 25oC onto nutrient agar other half of the PDA plates for 24 hours for one week

Test pathogen streaked Test pathogen (cylindrical piece) 20 µl of the ethyl acetate extract of onto nutrient agar placed on the other half of the fungi isolates added to each well of PDA plates nutrient agar and PDA plates

Cylindrical pieces (1cm x 1cm) of fungi isolates placed on the agar (nutrient agar and PDA) Antibacterial assay – Incubation 24 hrs

Antifungal assay – Incubation 1 week Antibacterial assay – Incubation 24 hrs

Antifungal assay – Incubation 1 week

Figure 21: Primary and Secondary screening of antimicrobial activity P a g e | 44

PREPARATION CYTOTOXICITY ASSAY

Eggs of the brine shrimp hatched in A 96-microtitre artificial seawaterfor 48 hours plate was used

Ethyl acetate extract of fungi isolates 50µl of each extracts of different (Figure 35) dissolved with 10ml of DMSO concentrations were placed into the wells of the plates

DMSO Extract diluted with artificial seawater to obtain different concentrations 10 matured shrimps (0.5, 5, 50 and 500ppm) were added to each of those wells

Number of brine shrimps that died after 24 hours were counted using stereomicroscope

LC50 was determined

Figure 22: Cytotoxicity assay P a g e | 45

2.7 Heavy Metal Analysis

2.7.1 Determination of heavy metal-resistant fungi Tolerance 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

DETERMINATION OF HEAVY HEAVY METAL BIOSORPTION METAL RESISTANT FUNGI

Cylindrical pieces (1cm x 1cm) of fungi Cu and Zn ions were added separately to isolates were added into the PDB PDB at 50 to 200 µg/ml concentration Incubated at 25oC for one Cylindrical pieces (1cm x 1cm) of fungi week under static condition isolates were added into the broth mixture The broth was filtered and the biomass obtained was dried at 60oC Incubated at 25oC for one week under static condition Dried biomass grind and passed through a 0.45 µm sieve The broth was filtered

0.1g of the powdered biomass was inoculated into The biomass obtained the 50 µg/ml Cu and Zn ion solutions was dried at 60oC

Incubated at 150 rpm and 30oC for 72 hours The percentage (%) of biomass dry weight was measured Solution filtered and measured with AAS

Figure 23: Determination of heavy metal resistant fungi using minimum inhibitory concentration (MIC) and heavy metal biosorption 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 %, as well as the source of the sample from which the isolate originates.

FUNGAL LOCATION / SOURCE IDENTITIES STRAINS CLOSEST RELATIVE ISLAND FRESHWATER VILLAGE [accession number]

Isolate 1 Penicillium dravuni 399 / 409 - Root - [AY494856] (98%) Isolate 2 Curvularia affinis isolate S255 469 / 469 - - Soil [HM770741] (100%) Isolate 3 Diaporthe sp. SAB-2009a 454 / 459 - - Leaves strain Q1160 [FJ799940] (99%) Isolate 4 Diaporthe sp. 138SD/T 471 / 473 - - Leaves [GU066697] (99%) Isolate 5 Penicillium citrinum strain 408 / 408 - Root - SGE29 [JX232276] (100%) Isolate 6 Aspergillus sp. Da91 501 / 501 - Root - [HM991178] (100%) Isolate 7 Guignardia mangiferae strain 426 / 439 Leaves - Leaves SCIW10 [HM150733] (97%) Isolate 8 Neosartorya stramenia isolate 349 / 357 Root Root Root NRRL 4652 [EF669984] (98%) Isolate 9 Eupenicillium sp. 5 JH-2010 447 / 449 - Root - culture-collection (99%) CBS:118134 [GU981610] Isolate 10 Penicillium dravuni 399 / 409 Leaves - - [AY494856] (98%) Isolate 12 Cladosporium 478 / 479 - - Root sphaerospermum strain (99%) SCSGAF0054 [JN851005] Isolate 13 Neosartorya hiratsukae strain 460 / 464 - - Root KACC 41127 [JN943580] (99%) 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 that showed 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 7.00 0 + 0 0 + 0 0 + 0 0 + 0 7.67 0 + 0 0 + 0 0 + 0 + 0.58 + 1.00 + 0.58

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 + 0 0 + 0 + 0.58

5mm 5mm

(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)). 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 that showed 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 + 13.00 0 + 0 0 + 0 0 + 0 0 + 0 12.33 0 + 0 0 + 0 0 + 0 1.00 + 2.65 + 1.15

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 + 0 0 + 0 0 + 0 11.67 + 0 + 0 0 + 0 0.00 1.15 56

5mm 5mm

(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. 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

Amounts (in mg) for each fractions Strains 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. Compounds Analysis based on fractions Strains 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  23 known compounds -

 isox-brom-derivat  Sumiki’s acid  benzyl-pyridin A  benzyl-pyridin A  meta-Chloro-para-hydroxy-  trihydroxy tetralone phenyl-essigsaureamid  Amin.-Chlor.-Phe.-Essigsr.  citrinin hydrate  citrinin hydrate  quinolactacin  quinolactacin  8-hydroxy-4-Quinolone  butyl 2-(4-hydroxyphenyl)  new emericellin derivative acetate  altenusin  N-ethylene-renieron  citrinin  citrinin  8E-6-3-2  hydroxyanthranilic acid  bastadin 11  benzyl-pyridin B  benzyl-pyridin B  stevensin  3,4,5-Tribromo-3-(2,4-dibromo-  cyclopenin phenoxy)-phenol  bastadin 3  bastadin 3  graphislactone derivative  sclerotigenin  cyclopenol  Sumiki’s acid  Fatty Acid  Cladosporin  naamine F  sarasinside H2  renieron  8E-2-5-1  altenusin  22-Dehydrocampesterol  cladosporin  8E-2-5-1  16 unknown compounds  S16

 27 unknown compounds Isolate 6  Seventeen known compounds  9 known compounds -

 phenylacetic acid  dienone dimethoxyketal  hydroxysydonic acid  phenylacetic acid  PC 3.3.21.E  citrinin hydrate  Isofistularin-1  8-hydroxy-4-Quinolone  8E-6-3-3 Aurantiamine  11,19-deoxyfistularin  cyclopenol  2-Hydroxy-3-methylbenzoic  aureonitol acid  benzyl-pyridin A  PC 3.3.6.6.3.A  A new gamma-pyrone  sarasiniside K  di-iso-Octylphtalat  triterpene acetate (Weichmacher)  4,5Dibr.pyrrol2carba  4 unknown compounds  sarasiniside A  sarasiniside K  sarasinside 12  adenosine  benzyl-pyridin B 61

 22-dehydrocampesterol

 Five unknown compounds

Isolate 7 - - - Isolate 8 - - - Isolate 9  Four known compounds  3 known compounds -

 microsphaerone B  microsphaerone B  sclerotigenin  paxilline  3,4-Dihydromanzamine  manzamin JN-Oxid  naamine F  21 unknown compounds  13 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. Compounds Analysis based on fractions Strains Ethyl Acetate (mg) Methanol (mg) n-Hexane (mg) Isolate 7  Three known  Four known  Five known compounds compounds compounds

 Pavetannin A1Ac  Pavetannin A1Ac  Pavetannin A1Ac  Epicatechin  Rocaglamid A  Trimeric Catechin  9alpha-OH-  Salicifoliol  Helenalin Pinoresinol  Procyanidin B3 o.  Catechin B6  Rocaglamid A  23 unknown compounds  45 unknown  4 unknown compounds compounds

Isolate 13  Three known  Five known  Five known compounds compounds compounds

 Trimeric Catechin  CS-H2O-2  Pavetannin A1Ac  Epicatechin  9-OH-Pinoresinol  Catechin  Helenalin  Helenalin  Rocaglamid A  Triandrin  Helenalin  14 unknown  Trimeric Catechin compounds  1 unknown  45 unknown compound compounds 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). 64

3.3.7 Cerebroside Cerebrosides are neutral glycosphingolipids that contain a monosaccharide, a glucose or galactose, in 1-ortho-beta-glycosidic linkage with the primary alcohol of an N-acyl sphingoid (ceramide). They are also known as ceramide monohexosides (CMHs) as they contain one sugar unit, which differs from gangliosides in that the latter contain at least one sialic acid residue. Barreto-Bergter and colleagues (2004) also reported that cerebrosides seem to be present in almost all fungal species studied so far (for instance, Aspergillus sp., Penicillium sp., Fusarium sp., etc).

In this study, this compound, cerebroside was isolated from methanol extracts of fungal strain Isolate 2 (closely related to Curvularia sp.), which showed similarity towards the findings by Wang and colleagues (2009) in which the fungal endophytes responsible for this compound were both from the sediment samples of mangroves. For the study reported by Wang and colleagues (2009), three new cerebrosides compounds were isolated from the ethyl acetate extract of the halotolerant fungal strain, identified as Alternaria raphani (from sediment in the Hongdao sea salt field, China). The cerebrosides belonging to the halotolerant fungal strain showed weak antibacterial activity against Escherichia coli, Bacillus subtilis, and Candida albicans. However, in this study, the ethyl acetate extract for the Isolate2 strain was not tested against these test pathogens as the secondary screening assay done was only to confirm the activity of the two selected fungal strains (Isolate 7 and Isolate 13) which displayed antimicrobial activity in the preliminary assay.

3.3.8 Cyclo(prolylvalyl) Cyclo(prolylvalyl) is classified as a diketopiperazine according to Smelcerovic and colleagues (2002), who isolated cyclo(prolylvalyl) from a marine actinomycete using high speed countercurrent chromatography (HCCC), which is a tool for separating natural products. This compound was isolated from ethyl acetate extract of fungal strain Isolate 3 (closely related to Diaporthe) and methanol extract of fungal strain Isolate 4 (closely related to Diaporthe). This compound was also reported by Kim and colleagues (2005) as one of the structures determined isolated from the methanol extract of the mushroom Sarcodon aspratus through ethyl acetate extraction where the compound showed 65

antioxidant activity by scavenging DPPH radical and superoxide radical which could be tested in the future for above mentioned isolates.

3.3.9 Kahalalide B Kahalalide B is a cyclic depsipeptide formed by seven different amino acids (Gly, thr, Pro, D-Leu, Phe, D-Ser, Tyr), and the fatty acid 5-methylhexanoic (5-MeHex), an aliphatic isoacid which is also present in the structure of other members of the series (Lopez-Macia et al. 2000). The finding of a compound with a similar structure from fungal strain Isolate 3 (closely related to Diaporthe sp.), which also showed cytotoxic activity against mature brine shrimps, is therefore highly promising and warrants further studies to isolate the compound and enumerate its structure.

3.3.10 Cyclo(tyrosylprolyl) Cyclo(L-tyrosyl-L-prolyl), known as a cyclic dipeptide, hasbeen reported in many studies with potential biological activity. Killian and colleagues (2011) reported that this particular compound possess antibacterial activity in vitro. Besides, this compound was also reported by Milne and colleagues (1998), with a potential to be used in muscle relaxants, anti- tumour compounds and antibiotics. This compound was isolated from fungal strain, Isolate 3 (which is closely related to Diaporthe).

3.3.11 Citreoisocoumarin and Diachlordiaportin Citreoisocoumarin, along with diachlordiaportin, [3-(3,3-dichloro-2-hydroxy-propyl)-8- hydroxy-6-methoxyisochromen-1-one] have been reported to be produced by has been reported to be produced by Penicillia related to Eupenicillium and other filamentous fungi (Frisvad et al. 2004; Brien et al. 2006). This compound was also reported to be the first isolated from a Phoma species by Sorensen and colleagues (2010). Both compounds were isolated from fungal strain Isolate 3 (which is closely related to Diaporthe) in this study lending support to previous findings. 66

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). 68

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 69

of seventeen subgenuses and found to possess potential herbicidal and anti-HIV activity (Frisvad et al. 2004).

3.3.23 Graphislactone derivative Graphislactones A-H and the structurally related ulocladol are highly oxygenated resorcylic lactones produced by lichens and fungi (Altemoller et al. 2009). Cudaj and Podlech (2010) werethe first to report on the synthesis of Graphislactone G by Cephalosporium acremonium. Graphislactone A was characterized as the most bioactive secondary metabolite of endophytic Cephalosporium sp. with free radical-scavenging and antioxidant activities (Song et al. 2005). These studies show the potential bioactivities possessed by these Graphislactones. In this study, Graphislactones derivatives compound was isolated from fungal strain Isolate 5 (which is closely related to Penicillium), however further studies would be required to identify the type of Graphislactone and its potential activity.

3.3.24 Phenylacetic acid

Phenylacetic acid is classified under phenolics (C6-C2), which is a compound needed by the plants for pigmentation, growth, reproduction, resistance to pathogens and for many other functions (Lattanzio, Lattanzio & Cardinali 2006). This compound is also known as an antifungal metabolite produced by endophytic bacteria, Burkholderia species (Mendes et al. 2007). A compound with similar structure was isolated from fungal strain Isolate 6 (which is closely related to Aspergillus) which did however not show antifungal activity.

3.3.25 Isofistularin-1 To date, there has been no literature citing the discovery of Isofistularin-1, but Isofistularin- 3 has been reported in several studies. Isofistularin-3 was reported as one of the brominated isoxazoline alkaloids found accumulated in Mediterranean marine sponge Aplysina aerophobaas part of a defensive mechanism against the polyphagous Mediterranean fish Blennius sphinx and also possibly as a protection from invasion of bacterial pathogens (Thoms et al. 2004). Acompound with similar structure to Isofistularin-1 was isolated from fungal strain Isolate 6 (which is closely related to Aspergillus) in this study but did not show any antimicrobial activity. 70

It is noteworthy that many of the compounds isolated in this study were similar in structure to compounds reported from marine sponges (Eg 3.3.65 4,5-dibromopyrrole-2-carboxylic acid and 3.3.69 Dienone dimethoxyketal) which shows the potential of marine life as a source of natural products for medicinal development purposes.

3.3.26 8E-6-3-3 Aurantiamine Aurantiamine was reported by Larsen and colleagues (1992) as a new substituted diketopiperazine, isolated from Penicillium aurantiogriseum. This compound was isolated from fungal strain Isolate 6 (which is closely related to Aspergillus).

3.3.27 Aureonitol This compound was first known as a fungal metabolite isolated from Chaetomium species and later found produced also by another endophytic fungus, Helichrysum aureo-nitens (Aly, Debbab & Kjer 2010). Aureonitol is now being isolated from fungal strain, Isolate 6 (which is closely related to Aspergillus).

3.3.28A new gamma-pyrone Gamme-pyrone compounds have been reported by Liou and colleagues in 1993 as potential anti-cancer drugs, showing inhibition towards cancer cell lines.

A new gamma-pyrone was reported to be isolated from dichloromethane extract of stems and roots of Hypericum brasiliense plant. This new gamma-pyrone compound was termed hyperbrasilone and found to possess antifungal properties (Rocha et al. 1994). Besides, many new gamma-pyrones have been reported, for instance, Carbonarones A and B obtained from the culture of the marine derived fungus, Aspergillus carbonarius, to which both compounds showed moderate cytotoxicity against KF62 cells. For this study, another new gamma-pyrone was also reported for a fungal strain that is also closely related to Aspergillus, Isolate 6. With that, this compound would require further structure elucidation to identify the new compound of interest. 71

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). 72

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. 73

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). 74

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. 76

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 77

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 Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate 1 2 3 4 5 6 7 8 9 10 12 13 MIC µg/ml Copper (Cu) 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

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 78

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 79

(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. 80

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 Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate 1 2 3 4 5 6 7 8 9 10 12 13 Q value Copper (Cu) 18 25 11 5 8 1 8 15 4 0 15 7

Q value Zinc (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. 81

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 82

quantitatively, according to the species used, the origin of the biomass, and its processing procedure (Raize et al. 2004). 83

4. PRELIMINARY RESULTS OF SCREENING OF MANGROVE ENDOPHYTIC FUNGI FOR BIOACTIVE COMPOUNDS May Ling ONN*, Po-Teen LIM2, AAZANI MUJAHID2, PETER PROKSCH3, AND MORITZ MÜLLER1

1Biotechnology, School of Engineering, Computing and Science, Swinburne University of Technology, Sarawak Campus, 93350 Kuching, Malaysia 2Department of Aquatic Science, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 93400 Kota Samarahan, Malaysia.

3Institut fürPharmazeutischeBiologie und Biotechnologie, Universität Düsseldorf, Germany.

*Email:[email protected]

Submitted to Journal of Basic Microbiology (Manuscript ID: jobm.201200752)

ABSTRACT Endophytic fungi are fungi that live inside the tissues of other organisms, such as mangrove plants. The plants provide protection to the fungi and the fungi often produce antimicrobial compounds to aid the host fighting off pathogens. These bioactive compounds are secondary metabolites which are often produced as waste- or by-products. In the present study, endophytic fungi isolated from mangrove plants and soils were characterized and their antimicrobial potential assessed. Twelve endophytic fungi were isolated and identified to belong to 7 families: Penicillium, Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya and Eupenicillium. Antimicrobial activities of these 12 fungal endophytes were tested againstgram positive bacteria (Bacillus subtilis and Staphylococcus aureus among others), gram negative bacteria (Escherichia coli among others), yeast (Saccharomyces cerevisiae) and fungi (Candida albicans and Aspergillus niger). Two strains; Isolate 7 and Isolate 13 (related to Guignardia sp. and Neosartoya sp., respectively) showed strong antimicrobial (and antifungal) activity whereas the rest showed no activity based on the formation of a clear zone of inhibition indicative of a positive activity. 84

Compounds were isolated from the extracts of both isolates and screened using HPLC. Both isolates displayed chemically very interesting chromatograms as they possess a high diversity of basic chemical structures and peaks over a wide range of polarities. In the ethyl acetate extract of Isolate 7, compounds with structures similar to Pavetannin A1Ac, Epicatechin, and 9alpha-OH-Pinoresinol were identified. In the ethyl acetate extract of Isolate 13, compounds with structures similar to Trimeric Catechin, Epicatechin, and Helenalin were identified.

Keywords: Mangroves; endophytic fungi; bioactive compounds; antimicrobial

INTRODUCTION Natural products have been gaining attention in the search for novel drugs. They are naturally derived bioactive compounds and by-products from microorganisms, plants or animals which pose no toxicity or harm in the prevention of diseases (Tenguria, Khan & Quereshi 2011).

Endophytic fungi reside within living tissues of plants without causing any adverse effects towards the host plant itself (Kaul et al. 2008; Tran et al. 2010). Mangrove endophytic fungi are increasingly recognized for their ability to produce bioactive compounds with anti-cancer, anti-diabetic, and antimicrobial properties which are of pharmacological importance (Strobel & Daisy 2003; Lu et al. 2010).

The genus Avicennia contains about 15 species and grows in the intertidal zone of coastal mangrove forests distributed widely throughout tropical and warm temperate regions of the world (Duke et al. 1998). Plants of this genus such as Avicennia marina were reported to display antimicrobial and cytotoxic activities against carcinoma cell lines, and were reported to be associated with endophytic fungi (Xu et al. 2005). Their adaptation to the unique mangrove environment and the production of bioactive compounds has been linked to their symbiotic relationship with the endophytes (Elavarasi & Kalaiselvam 2011). 85

With that, research on the bioactive compounds of endophytes can reveal the association between the endophytes and their host plant which would promise new discoveries of potentially life saving drugs. Thus the present study focuses on isolating, identifying and screening endophytic fungi for antimicrobial activity as they have been displaying great potential for discovery of new pharmacologically active metabolites. The endophytic fungi with activity were then selected to further evaluate theirbiological activity and to identify the bioactive compound which gives rise to the observed activity.

MATERIALS AND METHODS

Isolation of Endophytic Fungi Endophytic fungi were isolated from plant materials (Avicennia sp.) which were collected from Kampung Pasir Pandak, Sarawak. The protocol was adapted from Ebada and colleagues (2008). Plant and leaf samples were surface sterilized and cultured onto potato dextrose agar and incubated at 28oC for 1 week. After incubation period, hyphal tips of fungi growing out from the plant fragments were transferred to new PDA plates.

Identification of Endophytic Fungi The endophytic fungi were identified using molecular tools. Genomic DNA was extracted from 5-day old fungi cultures grown on plates using a modified thermolysis method (Zhang et al. 2010). Fungal DNA was amplified using universal primers of fungal DNA ITS1 (5’– TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’–TCC TCC GCT TAT TGA TAT GC-3’; 1st Base, Malaysia). Each sample ready for amplification contained 2 µl of 10X PCR buffer (Fermentas), 1.2 µl of dNTP mixture (2.5mmol l-1 each), 0.8 µl of -1 -1 formamidede ion, 0.4 µl of MgCl2 (25mmol l ), 0.8 µl of each primer (10µmol l ), 0.2 µl of Taq DNA polymerase (5 U µl-1) and 1 µl of genomic DNA in a total volume of 20 µl. PCR was carried out with denaturation at 94oC for 50 sec, annealing at 54oC for 50 sec and elongation at 72oC for 50 sec. This was conducted in 35 cycles and the final elongation reaction was set at 72oC for 10 min. PCR products were purified using PureLink PCR purification kit (Invitrogen, U.S.) and sent for sequencing to the Beijing Genomic Institute, BGI, China. 86

Antimicrobial Assay Test microorganisms included four Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus) and three Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Vibrio anguillarum) bacteria, one yeast (Saccharomyces cerevisiae) and two fungi (Candida albicans and Aspergillus niger). The bacteria and yeast were grown in nutrient broth and incubated at 30oC for 24 hours whereas the fungi were grown in potato dextrose broth and incubated at 25oC for 1 week.

The agar plug assay (Alias et al. 2010) was used to evaluate the antimicrobial activity where cylindrical pieces of 1 x 1 cm size (agar plugs), cut from well grown and sporulated cultures of one week old fungi strains were used. These pieces were placed on the agar previously streaked with test organisms. For the antibacterial activity, plates were incubated for 24 hours. For antifungal activity, agar plugs of the investigated fungi strains were placed opposite of the fungi test pathogen and incubated for one week at 25oC. Inhibition zones were measured after the incubation period. All tests were done in triplicates.

Cytotoxic assay The 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 88

(Bhimba et al. 2011; Xia et al. 2009; Silva et al. 2011). This species was reported to be isolated for the first time from the plant Undaria pinnatifida, having antibacterial and antifungal activity (Wang 2012). Hence, the findings showing that Guignardia with antibacterial activity supported the results of this study showing Isolate 7 having antibacterial activity against Gram positive bacteria.

Isolate 13 (related to Neosartorya sp.) showed comparatively stronger antibacterial activity against Gram positive bacteria (Micrococcus luteus, inhibition zone of 9.67 mm) and antifungal activity against fungi (Candida albicans, inhibition zone of 10.67 mm; see Table 12 and Figure 28b). The genus Neosartorya belongs to the family Trichocomaceae (Varga et al. 2000) and Galgoczy and colleagues (2011) reported on a novel antifungal peptide isolated from the Neosartorya fischeri and this antifungal peptide exhibited high antifungal activity against filamentous fungi within broad pH and temperature ranges. This finding by Galgoczy and colleagues again supports the results of this study where Isolate 13 (related to Neosartorya sp.) was found to show strong antifungal activity against Candida albicans.

Besides, the antimicrobial results of this study showed that the antibacterial activity of the isolates was more common towards Gram positive bacteria compared to Gram negative bacteria. The higher resistance level of the Gram negative bacteria compared to Gram positivecan be attributed to the differences in cell wall structure of Gram-positive bacteria which are less complex compared to the outer membrane present in Gram-negative bacteria thought to act as an additional barrier against antibiotics as also reported by Alias and colleagues (2010).

Cytotoxic assay The cytotoxic assay was done by testing the ethyl acetate extracts for each isolate obtained after 1 week incubation and extraction against matured shrimps at different concentrations. Table 13 shows that two fungal strains, Isolate 3 (related to Diaporthe sp.) and Isolate 9 (related to Eupenicillium sp.) displayed toxicity against the matured brine shrimps at concentrations of 500 ppm after 24 hours incubation. Isolate 3 showed a significantly 89

stronger cytotoxicity and was able to kill 100% of brine shrimps, whereas Isolate 9 only killed 10% (at 500ppm). The lethal concentration at which 50% of the nauplii die (LC50) could not be determined.

Brine shrimps lethality assay is said to be a rapid and useful method for preliminary screening of cytotoxic activity as it has been used in detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing (Carballo et al. 2002; Manilal et al. 2009). This was supported by Lin et al. (2005) who reported in their study on Diaporthe sp.-an endophytic fungus isolated from leaves of Kandelia candel plant of the mangroves in China-cytotoxic activity against lymphoma cell lines. Eupenicillium sp. was reported by Davis et al. (2008) to exhibit strong cytotoxic activity against human colorectal carcinoma and human lung carcinoma cells through the production of a bioactive compound known as trichodermamide C.

Besides, extraction of bioactive compounds was performed for extracts of all fungal strains (from Isolate 1 till Isolate 13). Particularly, for these two fungal strains, Isolate 3 (related to Diaporthe sp.) and Isolate 9 (related to Eupenicillium sp.) which displayed toxicity against the matured brine shrimps, the compounds extracted showed great interest. In this study, a compound of a similar structure with Kahalalide B was extracted from fungal strain Isolate 3 (closely related to Diaporthe sp.). This compound has been reported in marine molluscs but not yet been reported to be isolated from fungal endophytes. So far, only Kahalalide F, a new marine-derived compound, was reported as a novel antitumor drug which showed potent cytotoxicity activity against a panel of human prostate and breast cancer cell lines (Suarez et al. 2003) and not Kahalalide B. Hence, itis therefore highly promising and warrants further studies to isolate the compound and enumerate its structure, as this compound might be responsible for the cytotoxicity activity of the fungal strain Isolate 3 (closely related to Diaporthe sp.).

Besides, fungal strain Isolate 9 (closely related to Eupenicillium) also displayed toxicity and a compound with a structure similar to 3,4-dihydromanzamine was isolated. This compound might be responsible for the cytotoxicity activity of the fungal strain as further 90

supported with a finding by Kobayashi and colleagues (1994) who reported on the compound, 3,4-dihydromanzamine being isolated from the marine sponge, Amphimedon sp collected from the Kerama Islands, Okinawa, Japan, and showed cytotoxic activity against L1210 and KB cell lines. Yet, this is another one of the literatures that cited on compounds isolated from marine sponges.

Extraction of Bioactive Compounds Bioactive compounds from the extracts of both fungal isolates were screened using HPLC. Both isolates displayed chemically very interesting chromatograms as they possess a high diversity of basic chemical structures and peaks over a wide range of polarities (see Figure 29). In the ethyl acetate extract of Isolate 7, three compounds with structures similar to Pavetannin A1 Ac (with a retention time of 2.56 min, Figure 30a), Epicatechin (with a retention time of 38.77 min, Figure 30b), and 9alpha-OH-Pinoresinol (with a retention time of 37.50 min, Figure 30c) were identified. The other 23 compounds found in the spectrum were not identifiable and require further analyses by nuclear magnetic resonance spectroscopy. It is noteworthy that the spectrum contained not only one major compound but a few and over a wide range of polarity. This general picture might help explain why Isolate 7 shows activity towards a wide range of organisms (Gram positive and Gram negative bacteria).

A similar spectrogram was observed for Isolate 13 with several major compounds over a wide range of polarity and the majority of compounds of an unknown nature. Again, this might explain why Isolate 13 was able to inhibit the growth of Gram positive bacteria as well as fungi (see Table 12).

Epicatechin is a flavanoid that has been reported to be responsible for antibacterial activity against Gram-positive and Gram-negative bacteria. This compound was isolated by Masika and colleagues (2004) from Schotia latifolia, a plant commonly used in folkloric medicine. The fungal metabolite with a similar structure to Epicatechin most likely possesses a similar chromophore and could be responsible for the observed antibacterial activity against Gram-positive (Bacillus cereus and Bacillus subtilis) and Gram-negative (Vibrio 91

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 92

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. 93

TABLES Table 11: 18S rRNA phylogenetic results for endophytic fungi

PHYLOGENETIC FUNGAL STRAINS CLOSEST RELATIVE IDENTITIES DIVISION Isolate 1 Penicillium dravuni 399 / 409 (98%) Dikarya [AY494856] Isolate 2 Curvularia affinis isolate 469 / 469 (100%) Dikarya S255 [HM770741] Isolate 3 Diaporthe sp. SAB-2009a 454 / 459 (99%) Dikarya strain Q1160 [FJ799940] Isolate 4 Diaporthe sp. 138SD/T 471 / 473 (99%) Dikarya [GU066697] Isolate 5 Penicillium citrinum 408 / 408 (100%) Dikarya strain SGE29 [JX232276] Isolate 6 Aspergillus sp. Da91 501 / 501 (100%) Dikarya [HM991178] Isolate 7 Guignardia mangiferae 426 / 439 (97%) Dikarya strain SCIW10 [HM150733] Isolate 8 Neosartorya stramenia 349 / 357 (98%) Dikarya isolate NRRL 4652 [EF669984] Isolate 9 Eupenicillium sp. 5 JH- 447 / 449 (99%) Dikarya 2010 culture-collection CBS:118134 [GU981610] Isolate 10 Penicillium dravuni 399 / 409 (98%) Dikarya [AY494856] Isolate 12 Cladosporium 478 / 479 (99%) Dikarya sphaerospermum strain SCSGAF0054 [JN851005] Isolate 13 Neosartorya hiratsukae 460 / 464 (99%) Dikarya strain KACC 41127 [JN943580] 94

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 that showed 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 7.00 0 + 0 0 + 0 0 + 0 0 + 0 7.67 0 + 0 0 + 0 0 + 0 + 0.58 + 1.00 + 0.58

Isolate 13 0 + 0 0 + 0 0 + 0 9.67 + 0 + 0 0 + 0 0 + 0 10.67 0 + 0 0 + 0 1.53 + 0.58

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% 95

FIGURES

Figure 27: 18S gene-based phylogenetic tree representing the twelve endophytic fungal isolates. The phylogenetic tree was generatedwith distance methods, and sequence distances were estimated with the neighbor-joining method. Bootstrap values ≥50 are shown and accession numbers for the reference sequences are indicated. 96

5mm 5mm

(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. 97

(a)

(b)

Figure 29: HPLC chromatograms of Ethyl Acetate extracts of (a) Isolate 7 and (b) Isolate 13 recorded at 235 nm. 98

(a)

(b)

(c)

Figure 30: HPLC chromatograms of compounds from Isolate 7 that had similar structures to (a) Pavetannin A1 Ac, (b) Epicatechin, and (c) 9alpha-OH-Pinoresinol. Chromatograms were recorded at 235 nm and library hits are indicated at the top right of the picture. 99

(a)

(b)

(c)

Figure 31: HPLC chromatograms of compounds from Isolate13 that had similar structures to (a) Trimeric Catechin, (b) Epicatechin, and (c) Helenalin. Chromatograms were recorded at 235 nm and library hits are indicated at the top right of 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, Malaysia 2Department 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. 103

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 -1 each), 0.8 µl of deioned formamide, 0.4 µl of MgCl2 (25mmol l ), 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

TABLES Table 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 least resistant species are highlighted in bold, as are their respective MIC values.

Species Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate 1 2 3 4 5 6 7 8 9 10 12 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 Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate 1 2 3 4 5 6 7 8 9 10 12 13 Q value 18 25 11 5 8 1 8 15 4 0 15 7 (Cupper) Q value 3 6 8 15 16 21 16 24 14 16 14 24 (Zinc) 111

FIGURES

Figure 32: 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. 112

(a) Isolate1 (b) Isolate10

Figure 33: Two fungal strains: (a) Isolate 1 and (b) Isolate10 closely related to Penicillium dravuni but having different morphological characteristics and growth patterns where Isolate 10 grows at a faster rate within a week compared to Isolate 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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