The Diversity and Bioactivity of Endophytes Associated With

Home , Actinopolymorpha, Eora, Eremophila alternifolia, Gweagal, Koori

The Diversity and Bioactivity of Endophytes Associated with

Medicinal Plants Used by the Dharawal People of

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Shane David) Ingrey

B.Sc (Hons.) University) of New South Wales

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The School of Biotechnology:93*+#6!-)!73'%(3% and Biomolecular! Sciences !

The University of !New South Wales

Sydney,"*1*&#!;<=> Australia

! ! March 2014

Supervisor

Professor Brett Neilan

The School of Biotechnology and Biomolecular Sciences

iii

Thesis-nhay dedicated-ri ngabuwulawala, babawulawala, ngabawulawala, babaawulawala, djadjawulawala mamawulawala nagun knowledgeyanha ‘mirra ngayamayangay’.

Translates literally to:

This thesis is dedicated to the grandmothers, grandfathers, mothers, fathers, brothers and sisters who without their knowledge ‘I would have had nothing’. (Dharawal Language).

vii Abstract

Aboriginal Australians have one of the oldest surviving cultures in the world today. The Dharawal people of Botany Bay were the first to encounter the British first in 1770 and again in 1788. The Dharawal use plants for a variety of reasons that also includes medicinal properties. This thesis investigates ethnobotanical information of seven medicinal plants used by the Dharawal people of Botany Bay that were provided by the Dharawal elders group and community members of the La Perouse Aboriginal community who engaged in continual cultural practice. Endophytic microorganism communities that reside within the plant tissue of each medicinal plant were organised phylogenetically based on 16S rRNA and 18S rRNA genes. The culturable endophytes that were identifiable were phylogenetically grouped within ten orders of fungi and three classes of bacteria. These endophytes were also further investigated for biosynthetic potential of natural products via genetic screening targeting enzyme encoding genes, non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Endophytes that possessed NRPS or PKS encoding genes were subjected to preliminary bioassay tests against a range of bacterial and fungal test organisms. All but one showed activity against at least one test organism. Three endophytes were selected for further compound isolation. Isolation and structure elucidated of bioactive compounds from an endophytic Aspergillus sp. revealed known compounds such as triacylglycerides (SDI-63), bisabolane sesquiterpenoids (SDI-66) and a novel bioactive polyketide (SDI-69) via 1H, 13C, and 2D NMR experiments. Compound SDI-69 possessed anti-bacterial properties against S. aureus, P. aeroginosa, E. coli, C. diptheriae and B. subtilis at concentrations of 10 µg/mL or 100 µg/mL. The traditional knowledge present in today’s society has revealed that these medicinal plants provide a habitat for a diverse range of endophytic microorganisms. These endophytes contain NRPS and PKS encoding genes and produce bioactive compounds, leading to the inference of the role of endophytes and their medicinal properties in various indigenous plant species. This approach to natural product discovery will allow conservation of these medicinal plant species and the associated traditional practices to provide continual access to novel bioactive natural products produced by endophytes.

Acknowledgements

I would firstly like to acknowledge all the Dharawal people of the Sydney and Botany Bay area, both past and present, whose knowledge, resilience and persistence in the face of massive change and adversity have kept our culture alive. In regards to this study, my grandmother, a proud Dharawal and Dunghutti elder, whose knowledge was an integral part of this study, not only her knowledge of medicinal plants but her traditional values and pride in continuing our culture. This shown through my father and in my brothers, sister, cousins, nieces and nephews, made this study one of the most proudest moments of my life. It’s just a shame you are not physically here to see it completed. I would also like to thank the Dharawal Elders Group for their input into this project. I would also like to pay my respects and thanks to the Aunties, my grandmother’s sisters and cousins that had been involved in the project but had passed away. I hope I did you all proud. I would like to thank my supervisor Brett Neilan for letting me hang around so long. Your supervision, friendship and mentorship during this study has not only made my time in the lab most enjoyable, but also showed me the importance of a balance of work and play. It was a privilege to work and learn from you. Co-supervisor John Kalaitzis, thank you for all your help and guidance of the years, especially in recent times with chemistry and manuscript prepping and tough topic of Rugby League. Souths vs Parra grand final? A big thank you to Professor Nakata and the team at Nura Gili for all the support and advice that you have given during this time. Alex and Alper… well I did it. We’re just waiting for you now Alper. Thanks for being such good mates during our time here. Current and former endophyte/symbiont people Kristin, Alvin, Jeff, Toby and Rocky, thanks for all the support over the years with culturing, NMR, chemistry and general advice and friendship. To all the lab members of the BGGM, past and present, thank you for all your help and all the fun memories. You have made this time a most memorable one.

viii Mum and Dad thank you for all your support and patience. I really appreciate all the help and support you have always given me. Ditto Glenis and Van. I have finally finished. Yeano, Ray, Mick, Boote and Wana thanks for all the input especially from the cultural perspective. You’re all doing a good job in keeping our culture alive and moving forward. I’m very proud to have you as my siblings. Also thanks to Kez, Kobi, Shal, Clare and Aaron, Fleur, Chloe, Gordon, Dean and Vanessa. All the kids, lets hope some of you get to write one of these one day. Grandma. YAY! I finished this in your lifetime. Thank you for all your support that you have given us. Your kindness and love have made this journey much easier and I will always be grateful for everything you have done. Renee, a massive thank you for being the most patient, understanding, beautiful and loving wife a person could ask for. Without you I would not have been able to finish this. Maybe one day Olive could be doing this as well. M-A-Y-B-E. Olive I love you. (Post hand in: David I love you too) If I have forgotten anyone I am sorry and would like to apologise and thank you. My lack of sleep and excitement to hand in has got the better of me. Since I have finally finished and bought this long journey to a close, let’s hope the Rabbitohs can do the same thing and win the comp this year. (and they did!)

ix List of Publications and Presentations Refereed papers • Shane D. Ingrey, John A. Kalaitzis and Brett A. Neilan. Gooris, Invaders and Modern Ethnobotany: The Evolution of Traditional Medicines from Australia. (Submitted to Journal of Ethnobiology and Ethnomedicine). • Miller K, Ingrey S, Alvin A, Sze D, Roufogalis DA, Neilan BA (2010). Endophytes and the microbial genetics of traditional medicines. Microbiology Australia (May edition): 60-3. Conference procedings • Ingrey SD, Neilan BA (2008) (Abstract). The genetic basis for bioactivity in the traditional medicine plants of Australia. Planta Medica 74:908. (DI0668388)* This abstract was accepted for a short conference talk at the 7th meeting of AFERP, ASP, GA, PSE and SIF, 2008 in Athens, Greece. This was a joint meeting of top natural products societies of Europe and America. • Miller, K. I., Ingrey, S., Sze D.M., Roufogalis, B., Qing, C., Neilan, B. (2008) Bioprospecting for Endophyte Related Natural Products in TCM Anti-Cancer Herbs. Natural Products Conference, 7th Joint Meeting of AFERP, ASP, GA, PSE and GIF, Athens, Greece. Other UNSW TV. Cloning bush medicine (2010). http://tv.unsw.edu.au/video/cloning-bush- medicine.

Papers in preparation 1. Ingrey, S.D., Dharawal elders group, Miller, K., Kalaitzis, J.A., Neilan, B.A. The Microbial Diversity of Endophytes from Traditional Medicine Plants Used by the Dharawal People of Botany Bay, Sydney, Australia. 2. Ingrey, S.D., Sreekanth, D., Kalaitzis, J., Neilan, B.A. Bioactivity of Endophytes Isolated from Traditional Medicine Plants of Sydney, Australia. 3. Ingrey, S.D., Kalaitzis, J., and Neilan, B.A. Novel Polyketide Isolated from an Endophytic Aspergillius sp. and its Anti-bacterial Activity. (Proposed)

x List of Abbreviations

A adenylation AGA arginine/glycerol agar BLAST basic local alignment search tool bp base pairs C condensation CD3OD deuterated methanol CDCl3 deuterated chloroform COSY correlated spectroscopy DCM dichloromethane DH dehydratase DMSO dimethyl sulfoxide DMSO-d6 deuterated DMSO DNA deoxyribonucleic acid E epimerisation EDTA ethylenediamineteraacetic acid ER enoylreductase GTR general time reversible h hours HMBC heteronuclear multiple bond coherence HPLC high performance liquid chromatography HSQC heteronuclear single quantum coherence HR-ESI-MS high resolution electrospray ionisation mass spectrometry KR ketoreductase KS ketosynthase KSα ketoacyl synthase KSβ ketoacyl synthase m/z mass to charge ratio MHz megahertz MPLC medium pressure liquid chromatography MS mass spectrometry NA not available NCBI National Centre for Biotechnology Information NT not tested NMR nuclear magnetic resonance NRP non-ribosomal peptide NRPS non-ribosomal peptide synthetase OD optical density PCP peptidyl carrier protein PCR polymerase chain reaction PDA potato dextrose agar PK polyketide PKS polyketide synthase POP palmitic oleic palmitic PS plant species PUFA polyunsaturated fatty acid RDBE ring and double bond equivalent RP-HPLC reverse phase HPLC s seconds

xi rRNA ribosomal ribonucleic acid SDS sodium dodecyl sulphate SEM standard error of the mean sp. species (singular) spp. species (plural) TAE tris-acetate EDTA buffer TAG triaclyglycerides TE thioester TFA trifluoroacetic acid TLC thin layer chromatography TrN Tamura-Nei U units UV ultraviolet WA water agar XO xanthine oxidase YES yeast extract sucrose agar

xii TABLE OF CONTENTS ABSTRACT ...... V ACKNOWLEDGEMENTS ...... VIII LIST OF PUBLICATIONS AND PRESENTATIONS ...... X LIST OF ABBREVIATIONS ...... XI LIST OF FIGURES ...... XVI LIST OF TABLES ...... XIX 1 CHAPTER 1 INTRODUCTION: GOORI’S, INVADERS AND MODERN ETHNOBOTANY: THE EVOLUTION OF TRADITIONAL MEDICINE OF AUSTRALIA ... 20 1.1 INTRODUCTION ...... 20 1.2 INDIGENOUS PEOPLE AND PLANTS ...... 22 1.3 MEDICINAL USE OF TRADITIONAL KNOWLEDGE ...... 24 1.4 RECENT RESEARCH ON AUSTRALIAN ABORIGINAL MEDICINE PLANTS ...... 25 1.5 TRADITIONAL MEDICINE IN THE MODERN WORLD ...... 31 1.6 A MICROBIOLOGICAL APPROACH TO TRADITIONAL MEDICINE ...... 31 1.7 ENDOPHYTES FROM NATIVE AUSTRALIAN PLANTS ...... 34 1.8 ENDOPHYTES AND AUSTRALIAN MEDICINAL PLANTS ...... 35 1.9 NON-RIBOSOMAL PEPTIDE SYNTHETASE AND POLYKETIDE SYNTHASE ...... 36 1.10 STRUCTURE OF NON-RIBOSOMAL PEPTIDE SYNTHETASES AND MECHANISM OF BIOSYNTHESIS ...... 37 1.11 STRUCTURE OF POLYKETIDE SYNTHASE AND MECHANISM OF BIOSYNTHESIS ...... 39 1.12 STRUCTURE OF NRPS/PKS HYBRIDS ENZYME COMPLEXES ...... 40 1.13 CONCLUSION, SIGNIFICANCE AND FUTURE PROSPECTS FOR DRUG DISCOVERY ...... 41 1.14 STUDY AIMS AND REASONING ...... 44 1.15 THE KEY STEPS IN THIS STUDY ...... 45 2 CHAPTER 2 THE ETHNOBOTANICAL SURVEY-BASED COLLECTION OF MEDICINAL PLANTS USED BY THE DHARAWAL PEOPLE OF BOTANY BAY ...... 46 2.1 INTRODUCTION ...... 46 2.1.1 The Dharawal People ...... 46 2.1.2 Ethnobotany and Ethnomedicine of the Dharawal ...... 47 2.2 MATERIALS AND METHODS ...... 52 2.2.1 Study Area ...... 52 2.2.2 Ethnobotanical Investigation ...... 52 2.2.3 Collection Licence Application ...... 53 2.2.4 Plant Collection ...... 54 2.3 RESULTS ...... 55 2.3.1 Ethnobotanical Investigations ...... 55 2.3.2 Plant Selection ...... 55 2.3.3 Plant Collection ...... 58 2.4 DISCUSSION ...... 59 2.4.1 Ethnobotanical Investigation ...... 59 2.4.2 Dharawal tribal area ...... 63 2.5 CONCLUSION ...... 65 3 CHAPTER 3 THE MICROBIAL DIVERSITY OF ENDOPHYTES FROM ESSENTIAL ESSENTIAL MEDICINE PLANTS OF SYDNEY, AUSTRALIA ...... 68 3.1 INTRODUCTION ...... 68 3.2 MATERIALS AND METHODS ...... 70 3.2.1 Plant Collection ...... 70 3.2.2 Sample Preparation and Surface Sterilisation ...... 71

xiii 3.2.3 Endophyte Isolation ...... 71 3.2.4 DNA Isolation ...... 72 3.2.5 DNA Amplification and Phylogenetic Analysis ...... 73 3.2.6 Phylogeny ...... 75 3.3 RESULTS ...... 75 3.3.1 Surface Sterilisation and Endophyte Isolation ...... 75 3.3.2 16S and 18S rRNA Gene Amplification and Sequence Analysis ...... 76 3.3.3 Phylogeny ...... 80 3.4 DISCUSSION ...... 84 3.4.1 PCR Screening ...... 85 3.4.2 Phylogeny ...... 86 3.5 CONCLUSION ...... 89 4 CHAPTER 4 SCREENING OF ENDOPHYTES FROM MEDICINAL PLANT FOR POLYKETIDE AND NON-RIBOSOMAL PEPTIDE BIOSYNTHETIC GENES AS AN INDICATOR FOR BIOACTIVE NATURAL PRODUCTS ...... 91 4.1 INTRODUCTION ...... 91 4.2 MATERIALS AND METHODS ...... 95 4.2.1 DNA Isolation ...... 95 4.2.2 Polymerase Chain Reaction (PCR) ...... 95 4.2.3 Liquid broth culturing and compound extraction ...... 97 4.2.4 Disc Diffusion Anti-Microbial Activity Assays ...... 98 4.3 RESULTS ...... 98 4.3.1 NRPS and PKS Screening by PCR ...... 98 4.3.2 Preliminary Crude Extract Anti-Microbial Assays ...... 110 4.4 DISCUSSION ...... 113 4.4.1 Screening for NRPS and PKS Encoding Genes ...... 113 4.4.2 Taxonomy of Bioactive Endophytes ...... 117 4.4.3 Screening of Endophytic Penicillium Species ...... 118 4.4.4 Screening of Endophytic Botryosphaeria and Neofusicoccum Species ...... 119 4.4.5 Screening of Endophytic Phomopsis Species ...... 119 4.4.6 Screening of Endophytic Aspergillus Species ...... 120 4.4.7 Rationale for Selection of Endophytes for Up-scaled Fermentation ...... 121 4.5 CONCLUSION ...... 122 5 CHAPTER 5 INVESTIGATION INTO THE BIOACTIVITY OF NATURAL PRODUCTS ISOLATED FROM ENDOPHYTIC PENICILLIUM, PHOMOPSIS, NEOFUSICOCCUM/BOTRYOSPHERIA AND ASPERGILLUS SPECIES ...... 124 5.1 INTRODUCTION ...... 124 5.2 MATERIALS AND METHODS ...... 127 5.2.1 Culturing and Extract Isolation ...... 127 5.2.2 Thin Layer Chromatography ...... 127 5.2.3 Fractionation and Compound Isolation from Endophytic Penicillium (PS1-3) 127 5.2.4 Fractionation and Compound Isolation of PS1-3 Fractions ...... 128 5.2.5 Fractionation and Compound Isolation from Penicillium (PS2-2), Aspergillus (PS2-19), Phomopsis (PS6-7) and Neofusicoccum/Botryospheria (PS6-31) ...... 128 5.2.6 High Pressure Liquid Chromatography (HPLC) ...... 129 5.2.7 NMR and MS Analysis ...... 129 5.2.8 Anti-bacterial and Anti-fungal Activity Bioassay ...... 130 5.3 RESULTS ...... 131 5.3.1 Isolation of Compounds from Endophytic Penicillium (PS1-3) ...... 131 5.3.2 Isolation of Compounds from an Endophytic Aspergillus (PS2-19) ...... 132 5.3.3 Isolation of Compounds from an Endophytic Phomopsis (PS6-7) ...... 133

xiv 5.3.4 Isolation of Compounds from Endophyte Neofusicoccum/Botryosphaeria (PS6-31) ...... 134 5.3.5 NMR and structure elucidation ...... 135 5.3.6 Biological Activity Assay Investigation ...... 145 5.4 DISCUSSION ...... 147 5.5 CONCLUSION ...... 154 6 GENERAL DISCUSSION ...... 156 APPENDIX 1 ...... 164 REFERENCE ...... 186

xv List of Figures FIGURE 1 DUNGHUTTI TRIBAL AREA AND THEIR SURROUNDING [11]...... 22 FIGURE 2 SIMPLIFIED DIAGRAM OF A NRPS MODULE [138]...... 38 FIGURE 3 SIMPLIFIED DIAGRAM OF A PKS MODULE [138]...... 40 FIGURE 4 BIOSYNTHETIC PATHWAY OF MICROCYSTIN [151]...... 41 FIGURE 5 TRIBAL BOUNDARIES OF THE DHARAWAL NATION AND SURROUNDING TRIBES [11]...... 48 FIGURE 6 18S RRNA GENE PHYLOGENETIC TREE ENCOMPASSING ISOLATED FUNGAL ENDOPHYTES AND RELATED ORGANISMS. FUNGAL ISOLATES ARE SHOWN IN BOLD. ACCESSION NUMBERS OF THE REFERENCE STRAINS ARE IN BRACKETS. BOOTSTRAP VALUES >50% ARE DISPLAYED.* KIRSCHSTEINIOTHELIA SP. HAS UNCLASSIFIED ORDER ...... 82 FIGURE 7 16S RRNA GENE PHYLOGENETIC TREE ENCOMPASSING ISOLATED BACTERIAL ENDOPHYTES AND RELATED ORGANISMS. BACTERIAL ISOLATES ARE SHOWN IN BOLD. ACCESSION NUMBERS OF THE REFERENCE STRAINS ARE IN BRACKETS. BOOTSTRAP VALUES >50% ARE DISPLAYED...... 83 FIGURE 8 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS1 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS1-2 (LANE 1), PS1-3 (LANE 2), PS1-5 (LANE 3), PS1-6 (LANE 4), PS1-7 (LANE 5), PS1-8 (LANE 6), PS1-9 (LANE 7), PS1-R (LANE 8) AND POSITIVE CONTROL (LANE 9)...... 99 FIGURE 9 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS1 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS1-11 (LANE 1), NON PS1 PKS SCREENING (LANE 2-8), POSITIVE CONTROL (LANE 9) AND NEGATIVE CONTROL (LANE 10)...... 99 FIGURE 10 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS2 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS2-1 (LANE 1), PS2-2 (LANE 2), PS2-3 (LANE 3), PS2-4 (LANE 4), PS2-4 (LANE 5), PS2-6 (LANE 6), PS2-6T (LANE 7), PS2-8 (LANE 8), PS2-9 (LANE 9), PS2-10 (LANE 10), POSITIVE CONTROL (LANE 12), NEGATIVE CONTROL (LANE 13)...... 100 FIGURE 11 AMPLIFICATION OF PKS KETOSYNTHESIS DOMAIN OF PS2 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS2-10 (LANE 1), PS2-11 (LANE 2), PS2-12 (LANE 3), PS2-13 (LANE 4), PS2-14 (LANE 5), PS2-15 (LANE 6), PS2-16 (LANE 7), PS2-17 (LANE 8), PS2-18 (LANE 9), PS2-19 (LANE 10), PS2-20 (LANE 11), PS2-21 (LANE 12), POSITIVE CONTROL (LANE 13), NEGATIVE CONTROL (LANE 14)...... 100 FIGURE 12 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS7 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS7-2 (LANE 1), PS7-3 (LANE 2), PS7-4 (LANE 3), PS7-5 (LANE 4), PS7-6 (LANE 5), PS7-7 (LANE 6), PS7-8 (LANE 7), PS7-9 (LANE 8), PS7-10 (LANE 9), PS7-11 (LANE 10), PS7-12 (LANE 11), POSITIVE CONTROL (LANE 12), NEGATIVE CONTROL (LANE 13)...... 100 FIGURE 13 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS7 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS-7 (LANE 1), PS7-13 (LANE 2), PS7-14A (LANE 3), PS7-15 (LANE 4), PS7-16 (LANE 5), PS7-17 (LANE 6), PS7-14B (LANE 7), PS7-14WF (LANE 8), PS7-17B (LANE 9), PS7-17C (LANE 10), NON PS7 PKS SCREENING SAMPLES (LANE 11-14), POSITIVE CONTROL (LANE 15), (NEGATIVE CONTROL IN ABOVE GEL)...... 100 FIGURE 14 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS6-1 (LANE 1), PS6-2 (LANE 2), PS6-3 (LANE 3), PS6-4 (LANE 4), PS6-5 (LANE 5), PS6-6 (LANE 6), PS6-7 (LANE 7), PS6-8 (LANE 8), PS6-9 (LANE 9), PS6-10 (LANE 10), PS6-11 (LANE 11), PS6-12 (LANE 12), PS6-13 (LANE 13), POSITIVE CONTROL (LANE 14) (NEGATIVE CONTROL BELOW 1A)...... 101 FIGURE 15 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS6-14 (LANE 1), PS6-15 (LANE 2), PS6-16 (LANE 3), PS6-26 (LANE 4), PS6-27 (LANE 5), PS6-28 (LANE 6), PS6-29 (LANE 7), PS6-30 (LANE 8), PS6-31 (LANE 9), PS6-32 (LANE 10), PS6-33 (LANE 11), PS6-34 (LANE 12), PS6-35 (LANE 13), POSITIVE CONTROL (LANE 14). L=LADDER, NEGATIVE CONTROL (LANE 1A)...... 101 FIGURE 16 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING DKF/DKR PRIMER SET. L=LADDER; PS6-36 (LANE 1), PS6-37 (LANE 2), PS6-38 (LANE 3), PS6-39 (LANE 4), PS6-40 (LANE 5), PS6-41 (LANE 6), PS6-42 (LANE 7), POSITIVE CONTROL (LANE 8), NEGATIVE CONTROL (LANE 9)...... 101

xvi FIGURE 17 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS2 ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS2-1 (LANE 1), PS2-2 (LANE 2), PS2-3 (LANE 3), PS2-4GN (LANE 4), PS2-4 (LANE 5), PS2-6 (LANE 6), PS2-6T (LANE 7), PS2-8 (LANE 8), PS2-9 (LANE 9), PS2-10 (LANE 10), POSITIVE CONTROL (VERY FAINT) (LANE 11), NEGATIVE CONTROL (LANE 12)...... 102 FIGURE 18 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-1 (LANE 1), PS6-2 (LANE 2), PS6-3 (LANE 3), PS6-4 (LANE 4), PS6-5 (LANE 5), PS6-6 (LANE 6), PS6-7 (LANE 7), PS6-8 (LANE 8), PS6-9 (LANE 9), PS6-10 (LANE 10), PS6-11 (LANE 11), PS6-12 (LANE 12), POSITIVE CONTROL (LANE 13), (NEGATIVE CONTROL BELOW)...... 102 FIGURE 19 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-13 (LANE 1), PS6-14 (LANE 2), PS6-15 (LANE 3), PS6-16 (LANE 4), POSITIVE CONTROL (LANE 5), NEGATIVE CONTROL (LANE 6)...... 102 FIGURE 20 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-26 (LANE 1), PS6-27 (LANE 2), PS6-28 (LANE 3), PS6-29 (LANE 4), PS6-30 (LANE 5), PS6-31 (LANE 6), PS6-32 (LANE 7), PS6-33 (LANE 8), PS6-34 (LANE 9), PS6-35 (LANE 10), PS6-36 (LANE 11), PS6-37 (LANE 12), POSITIVE CONTROL (LANE 13), (NEGATIVE CONTROL BELOW)...... 103 FIGURE 21 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET. L=LADDER, PS6-38 (LANE 1), PS6-39 (LANE 2), PS6-40 (LANE 3), PS6-41 (LANE 4), PS6-42 (LANE 5), POSITIVE CONTROL (LANE 6), NEGATIVE CONTROL (LANE 7)...... 103 FIGURE 22 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF UPSCALED ENDOPHYTES USING LC1/LC2C FUNGAL SPECIFIC PRIMER SET (CHAPTER 5). PS1-3 (LANE 1), PS2-2 (LANE 2), PS2-9 (LANE 3), PS2-19 (LANE 4), PS6-7 (LANE 5), PS6-7 (LANE 6), PS6-31 (LANE 7), PS6-31 (LANE 8), POSITIVE CONTROL (LANE 9), NEGATIVE CONTROL (LANE 10)...... 103 FIGURE 23 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC3/LC5C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-1 (LANE 1), PS6-2 (LANE 2), PS6-3 (LANE 3), PS6-4 (LANE 4), PS6-5 (LANE 5), PS6-6 (LANE 6), PS6-7 (LANE 7), PS6-8 (LANE 8), PS6-9 (LANE 9), PS6-10 (LANE 10), PS6-11 (LANE 11), PS6-12 (LANE 12), PS6-13 (LANE 13), POSITIVE CONTROL AND NEGATIVE CONTROL BELOW)...... 103 FIGURE 24 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC3/LC5C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-14 (LANE 1), PS6-15 (LANE 2), PS6-16 (LANE 3), PS6-16 (LANE 4), POSITIVE CONTROL (LANE 5), NEGATIVE CONTROL (LANE 6)...... 104 FIGURE 25 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF PS6 ENDOPHYTES USING LC3/LC5C FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-26 (LANE 1), PS6-27 (LANE 2), PS6-28 (LANE 3), PS6-29 (LANE 4), PS6-30 (LANE 5), PS6-31 (LANE 6), PS6-32 (LANE 7), PS6-33 (LANE 8), PS6-34 (LANE 9), PS6-35 (LANE 10), PS6-36 (LANE 11), PS6-37 (LANE 12), POSITIVE CONTROL (LANE 13), (NEGATIVE CONTROL ON ANOTHER GEL AND WAS NEGATIVE) ...... 104 FIGURE 26 AMPLIFICATION OF PKS KETOSYNTHASE DOMAIN OF UPSCALED ENDOPHYTES (CHAPTER 5) USING LC3/LC5C FUNGAL SPECIFIC PRIMER SET. L=LADDER, PS1-3 (LANE 1), PS2-2 (LANE 2), PS2-9 (LANE 3), PS2-19 (LANE 4), PS6-7 (LANE 5), PS6-7 (LANE 6), PS6-7 (LANE 7), PS6-7 (LANE 8), POSITIVE CONTROL (LANE 9), NEGATIVE CONTROL (LANE 10)...... 104 FIGURE 27AMPLIFICATION OF NRPS ADENYLATION DOMAIN OF PS1 ENDOPHYTES USING MTF2/MTR2 PRIMER SET. L=LADDER; PS1-2 (LANE 1), PS1-3 (LANE 2), PS1-5 (LANE 3), PS1-6 (LANE 4), PS1-7 (LANE 5), PS1-8 (LANE 6), PS1-9 (LANE 7), PS1-R (LANE 8), POSITIVE CONTROL (LANE 9)...... 105 FIGURE 28 AMPLIFICATION OF NRPS ADENYLATION DOMAIN OF PS1 ENDOPHYTES USING MTF2/MTR2 PRIMER SET. L=LADDER; PS1-11 (LANE 1), POSITIVE CONTROL (LANE 9 & 10), NEGATIVE CONTROL (LANE 11)...... 105 FIGURE 29 AMPLIFICATION OF NRPS ADENYLATION DOMAIN OF PS6 ENDOPHYTES USING RJ016F/RJ016R FUNGAL SPECIFIC PRIMER SET. L=LADDER; PS6-1 (LANE 1), PS6-2 (LANE 2), PS6-3 (LANE 3), PS6-4 (LANE 4), PS6-5 (LANE 5), PS6-6 (LANE 6), PS6-7 (LANE 7), PS6-8 (LANE 8), PS6-9 (LANE 9), PS6-

xvii 10 (LANE 10), PS6-11 (LANE 11), PS6-12 (LANE 12), PS6-13 (LANE 13), POSITIVE CONTROL (LANE 14) (NEGATIVE CONTROL SHOWN ON SEPARATE GEL AND WAS NEGATIVE)...... 106 FIGURE 30 EXAMPLES OF NATURAL PRODUCTS PRODUCED BY FUNGI. (1) PENICILLIN AND CEPHALOSPORIN [332]; (2) CYCLOSPORINE A [333]; (3) LOVASTATIN [334]; (4) A. CAMPTOTHECIN, B.10- HYDROXYCAMPTOTHECIN, C. 9-METHOXYCAMPTOTHECIN AND D. 10-METHOXYCAMPTOTHECIN [335]; (5) TAXOL [336]...... 126 FIGURE 31 ISOLATION AND PURIFICATION OF EXTRACTS OBTAINED FROM PS1-3 ...... 132 FIGURE 32 ISOLATION AND PURIFICATION OF EXTRACTS FROM PS2-19 ...... 133 FIGURE 33 ISOLATION AND PURIFICATION OF EXTRACTS FROM PS6-7 ...... 134 FIGURE 34 ISOLATION AND PURIFICATION OF EXTRACTS FROM PS6-31 ...... 135 FIGURE 35 PARTIAL STRUCTURE A OF SDI-139 DETERMINED FROM HSQC AND HMBC CORRELATIONS. ARROWS INDICATE HMBC CORREALTIONS...... 137 FIGURE 36 PARTIAL STRUCTURE B OF SDI-139 DETERMINED FROM HSQC AND HMBC CORRELATIONS. ARROWS INDICATE HMBC CORREALTIONS...... 137 FIGURE 37 FINAL STRUCTURE OF SDI-139 ...... 138 FIGURE 38 COMPARISON OF 13C NMR OF FATTY ACID SDI-05 (1) AND TAG SDI-63 (2). (A) REPRESENTS 3 CARBOXYL CARBONS AND (B) REPRESENTS CARBONS OF THE TAG BACKBONE. (A) REPRESENTS ONE CARBOXYL IN FATTY ACIDS AND (B) IS ABSENT IN FATTY ACIDS ...... 140 FIGURE 39 COMPARISON OF 1H NMR SPECTRA OF FATTY ACID SDI-05 (1) AND TAG SDI-63 (2). (B) REFERS TO PROTON SHIFTS OBSERVED FOR TAG BACKBONE. THESE ARE ABSENT IN FATTY ACID SDI-05 (1) ...... 141 FIGURE 40 STRUCTURE OF 1,3-DIPALMITOYL-2-OLEOYLGLYCEROL (POP) ...... 142 FIGURE 41 HR-ESI-MS POSITIVE MODE SPECTRA WITH PREDICTED STRUCTURES OF THE MIXTURE SDI-66 .... 143 FIGURE 42 TWO PARTIAL STRUCTURES OF SDI-69...... 144 FIGURE 43 CELL GROWTH INHIBITION OF STAPHYLOCOCCUS AUREUS AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS CIPROFLOXACIN (50 µG/ML). ALL DATA ARE MEAN +/- SEM N=3...... 147 FIGURE 44 CELL GROWTH INHIBITION OF CORYNEBACTERIUM DIPTHERIAE AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS CIPROFLOXACIN (50 µG/ML). ALL DATA ARE MEAN +/- SEM N=3...... 148 FIGURE 45 CELL GROWTH INHIBITION OF ESCHERICHIA COLI AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS CIPROFLOXACIN (50 µG/ML). ALL DATA ARE MEAN +/- SEM N=3. 148 FIGURE 46CELL GROWTH INHIBITION OF PSEUDOMONAS AERUGINOSA AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS CIPROFLOXACIN (50 µG/ML). ALL DATA IS MEAN +/- SEM N=3...... 149 FIGURE 47 CELL GROWTH INHIBITION OF BACILLUS SUBTILIS AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS CIPROFLOXACIN (50 µG/ML). ALL DATA ARE MEAN +/- SEM N=3. 149 FIGURE 48 CELL GROWTH INHIBITION OF CANDIDA ALBICANS AT EXTRACT CONCENTRATION OF 10 µG/ML AND 100 µG/ML. POSITIVE CONTROL IS NYSTATIN (50 µG/ML). ALL DATA ARE MEAN +/- SEM N=3...... 150

xviii List of Tables

TABLE 1 MEDICINAL PLANTS USED BY A VARIETY OF TRIBES OF AUSTRALIAN ABORIGINALS ...... 29

TABLE 2 ENDOPHYTES ISOLATED FROM MEDICINAL PLANTS WORLD-WIDE ...... 33

TABLE 3 EXAMPLES OF NRPS AND PKS SYNTHESISED COMPOUNDS ...... 37

TABLE 4 MEDICINAL PLANTS USED BY THE DHARAWAL PEOPLE OF BOTANY BAY ...... 50

TABLE 5 PLANTS SECTIONS COLLECTED AND LOCATION WITHIN BOTANY BAY ...... 58

TABLE 6 ENDOPHYTE ISOLATION AND SURFACE STERILISATION RESULTS ...... 76

TABLE 7 BLASTN ANALYSIS OF 18S RRNA GENE SEQUENCES FROM ISOLATED FUNGAL ENDOPHYTES . 77

TABLE 8 BLASTN ANALYSIS OF 16S RRNA GENE SEQUENCE FROM ISOLATED BACTERIAL ENDOPHYTES

...... 79

TABLE 9 ISOLATED ENDOPHYTES AND THEIR BIOACTIVITY ...... 94

TABLE 10 PCR PRIMERS USED AND AMENDMENTS TO REACTION AND CYCLING CONDITION ...... 97

TABLE 11 BLAST COMPARISON OF MIXED SEQUENCE FROM NRPS AND PKS SCREENING ...... 107

TABLE 12 ENDOPHYTE NRPS AND PKS ENCODING GENE SCREENING AND BIOACTIVITY RESULTS .... 112

TABLE 13 NMR DATA FOR SDI-139 COLLECTED AT 600HZ (1H) & 150 MHZ (13C) IN METHANOL-D-4

...... 138

TABLE 14 GROWTH INHIBITION OF TEST ORGANISMS BY ENDOPHYTE FRACTIONS AT 100 µG/ML

(%+SEM) ...... 146

TABLE 15 GROWTH INHIBITION OF TEST ORGANISMS BY ENDOPHYTE FRACTIONS AT 10 µG/ML (%+SEM)

...... 146

xix 1 Chapter 1 Introduction: Goori’s, Invaders and Modern

Ethnobotany: The Evolution of Traditional Medicine of Australia

1.1 Introduction

Australian Aboriginals are the original inhabitants of the land now known as

Australia and have lived there since the era of creation, the Dreamtime. Australian

Aboriginals have one of the oldest surviving and continuing cultures in the world today.

Many scientists and academics agree that Australian Aboriginals settled the continent

40000 years ago [1] and that the first humans to reach Australia did so by sea from

South East Asia. The oldest archaeological evidence of human occupation in Australia is a skeleton that was uncovered at Lake Mungo in Western New South Wales (NSW) in 1974. Dating techniques used on the skeleton and sand from the burial site suggest that the skeleton was 57,000–71,000 years old [2]. The latest theory to back these findings, comes from genomic studies of Australian Aboriginals, which suggest that they were one of the first modern human groups to disperse from Africa 65000-75000 years ago, and are one of the oldest continuous populations outside of Africa [3].

There is a misconception in the way that modern society views Aboriginal

Australia. It is often thought that there is one type of Aboriginal person, with uniform tradition and culture across Australia, however this is far from reality. Before European invasion of Aboriginal Australia, there were an estimated 300 tribes (Figure 1)

Australia-wide, with over 200 languages spoken, comprising about 600 dialects [4, 5].

Early population estimates by the first Europeans ranged from 150,000 to 1,000,000 people, as reported by the first governor of NSW [6]. However, the most commonly accepted estimates are around 300,000.

Each tribe across Australia had boundaries that separated their land from those of their neighbouring tribes. Tribal areas are like nations or countries, with each tribe

20 speaking a different language or dialect, and having different beliefs and ways of life

[7]. For example, the Dunghutti people (also pronounced Dhanggati, Dungatti,

Daingatti, Thunghutti, Thangatti) are a tribal group located on the Northern NSW coast of Australia. The Dunghutti tribal boundaries range from roughly the Wilson River near

Port Macquarie, westward to Walcha, northward to Ebor and back through Bowraville to the coast near Nambucca. The neighbouring tribes of the Dunghutti are the Biripai to the south, Gumbainggir to the north and the Aniwan to the west [5, 7-10]. Each of these tribes spoke different languages, and had variations in regards to their rules and lore.

Each tribe consists of clans or sub-tribes and these usually consisted of family groups that may have spoken different dialects of the same language. This could also account for the modern misconception or overestimation of the number of language and tribal groups that were present at the time of European arrival.

As tribal boundaries change from place to place, so do geographical features, weather, seasonal patterns, and availability of fauna and flora. Plants that grow in certain biomes will not grow in others; for example, plants that grow in coastal areas will differ from plants growing in tropical areas and more than likely will not grow in arid central Australia. Plants can therefore be exclusive to certain tribal areas and, therefore, be used as a valuable commodity. Given this, different plants may be used to cure the same disease in different tribes depending its location.

21 Figure has been removed due to Copyright restrictions.

Figure 1 Dunghutti tribal area and their surrounding [11].

1.2 Indigenous People and Plants

The term ‘ethnobotany’ was first used by the American botanist John W

Harshberger in 1895 to describe the studies of “plants used by primitive and Aboriginal people” [12]. Over time ‘ethnobotany’ has been redefined multiple times and is now defined as all studies concerning the mutual relationship between plants and traditional people [13]. Ethnobotany encompasses a number of scientific disciplines, including botany, anthropology, archaeology, phytochemistry, pharmacology, medicine, history, religion and geography [14]. This range of disciplines has rendered the study of ethnobotany one of the most intriguing fields of research for many centuries. European explorers have documented and “discovered” certain plants that have come from their newly explored lands. For example, Christopher Columbus discovered tobacco in Cuba in 1492 and almost 350 years later, British explorer Richard Spruce first noted the psychoactive properties of the South American vine Banisteriopsis caapi, both resulting from the observation of plants used by local, native people [13].

Sir Joseph Banks, a botanist present on the first voyages of the British led by

Lieutenant James Cook, observed and documented use of plants by indigenous people. 22 He noted in his journal how the people of Tierra del Fuego, South America, used various plants for shelters, Marais, boats and canoes, weapons, ropes to climb cliffs and local food. On his further voyage, he observed the Maori of New Zealand using their plants for building the beautifully carved and decorated Marais, canoes, haircombs, clothes, bedding and mats, baskets, burial chambers, food, fishing nets and weapons

[15]. Banks also observed plants used by the Dharawal people of Botany Bay,

Australia, to construct shelters, weapons, canoes, fishing lines, baskets and scarred trees used to catch prey. Several other accounts of plants used by the Dharawal people of

Sydney and surrounding tribes were also recorded in journals by first fleet personnel including Governor Arthur Phillip [16], Surgeon General of the settlement (Port

Jackson) John White [17], convict George Barrington [18], Lieutenant Phillip Gidley

King [19], surgeon of the Sirius, George Worgen [20] and Judges Advocate and secretary of the colony, David Collins [21].

Indigenous people have been using plants as a major part of their culture and survival since the beginning of their existence and continue to do so in the present day.

Indigenous people from Australia utilise different plants for a variety of applications including tools and weapons, for example, constructing spears from grass trees

(Xanthorrhoea species) [22], woomeras from the mulga tree (Acacia aneura) [23] and shields, boomerangs and buundis (clubs) from Avicennia marina and Aegicerias corniculatum [24]. Bark of the stringy bark tree (Eucalyptus agglomerata) is used to construct canoes [22, 25] and people built shelters by using whole bark pieces of eucalypt or melaleuca as a roof [7]. Plants were used for food, such as the pig face

(Carpobrotus glaucescens), bracken fern root (Pteridium esculentum) [22] and numerous other examples including native fruit, nuts, flowers, seeds and roots. Certain plants were also used for fire preparation, using a hardwood plant and the stalk of the

23 grass tree, Xanthorrhoea species, to create firesticks; poisons for hunting food; such as

Acacia sp. used as emu and fish poisons or plants used for their narcotic properties, for example Duboisia sp. (corkwood) that was used for social and ceremonial purposes and most importantly for medicinal properties [7, 9].

1.3 Medicinal Use of Traditional Knowledge

Traditional knowledge has influenced and shaped the modern medicines of today. Many pharmaceutical or prescription medicines contain an active ingredient that was originally derived from a plant. Some examples include the precursor for aspirin, salicylic acid, the analgesic compound morphine, the anti-malarial drug quinine and the bronchodilator ephedrine [12]. Indigenous people of Australia have used plants as medicines for tens of thousands of years and have a vast knowledge of Australia’s unique flora [7]. All tribes had a special person of significance that retained medicinal knowledge. The knowledge included what plants to use for which disease, when the plants were available, where to find the plants and how to prepare the medicines. This knowledge of what plants to use for healing purposes was passed down from generation to generation, particularly to people who reach a level of social importance within the tribe.

There are some very interesting examples of the creation of modern medicine from the traditional knowledge of plants worldwide including from the indigenous people of Australia. The most notable commercial example of traditional Australian medicine is tea tree oil. The main source of extracted tea tree oil is the plant Melaleuca alternifolia. Tea tree is traditionally prepared by crushing the leaves and soaking them in water to create a poultice or a liquid. This is then applied to wounds or used as a rinse to treat and wash-out bacterial infection, to remove lice or ticks and to treat fungal infection. In modern times, tea tree oil was first extracted on a large-scale in 1923 and

24 was a standard issue in all Australian medical kits for soldiers and sailors during World

War II because of its antiseptic properties [26]. There are over 100 monoterpenes, sesquiterpenes, hydrocarbons, and their alcohols that constitute tea tree oil, with the main anti-microbial constituents being the terpenes, terpin-4-ol, g-terpinene, a- terpinene, 1,8-cineole and a-pinene [27-29]. Tea tree oil has a range of activities including anti-bacterial [30], anti-fungal, anti-viral [31] and possibly anti-cancer [32,

33] to name a few.

1.4 Recent Research on Australian Aboriginal Medicine Plants

Numerous studies over the last century have led to the discovery and characterisation of bioactive compounds from plants worldwide, with activities including anti-bacterial, anti-inflammatory, anti-fungal, anti-viral and anti-cancer effects. Plants traditionally used by Indigenous people cure a range of illnesses; however, in the past little work focused on what bioactive compounds are present in the

Australian Aboriginal medicinal botany [34]. Over the last 10-20 years, there has been an increased interest in the study of these plants, with most of the studies focussing on plants from the tribal areas of Central, North and Western Australia. The investigated plants have been tested for their anti-microbial, anti-viral, anti-inflammatory and anti- fungal activities, and it has been hypothesised that some plants showed activity against diabetes, heart problems and even cancer. The following examples are investigations into the activity of plants used by Aboriginal people against some common illnesses.

A commonly studied plant is from the Eremophilia genus. In one study, thirty- nine native plants were tested for anti-bacterial activity against eight common Gram- positive and Gram-negative bacteria. The extracts exhibiting the greatest activity were from the leaves of a plant called Red Poverty Bush (Eremophilia duttonii) [35], which is a shrub found in Central Australia. In traditional medicine, it is used to treat

25 respiratory tract infections, sore throats and painful ears [36]. Extracts of the leaf showed significant growth inhibition of Bacillus cereus, Entercocci faecalis, and

Staphylococcus aureus within 1 hour, and within 2 hours in cultures of Streptococcus pyogenes, in time course growth assays [36]. In a separate study of Eremophilia duttonii, two diterpenes, serrulat-14-en-7,8,20-triol and serrulat-14-en-3,7,8,20-tetraol, were isolated and exhibited anti-bacterial activity against S. aureus, S. epidermidis and

S. pneumoniae [37]. Other species of Eremophilia also showed cardioactive effects in rats, while the compound verdascoside, isolated from methanol extracts of the leaf, increased heart rate, contractile force and Coronary Perfusion Rate (CPR) [38, 39].

Interestingly, geniposidic acid was isolated from the same plant and it showed the opposite cardiac effects to verdascoside. From another Eremophilia species,

(Eremophilia serrulata) came two new compounds, o-naphthoquinone, 9-methyl-3-(4- methyl-3- pentenyl)-2,3-dihydronaphtho[1,8-bc]pyran-7,8-dione and a serrulatane diterpenoid, 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid. The o-naphthoquinone showed anti-bacterial activity against the Gram-positive S. aureus, S. pneumoniae and

S. pyogenes; however, no activity was observed in any Gram-negative microorganisms

[40]. Two serrulatane diterpenes, 3,8-dihydroxyserrulatic acid and serrulatic acid, were isolated from Eremophilia sturtii that is traditionally used as a wash for sores and cuts as a disinfectant. The two compounds show anti-bacterial activity against S. aureus, as well as anti-inflammatory activity. Cyclooxygenase and lipoxygenase inhibitor screening resulted in the inhibition of COX-1 and COX-2 by 57% and 14% for 3,8- dihydroxyserrulatic acid and 99% and 97% for serrulatic acid, respectively [41].

In another study twenty-four native Australian plants were tested for activity against COX-1 enzymes and the arachidonic acid metabolites PGE2 and PGD2, which are known to be involved in inflammatory responses [42]. Whipstick wattle (Acacia

26 adsurdgens) is a rigid shrub that has been used by Aboriginal people to treat inflammatory-related illnesses [35]. Acacia adsurdgens and Acacia ancistrocarpa showed more than 70% inhibition of COX-1, 84% of inhibition of PGE2 and 92% inhibition in the PGD2 assay [43].

In another investigation, sixteen other native plants were tested for activity against the enzyme xanthine oxidase (XO). XO is responsible for production of uric acid from purines and causes gout, a painful arthritic joint condition. Lolly bush

(Clerodendrum florrbundum) [44] extracts inhibited XO activity by 84% with 6 other plants showing greater than 40% inhibition [45].

Research by von Martius et al., showed the bioactivity of kino, the sap that is extruded from the wood of Eucalyptus species. Kino was collected from 19 varieties of eucalypt trees in Kings Park Botanical gardens, Perth, Western Australia. The study showed that the majority of kino is rich in tannin and phenolic compounds, while kino collected from Corymbia maculata and Eucalyptus ficifolia showed the strongest anti- bacterial activity against a number of Gram-positive indicator organisms [46].

Euphorbia australis was included as one of the thirty nine plants in the study mentioned above and had been the only medicinal plant to show activity against Gram-negative bacteria, with all other plants showing activity against Gram-positive by using diffusion assays and time course growth assays [36].

Apple bush (Pterocaulon sphacelatum) [35], a perennial herb, has been used to treat respiratory infections, particularly colds, as well as fever and headaches in traditional medicine. In recent studies P. sphacelatum extracts were tested using the poliovirus assay and yielded an anti-viral flavonoid chrysosplenol C [34].

Chrysosplenol C belongs to a group of compounds that are responsible for the anti-

27 replication activity against picornaviruses and rhinoviruses, which cause pulmonary infection and common cold.

Smoke bush is a plant that only grows in Western Australia and was the subject of a study conducted by the US National Cancer Institute. Though inactive against cancer, extracts from certain parts of the bush were tested against the HIV virus. The active compound present was named conocurvone and shown to be effective at concentrations of 0.02 µΜ in preventing HIV from killing infected cells [47, 48].

A study conducted into compounds of the medicinal plant Dodonaea polyandra, collected from Northern Queensland around the Cape York Peninsula area, also showed significant anti-inflammatory activity in the mouse ear oedema model of acute inflammation induced by croton oil and 12-o-tetradecanoylphorbol-13-acetate (TPA)

[49].

Finally, Duboisia species are native to Queensland and Northern NSW and contains an active compound, hyoscine, which has been used as a sedative in treating motion sickness. It is used in the medicine Buscopan® and can also be used as a truth serum [50, 51].

These selected examples illustrate the broad application of Australian native plants as sources of medicines, some of which have been used by Indigenous

Australians for many years. Table 1 contains further examples of traditional medicine plants used by Indigenous Australians to treat various conditions, as well as details of their medicinal preparations.

28 Table 1 Medicinal plants used by a variety of tribes of Australian Aboriginals

Plant species Common name Geographical Traditional use and Biological Reference region preparation Activity Acacia implexa Hickory Sydney region Used as a fish poison. ND [22] NSW Also bark was soaked in water to treat skin infections. Dendrobium Rock orchid East coast of NSW Stems were chewed and ND [23, 52, 53] speciosum from Newcastle to rubbed onto sores, Victoria and inland wounds and burns to the upper Hunter Valley. Duboisia Emu plant, Western NSW, Leaves chewed or dried Narcotic, [44, 54, 55] hopwoodii camel poison, QLD, Central leaves and sticks stimulant poison bush Australia to NW ground and smoked coast of WA. Smilax Native NSW coast and Leaves brewed into a Antioxidant [22, 24, 56] glyciphylla Sarsaparilla Northern tea to help with colds Tablelands and up and flu, unsettled into QLD stomach, for sores and wounds, blood cleansing, diabetes and for general well being Planchonia Cocky Apple Northern WA, NT Pulped leaves used to Anti-bacterial [44, 57] careya and Northern QLD cure sores and ulcers. Bark soaked in water and tipped into wound for healing. Roots mashed and applied to burns and sores. Eremophilia Kangaroo Central Western Leaf decoctions used as Anti-bacterial [37, 44, 58, duttonii bush, red WA, Central a wash for colds and 59] poverty bush southern NT, SA, flus and as an antiseptic South Western for skin infections, open QLD and North wounds, sore ears and Western NSW eyes and drunk to help sore throats and respiratory infections. Melaleuca Swamp tea North QLD, NT Leaves crushed and ND [44] cajuputi subsp. tree, paperbark and Northern WA rubbed on to treat aches cajuputi tea tree and pains. Also soaked in water and bathed in to relieve pain and headaches Corymbia Northern NT and Kino was sucked or ND [44] dichromophloia WA made into a weak drink to treat colds and bronchial infections, mouthwash or used to plug holes in tooth. Gleichenia Pouched coral Coast and ranges of Stems/leaves used to ND [24] dicarpa fern East Australia deter insects and flies

Dodonaea Hop bush Cape York Plant material applied Anti-bacterial [49] polyandra Peninsula directly to hole after removal of rotten tooth.

29 Tinospora Snake vine Northern regions of Mashed stem applied to Anti- [44, 60, 61] smilacina Australia and also ease pain of stomach inflammatory Northern NSW ache, rheumatism, cytotoxic headaches and stingray/stonefish stings. Ipomoea pes- Goat's foot North coast NSW Used to ease the pain of Anti- [62-64] caprae and coastal QLD stingray and stonefish inflammatory, stings anti-spasmodic, antinociceptive, anti-bacterial and resistance modifying, cytotoxic. Phytolacca Inkweed Naturalised Leaves boiled in water Anti-fungal [24, 65] octahedra Southern and and applied to Eastern Australia. ringworm, feet soaked in that water to cure tinea and also water used as a wash for scabies. Also used in preparation of a plant tonic mix. Dodonaea Hop bush Cape York Plant material applied Anti-bacterial [49] polyandra Peninsula directly to hole after removal of rotten tooth. Conospermum Smokebush Western/Central Anti-HIV [66] sp. Australia Grevillea Red grevillea North Western WA Inner bark and leaves ND [44, 67] heliosperma and NT boiled to make an antiseptic wash for scabies, boils, infected sores. Castanospermum Black bean or Seeds split and soaked Anti-HIV [68, 69] australe Morton bay in running water. Used chestnut to make damper (bread). Melaleuca Tea tree Coastal QLD into Prepared by crushing Cytotoxic, anti- [30-33] alternifolia Northern NSW and soaking the leaves bacterial, anti- using the resulting fungal, anti- poultice/liquid extract viral, to apply to wounds or used as a rinse to treat/wash out infections, sore throats, removal of lice/parasites, treat fungal infections, to treat cold and flu symptoms

30 1.5 Traditional Medicine in the Modern World

Cultural practice in Australia was always taught and passed on by word of mouth in the form of stories and songs usually accompanied by dance, rock art or rock engravings, with nothing ever being documented in written words. It has become an increasing trend in research today to study and document traditional knowledge from all cultures around the world. An example of a published Australian Aboriginal ethnobotanical survey is the first comprehensive survey carried out on the tribal area of the Yaegl Aboriginal community in Northern NSW [70]. The interviews were conducted in 2004-05 and 2008-09 with a total of nineteen elders. The outcomes of the survey were the revelation of fifty-four plants that have been prepared and used for a variety of illnesses and conditions by the local people. This traditional knowledge was published as a medicinal handbook and returned to the community for local use.

Although plants used as medicinal remedies for a wide range of different aliments are often considered to be solely responsible for the healing properties, the popular view is that there maybe a symbiotic microbe that may contribute, directly or indirectly to the production of the active compounds.

1.6 A Microbiological Approach to Traditional Medicine

The ethnobotanical study of the medicinal use of plants has led to the discovery of a large number of pharmaceutically important compounds or products used in modern pharmacy. As mentioned above, these include aspirin (analgesic) [71], curare

(anaesthetic) [72], tea tree oil (anti-microbial, disinfectant) [26], quinine (anti-malarial)

[12], digitalis (anti-arrhythmic) [12], and hyscoine (stomach relaxant) [51], just to name a few examples. These compounds may not be produced by the plant itself but could rather be a result of the production of secondary metabolites from microorganisms that live within the plant tissue, a plant response to the presence of microbes as a defence

31 mechanism, or a partial involvement of the microbe in the steps of metabolite production. Such plant-associated microbes are known as endophytes.

Endophytes are either fungi or prokaryotic microorganisms, including bacteria and archaea, which are found to reside within the tissue of a host plant and may have a range of effects from beneficial to asymptomatic to pathogenic [73, 74]. An endophyte can be found in every plant on Earth and are known to produce a number of effects on the host [74]. It is not fully known what role endophytes play in most plants, with suggestions that they help with plant defence against predators [75], help promote plant growth in harsh environments [76-79] or contribute a substrate or metabolic steps in the production of metabolites that improve plant growth [80].

Endophytes are also now known to produce a number of secondary metabolites that are bioactive against a number of pathogenic organisms and indicator cell lines.

These molecules have the potential to be commercially produced as drugs or for other applications, for example as pesticides. A well known example of an endophyte that produces a pharmaceutically important compound involved the isolation of Taxomyces andreanae, from the Yew tree, Taxus brevifolia, that producs the highly valued anti- cancer compound taxol [81]. Other examples of endophytes and their associated bioactive compounds include Cladosporium cladoporioides, also producing the anti- cancer compound taxol [81, 82]; Lophodermium sp. which produces the anti-fungal pyrenophorol and related sesquiterpenes [83]; Muscodor crispans which synthesises a number of anti-bacterial and anti-fungal volatile organic compounds [84]; and

Stemphylium globuliferum that produces alterporriol G and its atropisomer alterporriol

H with cytotoxic activity against leukaemia L5178Y cells [85].

In recent times, the search for commercially significant endophytes has turned to the use of ethnobotanical information from Indigenous people and their traditional

32 medicine (Table 2). From this information, knowledge of the specific activity resulting from the use of a particular plant determines the selection criteria for the isolation of endophytes and increases the potential that the endophyte will produce or be involved in producing a bioactive compound. Table 2 summarises some of the endophytes that have been isolated from traditional medicinal plants and the bioactivity of the endophyte extracts.

Table 2 Endophytes isolated from medicinal plants world-wide

Endophyte Plant name Country Traditional use of plant Activity of Reference endophyte Phoma sp. Arisaema China Remove phlegm, arrest Anti-fungal (plant [86, 87] ZJWCF006 erubescens convulsions, dispel wind and pathogen), reduce swelling cytotoxic Chaetomium Curcuma China Eliminate blood stasis, Anti-fungal, [87, 88] globosum strain L18 wenyujin stimulate menstrual discharge cytotoxic (Erzhu) and relieve pain Aspergillus sp. Garcinia Malaysia Treat peptic ulcer and Anti-bacterial, [89] HAB10R12 scortechinii postpartum care cytotoxic Chloridium sp. Azadirachta India Treat fever, pain, leprosy, and Anti-bacterial, anti- [90, 91] indica A. Juss. malaria fungal, Insecticidal (Neem) Stemphylium Mentha Morocco Treat common colds, Cytotoxic [85] globuliferum pulegium disorders of the liver and gall- bladder, as a carminative, as a diuretic, and to stimulate digestive action Fusarium sp. Tlau3 Thunbergia Thailand Detoxification, anti- Anti-acanthamoeba [92, 93] laurifolia Lindl. inflammatory and anti-pyretic Ampelomyces sp. Urospermum Egypt Anti-inflammatory properties Anti-bacterial, [94] picroides cytotoxic Streptomyces sp. Kennedia Australia Treat cuts, wounds, and Anti-bacterial, anti- [95] strain NRRL 30562 nigriscans infections Plasmodium Preussia minima Eremophilia Australia Respiratory complaints, Industrial [96] longifolia headaches, infection healing, important enzymes viral infections Cladosporium Ocimum India Cure common colds, Anti- [97] cladosporioides sanctum L. headaches, stomach disorders, cancer/cytotoxic inflammation, heart disease, various forms of poisoning, and malaria Xylaria sp. (PR6) Emblica India Treatment of diabetes Anti-microbial, [98, 99] officinalis mellitus, chronic diarrhoea, antioxidant (Amla) anti-inflammatory and antipyretic Endophyte MD-09 Ophiopogon China Coughing, phlegm and lung Anti-bacterial [100] japonicus infections Phaeoacremonium Aquilaria China Leaves used for inflammation Anti-bacterial, anti- [101, 102] rubrigenum sinensis and anaphylaxis fungal, anti-tumor

1.7 Endophytes from Native Australian Plants

Australia has one of the most unique range of environmental niches in the world, with a great amount of diversity among plant species across the country. Numerous studies have been conducted on Australian native plants, including grasses, of the medicinal and non-medicinal kind. Studies on the presence and population of endophytes have also been reported from plants across Australia.

Seven new species of the fungi family Botryosphaeriaceae, namely Dothiorella longicollis, Fusicoccum ramosum, Lasiodiplodia margaritacea, Neoscytalidium novaehollandiae, Pseudofusicoccum adansoniae, P. ardesiacum and P. kimberleyense were isolated from the only endemic species of Adansonia (Adansonia gibbosa)

(Baobab tree) found in northern Western Australia [103]. A further eight

Botryosphaeriaceae species were isolated from nine different species of tree from the

Yalgorup National Park and Woodman Point Regional Park on the south western coast of Western Australia. Four new species were described as Dothiorella moneti,

Dothiorella santali, Neofusicoccum pennatisporum, and Aplosporella yalgorensis [104].

Kaewkla and Franco conducted research on 4 native treesfrom which 576 endophytic isolates were obtained and classified into 17 different genera, with the majority of the isolates being actinobacteria (Streptomyces sp.) [105]. The trees were the native pine tree, Callitris preissii, a Red Gum tree, Eucalyptus camaldulensis, a

Grey Box tree, Eucalyptus microcarpa and the native apricot tree, Pittosporum phylliraeoides that also have revealed several novel endophytic species [106-112]. In similarly extensive survey, 281 endophytes were isolated from four species of the native plant Gossypium. Of the 17 genera identified in this study, the most commonly isolated were Phoma sp., Alternaria sp. and Fusarium sp.[113].

34 1.8 Endophytes and Australian Medicinal Plants

Using traditional knowledge to target plants for endophytic surveys is an increasingly common practice when searching for microorganisms that produce bioactive compounds. The fern leaf Grevillea, Grevillea pteridifolia, is traditionally used as a food where the nectar is sucked out or collected from the flowers. The bacterium Streptomyces sp. 30566, isolated from the stem of that species, produced the novel antibiotics (kakadumycins with kakadumycin A) that show both anti-bacterial and anti-malarial activities [114].

Melaleuca quinquenervia is a plant used traditionally to cure colds and chest infections, wound healing and skin infections and internal complaints, and is also included in a concoction as a skin wash. A fungal endophyte identified as a

Pestalotiopsis sp. was isolated and shown to produce three novel caprolactams, pestalactams A-C. Pestalactam A and B, tested for anti-malarial and anti-cancer activity, had modest activity in the assays used [115].

Acacia sp. is a plant commonly used by Indigenous people all over Australia, not only for medicinal purposes but also for the construction of shelter, tools, weapons and for fish and emu poisons. Six fungal endophytes of the genera Aureobasidium,

Chaetomium and Sordariomycetes were isolated from the phyllodes of three Acacia sp.

All but one endophyte showed activity against Escherichia coli and Candida albicans, among other target species. Three of the endophytes also showed amylase activity that has proposed industrial applications [116].

Snakevine (Kennedia nigriscans) is a vine traditionally used as a remedy for wound and skin infection. An endophytic Streptomyces was isolated from the stem of the vine and shown to produce four novel antibiotics munumbicins A-D. Munumbicins

A-D are active against a range of selected plant pathogens, with munumbicins B and D

35 showing the highest anti-bacterial activity. Munumbicins B had activity against multidrug resistant Mycobacterium tuberculosis, while all isoforms had anti-fungal activity against at least one human pathogenic strain as well as against the causal agent of malaria, Plasmodium falciparum. Munumbicin C and D were shown to be more potent against P. falciparum than the standard anti-malarial drug chloroquine [95]. Further studies of Streptomyces NRRL 30562, revealed two other novel antibiotics, munumbicin E4 and E5, that both showed activity against Gram-negative and Gram- positive bacteria, as well as, the plant pathogen Pythium ultimum (Oomycota) and also had anti-malarial activity [117].

1.9 Non-ribosomal Peptide Synthetase and Polyketide Synthase

In the early twentieth century, the official discovery of antibiotics produced by microorganisms, and their large-scale production, changed the way the world dealt with infection and treatment of illnesses. However, with the emergence of antibiotic-resistant microorganisms and re-emergence of diseases such as TB, the search for novel anti- infective agents has become a priority for the future health and well being of humans worldwide. The majority of these pharmaceutically important natural compounds and their semi-synthetic derivatives are non-ribosomal peptides, polyketides or a hybrid of both. These compounds are produced by the enzyme complexes non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). Table 3 shows examples of compounds that are biosynthesised by NRPS and PKS enzyme complexes.

36 Table 3 Examples of NRPS and PKS synthesised compounds

Compound Enzyme complex Organism Bioactivity Reference B Daptomycin NRPS Streptomyces roseosportus Antibiotic [118] F Cyclosporin A NRPS Tolypocladium nivem Immunosuppressant [119] B Laxaphycin NRPS Anabaena laxa Anti-fungal [120] B Erythromycin A PKS Saccharopolyspora erythraea Anti-bacterial [121] B Oxytetracycline PKS Streptomyces rimosus Anti-bacterial [121] Bryostatin PKS Bugula neritina Anti-cancer [122] B Albicidin Mixed Xanthomonas albilineans Anti-bacterial [123] NRPS/PKS B Leinamycin Mixed Streptomyces atroolivaceus S-140 Anti-tumour [124] NRPS/PKS

B Bleomycin Mixed Streptomyces verticillus Anti-tumour [125] NRPS/PKS B Cephabacin Mixed Lysobacter lactamgenus Anti-bacterial [126] NRPS/PKS B Microcystin Mixed Microcystis aeruginosa Heptotoxin [127, 128] NRPS/PKS B Cylindrospermopsin Mixed Cylindrospermopsis raciborskii Heptotoxin, [129] NRPS/PKS Neurotoxin and Cytotoxin

1.10 Structure of Non-ribosomal Peptide Synthetases and Mechanism of

Biosynthesis

Non-ribosomal peptides are some of the most important pharmaceutically important compounds that include penicillin [130] and vancomycin [131, 132].

NRPS have been discovered in a number of bacteria and fungi, producing compounds that show a range of bioactivity including anti-bacterial, anti-tumour and anti-fungal compounds (Table 3). NRPS use amino acid to synthesis peptides without the use of ribosomes. The NRPS complex does not only incorporate the conventional 20 amino acids but also non-proteinogenic amino acids to add more variance to the synthesised compound. For example, L-α-aminoadic acid is a non-proteinogenic amino acid that is incorporated into the tripeptide d-(L-α-aminoadipyl)-L-cysteinyl-D-valine, a precursor to the production of the β-lactam antibiotics penicillin and cephalosporin family [130,

133]. Non-ribosomal synthetases are comprised of three basic domains that are present

37 in all modules, an adenylation domain (A), a peptidyl carrier protein (PCP) and a condensation domain (C). The adenylation domain is responsible for the recognising and activation of the required amino acid. Once attached to the A domain, it is the covalently attached to the peptidyl carrier protein via a 4’-phosphopantethein binding co factor, which is involved in the binding and transportation of the activated amino acid from the A domain to the C domain. The C domain then catalyses the peptide bond formation between the activated amino acid on the first module with the neighbouring

2nd module’s amino acid. This process continues until the required peptide is formed and is released from the enzyme complex by the thioesterase (TE) domain.

Other mechanisms of compound variety include enzyme domains for epimerisation (E) of amino acids to D-amino form, heterocyclisation, N-methylation and/or N-formylation [134-137]. A general representation of a PKS enzyme complex is shown in Figure 2.

Figure has been removed due to Copyright restrictions.

Figure 2 Simplified diagram of a NRPS module [138].

38 1.11 Structure of Polyketide Synthase and Mechanism of Biosynthesis

Polyketide synthases (PKS) are another multi-enzyme complex that is involved in biosynthesis of natural pharmaceutical products drugs, such as erythromycin A [139,

140] and tetracycline [141]. The polyketide synthesis starter molecule is malonyl-CoA, however, some PKS enzymes can use a more diverse range of starter and extender molecules including methyl-, ethyl-, propylmalonyl- and chloroethylmalonyl-CoA [142,

143]. A PKS enzyme complex is an assembly line that contains, as a minimum, an acyl transferase module (AT), an acyl carrier protein (ACP) and a ketosynthase (KS).

Polyketide synthesis starts by the selection and activation of a short starter unit from the

AT domain where it is then covalently attached to the ACP via the 4’- phosphopantethein binding cofactor and transported to the KS domain where it condensed to the neighbouring extender unit to form a small ketide chain. This continues until the desired polyketide is synthesised and transported to the thioesterase domain (TE) where it is released from the enzyme complex. PKS enzyme complexes also achieve variance in the molecule’s structure through the presence of extra enzyme domains that are incorporated into the PKS in different positions along the assembly line and include ketoreductase (KR), dehydratase (DH) and enoylreductase (ER).

There are three types of polyketide synthase enzymes, type 1, 2 and 3.

Type 1 PKS can be divided into two groups, modular and iterative. Modular

PKS consist of a number of modules that repeat along an assembly line. Iterative PKS is a single module that is used repeatedly to synthesis the PK molecule. Examples of a modular PK is aureothin [144] and an iterative PK is lovastatin [145]. Type 2 PKS are similar to PKS 1 however differ by containing a minimal ketoacyl synthase (KSα), a chain length factor (KSβ) and an acyl carrier protein (ACP) [146, 147]. Type 2 PKS produce aromatic polyketides, such oxytetracycline [148] and other tetracycline

39 antibiotics. Type 3 PKS lack multi-enzyme domains and catalyse reactions in a single step using coenzyme A thioesters directly as substrates [149]. Type 1 and type 2 PKS are usually only found in bacteria and fungi with type 3 PKS usually only found in plants, although a small number of type 3 PKS have been found in microorganisms

[142]. A general representation of a PKS enzyme complex is shown in Figure 3.

Figure has been removed due to Copyright restrictions.

Figure 3 Simplified diagram of a PKS module [138].

1.12 Structure of NRPS/PKS Hybrids Enzyme Complexes

Although these enzyme complexes exist and produce antibiotic compound as two separate and independent entities, there are instances in nature where the two enzyme complexes are involved in the biosynthesis of a NRP/PK hybrid molecule. The hybrid enzyme complex can be comprised predominantly of PKS and a few NRPS or vice versa. Production of the anti-fungal, immunosuppressive, anti-tumour, neuroprotective and anti-aging compound, rapamycin, involves 14 PKS modules spread over 3 proteins and 1 NRPS module that attaches a pipecolate moiety to the polyketide to create pre- rapamycin [150]. The assembly line of the anti-tumour compound, bleomycin, consists

40 of 10 NRPS modules and 1 PKS module [125]. The biosynthetic pathway of the hepatotoxic microcystin is also an integrated pathway [151] and is shown in Figure 4.

Figure has been removed due to Copyright restrictions.

Figure 4 Biosynthetic pathway of microcystin [151].

1.13 Conclusion, Significance and Future Prospects for Drug Discovery

Research on medicinal plants using a basis of ethnobotanical information has the potential to find cures for the ever-more resistant microbes that cause many of the diseases and illnesses of the modern world. Discoveries of the anti-cancer properties of taxol from the yew tree, anti-HIV activity of the smoke bush plant of Central Australia and the production of a multi-purpose antiseptic with possible anti-cancer properties in tea tree oil were made by observing the knowledge of these plants by traditional people

[33]. Studies of ethnobotany on Australian medicinal plants have already revealed novel antibiotics produced by endophytic Streptomyces spp. with activity against drug resistant strains of Mycobacterium tuberculosis and Staphylococcus aureus [95].

However, a great deal of traditional knowledge and traditional teachings has been, and will continue to be, diminished in most areas of the world. This is even more evident in

41 Aboriginal Australia with the knowledge available to today’s Aboriginal people becoming critically limited. A number of factors have contributed to this demise of traditional knowledge, including dispossession of the land from Aboriginal people. The dispossession of land has prevented the people of that area from practicing their culture on their land. Land that was cleared for farming or building townships has resulted in sacred sites being destroyed and removed. Animals of the local area and waterways have been forced away and over-fishing and over-collection of sea animals have depleted the aquatic life in coastal and river areas. Native plants that were protected and to some extent propagated grew in abundance and were readily available, but have now gone. This means that customs and traditional teachings were interrupted and not passed on to the next generation, no longer teaching them, in the traditional way, their relationship to land, the animals and special places within their tribal boundaries and beyond [7].

Another reason was the introduction of new government and missionary representatives that came to Australia to enforce new foreign laws and to introduce foreign religion to the Aboriginal people that conflicted with the lore and religion that the local people followed. This, coupled with forced assimilation into white Australia including the removal of children and of “half caste” children by the government and missionaries, made it impossible to continue traditional knowledge and practices. For example, in the 1800’s to as late as 1967, Aboriginal people that were caught talking their language or teaching/practicing traditional ways and culture, were threatened with the removal of their children by the government [7].

Depletion of the population through diseases such as smallpox, pneumonia, tuberculosis, the common cold, and to a lesser extent venereal diseases, accounted for many deaths among the Aboriginal population, especially in the Sydney region, where

42 first contact of Aboriginals and Europeans occurred. Within a year of settlement over half of the Aboriginal population living in the Sydney basin had died from smallpox

[152, 153]. People of all generations were lost, with the greatest loss of life in the middle aged and elderly whose knowledge was the greatest. This interrupted social rule and kinship, leaving the survivors deprived of ceremonial and oral traditions, not to mention leadership [7, 154].

In addition to these atrocities caused by the European invasion, a person or persons that guarded the knowledge of these medicinal plants, the uses and preparation, were usually kept secret, and only taught by word of mouth and demonstration. Thus little information was recorded [155]. Culturally, if knowledge holders thought that the people or persons would not be suitable or that the information would not be used in the correct way, the knowledge was not passed on to the next generation, and was sometimes lost [7]. However, some traditional knowledge that was held by different people in the tribe was passed down to the next generation, allowing the traditional knowledge and culture practices to survive into the modern day. This knowledge was and still is taught by traditional teaching methods, stories, and in extreme cases, secret meetings. This ensured that the culture and knowledge was not lost and can still being practiced across the country in today’s world.

As mentioned before, learning and education was key to ensure the survival of culture and traditional knowledge. In modern times, education and learning, especially for Indigenous people, is still important not only for continuation of culture but also as a tool for Indigenous people to take part and understand factors of their traditional knowledge that were unavailable to them without modern research techniques. This can be applied to education and training in areas such as molecular biology and microbiology that will allow Indigenous people to understand and build on their

43 knowledge. When this is applied, for example, to medicinal plants, endophyte isolation and DNA screening for genes that encode secondary metabolite production will reveal a more comprehensive scientific and in-depth understanding of the application of these important medicinal plants. Accessing only small amounts of plant material to isolate endophytes or obtain plant DNA, will aid in the conservation of possibly endangered plants and plant ecosystems so that they survive for future generations to utilise. There is a timely need to place greater emphasis on the importance of conserving the remaining rainforests and bushlands, and most importantly, the ones that have been utilised for millennia by Indigenous people. This will allow for the access of Indigenous people to these plants and plant communities so that they can ensure that these unique cultures and traditional practices can be preserved and passed on to future generations.

Hopefully then, the discovery of new medicines or drug lead compounds will continue the fight against antibiotic resistant strains of bacteria, incurable viral infections and other diseases, such as asthma, diabetes, and cancer, for the benefit of all.

1.14 Study Aims and Reasoning

This study sets out to utilise parts of traditional knowledge of the Dharawal people of Botany Bay to utilise medicinal plants and isolate endophytes and potentially bioactive natural products produced by these endophytes.

Firstly, a general introduction into ethnobotany, the use of traditional knowledge and the biosynthetic pathways of the enzyme complexes NRPS and PKS (Chapter 1) has been provided. Secondly, the ethnobotanical information collected and the medicinal plant species chosen for further study is reported (Chapter 2). This will be followed by microbial diversity studies of the endophyte community (Chapter 3). The genetic screening for NRPS and PKS genes encoding natural products along with preliminary bioassays (Chapter 4) will be presented. The isolation and characterisation

44 of compounds produced by selected endophytes is then described (Chapter 5). A general discussion and future direction of the study will be presented as the last chapter of the research topic.

1.15 The Key Steps in this Study

1. Collect and document the use of traditional medicine plant species of Botany Bay in coordination with the Dharawal elders and community members;

2. Isolate endophytes from the selected medicinal plant candidates into pure cultures and analyse these phylogenetic relationships;

3. Use molecular biology techniques to screen endophyte DNA for genes encoding for secondary metabolite-producing enzymes such as non-ribosomal peptide synthetases

(NRPS) and polyketide synthases (PKS). Use biochemical inference bioinformatics to characterise the uniqueness of these biosynthetic genes and perform bioassays to determine the bioactivity of these de-replicated endophyte extracts;

4. Apply chromatographic and spectroscopic techniques, such as TLC, HPLC and NMR and MS to identify and solve the structure of the bioactive compounds present.

45 2 Chapter 2 The Ethnobotanical Survey-Based Collection of

Medicinal Plants Used by the Dharawal People of Botany Bay

2.1 Introduction

2.1.1 The Dharawal People

The Dharawal tribe people, more specifically the Gweagal clan, were the first tribe to encounter the British, first in 1770 with the expedition led by Lieutenant James

Cook and later with the first fleet in 1788 led by Admiral Arthur Phillip.

Contact with Europeans first took place on the southern shores of Botany Bay as then Lieutenant James Cook sailed in on April 29 1770. The European presence and landing in Botany Bay was a hostile encounter, with the local Gweagal clan showing their displeasure towards Cook and his crew. To this end, the first ever exchange between the English and Aboriginal people was Cook’s crewmember firing a musket upon two warriors on the shore of what is now known as Kurnell. As written in the endeavour journal of Sir Joseph Banks on the boat approaching the landing site at

Kurnell:

“…for as soon as we aproachd the rocks two of the men came down upon them, each armd with a lance of about 10 feet long and a short stick which he seemd to handle as if it was a machine to throw the lance. They calld to us very loud in a harsh sounding Language of which neither us or Tupia understood a word, shaking their lances and menacing, in all appearance resolvd to dispute our landing to the utmost tho they were but two and we 30 or 40 at least. In this manner we parleyd with them for about a quarter of an hour, they waving to us to be gone, we again signing that we wanted water and that we meant them no harm. They remaind resolute so a musquet was fird over them, the Effect of which was that the Youngest of the two dropd a bundle of lances on the rock at the instant in which he heard the report; he however snatchd them up again and both renewd their threats and opposition. A Musquet loaded with small shot was now fird at the Eldest of the two who was about 40 yards from the boat; it struck him on the legs but he minded it very little so another was immediately fird at him; on this he ran up to the house about 100 yards distant and soon returnd with a sheild. In the mean time we had landed on the rock. He immediately threw a lance at us and the young man another which fell among the thickest of us but hurt nobody; 2 more musquets with small shot were then fird at them on which the Eldest threw one more lance and then ran away as did the other.” [15].

The Dharawal people occupy the land roughly from the south heads of Sydney

Harbour, in a westerly direction out to the area around Liverpool and Camden, and

46 directly south to the Shoalhaven River including the city of Sydney, the eastern beaches of Sydney, Botany Bay, Kurnell, Wollongong, Kiama, Gerringong, and Coolangatta.

The Dharawal had differing dialects within the tribal boundary: mainly a northern dialect spoken in Port Jackson, Botany Bay and Illawarra and a southern dialect spoken south of Wollongong to the area of the Shoalhaven [5, 7-9, 156]. Family or clan groups also existed within tribal boundaries and some of the known Dharawal clans in the

Sydney area include the Gadigal (Sydney Harbour), Birrabirragal (Rosebay, South Head and Bondi), Bideagal (St George, Peakhurst and Cooks River) and Gweagal (Kurnell and Sutherland) clans. The neighbouring tribes that surround the Dharawal are the

Kuring-gai tribe on the northern heads of Sydney Harbour, the Dharug tribe to the

Northwest, the Gundungarra tribe to the west and the Dhurga tribe to the South. All of these tribes, despite the close proximity and some common grounds they share, exhibited many differences from each other in regards to culture, language and knowledge. Figure 5 shows the tribal areas of the Dharawal and neighbouring tribes.

2.1.2 Ethnobotany and Ethnomedicine of the Dharawal

From the first discoverers setting sail in search of new lands, exotic plants have been widely sought as a source of food, medicines and economic gain. From

Christopher Columbus’ observation of tobacco use by the Cubans [13] to the uncovering of anti-microbial compounds in traditional medicine plants in modern times

[37, 40, 95, 157], the use of ethnobotanical information has introduced the rest of the world to the new and exciting topic of bioprospecting.

47 Figure has been removed due to Copyright restrictions.

Figure 5 Tribal boundaries of the Dharawal nation and surrounding tribes [11].

All Australian Aboriginal people have accumulated the knowledge of medicinal plants in Australia since the beginning of time. They have a vast knowledge of the unique flora used within their tribal area as medicines [34]. The study of ethnobotany and anthropology of Australian Aboriginals was keenly pursued by the early Europeans.

Samples were collected and documented by Sir Joseph Banks in 1770 and other

European persons that followed the first fleet in the years following 1788. Amongst the collection were canoes, spears, shields, boomerangs, ornaments, ceremonial objects and plants used for food and medicinal purposes. Still to this day 4 spears are in existence that were taken from the Gweagal by Sir Joseph Banks [158].

However, long before the arrival of the Europeans, Aboriginal people of

Australia used their vast knowledge of the use of all the plants available to them for healthy nutritional value, cultural practices, shelter, transport, general day-to-day survival and healing properties. An example of a traditionally important and useful plant is the flowering tree of the Myrtaceae family and the genus, Eucalyptus. Eucalyptus is derived from the Greek language Eu meaning well and kalyptos meaning covered (that

48 refers to the calyptra that protects the unopened bud). There are over 500 different species mostly native to Australia, and they are the dominant vegetation over much of

Australia [22]. In modern times, Eucalyptus species are used commercially in construction, fuel, pulp and for its essential oils, making it one of the most valuable plants used today [159]. Eucalyptus genera have closely related genera, namely

Angophora and Corymbia, and are all generally named ‘Eucalypts’. Eucalyptus species are often identified based on physical traits and separated into four main categories: smooth barks, part barks, full barks and mallees. The full barks are further divided into crumbly barks, stringy barks and ironbarks. The various species of Eucalyptus trees or shrubs have been used by most of the Australian Aboriginals continent-wide and have a number of different uses.

The Dharawal people utilised eucalyptus species for a range of activities.

Stringy bark Eucalyptus trees are used to make canoes, leaves from the Eucalyptus are crushed or smoked over a fire to treat colds and chest infections, bark from specific

Eucalyptus trees are used to treat infections of skin and the resin sap extruded from the tree can be prepared in a specific way to combat infections, stomach complaints with certain sap also being used to tan or stop fishing nets from fraying or becoming weak

[7, 22, 24].

Other examples of common plants traditionally used include the following:

Xanthorrhoea sp. is a grass tree that has a trunk with varying size from underground to up to 4 metres tall with a dense flower spike on top of a tall stout scape. Traditionally this plant has a number of useful purposes, and the whole plant is used. The flower is mixed with water to make a sweet drink, the flower spike was used to make spear shafts and firesticks, and the resin within the tree was used to fasten spearheads and bind string tied objects, like a glue. Melaleuca sp. or paperbarks, are a common plant in and

49 around the Sydney area. Traditionally, the young leaves can be crushed or soaked to be used to treat infected wounds (some species leaves contain tea tree oil), cold and flu symptoms and also relieve headaches. The bark from the tree is used to wrap around food before going into the fire and it was also used as patching to plug holes that may have appeared in a canoe [7]. Avicennia marina and Aegicerias corniculatum, mangrove trees, are traditionally utilised by choosing bends in the tree branches that were of suitable shape and size, and then cut and shaped to make boomerangs, shields and clubs [7, 24].

Medicinal plants are a critical and essential part of survival for all Australian

Aboriginals. Since arrival of Europeans and the increasing interest in finding new medicines and novel drugs there has been an increased interest in the use of Australia’s unique native plants for their medicinal properties. Table 4 shows medicinal plants from literature and other sources that grow within the boundaries of and are utilised by the

Dharawal people.

Table 4 Medicinal plants used by the Dharawal people of Botany Bay

Plant species Common Geographical Part used Traditional use Preparation Reference name region Acacia Hickory NSW coastal Sap, Used for Sap from A. decurrens was [22, 156] implexa area leaves, treating skin either chewed of dissolved bark diseases, in water and drunk for diarrhoea diarrhoea, bark preparations used to cure skin diseases. Crinum Swamp lily NSW coast, Leaves Used to relieve Leaves crushed or broken [22, 24, pedunculatum QLD and the pain of and juice applied to marine 156] Lord Howe marine stings animal stings to ease the Is. pain. Smilax Native NSW coast Leaves, Used for Leaves brewed into a tea [22, 24, glyciphylla sarsaparilla and Northern berries general well and drunk warm. 56, 156] Tablelands being, infected and into Qld sores, colds, coughs, arthritis, blood cleansing, diabetes, unsettled stomach and infected eye

50 wash. Eucalyptus Bloodwood Victoria Leaves, Used for upset Nectar from flowers [22, 24, gummifera border to bark, sap, stomach, sucked, sap dissolved in 156] southern Qld flowers antiseptic, sores water to make a solution to that won’t heal drink of wash sores, bark and some used to heal open wounds. cancers. Melaleuca Snow in NSW coast Leaves Used for colds, Leaves crushed and used as [22, 156] lineariifolia summer and ranges coughs and flus a poultice on infected sores and as and wounds. Also leaves antiseptic crushed and soaked in water and used as an antiseptic wash. Dodonaea Hopbush NSW coast, Leaves, Used for Leaves chewed to relieve [22, 156] triquetra Blue root toothache, toothache, a poultice of Mountains, stingray and leaves applied to stingray Qld and Vic stonefish wounds to ease pain, roots wounds, fever soaked in water and used relief and as a wash on skin, sores antiseptic and open wounds. Melaleuca Broad leaf From Botany Leaves, Used for colds, Young leaves chewed on [22, 156] quinquenveria paperbark Bay to Cape bark coughs and flu, when a cold is present, York infected sores leaves boiled and drunk as and wounds and a tea, bark used to wrap internal infected sores of wounds to cleanser. heal. Pteridium Bracken Most parts of Rhizome, Used to stop Rhizomes were cooked up [22, 24, esculentum fern Australia young mosquito bites and eaten. Young fonds 156] fonds or insect bites and stems are crushed and from irritating. applied to insect bites to stop Phytolacca Inkweed Naturalised in Leaves, Used to stop Leaves boiled in water and [24, 156] octandra Southern and berries scabies, applied to ringworms. Feet Eastern ringworm and soaked in boiled juices for Australia. tinea. Also as a tinea or as a wash for mix tonic. scabies. Also used with other plant extracts and drunk as a tonic Duboisia Corkwood North from Leaves, Used as a Holes drilled in the trunk [22, 156] myoporoides Clyde stem, sap narcotic and filled with water and Mountain to later drunk. Leaves also Qld chewed on. Melaleuca Tea tree Coastal Qld Leaves Used as a Prepared by crushing and [156] alternifolia into coastal general soaking the leaves using Northern antiseptic the resulting poultice/liquid NSW and extract to apply to wounds or used as a rinse to treat/wash out infections, sore throats, removal of lice/parasites, treat fungal infections, to treat cold and flu symptoms.

As the Dharawal were the first to encounter both Lieutenant Cook in 1770 and

later Captain Phillip and the first fleet in 1788, traditional knowledge, culturally

51 important bushland and cultural practices of the Dharawal had diminished to near extinction due to disease, dispossession of land, modernisation and urbanisation.

However, Dharawal people still possess traditional information and practice culture in tandem with living in the modern world including using plants as medicines.

In this study, ethnobotanical knowledge of Dharawal elder and Community members of the La Perouse Aboriginal Community has been accessed through interviews to select medicinal plants for further investigation. This chapter describes the interview processes, plants selected and the collection of the plants from different areas of Botany Bay.

2.2 Materials and Methods

2.2.1 Study Area

The Dharawal tribal land covers a large amount of landmass: however, the study area is limited to the Kamay/Botany Bay National Park on both the La Perouse (33.9898° S,

151.2390° E) and Kurnell side (34.0080° S, 151.2229° E) of the Bay. Botany Bay still has a high percentage population of Aboriginal people as it encompasses the La Perouse

Aboriginal community. The La Perouse Aboriginal community and surrounding suburbs still have Dharawal people along with people of many other tribes residing in the area.

2.2.2 Ethnobotanical Investigation

Selected Dharawal elders and La Perouse Aboriginal community members were approached and asked to participate in discussions about medicinal plants they had used or knew of around the Sydney area. This group of elders and community members were respected in the community, and possessed credible and continual traditional knowledge. The interviews were set up initially as an informal conversation about this

52 project and the aims and scope of the research. During this time, other people were recommended and referred as additional sources of information about medicinal plants.

These people were informed about the project as well and agreed to participate in the study. After initial discussions it was decided that no formal surveys or questionnaire were to be used in the collection of data rather, using an interview style in general conversation and informal talks. Interviews were conducted both individually and in groups of two or three. The project aims were then explained to the participants and a discussion about plant and plant use followed. The participants were also revisited at a later date to allow for some time to remember any other details for further discussion.

Permission was requested from the interviewees to use the information shared for this project. The information was then collected and collated and the participants again confirmed that this study could proceed with the information that was shared.

It was also decided that the identification of the plant species was to stay somewhat private to protect this knowledge at this time and the plant material that is collected will be referred to as plant species 1, plant species 2 up to plant species 7. The classification level of family is provided for each plant as well as a description of the plant species used in this study.

2.2.3 Collection Licence Application

An application for the scientific collection of flora was sent to the DECCW on the February, 2009 and granted in May, 2009. This licence allowed for the collection of leaf, stem and root of the plant species used in this study. The plant samples were collected under licence number S12778. An extension of the licence until 31st January

2012 was later granted.

53 2.2.4 Plant Collection

With permission given to use information about particular plants, a guide was used to demonstrate the collection and preparation methods of the plants as well as plant location. If it was not possible to use a guide, the plant sample were collected, with photos, and taken back to the interviewee for positive identification. Botanical identification books were also used to confirm the identification of plant species and obtain the correct scientific name. All plant samples were collected from the

Kamay/Botany Bay National Park. The plant material was collected according to how or what part of the plant (leaf, stem and/or root) is used in the preparation of the medicine. Plant species being located in a National Park, and protected from development and urbanisation, allows for the environment to remain near enough to the original conditions that would have existed pre European contact.

The plant samples were collected in accordance set out in the licencing agreement. Before entering the National Park, shoes that were to be worn were first scrubbed with a small scrubbing brush and disinfectant. The plant sections were removed from the main plant with most minimal contact. Collected plant sections were placed into sterile falcon tubes or zip-lock bags and transported back to the laboratory, for further experimentation.

Collection of plant species 5 was not possible at the time as this plant is considered a weed by the National Parks and had been sprayed with herbicide and no plant was growing at the time of collection.

Voucher specimen samples are lodged with the Dharawal elders group and have not been lodged with the University of NSW herbarium, as yet, until further discussions and decisions on lodging specimens are resolved in regards to confidentiality and the level of protection of the traditional knowledge of the plant species.

54 2.3 Results

2.3.1 Ethnobotanical Investigations

A total of eleven elders and community members, including members of the

Dharawal Elders Group and the La Perouse Aboriginal Community, were interviewed to about this project and medicine plants from the Sydney area. In this study, seven of the eleven (64%) interviewees were respected women elders and the other four were men (36%), representatives of the La Perouse community. Eight of the eleven interviewee (73%) were over the age of 50 and three, all men, were under the age of thirty. Sadly, four of the seven women that participated in the interviews passed away during the time of this study.

2.3.2 Plant Selection

Seven plant species were chosen for further study. The plants were chosen for this study on the basis that they were spoken about most commonly, that is, by more than 50% of the participants during the interviews or direct information provided by the elders. The medicine plants used in this study are from the Myrtaceae (four species),

Smilacaceae (one species), Sapindaceae (one species) and Phytolaccaceae (one species).

These plants were discussed in the context of using them medicinally to treat skin infections both bacterial and fungal skin infections (seven species), for digestive system problems (seven species), internal wellbeing (five species), respiratory system (three species), toothache and infection (two species), and lice or tick removal (two species).

Plant species 1: from the Smilacaceae family, is a small climbing vine that grows in or near rainforests and moist eucalypt forest, but also is found on sand dunes. It has leaves that are alternate, ovate, shiny green on top and a dull ash grey underneath with 3 prominent longitudinal veins. Flowers are borne in umbels and are yellowish in colour.

55 The fruit are small clusters of black berries. It is distributed in coastal areas of NSW and north as far as Qld. [22, 24, 67, 160, 161]. This plant is traditionally prepared by soaking the leaves in hot/boiling water and drunk as a tea. The leaves are also directly chewed. This plant was used medicinally to relieve colds, flu, sore throats, managing diabetes, and also in combination with other medicine plants as a “good blood” medicine [24, 156].

Plant species 2: from the Myrtaceae family, is a medium to large tree growing up to 30 m but is in mallee form around coastal heaths. It has an irregularly cracked rough, short- fibred, flaky bark with leaves that are tapered at both ends with a paler underside. The lateral veins are parallel, very close together and form an angle of about 60o with the midrib. The flowers are in 7 flowered clusters in a terminal corymbose arrangement and the fruit are large, woody and urn-shaped [22, 24, 67, 160, 161]. This plant is used traditionally by obtaining the sap or kino from the tree and using it in a number of ways.

The kino was mixed in with water and drunk to alleviate stomach complaints or put on sores that wouldn’t heal or were otherwise infected. The sap was also directly used from the tree without mixing it with water. The kino was included in a lot of “mixture” medicines where multiple plants were used for healing an ailment [156]. The sap was also reported to heal a cancer growth on a dog [24]. The leaves of the bloodwood were used to treat headaches, coughs and colds by steaming the leaves over a fire and inhaling the smoke/steam. Lining the floor where you slept with leaves also aided via inhaling of the aromatic smell.

Plant species 3: from the Myrtaceae family, is a tall shrub that grows thickly on sand dunes around coastal systems and is extremely important in coastal ecology. The leaves have a grey-green colour, obovate with a short blunt tip. New fruit is a non-woody

56 capsule with a flat top that splits into 8-12 compartments. The flowers have 5 white petals with a distinct reddish ring around the stamen [22, 160]. The traditional preparation is to boil the leaves into a tea and drink while hot. It has also been used as a skin and eye wash. It is believed to be very high in vitamin C.

Plant species 4: from the Myrtaceae family, is a shrub to small tree with papery bark mainly growing in or near swamps near the NSW coast and ranges. The leaves are opposite, narrow lanceolate approximately 3 mm wide. The flowers are white and feathery, growing in dense spikes around the end of the stems [22, 67, 160].

Traditionally this plant is used the same way as tea tree (Melaleuca alternifolia). The leaves are crushed into a poultice or crushed and soaked in water and applied to infected sores, fungal infections, mouth infections, and for lice removal. Similarly to Eucalyptus leaves, they can be steamed over a fire and used to clear headaches, as well as cough and cold symptoms [156].

Plant species 5: from the Phytolaccaceae family, is a perennial herb growing about 1 m high and as wide, with an often woody base that is green to pink. The leaves are large and oval-shaped coming to a point at the tip. The flowers are green to pink with the fruit being an 8 lobed berry that is red in colour and turns black when ripe with 8 seeds per fruit. This is not a native plant to Australia but has naturalised on the eastern coast of

NSW [162]. Traditionally, it is prepared by boiling the plant in water (without the berries) and can be used to treat fungal, scabies and other mite infections. It has also been used as a tonic to help with internal complaints [156].

Plant species 6: from the Sapindaceae family, an erect shrub that grows in open and low forest and is especially abundant after bushfires. The leaves are bitter tasting, elliptic to lanceolate, thin, soft and hairless with a pointed apex and narrowing to a short

57 stalk. The fruit is a three-winged capsule that is green or reddish and becomes papery on maturation. Traditionally this plant is used by chewing on leaves for toothaches or soaked in water to relieve fevers. The roots were liquefied and applied to open and infected sores [22, 67, 156, 160, 161].

Plant species 7: from the Myrtaceae family, common name broad leaf paperbark, is a tall paperbark that grows around swampy, brackish and lagoon habitats. The leaves are alternate, stiff and tapered at both ends with 5 main longitudinal veins. The flowers are dull-white to creamy formed in terminal spikes [22, 67, 160, 161]. Traditional use of this plant is to crush young leaves and sniffed for a period to relieve colds, coughs and headaches. The young leaves can also be crushed and soaked in water to be drunk for relief of internal complaints, as well as the symptoms of a cold [67, 156].

2.3.3 Plant Collection

Plant material was collected according to the methods of traditional preparation shared during the interview process. Table 5 summarises the collection of medicinal plants, sections of the plant material collected, type of plant, area collected and description of the plants habitat.

Table 5 Plants sections collected and location within Botany Bay

Plant Plant familyb Section Plant Time of Description of the plant Speciesa collectedc typed collectione communityf

Plant Smilacaceae Leaf Vine Autumn Wet eucalyptus forest (in species 1L Stem sheltered valleys of the La Root Perouse sand dune)

Plant Myrtaceae Leaf Tree Autumn Wet eucalyptus forest (in species 2L Branch sheltered valleys of the La Perouse sand dune)

Plant Myrtaceae Leaf Tree Autumn Coastal heathland species 3L Branch

Plant Myrtaceae Leaf Tree Autumn Wet heathlands

58 species 4L Branch

Plant Phytolaccaceae N/A N/A N/A N/A species 5N

Plant Sapindaceae Leaf Shrub Autumn Grassy woodland of Kurnell species 6K Stem dune forest. Root

Plant Myrtaceae Leaf Tree Autumn Grassy woodland of Kurnell species 7K Branch dune forest. a Name given to plant species, LCollected from the Kamay/Botany Bay National Park, La Perouse and KCollected from the Kamay/Botany Bay National Park, Kurnell, NNot collected due to herbicidal spraying), bFamily classification, cSection of plant tissue collected, dplant type description (Tree, vine, shrub, etc), eSeason the plant tissue was collected, fDescription of the plant community where the plant was collected.

2.4 Discussion

2.4.1 Ethnobotanical Investigation

2.4.1.1 Interview Process

This survey was carried out to research the ethnobotanical information that is available in the Botany Bay region. A total of eleven Elders and community members from the La Perouse Aboriginal Community were informed about this project and questioned about their medicinal plants from the Sydney area. In this study, the consultation process was in the form of casual face-to-face informal talks or over the phone conversations. In Australian Aboriginal culture, nearly everything is taught by word of mouth including story telling, song, and dance, accompanied by rock art and rock engravings. Nothing was written down or documented until the arrival of the

European people. Talking with people in the community rather than sitting with a pen and paper recording was a more comfortable and culturally appropriate experience for everyone involved. Some of the interviewees were reluctant in talking about certain

59 topics, with one person, a known medicine woman with ties to the community, not comfortable in sharing any information about the topic at all.

Certain information that is passed down from generation to generation is only for selected people to know and only available to other people when the time is right to pass the information on. This is a key component of keeping Aboriginal traditional ways and culture alive today. The information that was used in this study received permission by Dharawal elders to continue. Once the information was collected, some of the interviewees, particularly the elders, were too sick or too old to come out on a collection expedition. The names, descriptions and location of the plants were discussed and the plants were collected and taken back to the interviewee to confirm if it was the correct plant species or not. On some of the sample location walks, family and community members also came along and offered location advice and help with identification. Supplementary to the above, botanical identification books were used to confirm what the correct species was and to give more scientific information on the selected plant [22]. However, in this study not the whole community was consulted.

This was because the people interviewed had known continual cultural practice of their culture and had credible information on this topic. Future work will include a full comprehensive ethnobotanical survey of the whole La Perouse Aboriginal community and the wider Dharawal communities as well.

The key to this study was to receive permission to use any particular plant. This was demonstrated during the interviews that certain plants and information were not permitted for use in this study. Permission was granted to use only seven medicinal plants from the Botany Bay area. Other medicinal plant species were also mentioned during the interview; however, they were not included in this study because permission

60 was not granted by one or more of the participants or they were not mentioned or unknown by other interviewees.

2.4.1.2 Plant Collection

The plant species used in this study were collected from the Kamay/Botany Bay

National Park, both at La Perouse (Northern) and Kurnell (Southern) Parks. Both La

Perouse and Kurnell fall within the tribal boundaries of the Dharawal.

Plant species being located in a National Park, and protected from development and urbanisation, allows for the environment to remain close to the original conditions that would have existed pre-European contact. The sites of plant collection within the

Kamay/Botany Bay National Parks were chosen on the basis that they were growing in close proximity to known pre-European Dharawal occupation sites. These are known sites to the local Aboriginal community and have also been uncovered through archaeological investigations. The areas include the foreshore at Kurnell, that contains midden, burials, stone tools, artefacts; inscription point that has a burial cave; an ochre pit that is a very important site to the Dharawal people; rock engravings at La Perouse; a midden at Congwong Bay beach; and stone artefacts found in and around the same area of the National Park. These sites are of cultural importance and indicate that the

Dharawal people continually used the area for long periods of time, and who might have used medicinal plants from the surrounding bushland. The plants that grow in the environment around the occupation sites have a higher chance of very similar growing conditions to the plants that were growing within the same area pre-European contact.

This provides a more accurate representation of wild native plants when compared to plants from nurseries or greenhouses.

61 2.4.1.3 Selected Plant Species

The plant species and related plants species of those used in this study have been documented and utilised for both medicinal and non-medicinal uses by the Aboriginal people of Australia, early settlers, as well as modern day scientists and nutritionists.

As mentioned in the introduction, eucalypt species are one of the most commonly used and abundant plant species available to Aboriginal people. They are used for a number of reasons including medicines and tonics made from the kino of the bloodwood trees, bark used to make canoes, and the leaves used to relieve cold and flu symptoms to name a few. Essential oils have been isolated from Eucalyptus spp. and used for a host of different reasons, including aromatherapy and fragrance products, parasite and insect deterrents, for their anti-inflammatory and antioxidant activity and as anti-bacterial, anti-viral and anti-fungal compounds [163-166].

Similarly, Melaleuca spp., which include the paperbark melaleucas, are also utilised for medicinal use of the bark, leaves and sap, for construction of shelter and tools and for food and food preparation [7, 156].

The most famous and commercial example of Melaleuca used traditionally, is the extraction of tea tree oil from the Melaleuca alternifolia plant. Traditionally, tea tree oil is prepared by crushing and soaking the leaves of the paperbark tree using the resulting poultice/liquid extract to apply to wounds or used as a rinse to treat/wash out infections. In modern times, tea tree oil was manufactured in 1923 and was a standard issue in all Australian medical kits for soldiers and sailors during World War II for its antiseptic properties [26]. There are over 100 monoterpenes, sesquiterpenes hydrocarbons and their alcohols with the main anti-microbial constituents of tea tree oil being the terpenes, terpin-4-ol., γ-terpinene, α-terpinene, 1,8-cineole and α-pinene [27-

62 29]. Tea tree oil has a range of activities including anti-bacterial [30], anti-fungal, anti- viral [31] and possibly anti-cancer [32, 33].

Smilax glyciphylla, is a medically important plant, is used traditionally by breaking up the leaves and soaking them in warm water to make a tea like drink. This concoction is drunk whilst still warm to treat sore throats, stomach complaints or can be used as a wash for skin/eye infections. This method was also used by the first fleet as a tonic type tea for general health and wellbeing of the new people. Smilax extracts were tested for its antioxidant properties [56] and showed that a hot water extract inhibited peroxidation of phosphatidylcholine liposomes, inhibited deoxyribose degradation and quenched chemically generated superoxide anion at levels comparable to 50mM of

TROLOX, a standard method for measuring antioxidant strength. It was also reported that glycyphyllin, when metabolised and absorbed as phloretin, has potential for possible anti-cancer properties. In a separate study, it was again tested for antioxidant properties and three phenolic compounds isolated, glycyphyllin B, catechin and quercetin-3-O-β-D-glucopyranoside showed potent antioxidant activity [167].

The information shared by the interviewees on the traditional knowledge of medicinal plants was supported by information reported in the literature on the plants used in this study or closely related plant species [22, 44, 160-162].

2.4.2 Dharawal tribal area

2.4.2.1 Collection Area Clarification

The Dharawal tribal area encompasses a small part of the South Eastern coast of

New South Wales. The tribal boundaries in this study of the Dharawal people were determined by traditional teachings and traditional knowledge. However, there is a lot of dispute in modern day academia and the wider communities about where actual tribal boundaries start and finish. This has mainly come about by information that has been

63 inaccurately recorded or misinterpreted by early Europeans in the settlement period or by anthropologists, who published material in the late 1800’s to mid 1900’s.

Unfortunately, these archived materials are received as ‘gospel’ and have not yet been interpreted from an Aboriginal perspective. As mentioned by Sir Joseph banks in describing the Sydney language:

‘….consequently the list of words I have given could be got no other manner than by signs enquiring of them what in their Language signified such a thing, a method obnoxious to many mistakes: for instance a man holds in his hand a stone and asks the name of [it]: the Indian may return him for answer either the real name of a stone, one of the properties of it as hardness, roughness, smoothness etc., one of its uses or the name peculiar to some particular species of stone, which name the enquirer immediately sets down as that of a stone.’ [15] Here Banks acknowledges that interpretation and translation could lead to problems of accurate information. In recent times, Aboriginal history has been presented in a way that is misleading.

For example, the tribal group and clans from the city of Sydney and surrounding areas, belong to the Dharawal tribe and associated clan groups; however, this area is still being disputed as to which tribe occupied that area. A number of individuals and community groups today believe that the Sydney tribe is known as the ‘Eora nation’ or coastal Dharug.

As quoted on an information map from the Land and Property website of the

NSW government, and as informed by NSW Aboriginal knowledge-holders, organisations and communities , “Eora is a term that has come into recent use by some of the Sydney metropolitan Aboriginal communities to provide them with a form of discrete geographic identity. The term is derived from two words of the broader

D’harawal language of the region and literally translates to mean “Yes, People ”. [11].

Separately, an Aboriginal informant, Jim Lowndes, considered to be the last Darug man, was recorded by anthropologist Robert Matthews in saying ‘The Dharook dialect,

64 very closely resembling the Gundungurra, was spoken at Campbelltown, Liverpool,

Camden, Penrith, and possibly as far east as Sydney, where it merged into the

Thurrawal (Dharawal).’ [168]. Years later, it was then mentioned and quoted in a book written by Jim Kohen, that ‘The Dharook dialect, very closely resembling the

Gundungurra, was spoken at Campbelltown, Liverpool, Camden, Penrith, and possibly as far east as Sydney…’ leaving off ‘where it merged with the Thurrawal’ (Dharawal).

This has since been quoted and regurgitated giving out false representations of the tribal people of Sydney. This is only one example from editing and quoting misrepresentation through reading historical records, and shows how people can be mislead.

Watkin Tench, a British marine officer, documented another early account that the

Dharawal people spoke a separate language from their neighbours. Tench recounts an expedition to the west of Sydney, two locals from the Sydney tribe, Colbee and

Boladeree, were used as guides to help the officers explore new lands. The two

Aboriginal men reached an area now known as Parramatta/Rose Hill and was observed by Tench as:

‘At a very short distance from Rose Hill, we found that they were in a country unknown to them, so that the farther they went the more dependent on us they became, being absolute strangers inland. To convey to their understandings the intention of our journey was impossible. …..We asked Colbee the name of the people who live inland, and he called them Boorooberongal; and said they were bad, whence we conjectured that they sometimes war with those on the sea coast, by whom they were undoubtedly driven up the country from the fishing ground, that it might not be overstocked; the weaker here, as in every other country, giving way to the stronger’ [169].

The examples above and the traditional knowledge shared by the interviewees indicate that the Sydney area and surrounding areas are all recognised as Dharawal land.

The plant species collected were all from within the tribal boundaries of the Dharawal.

2.5 Conclusion

Ethnobotanical information in Australia will no doubt be both of national and international focus in the coming years with increased importance in the search for 65 novel anti-infective compounds. Various research papers on bioactivity studies that access ethnopharmalogical information of Aboriginal Australia as a basis have been published in the past but mainly concentrate on areas such as Northern Territory,

Central Australia, North Western Australia [39, 43, 46, 60, 66, 95, 170-173] with one published comprehensive ethnobotanical survey that has been carried out on the Yaegl people of Northern New South Wales [174].

With the ever increasing threat of loss of traditional knowledge, it is important to learn and find ways to keep this knowledge alive. In this study, permission was given to use only seven species of plants from four different families, a very small amount of information of medicinal plant species from Botany Bay. Other plant species were discussed; however, they were exempt from being used. The low number of plant species discussed may be an indication of how traditional knowledge of the area is diminishing to an alarming point but also indicates that there is still precious information that exists today. Another disturbing indicator as to why this precious knowledge is being lost is that four of the seven women in the discussion group passed away during this study, reiterating the importance of accessing and documenting this precious information.

Although the Sydney area was the place of first contact with the Europeans and therefore the most culturally interrupted place in Aboriginal Australia, there are still people alive today who have direct family bloodlines to the area and possess traditional knowledge that allow for the continuation to practice and teach their culture as it has been done since the time of creation. However, the remaining traditional information is under threat of being lost through the aging and passing of the elderly people of the community. It is also extremely important that there is respect shown and full consultation with local Aboriginal people and Indigenous communities when

66 conducting any ethnobotanical investigations or accessing traditional knowledge for research purposes. This will be beneficial for both Indigenous and non-Indigenous people when accessing this precious information for the benefit of all.

67 3 Chapter 3 The Microbial Diversity of Endophytes from Essential

Essential Medicine Plants of Sydney, Australia

3.1 Introduction

The study of microbes from distinctive environments has driven research to new and exciting heights. Endophytes are microbes that reside in living, internal tissues of plants without causing any immediate, overt negative effects [175, 176]. They are usually considered to be non-pathogenic, mutualistic bacteria, cyanobacteria or fungi that grow within plant tissues. It is believed that all of the approximate 300,000 plant species world-wide contain one or more microbial symbionts [177], but the exact diversity and distribution of these microbes is unknown.

Studies have shown that the level of endophyte symbiosis and diversity varies drastically and could be due to a number of factors, both abiotic and biotic, including specific host plant-microbe interactions, climate and seasonal variation, and the biogeographic position of plant habitation [178-182]. It is not yet fully known what role endophytes play in their host plants, with suggestions that they contribute to defence against grazers or pathogens [75], promote growth in harsh environments [76-78] and/or facilitate the production of metabolites that improve plant growth in general [80].

The initiation of the plant-endophyte association is not yet fully understood. The most studied example of endophyte-plant symbiosis is that between Epichloë endophytes and grasses. These epichloë endophytes consist of Epichloë and

Neotyphodium species, which are arguably the most economically important endophytes associated with cold season grasses [183]. The microbes provide several advantages to their hosts: they assist with growth promotion and survival in harsh conditions; they produce alkaloids, which deter herbivorous insects and other grazing animals; and they inhibit infection by plant pathogens [183-188]. Studies have shown that epichloë

68 endophytes can be vertically transmitted through infiltration of the seed during seed production or horizontally transmitted via mitotic spore transfer from plant to plant

[189, 190].

Endophytes are now recognised for their potential commercial value. For example, they have proven to be a rich source of useful enzymes [96] such as cellulases for biofuel production [191], as well as indole acetic acid for plant growth promotion, anti-oxidants and hydrolytic enzymes for use in the food and health industries [192], natural plant pathogen control [193-199] and novel pharmaceuticals (See Chapter 4).

Diversity studies have revealed a huge variety of endophytes within different plant species. Some commonly encountered endophytes include species of the fungal genera Aspergillus, Penicillium, Phomopsis, Cladosporium, Colletotrichum, Fusarium,

Guignardia, Xylaria and Botryosphaeria, as well as species of the bacterial genera

Streptomyces, Bacillus, Rhizobium and Agrobacterium to name a few. Interestingly, a few of these genera, including Botryosphaeria and Guignardia also contain pathogenic species. Endophyte diversity studies have been conducted on ginseng (Panax ginseng)

[200], soy bean (Glycine max (L.) Merril) [201], and coffee (Coffea arabica L.) plants

[202], traditional Chinese medicine (TCM) herbs [203] and the South American medicinal plant Trichilia elegans (Meliaceae) [204].

Endophytes have been isolated from a number of native Australian plants and grasses, including both medicinal and non-medicinal plants. Briefly, examples include four native plants, the native pine tree (Callitris preissii), red gum (Eucalyptus camaldulensis), grey box (Eucalyptus microcarpa) and native apricot tree (Pittosporum phylliraeoides) from which 576 endophytes have been isolated, including several novel species. The diversity of the endophyte community within these native plants encompasses 17 different actinobacteria genera, including Streptomyces sp. (the most

69 commonly encountered genus) [105], Pseudonocardia, Nocardia, Actinopolymorpha and the novel genus Flindersiella [105-112].

Other native Australian plants known to harbour endophytes include Eucalyptus sp., Acacia sp., Adansonia gibbosa, Eremophilia longifolia, Gossypium sp., Banksia grandis, Allocasuarina fraseriana, Agonis flexuosa, and several dry rainforest species from South Eastern Queensland [96, 103, 104, 205, 206].

In light of these findings, and further driven by the traditional knowledge of the

Dharawal people of Botany Bay, Sydney, this study investigates the diversity of endophytes residing in traditional medicinal plant species native to the Eastern coast of

NSW. Isolation and identification of endophytes was carried out for six plant species, four classified within the Myrtaceae, and one each of Smilacaceae, Phytolaccaceae and

Sapindaceae. These plants are used traditionally and their anti-bacterial, anti-fungal, anti-viral and anti-inflammatory properties are described. Bacterial and fungal endophytes were classified according to 16S and 18S rRNA gene sequencing and subsequent phylogenetic analysis.

3.2 Materials and Methods

3.2.1 Plant Collection

Information on medicinal plants of Botany Bay was collected through a series of interviews conducted with Dharawal elders and members of the La Perouse Aboriginal community. Plant material was collected for this study as described in Chapter 2. The plant samples were collected according to traditional collection methods. Individual leaf, stem, inner bark or root sections were selected and were taken from 3 different plants within the site of the collection area.

70 3.2.2 Sample Preparation and Surface Sterilisation

All samples collected were processed on the same day of collection. Plant samples consisted of leaves, stems and roots, according to traditional use. Cut ends of plant sample were sealed with Parafilm prior to the sterilisation treatment. This process was modified from a surface sterilisation protocol from Hata, et al 1998 [207]. The following protocol has also been successfully utilised in the isolation of endophytes from TCM [203]. The samples were initially rinsed with sterile Milli-Q water to remove surface debris then transferred to Falcon tubes containing 30 mL 30% hydrogen peroxide and agitated by hand 20-30 min. Samples were then aseptically transferred to fresh 50 mL falcon tubes containing 20 mL sterile Milli-Q water and agitated for 1 min.

The rinsed samples were finally transferred to 50 mL Falcon tubes containing 20 mL of

70% ethanol and agitated for an additional minute. The sterilised plant samples were then air dried in sterile Petri dishes. In order to verify surface sterility, a section of each treated plant sample was placed into Heart Infusion Broth, and incubated at 30oC and room temperature for 48-72 h. If no growth was observed, the remaining sample was used as the inoculum for endophyte isolation as described below.

3.2.3 Endophyte Isolation

Surface sterilised plant samples were aseptically sectioned into ~1cm squares and scored with a scalpel to expose inner tissues. Exposed tissues were placed onto water agar (WA), potato dextrose agar (PDA) and arginine/glycerol agar (AGA) plates all in triplicate. In the case of woody samples, the outer layer of bark was carefully removed and the exposed inner bark placed directly onto the isolation medium. The plates were incubated at room temperature and were checked morning and night for 1-2 weeks.

Once growth was observed, the organism was sub-cultured onto fresh PDA or

ARG/GLY until pure cultures were obtained. Isolates growing in pure culture were

71 characterised according to visual culture morphology and cryogenically preserved as glycerol stocks at -80oC.

3.2.4 DNA Isolation

Genomic DNA from isolated endophytes was extracted using the XS buffer method [208]. Approximately 100 mg of fungal or bacterial biomass, scraped from the surface of agar plates, was placed in 1.5 ml tubes containing 500 µl of XS buffer (1% potassium ethyl xanthogenate, 800 mM NH4OAc, 100 mM Tris-HCl (pH 7.4), 20 mM

EDTA and 1% sodium dodecyl sulphate (SDS)). Samples were incubated at 65oC for 2 h, with brief vortexing after 1 h. The lysed cell suspensions were then incubated on ice for 10 min and centrifuged at 12,000 g for 10 min. The resulting supernatants, containing the DNA, were aspirated and transferred to fresh 1.5 mL Eppendorf tubes containing 500 µL of phenol/chloroform/isoamylalcohol (25:24:1). Samples were mixed via inversion then centrifuged as before. Aqueous layers were transferred to fresh

1.5 mL Eppendorf tubes and 2 volumes of 100% ethanol and 1/10th of a volume of 3 M sodium acetate were added. The tubes were incubated for 15 min at 4oC and precipitated

DNA was pelleted by centrifugation at 14,000 g for 20 min. DNA pellets were washed with 70% ethanol and centrifuged at 14,000 g for 10 min. Washed DNA pellets were air dried then re-suspended in 50 µL of sterile milli-Q water. DNA yield and quality were checked using a Nanodrop® spectroscopy system (Thermo Scientific) and final DNA concentrations were adjusted to ~20 ng/µL with sterile milli-Q water.

72 3.2.5 DNA Amplification and Phylogenetic Analysis

3.2.5.1 Polymerase Chain Reaction (PCR)

3.2.5.1.1 16S and 18S rRNA Gene Amplification Amplification of 18S rRNA gene fragments was performed via PCR using the fungal-specific primers nu-ssu-0817 5’-(TTAGCATGGAATAATRRAATAGGA)-3’ and nu-ssu-1536 5’-(ATTGCAATGCYCTATCCCCA)-3’ [209]. PCR reactions were carried out in a total volume of 20 µl and contained 1× reaction buffer (Promega), 2.5 mM MgCl2, 0.25 mM dNTPs (Sigma), 0.5 pmol of each primer, 0.2 U of Taq polymerase and 10-20 ng of DNA template. Thermocycling was carried out in a

BIORAD MyCycler thermocyler (Biorad) and consisted of an initial denaturation step of 94oC for 2 min, followed by 35 cycles of 94oC for 30 s, 56oC for 30 s and 72oC for

30 s. A final extension step of 72oC for 7 min was also included. This protocol was adapted from Borneman and Hartin, 2000 [209].

Amplification of 16S rRNA gene fragments was performed via PCR using the bacterial-specific primers 27FI 5’-(AGAGTTTGATCCTGGCTCAG)-3’ and 1494Rc

5’-( TACGGCTACCTTGTTACGAC)-3’ [210]. PCR reactions were carried out in a total volume of 20 µl and contained 1× reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTPs,

0.5 pmol of each primer, 0.2 U of Taq polymerase and 1-10 ng DNA template.

Thermocycling was carried out in a BIORAD MyCycler thermocyler and consisted of an initial denaturation step of 94oC for 5 min, followed by 35 cycles of 94oC for 20 s,

55oC for 30 s and 72oC for 1 min. A final extension step of 72oC for 7 min was also included. This protocol was adapted from Neilan et al, 1997 [210].

3.2.5.2 PCR Product Purification and Sequencing

16S and 18S rRNA gene PCR products were purified via ethanol precipitation as follows. PCR products were transferred to 1.5 ml eppendorf tubes prior to the addition

73 of two volumes of 95% ethanol and 1/10th of a volume of 3M sodium acetate. Tubes were incubated at room temperature for a 15-30 min then centrifuged at 14,000 g for 15 min. Supernatants were removed and pellets washed with 70% ethanol and centrifugation at 14,000 g for 10 min. Pellets were then air dried and resuspended in the original reaction volume of sterile milli-Q water. The purity and concentration of PCR products was verified visually via electrophoresis through 1% agarose gels in TAE buffer and ethidium bromide staining and transillumination on a GelDoc UV transluminator System (BioRad).

3.2.5.3 Sequencing

PCR products were uni directionally sequenced using the automated sequencing

Big Dye cycle sequencing protocol. Reactions were carried out in a total volume of 20

µl, consisting of 1 µL of Big Dye, 20-50 ng of purified PCR product, 3.2 pmol of primer, 3.5 µL of 5× sequencing buffer and milli-Q water to a final volume of 20 µL.

Thermal cycling was carried out on a BIORAD MyCycler thermocyler and consisted of an initial denaturation step of 96oC for 3 min, followed by 30 cycles of 96oC for 10 s,

50oC for 5 s and 60oC for 4 min.

3.2.5.4 Sequencing Product Clean Up

Completed sequencing PCR reactions were transferred to 1.5 ml eppendorf tubes to which 16 µL of sterile milli-Q water and 64 µL of 95% ethanol were added. Tubes were vortexed briefly then incubated at room temperature for 30 min and centrifuged at

14,000 g for 20 min. Supernatants were removed and pellets washed with 70% ethanol and air-dried as described above. Purified sequencing reaction products were submitted to the Clive and Vera Ramaciotti Centre for Gene function analysis (UNSW) for sequencing.

3.2.5.5 Sequence Analysis

Returned sequence data were visually edited for quality via trimming low quality ends and were compared to sequences in the GenBank database using the

BLASTn search tool (http://www.ncib.nlm.nih.gov/BLAST/).

3.2.6 Phylogeny

Phylogenetic analysis of bacterial and fungal isolates was based on 16S and 18S rRNA genes sequences, respectively. Sequences obtained from Genbank via BLASTn and various reference strains were aligned using ClustalX [211]. The most suitable models for the phylogenetic analyses as determined via FindModel [212] (http://www. hiv.lanl.gov/content/sequence/findmodel/findmodel.html) were the Tamura-Nei (TrN) + gamma model for the fungal data and the general time reversible (GTR) + gamma model for the bacterial data. Maximum likelihood trees were constructed using PHYML

(http:// www.atgc-montpellier.fr/phyml/) with bootstrap values obtained from 1,000 replicates [213].

3.3 Results

3.3.1 Surface Sterilisation and Endophyte Isolation

Surface sterilisation of plant material used to isolate endophytes was verified by incubation in HIB for 3 days. Under the described conditions, no contaminating surface microorganisms were detected. The initial selection of endophytes was based on differential growth rates, morphologies, pigments and odours. Using this method, 122 potentially different endophytes were isolated from six plant species (Table 6).

However, using the 16S and 18S sequence data together with further visual comparison of the morphology of mature colonies on the media plates, the endophytes from each plant species were de-replicated. Only endophytes that could be distinguished by 16S

75 and 18S rRNA sequence data were included in subsequent experiments. Unfortunately most of the originally isolated endophytes were excluded because they could not be amplified or became comtaminated with dust mites and contaminating microbes. This gave a revised total of 48 morphologically unique endophytes, including 36 fungal and

12 bacterial isolates.

Table 6 Endophyte isolation and surface sterilisation results Plant species Plant family Section Incubation temperature1 Estimated no. of strains2 number collected 30oC RT Morphology- Sequence- based based Plant species 1 Smilacaceae Stem Clear Clear 3 3 Root Clear Clear 8 4 Plant species 2 Myrtaceae1 Leaf Clear Clear 4 0 Branch Growth from Growth from 17 6 within leaf within leaf (1st attempt); (1st attempt); Clear (on Clear (on repeat) repeat) Plant species 3 Myrtaceae2 Leaf Clear Clear 5 1 Branch Clear Clear 12 5 Plant species 4 Myrtaceae Leaf Clear Clear 0 0 Branch Clear Clear 11 0 A A A A A A A Plant species 5 N/A N/A N/A N/A N/A N/A Plant species 6 Sapindaceae Leaf Clear Clear 4 4 Stem Clear Clear 10 7 Root Clear Clear 28 12 Plant species 7 Myrtaceae3 Leaf Clear Clear 7 4 Branch Clear Clear 13 2 Total 122 48 A Plant species 5 was not collected as it was unavailable due to herbicide spraying and no further information is available, 1Surface sterilised plant material was incubated in HIB at 30OC or room temperature for 3 days to check for contaminating microorganisms. 2Endophytes were initially distinguished via colony morphology and gross physical traits prior to rRNA gene sequence analysis.

3.3.2 16S and 18S rRNA Gene Amplification and Sequence Analysis

PCR screening, targeting 16S and 18S rRNA genes, was carried out on the 48 morphologically distinct endophytes, isolated on WA, PDA or AGA plates. The amplified genes were sequenced for further identification and phylogenetic analysis.

76 The fungal 18S rRNA gene analysis revealed that the fungal isolates were most closely matched to strains within the genera Penicillium (5 isolates),

Botryosphaeria/Neofusicoccum (5 isolates), Acremonium (3 isolates),

Cladosporium/Davidella (1 isolate), Colletotrichum (4 isolates), Helicoon (1 isolate),

Phaeomoniella (1 isolate), Aspergillius (1 isolate), Phomopsis (5 isolates),

Tetrachaetum/Graphium (5 isolates), Chrysoporthe (1 isolate), Collophora (1 isolate),

Phyllosticta (3 isolates) and Rhodotorula (1 isolate) (Table 7). Bacterial 16S rRNA gene analysis revealed that the bacterial isolates were most closely matched to strains within the genera Bacillus (5 isolates), Burkholderia (5 isolates), Agrobacterium and

Rhizobium (2 isolates) (Table 8). Sequnce data was submitted to NCBI and accession numbers were obtained for the 18S rRNA gene for the isolated fungal endophytes

(KM659040-KM659040) and for the 16SrRNA gene for the bacterial isolates

(KM659076-KM659087) shown in the Appendix.

Table 7 BLASTn analysis of 18S rRNA gene sequences from isolated fungal endophytes Isolate Plant family Accession Coverage Identity numbera Originb Closest matchc numberd (%) (%) PS1-3 Smilacaceae Stem Penicillium sp. C-5 KC405608.1 100 99

Penicillium purpurogenum strain 6-16P KC143068.1 100 99 PS2-2 Myrtaceae1 Bark Penicillium sp. 8-12c KC790520.1 100 99 section Penicillium sp. MAU-4 JQ824839.1 100 99 PS2-6 Myrtaceae1 Bark Penicillium sp. 8-12c KC790520.1 100 99 section Penicillium sp. MAU-4 JQ824839.1 100 99 PS2-9 Myrtaceae1 Bark Penicillium sp. 8-12c KC790520.1 100 99 section Penicillium sp. MAU-4 JQ824839.1 100 99 PS2-17 Myrtaceae1 Branch Cladosporium cladosporioides JX982602.1 100 100

Davidiella sp. N5 HQ600972.1 100 100 PS2-18 Myrtaceae1 Branch Chrysoporthe cubensis DQ862047.1 100 99 Endothia gyrosa L42443.1 100 99 PS2-19 Myrtaceae1 Branch Aspergillus sp. CB-6 JQ256469.1 100 100

Fungal sp. PhSW1 JX262555.1 100 100 PS3-3 Myrtaceae2 Branch Acremonium sp. 1 FW2SW3 JX273063.1 100 99

Acremonium radiatum HQ232205.1 99 96 PS3-5 Myrtaceae2 Leaf Phyllosticta sp. L79 JN543168.1 100 100

Guignardia sp. IFB-GLP-4 GU380271.1 100 100 Rhodotorula mucilaginosa isolate ZH9 JQ838010.1 100 100

77 PS3-7 Myrtaceae2 Branch Rhodotorula mucilaginosa strain RM-1 KC186124.1 100 100

Rhodotorula mucilaginosa isolate ZH9 JQ838010.1 100 100 PS3-14 Myrtaceae2 Branch Acremonium sp. 1 FW2SW3 JX273063.1 100 99

Acremonium radiatum HQ232205.1 99 96 PS3-16 Myrtaceae2 Branch Acremonium sp. 1 FW2SW3 JX273063.1 100 99

Acremonium radiatum HQ232205.1 99 96 PS3-17 Myrtaceae2 Branch Penicillium sp. MZ4-12 JQ074031.1 100 100

Penicillium charlesii strain MI6 HQ600969.1 100 100 PS6-1 Sapindaceae Stem Phomopsis mali AB665315.1 100 100

Phomopsis sp. U4A2-A AB665312.1 100 100 PS6-2 Sapindaceae Stem Neofusicoccum parvum strain MFLUCC 11-0184 JX646828.1 100 99 Botryosphaeria sp. MUCC0098 AB454225.1 100 99 PS6-3 Sapindaceae Stem Phomopsis mali AB665315.1 100 100

Phomopsis sp. U4A2-A AB665312.1 100 100 PS6-4 Sapindaceae Stem Neofusicoccum parvum strain MFLUCC 11-0184 JX646828.1 100 99 Botryosphaeria sp. MUCC0098 AB454225.1 100 99 PS6-5 Sapindaceae Stem Neofusicoccum parvum strain MFLUCC 11-0184 JX646828.1 100 99 Botryosphaeria sp. MUCC0098 AB454225.1 100 99 PS6-6 Sapindaceae Stem Phomopsis mali AB665315.1 100 100

Phomopsis sp. U4A2-A AB665312.1 100 100 PS6-7 Sapindaceae Stem Neofusicoccum parvum strain MFLUCC 11-0184 JX646828.1 100 99 Botryosphaeria sp. MUCC0098 AB454225.1 100 99 PS6-11 Sapindaceae Leaf Colletotrichum gloeosporioides isolate CG0804 FJ227900.1 100 99 Colletotrichum truncatum AJ301945.1 100 99 PS6-12 Sapindaceae Leaf Colletotrichum gloeosporioides isolate CG0804 FJ227900.1 100 99 Colletotrichum truncatum AJ301945.1 100 99 PS6-13 Sapindaceae Leaf Colletotrichum gloeosporioides isolate CG0804 FJ227900.1 100 99 Colletotrichum truncatum AJ301945.1 100 99 PS6-14 Sapindaceae Leaf Colletotrichum gloeosporioides isolate CG0804 FJ227900.1 100 99 Colletotrichum truncatum AJ301945.1 100 99 PS6-15 Sapindaceae Root Helicoon richonis strain CBS 282.54 AY856952.1 100 99 Kirschsteiniothelia elaterascus strain A22-11B AF053728.1 100 99 PS6-16 Sapindaceae Root Tetrachaetum elegans strain FI-IIII- FF1 JX967531.1 100 99 Graphium sp. UM73-75 HQ595739.1 100 99 PS6-29 Sapindaceae Root Phomopsis mali AB665315.1 100 99

Phomopsis sp. U4A2-A AB665312.1 100 99 PS6-31 Sapindaceae Root Phomopsis mali AB665315.1 100 100

Phomopsis sp. U4A2-A AB665312.1 100 100 PS6-34 Sapindaceae Root Collophora rubra CBS:120873 GQ154627.1 100 99

Graphium sp. UM73-75 HQ595739.1 100 99 PS6-39 Sapindaceae Root Tetrachaetum elegans strain FI-IIII- FF1 JX967531.1 100 99

78 Graphium sp. UM73-75 HQ595739.1 100 99 PS6-40 Sapindaceae Root Tetrachaetum elegans strain FI-IIII- FF1 JX967531.1 100 100 Graphium sp. UM73-75 HQ595739.1 100 100 PS6-41 Sapindaceae Root Tetrachaetum elegans strain FI-IIII- FF1 JX967531.1 100 100 Graphium sp. UM73-75 HQ595739.1 100 100 PS7-14WF Myrtaceae3 Leaf Phyllosticta sp. L79 JN543168.1 100 100

Guignardia sp. IFB-GLP-4 GU380271.1 100 100 PS7-14B Myrtaceae3 Leaf Phyllosticta sp. L79 JN543168.1 100 100

Guignardia sp. IFB-GLP-4 GU380271.1 100 100 PS7-15 Myrtaceae3 Leaf Phaeomoniella dura CBS:120882 GQ154638.1 100 99

PS7-17 Myrtaceae3 Leaf Neofusicoccum parvum strain MFLUCC 11-0184 JX646828.1 100 99 Botryosphaeria sp. MUCC0098 AB454225.1 100 99 aName assigned to endophyte isolate, bSection of plant from which endophyte was isolated, cClosest sequence similarity in GenBank based on 18S rRNA gene sequence, dGenbank accession number. Where two or more matches were returned with the same identity and coverage score, the top two organisms were tabulated or organisms (with the same match) that have been isolated as endophytes or symbiotic organisms were recorded instead.

Table 8 BLASTn analysis of 16S rRNA gene sequence from isolated bacterial endophytes Isolate Accession Covera Identity numbera Plant familyb Originc Closest matchd numbere ge (%) (%)

PS1-5 Smilacaceae Root Bacillus sp. FJAT-17278 KF040591.1 100 100 Bacillus sp. UTPF23b AB769177.1 100 99

PS1-6 Smilacaceae Root Burkholderia sp. I2 JF763857.1 100 99 Burkholderia nodosa HQ849091.1 100 99

PS1-7 Smilacaceae Root Lactobacillus sp. TLMP4 KF406347.1 100 100 Bacillus cereus strain DRMS KF258874.1 100 100

PS1-8 Smilacaceae Root Lactobacillus sp. TLMP4 KF406347.1 100 100 Bacillus cereus strain DRMS KF258874.1 100 100

PS1-10 Smilacaceae Stem Lactobacillus sp. TLMP4 KF406347.1 100 100 Bacillus cereus strain DRMS KF258874.1 100 100

PS1-11 Smilacaceae Stem Lactobacillus sp. TLMP4 KF406347.1 100 100 Bacillus cereus strain DRMS KF258874.1 100 100

PS6-18 Sapindaceae Root Burkholderia phytofirmans PsJN NR_102845.1 100 99 Burkholderia phytofirmans HQ849096.1 100 99

PS6-22 Sapindaceae Root Burkholderia phytofirmans PsJN NR_102845.1 100 99 Burkholderia phytofirmans HQ849096.1 100 99

PS6-24 Sapindaceae Root Burkholderia phytofirmans PsJN NR_102845.1 100 99 Burkholderia phytofirmans HQ849096.1 100 99

PS6-25 Sapindaceae Root Burkholderia phytofirmans PsJN NR_102845.1 100 99 Burkholderia phytofirmans HQ849096.1 100 99 PS7-2 Myrtaceae3 Branch Agrobacterium sp. MS2 KF021239.1 100 100

79 Rhizobium sp. M10 KC787583.1 100 100

PS7-11 Myrtaceae3 Branch Agrobacterium sp. L-1 EU293211.1 100 99 Agrobacterium sp. MS2 KF021239.1 100 99 a Name assigned to endophyte isolate, bPlant family, cSection of plant from which endophyte was isolated, dClosest sequence similarity in GenBank based on 16S rRNA gene sequence, eGenbank accession number. Where two or more matches were returned with the same identity and coverage score, the top two organisms were tabulated or organisms (with the same match) that have been isolated as endophytes or symbiotic organisms were recorded instead. 3.3.3 Phylogeny

3.3.3.1 Phylogenetic Analysis of Fungal Isolates

A phylogenetic tree encompassing 18S rRNA gene sequences from the fungal isolates and related reference strains (from GenBank) was constructed (Figure 6). The fungal endophytes grouped within 10 orders: Sporidiales, Diaporthales, Capnodiales,

Hypocreales, Glomerellales, Eurotiales, Botryosphaeriales, Helotiales and Microascales.

If the order was unable to be determined other classification level names were assigned.

The endophytes isolated from plant species 2, which is classified within the

Myrtaceae family, all grouped within the Eurotiales, with the exception of PS2-17 and

PS2-18, which grouped within the orders Capnodiales (genus Cladsporium) and

Diaporthales (genus Chrysoporthe), respectively. The only fungus isolated from plant species 1 (Smilacaceae) also grouped within the order Eurotiales. The PS1-3 endophyte and most of the plant species 2 endophytes (PS2-2, PS2-6 and PS2-9), clustered within the Penicillium genus of the Eurotiales, with the exception of PS2-19, which clustered within the Aspergillus (Eurotiales).

The endophytes isolated from plant species 3, (Myrtaceae), were spread across the tree; however the majority (PS3-3, PS3-14 and PS3-16) clustered within the

Hypocreales near Acremonium sp. Interestingly, these endophytes were all isolated from stem sections. PS3-7 grouped within the Sporidiales (Rhodotorula), PS3-17 grouped within the Eurotiales (Penicillium), and PS3-5 grouped within the Botryosphaeriales

(Guignardia/Phyllosticta).

80 The endophytes isolated from plant species 6 (Sapindaceae) were distributed among the Diaporthales, Glomerellales, Helotiales and especially Botryosphaeriales orders. The majority of endophytes isolated from stem tissue (PS6-1 to PS6-7) and endophytes isolated from root tissue (PS6-15 to PS6-41) grouped within the

Botryosphaeriales (Botryosphaeria/Neofusicoccum genera), Diaporthales (Phomopsis genus) and Helotiales (Tetrachaetum genus). All endophytes isolated from the leaf tissue clustered in Colletotrichum (Glomerellales).

Four fungal endophytes were isolated from the leaf tissue of plant species 7

(Myrtaceae). Of these, P7-14WF, P7-14B and P7-17 grouped within the

Botryosphaeriales, while P7-15 was only identified as mitosporic Ascomycota. PS7-

14B and PS7-14WF shared a branch with Phyllosticta sp., PS7-17 with

Botryosphaeria/Neofusicoccum sp., and PS7-15 with Phaeomoniella sp.

3.3.3.2 Phylogenetic Analysis of Bacterial Isolates

The bacterial endophytes grouped into 3 major clades encompassing

α-proteobacteria (2 isolates), β-proteobacteria (5 isolates) and Firmicutes (5 isolates).

Five of the isolated endophytes from plant species 1 (Smilacaceae), were associated with Bacillus (PS1-5, PS1-7, PS1-8, PS1-10, PS1-11) within the Firmicutes, while the 6th PS1 isolate (PS1-6) was placed with Burkholderia within the

β-proteobacteria. PS1-5, PS1-7 and PS1-8, were isolated from root tissue while PS1-10 and PS1-11 were isolated from stem tissue.

Endophytes isolated from the root tissue of plant species 6 (Sapindaceae) all grouped within the β-proteobacteria (Burkholderia).

The two endophytes isolated from stem tissue of plant species 7, (Myrtaceae) grouped within the α-proteobacteria (Agrobacterium).

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82 Outgroup

Microcystis aeruginosa (AF139294.1)

Burkholderia phytofirmans (NR_102845.1) 63.7 Burkholderia phytofirmans (HQ849096.1) Burkholderia sp. (AB669480.1) Burkholderia nodosa (HQ849091.1) 86.5 !

PS1-6 (Smilacaeae-Root) - 68.2 proteobacteria 73.7 Burkholderia unamae (HQ023250.1) Burkholderia sp. (JF763857.1) Burkholderia sp. (JN872505.1) 100 PS6-22 (Sapindaceae-Root) PS6-25 (Sapindaceae-Root) PS6-24 (Sapindaceae-Root) 97 PS6-18 (Sapindaceae-Root) Burkholderia phytofirmans (JF494821.1) Rhizobium sp. (KC787583.1) 100

Agrobacterium tumefaciens (AY513489.1) " - Agrobacterium sp. (KF021239.1) proteobacteria Rhizobium sp. (JN871230.1) PS7-2 (Myrtaceae3-Branch) PS7-11 (Myrtaceae3-Branch) 67.5 Agrobacterium sp. (EU293211.1) Bacillus sp. (AB769177.1) 97.7 Bacillus luciferensis (JF496453.1) PS1-5 (Smilacaeae-Root) 68.3 Bacillus sp. (AB366327.1) 65.3 Bacillus sp. (KF040591.1) Bacillus cereus (AF155958.1) 99 Bacillus cereus (KF258874.1) Bacillus sp. (EF690408.1) Firmicutes Uncultured Bacillus sp. (FJ863113.1) Bacillus thuringiensis (FJ174643.1)

PS1-7 (Smilacaeae-Root) PS1-10 (Smilacaeae-Stem) PS1-11 (Smilacaeae-Stem) PS1-8 (Smilacaeae-Root) Bacillus sp. (AB330405.1) Bacillus cereus (EU326488.1) Lactobacillus sp. (KF406347.1)

0.05

Figure 7 16S rRNA gene phylogenetic tree encompassing isolated bacterial endophytes and related organisms. Bacterial isolates are shown in bold. Accession numbers of the reference strains are in brackets. Bootstrap values >50% are displayed.

83 3.4 Discussion

It is hypothesised that every plant hosts at least one endophyte species within its tissues [177]. To obtain a representation of the culturable endophytes that are present within any given plant sample, microbes must first be removed from the surface. In this study, the surface sterilisation step was adapted from a method used to isolate fungal endophytes from pine needles [207]. Like pine needles, most of the leaves from the

Myrtaceae have tough or waxy leaves that were able to withstand this harsh chemical treatment. However, plant species 1 (Smilacaceae) and plant species 6 (Sapindaceae) have fragile leaves, which were degraded by the initial H2O2 treatment. This may explain the bias in the number of endophytes that were isolated from the robust versus the fragile plant samples using this method. Alternative techniques that may be more suited to the isolation of endophytes from fragile plant materials include treatment with sodium hypochlorite [214], 70% ethanol and ethanol flaming [177] and calcium hypochlorite [215].

While the sterilisation method employed in this study was successful in most cases, on one occasion during the 3-day surface sterilisation confirmation step, microbial growth was observed below the bark and around the leaf mid vein of the

Eucalyptus sp. This growth was unlikely to be the result of failed surface sterilisation as it originated from the section point where the tissue was removed from the plant. A more likely explanation is that the culture was inoculated by an endophyte that escaped through the damaged tissue at the section point. In this case, plant material was re- collected, re-surface sterilised, and placed in HIB until no growth was observed. To validate the surface sterilisation protocol, unsterilised plant matter was placed into HIB and growth was seen within 24 hrs (data not shown). The surface sterilisation was validated if no growth was observed within 72 hrs. Post 72 hrs, if left in the right

84 nutrient conditions, the only organisms left to grow would be endophytic organisms from within the plant material, that is why the false positive was not considered and commented on. However, it must be noted that the interference by endophytic microorganisms cannot be ruled out.

Another limitation of this study was the use of only a few media types (WA, PDA and

AGA), which may have selected for certain endophytes at the expense of others. For example, AGA is a selective medium for aerobic actinomycetes (although no actinobacteria were isolated) [216]. Surface sterlised plant material was initially plated on PDA, WA and ARG/GLY agar plates. All endophytes were then restreaked onto

PDA until pure cultures were obtained in which all endophytes were eventually cultured on PDA plates. In this regard, the culturable endophytes were all classified as isolated/culturalable on PDA.

Future studies should incorporate a more diverse range of media to maximise the spectrum of culturable endophytes isolated.

3.4.1 PCR Screening

Although most DNA extractions performed in this study resulted in the isolation of clean and pure DNA, we were unable to isolate good quality (PCR amenable) DNA from twenty-three endophyte isolates. This may have been due to the presence of secondary metabolites, polysaccharides, DNA bound proteins or a number of other inhibitors that interfere with the PCR process.

The 16S and 18S PCR sequences were used as a source of de-replication according to BLAST sequence comparisons. However, in certain cases, even though isolates shared 100% sequence identity, they were considered unique if they had significant morphological or metabolic differences. For example, PS2-2 and PS2-9 were both identified via 18S rRNA sequencing to be Penicillium sp., however PS2-2 changed

85 the colour of the PDA agar plate to a deep red colour whereas PS2-9 did not. These results suggest that the two isolates are distinct strains. Cui et al (2011) also observed metabolic differences between two closely related Fusarium solani strains isolated from the same plant species in different areas of China. Although phylogenetically similar, the two F. solani strains had different antimicrobial and cytotoxic activities [102].

Penicillium endophytes have previously been isolated and described producing red pigment by Wang et al (2013). Penicillium kongii, an endophytic fungus isolated from the leaves of the Chinese plant, Cotoneaster sp., produced a characteristic soluble red pigment when grown on yeast extract sucrose agar (YES). Interestingly, morphologically and phylogenetically related species did not produce this pigment

[217].

3.4.2 Phylogeny

3.4.2.1 Phylogeny of Fungal Endophytes

Fungi comprise a large proportion of the endophytic microbial community and are recognised as a source of novel bioactive compounds. When phylogenetic grouped by order, the isolated endophytes from this study, together, are a unique data set. The grouping of the fungal endophytes are validated when comparing several phylogenetic analysis, by order, of the fungal community in medicinal plants [218, 219], marine sponges [220], tropical sea grass [221] and coffee plants [222]. Diversity studies also show that endophytic fungi usually represent many genera. In line with this, the results of our phylogenetic study suggest that the fungal endophytes isolated from our six medicine plant species belong to the genera Penicillium,

Botryosphaeria/Neofusicoccum, Acremonium, Cladosporium/Davidella,

Colletotrichum, Aspergillus, Phomopsis, and Phyllosticta. A similar study by Miller et al. (2012), investigating the endophyte diversity of 8 TCM plants, also resulted in the

86 isolation of fungi belonging to Cladosporium/Davidella, Colletotrichum, Phomopsis and Guignardia [203]. Members of these genera have also been isolated from coffee plants in Columbia, Hawaii, Puerto Rico and Mexico [182], rubber trees growing within the Amazon basin [223], tropical fruit and medicinal plants of Malaysia [224] and carnivorous plant species (Sarracenia) from North America [225]. Interestingly, we also isolated endophytes belonging to the genera Rhodotorula, but not Halotiales previously associated with Rhodotorula isolated from bryophytes growing in Antarctica

[226].

Previous studies suggest that fungal endophytes are a rich source of natural products. For example, Cladosporium, Botryosphaeria and a Penicillium species producing anti-bacterial saponins with activity against Staphylococcus aureus were identified by Wu et al. (2012) [227], while a Penicillium species producing an alkaloid with cytotoxic and anti-fungal activites was identified by Zheng et al. (2012) [228].

Anti-fungal compounds have also been associated with endophytic Phomopsis species isolated from Indian medicinal plants [98].

The endophytes isolated in this study are common to other Australian native plants at an order or genus level. For example, 273 fungal endophytes were isolated from four different Gossypium (cotton) species collected from central Northern

Territory, Queensland and South Australia. The most commonly isolated endophytes from these plants belonged to Botryosphaeria, Phomopsis, Phoma, Alternaria,

Fusarium and Dichomera [113]. In a similar study 154 out of 226 fungal endophytes isolated from nine native plants in Western Australia belonged to the family

Botryosphaeriaceae. The most commonly isolated species in this study was

Neofusicoccum austral, however Alternaria spp., Paraconiothyrium sp.,

Colletotrichium sp., Embellisia sp., Bipolaris sp., Phoma sp., Paraphaeosphaeria sp.,

87 Preussia sp., Stemphylium sp., Pestalotiopsis sp., and Pleospora sp. were also identified

[104]. Another study examining ten different Western Australian native trees isolated endophytes representing 8 genera of fungi, Lasiodiplodia, Diplodia, Dothiorella,

Neofusicoccum, Botryosphaeria, Macrophomina, Neoscytalidium and

Pseudofusicoccum, including seven new species of Botryosphaeriaceae from Adansonia gibbosa (the Boabab tree) [103]. Lastly, a search for industrial enzymes from the medicinal Australian native plant, Eremophilia longifolia, uncovered endophytes from the genera Cladosporium and Fusarium, as well as Leptosphaerulina, Nigrospora,

Alternaria, Phoma, Preussia and Stemphylium [96].

3.4.2.2 Phylogeny of Bacterial Endophytes

In this study, bacterial endophytes where isolated from three of the six medicinal plants, plant species 1, 6 and 7 (from Smilacaceae, Sapindaceae and Myrtaceae, respectively).

The results from our phylogenetic study suggest that these bacterial isolates belong to the genera Bacillus, Burkholderia and Agrobacterium. Species belonging to these genera have been isolated previously from various plant tissues particularly roots. For example, Miller et al (2012) isolated numerous endophytic Bacillus species from eight

TCM plants including a Bacillus sp. that showed very low cytotoxic activity [203].

Endophytic Bacillus have also been isolated from the Indian medicinal plant Ocimum sanctum, showing activity against phytopathogenic fungi and enhancing plant growth and essential oil production in the host plant [229]. Likewise, multiple Burkholderia sp. were isolated from the leaves of various Rubiaceae of the tribe Vanguerieae [230] and roots of Oryza sativa L. (rice). Bacillus and Burkholderia endophytes have also been isolated together from healthy and infected roots of Italian Malus domestica, Borkh

(apple plants) [231], as well as the roots of Brazilian Zea mays L. (Maize) [232, 233].

These examples are in agreement with our findings showing that both Bacillus and

88 Burkholderia species are common root endophytes. The literature also indicates that endophytes belonging to these genera have the potential to produce bioactive natural products, and we intend to investigate this in future studies.

3.5 Conclusion

This study is the first to be conducted on the diverse endophyte community within medicinal plants used by the Dharawal people of Botany Bay. A total of 48 endophytes, 36 fungi and 12 bacteria, were isolated and phylogenetically organised into

13 orders and 3 class-levels. The results of this phylogenetic study show the majority of fungal and bacterial genera of endophytes have been previously isolated from both medicinal and non medicinal plants of China, India, Malaysia, South America,

Antarctica and Australia. The above literature gives a strong indication that there is potential for these isolated endophytic fungi to produce bioactive natural products that may have biotechnological value. For example, we isolated a Penicillium sp. (PS2-9) from plant species 2 (Myrtaceae), which is traditionally used to treat bacterial infections of the skin, upset stomach and other internal complaints. Wu et al. (2012) also isolated a saponin-producing Penicillium sp. from Aralia elata, a medicinal plant used to treat bacterial infections in China, Japan and Russia [227]. Our study therefore serves as a platform for the future discovery of novel anti-microbials from the endophytes isolated from native Australian plants. While our results indicate that the endophytes residing in medicinal plants of Botany Bay are similar to those found in other plant species around the world, at a genus level, differences relating to natural product synthesis and other metabolic traits need to be studied and compared in more depth at the species level.

This may give a more accurate representation of the total culturable endophytic community of plants.

89 Although the mentioned species are commonly isolated as endophytes, their role in the host plants as individual endophytes and as a diverse community is unknown. The culturable endophyte community gives a narrow insight into the total diversity and range of environments that endophytes thrive in. To further investigate the total endophyte community of the studied medicinal plants, future studies may include the use of different isolation media and isolation conditions, different surface sterilisation protocols, alternate DNA extraction methods and employing the use of metagenomic studies of the total plant meta-genome.

Further screening of endophyte DNA can also give insight into possible secondary metabolite and bioactive compound biosynthesis by using primers designed to target genes encoding biosynthetic enzymes, such as non-ribosomal peptide synthetases and polyketide synthases, as described in the next chapter.

90 4 Chapter 4 Screening of Endophytes from Medicinal Plant for

Polyketide and Non-ribosomal Peptide Biosynthetic Genes as an

Indicator for Bioactive Natural Products

4.1 Introduction

In the search for novel pharmaceuticals, the accepted approach to discover bioactive natural products is to typically randomly sample large quantities of plants in the environment and screen them for anti-microbial, anti-viral and anti-cancer activities, to name a few. For example, taxol is a complex diterpene that was isolated from the bark of the Pacific yew tree, Taxus brevifolia [234], and represents the most successful search for an anti-cancer compound. Originally, taxol was obtained by harvesting the bark of the yew tree, in effect killing the tree. To obtain 1 kg of taxol, 10,000 kg of bark is needed with the percentage of taxol harvested from bark ranging from 0.00014-

0.069% w/w [235]. Isolated from the same tree was an endophytic fungus, Taxomyces andreanae, that was found to produce the highly valued anti cancer compound taxol

[81].

The plant-microbe interaction is an example of a unique environment that has the potential to produce the next generation of antibiotics or anti-cancer compounds.

Endophytes are known to produce a number of secondary metabolites that are bioactive against a number of pathogenic organisms and indicator cell lines (Table 9). They have the advantage of being culturable and thus the potential to produce commercial amounts of the bioactive compounds. Endophyte-derived secondary metabolites could possibly be responsible, directly or indirectly, for the host plant’s medicinal properties and thus are a good target for future work. Examples of endophytes and their associated bioactive compounds include the anti-cancer compound taxol from Cladosporium cladoporioides [81, 82], anti-fungal pyrenophorol and related sesquiterpenes produced

91 by Lophodermium sp. [83], a number of anti-bacterial and anti-fungal volatile organic compounds biosynthesised by Muscodor crispans [84] and the cytotoxic alterporriol G and its atropisomer alterporriol H isolated from Stemphylium globuliferum [85]. As shown in Table 9, there are a range of endophytes that have shown promising potential for drug discovery of novel antibiotic compounds.

Recently, an increasing amount of endophyte research has been based on ethnobotanical information collected from the Indigenous people of South America

[236, 237], China [102, 238-241], and Africa [85, 94], while there have been limited reports involving the Australian Aboriginals [95, 117].

Endophyte research on Australia’s native medicinal and non-medicinal plants is very new. Numerous studies have been carried out on the bioactive compounds isolated from medicinal plants [34, 36, 43, 56, 172, 173, 242, 243], however, not on their endophyte community. Australia has many unique environments and numerous plants species that thrive in these ecosystems; thus the potential to find endophytes that produce bioactive compounds is immense. One of the first reports of endophytes from traditional medicine plants of Australia using traditional knowledge resulted from consultation with a Northern Territory community. Traditional knowledge of a plant used for treating infections and wound healing, called Snakevine (Kennedia nigriscans)

[244], formed the basis for further investigations of endophyte derived natural products.

Isolated from within the living plant tissue was a novel Streptomyces, Streptomyces munumbi. S. munumbi produced a novel class of antibiotics, munumbicins A-D [95,

245]. Munumbicins were tested for activity against Bacillus anthracis (anthrax), multidrug resistance strains of Mycobacterium tuberculosis (TB), Plasmodium falciparum

(malaria) and Staphylococcus aureus [246]. Disk bioassays (bacterial) and anti-malarial assays all showed that these organisms are sensitive to the munumbicins. Furthermore,

92 munumbicin B showed activity against multi-drug resistant M. tuberculosis, while munumbicins C and D displayed anti-malarial activity at levels 50% lower than the accepted anti-malarial drug, chloroquine [95].

Endophytic bacteria and fungi isolated from both medicinal and non-medicinal plants have been shown to produce anti-infective agents [114, 175]. Initial studies into endophyte research set out to a) isolate the endophytes, b) grow them in liquid culture and c) chemically screen the organism for anti-microbial, anti-cancer or immunosuppressive activities. To advance this type of research further, we have also screened the endophytes for genes involved in the biosynthesis of small bioactive compounds. Most endophyte-derived bioactive compounds are produced via a NRPS,

PKS or a NRPS/PKS hybrid enzyme complex (See Chapter 1). Compounds biosynthesised by NRPS, in part or full, include b-Lactam antibiotics such as nocardicin

A, penicillin and cephalosporin, the anti-tumour cyclic peptide echinomycin, the lipopeptide daptomycin, and the glycopeptide vancomycin [247-253]. PKS synthesised compounds include examples such as the anti-bacterial erythromycin and tetracycline

[139-141, 254] and in some cases NRPS and PKS hybrid enzymes are involved in the biosynthesis, such as for the antitumor compound bleomycin, the cholesterol lowering compound lovastatin, a streptogramin-type A antibiotic virginiamycin M, and the immunosuppressant rafamycin [125, 145, 250, 255, 256].

This study is focused on screening isolated endophytes from Dharawal medicinal plants for the presence of NRPS or PKS encoding genes and to test the extracts of microbes that possess the gene/s for bioactivity against a range of indicator test microorganisms.

93 Table 9 Isolated endophytes and their bioactivity

Plant Endophyte Species Compound Activity Reference Taxus Fungi Paraconiothyrium Paclitaxel Anti-cancer [257] brevifolia strain SSM001 Thunbergia Fungi Fusarium sp. Tlau3 Crude extract Anti - [92] laurifolia Acanthamoeba Lindl. Aquilaria Fungi Phaeoacremonium Crude extract Anti-bacterial [102] sinensis rubrigenum Anti-fungal Cytotoxic Curcuma Fungi Chaetomium Chaetoglobosin X Anti-fungal [88] wenyujin globosum L18 Cytotoxic Salvia Fungi Trichoderma Tanshinone I and Anti-cancer [258] miltiorrhiza atroviride D16 tanshinone IIA Arisaema Fungi Phoma sp. (3S)-3,6,7-trihydroxy- Anti-fungal [86] erubescens ZJWCF006 α-tetralone; (phytopathogen) Cercosporamide ; Cytotoxic Juniperus Fungi Aspergillus fumigatus Deoxypodophyllotoxin Anti-cancer [259] communis L. Horstmann Urospermum Fungi Ampelomyces sp. Altersolanol A Cytotoxic, Anti- [94] picroides bacterial Emblica Fungi Xylaria sp. Crude extract Anti-fungal [98] officinalis Anti-bacterial Aquilaria Fungi Xylaria mali Crude extract Cytotoxic [102] sinensis Licuala Fungi Xylaria sp. BCC 1α,10α-Epoxy-7α- Cytotoxic [260] spinosa 21097 hydroxyeremophil- Anti-malarial 11(13)-en-12,8β -olide Anti-fungal Melia Fungi Aspergillus fumigatus 12β-Hydroxy-13α- Anti-fungal [261] azedarach methoxyverruculogen (plant) TR-2 Leonurus Bacteria Pseudomonas sp. B42 Crude extract Anti-bacterial [203] heterophyllus Kennedia Actinobacteria Streptomyces Munumbicins Anti-bacterial [95] nigriscans munumbi Anti-malarial Ophiopogon Bacteria Bacillus Exopolysaccharides Anti-tumor [262] japonicas amyloliquefaciens Salvia Bacteria Pseudomonas Cyclopeptides Anti-fungal [263] miltiorrhiza brassicacearum Brine shrimp Bunge. subsp. Neoaurantiaca lethality Heracleum Bacteria Bacillus subtilis HC8 Lipopeptide fengycins Anti-fungal [264] sosnowskyi Manden Kandelia Actinobacteria Streptomyces sp. Kandenols Anti-bacterial [265] candel HKI0595 (eudesmenes-like sesquiterpenes) Myoporum Actinobacteria Streptomyces sp. MA- 7,30-di-(γ,γ- Anti-fungal [266] bontioides A. 12 dimethylallyloxy)-5- Gray hydroxy-40- methoxyflavone Glycine max Actinobacteria Streptomyces sp. Borrelidin Anti-fungal [267] (L.) Merr neau-D50

94 4.2 Materials and Methods

4.2.1 DNA Isolation

Genomic DNA from the pure cultured endophytes is isolated using the XS buffer method [208], previously described in Chapter 3 section 3.2.4.

4.2.2 Polymerase Chain Reaction (PCR)

4.2.2.1 Screening for Polyketide Synthases (PKS)

Amplification of PKS gene fragments was performed via PCR using three sets of primers. The primer set DKF 5’-(GTGCCGGTNCC(A/G)TGNG(T/C)(T/C)TC)-3’ and DKR 5’-(GCGATGGA(T/C)CCNCA(A/G)CA(A/G)(C/A)G)-3’ targets the ketosynthase domain of the PKS encoding gene in bacteria [268]. This PCR reaction was carried out in a total volume of 20 µL and contained 1X reaction buffer (Promega),

2.5 mM MgCl2, 0.2 mM dNTPs (Sigma), a total of 2.5 pmol of each primer, 0.2 U Taq polymerase and 10-30 ng DNA template. The primer sets

LC1 5’-(GAYCCIMGITTYTTYAAYATG)-3’ and LC2c 5’-

(GTICCIGTICCRTGCATYTC)-3; LC3 5’-(GCIGARCARATGGAYCCICA)-3 and

LC5c 5’-(GTIGAIGTICRTGIGCYTC)-3’ [269], target fungal specific PKS and amplify the ketosynthase domain of the PKS encoding gene. Primers set LC1/LC2c target genes involved in the biosynthesis of non-reduced PKS and the LC3/LC5c target genes that are involved in the biosynthesis of reduced polyketides. The LC primer set

PCR reactions were performed in a total volume of 20 µL containing 1X reaction buffer

(Promega), 2.5 mM MgCl2, 0.25 mM dNTPs (Sigma), a total of, 0.8 pmol (LC1), 11.2 pmol (LC2c), 2.4 pmol (LC3) and 33.6 pmol (LC5c) primers, 0.2 U Taq polymerase and 10-30 ng DNA template. The positive control was Cylindrospermopsis raciborskii

95 AWT205 DNA that is known to possess PKS encoding genes [270]. The fungal PKS

PCR positive is one of the original isolates, PS2-6.

Thermocycling was carried out in a BIORAD MyCycler thermocyler with all PCR cycling conditions contained in Table 10.

4.2.2.2 Screening for Non-Ribosomal Peptide Synthetases (NRPS)

Amplification of NRPS gene fragments was performed via PCR using two sets of primers. The primer set MTF2 5’-(GCNGGYGGYGCNTAYGTNCC)-3’ and MTR2

5’-(CCNCGDATYTTNACYTG)-3’ are designed to target the gene encoding the adenylation domain of the NRPS gene cluster in bacteria [271]. This PCR reaction was carried out in a total volume of 20 µL and contained 1X reaction buffer (Promega), 2.5 mM MgCl2, 0.2 mM dNTP’s (Sigma), a total of 2.5 pmol of each primer, 0.2 U Taq polymerase and 10-30 ng of DNA template. The primer set RJ016F 5’-

(TAYGGNCCNACNGA)-3’ and RJ016R 5’-(ARRTCNCCNGTYTTRTA)-3’ targets the adenylation domain encoding gene [272]. This PCR reaction was carried out in a total volume of 20 µL and contained 1X reaction buffer (Promega), 3.25 mM MgCl2,

0.2 mM dNTPs (Sigma), a total of 2 pmol of each primer, 0.3 U Taq polymerase and

10-30ng DNA template. The positive control used was Microcystis aeuriginosa 7806

DNA that are known to possess NRPS encoding genes [271]. The fungal NRPS PCR positive is one of the original isolates, PS2-6.

Thermocycling was carried out in a BIORAD MyCycler thermocyler with all

PCR cycling conditions contained in Table 10.

4.2.2.3 NRPS and PKS sequencing and sequencing clean up

NRPS and PKS amplified genes were sequenced according to Chapter 3 sections

3.2.5.2 to 3.2.5.5. In any case of multiple band amplification, correct band was excised

96 for gel purification was performed using the UltraClean GelSpin DNA Extraction Kit

(MoBio) in accordance with the manufacturer’s instructions.

4.2.3 Liquid broth culturing and compound extraction

Endophytes that tested positive for a PKS or NRPS gene by PCR amplification were grown in small liquid culture flasks. One hundred millilitres of potato dextrose broth (PDB) was autoclaved in cotton wool-plugged 500 mL conical flasks. A loop full of biomass from the agar plate or frozen stocks was added to the flasks under sterile conditions. The flasks were incubated at room temperature on a shaker at 100 rpm until required biomass is obtained.

The flasks were removed from the shaker and exhaustively extracted with equal volume of ethyl acetate, the organic layer was removed and dried over sodium sulphate

(anhydrous) or magnesium sulphate (anhydrous) to remove residual water. The solution was filtered using Whitman filter paper number 4 and collected into a round bottom flask. The ethyl acetate extract was dried via evaporation and weighed.

Table 10 PCR primers used and amendments to reaction and cycling condition a c f Primer Target (expected Primer sequence Reaction Thermocycling Reference d e product size in conditions conditions b bp) MTF2 Adenylation gcnggyggygcntaygtncc 94°C for 10 s, [271] domain bacterial 52°C for 30 s, MTR2 NRPSs (~1000) ccncgdatyttnacytg 72°C for 1 min

DKF Ketosynthase gtgccggtnccrtgngyytc 94°C for 20 s, [268] domain of 55°C for 30 s, DKR bacterial PKSs gcgatggayccncarcarmg 72°C for 1 min (~650-700) RJ016-R Adenylation arrtcnccngtyttrta [272] domain of fungal RJ016-F NRPSs (~300) tayggnccnacnga

LC1 Ketosynthase gayccimgittyttyaayatg Primers (pmol): 94°C for 1 min, [269] domain of fungal 55°C for 1 min LC2c PKSs (~700 for gticcigticcrtgcatytc LC1: 50; 72°C for 3 min both primer sets)

97 LC3 gcigarcaratggayccica LC2c: 8;

LC5c gtigaigticrtgigcytc LC3: 24;

LC5c: 16 aPrimer set name, bGene fragment target of the primer, cPrimer sequence 5’à3’, dAmendments to the original reaction conditions, eAmendments to the original thermocyling conditions, freferences.

4.2.4 Disc Diffusion Anti-Microbial Activity Assays

Test strain organism for the anti-microbial activity assays were: Enterococci faecalis, Escherichia coli (NCTC 10418), Pseudomona aeruginosa (NCTC 10490),

Staphylococcus aureus (NCTC 6571), Bacillus subtilis (ATCC 6633), Cornyebacterium diphtheriae, and Cryptococcus albidus (ATCC 10666). Disc diffusion assays were set up by growing the required culture to OD 0.6 at 600nm and spread plating 100 mL of the culture onto nutrient agar plates. Sterile discs were placed onto the plates and 50 µg

(5 mL of 10 µg/mL) of extract dissolved in DMSO was added. The plates were then placed in either at 30oC or 37oC for 24 h incubation. Ampicillin (100 µg/mL) and cycloheximide (100 µg/mL) were used as a positive control for the bacterial and fungal assay, respectively and DMSO as the negative control.

Activity was measured by the distance from the centre of the disc to the outermost boundary of the zone of clearance. Each assay was performed in triplicate and the average distance was recorded.

4.3 Results

4.3.1 NRPS and PKS Screening by PCR

The NRPS and PKS screening was only used in this study as an indicator for the presences of potential bioactive encoding genes for selection of endophyte candidates for further study.

98 4.3.1.1 Screening for the PKS Encoding Gene

PCR was undertaken to screen the endophyte DNA in the search for PKS encoding genes using the primers DKF/R and the LC1/2c and LC3/5c. Amplification of the PKS encoding gene was shown to be positive in 31 of the 48 identifiably unique endophytes by 16S and 18S rRNA gene sequencing (Chapter 3). For endophytes isolated from plant species 1 (labelled PS1-#), 3/7 tested positive, with PS1-3 amplifying the PKS using all three DKF/R, LC1/2c and LC3/5c primer sets. Endophytes from plant species 2 (labelled PS2-#) had a positive PKS amplification for 6/6 of the total identifiable isolates. Again, PS2-2 and PS2-9 had positive amplification for all of the three primer sets. PS3-17 was the only endophyte from plant species 3 (labelled

PS3-#) that showed amplification of the PKS encoding gene. Plant species 6 endophytes

(labelled PS6-#) had positive amplification for 18/23 endophytes. Only PS7-2 and PS7-

17 had a positive PKS result from plant species 7 endophytes (PS7-#) (Table 12).

Representitive gel photos of PKS screening are presented below.

L 1 2 3 4 5 6 7 8 9

Figure 8 Amplification of PKS ketosynthase domain of PS1 endophytes using DKF/DKR primer set. L=ladder; PS1- 2 (Lane 1), PS1-3 (Lane 2), PS1-5 (Lane 3), PS1-6 (Lane 4), PS1-7 (Lane 5), PS1-8 (Lane 6), PS1-9 (Lane 7), PS1-R (Lane 8) and positive control (Lane 9).

L 1 2 3 4 5 6 7 8 9 10

Figure 9 Amplification of PKS ketosynthase domain of PS1 endophytes using DKF/DKR primer set. L=ladder; PS1- 11 (Lane 1), non PS1 PKS screening (Lane 2-8), positive control (Lane 9) and negative control (Lane 10).

99 L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 10 Amplification of PKS ketosynthase domain of PS2 endophytes using DKF/DKR primer set. L=ladder; PS2-1 (Lane 1), PS2-2 (Lane 2), PS2-3 (Lane 3), PS2-4 (Lane 4), PS2-4 (Lane 5), PS2-6 (Lane 6), PS2-6t (Lane 7), PS2-8 (Lane 8), PS2-9 (Lane 9), PS2-10 (Lane 10), positive control (Lane 12), negative control (Lane 13).

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 11 Amplification of PKS ketosynthesis domain of PS2 endophytes using DKF/DKR primer set. L=ladder; PS2-10 (Lane 1), PS2-11 (Lane 2), PS2-12 (Lane 3), PS2-13 (Lane 4), PS2-14 (Lane 5), PS2-15 (Lane 6), PS2-16 (Lane 7), PS2-17 (Lane 8), PS2-18 (Lane 9), PS2-19 (Lane 10), PS2-20 (Lane 11), PS2-21 (Lane 12), positive control (Lane 13), negative control (Lane 14).

L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 12 Amplification of PKS ketosynthase domain of PS7 endophytes using DKF/DKR primer set. L=ladder; PS7-2 (Lane 1), PS7-3 (Lane 2), PS7-4 (Lane 3), PS7-5 (Lane 4), PS7-6 (Lane 5), PS7-7 (Lane 6), PS7-8 (Lane 7), PS7-9 (Lane 8), PS7-10 (Lane 9), PS7-11 (Lane 10), PS7-12 (Lane 11), positive control (Lane 12), negative control (Lane 13).

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 13 Amplification of PKS ketosynthase domain of PS7 endophytes using DKF/DKR primer set. L=ladder; PS- 7 (Lane 1), PS7-13 (Lane 2), PS7-14a (Lane 3), PS7-15 (Lane 4), PS7-16 (Lane 5), PS7-17 (Lane 6), PS7-14b (Lane

100 7), PS7-14wf (Lane 8), PS7-17b (Lane 9), PS7-17c (Lane 10), non PS7 PKS screening samples (Lane 11-14), positive control (Lane 15), (negative control in above gel).

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 14 Amplification of PKS ketosynthase domain of PS6 endophytes using DKF/DKR primer set. L=ladder; PS6-1 (Lane 1), PS6-2 (Lane 2), PS6-3 (Lane 3), PS6-4 (Lane 4), PS6-5 (Lane 5), PS6-6 (Lane 6), PS6-7 (Lane 7), PS6-8 (Lane 8), PS6-9 (Lane 9), PS6-10 (Lane 10), PS6-11 (Lane 11), PS6-12 (Lane 12), PS6-13 (Lane 13), positive control (Lane 14) (negative control below 1a).

L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 L 1a

Figure 15 Amplification of PKS ketosynthase domain of PS6 endophytes using DKF/DKR primer set. L=ladder; PS6-14 (Lane 1), PS6-15 (Lane 2), PS6-16 (Lane 3), PS6-26 (Lane 4), PS6-27 (Lane 5), PS6-28 (Lane 6), PS6-29 (Lane 7), PS6-30 (Lane 8), PS6-31 (Lane 9), PS6-32 (Lane 10), PS6-33 (Lane 11), PS6-34 (Lane 12), PS6-35 (Lane 13), positive control (Lane 14). L=ladder, negative control (Lane 1a).

L 1 2 3 4 5 6 7 8 9

Figure 16 Amplification of PKS ketosynthase domain of PS6 endophytes using DKF/DKR primer set. L=ladder; PS6-36 (Lane 1), PS6-37 (Lane 2), PS6-38 (Lane 3), PS6-39 (Lane 4), PS6-40 (Lane 5), PS6-41 (Lane 6), PS6-42 (Lane 7), positive control (Lane 8), negative control (Lane 9).

101 L 1 2 3 4 5 6 7 8 9 10 11 12

Figure 17 Amplification of PKS ketosynthase domain of PS2 endophytes using LC1/LC2c fungal specific primer set. L=ladder; PS2-1 (Lane 1), PS2-2 (Lane 2), PS2-3 (Lane 3), PS2-4gn (Lane 4), PS2-4 (Lane 5), PS2-6 (Lane 6), PS2- 6t (Lane 7), PS2-8 (Lane 8), PS2-9 (Lane 9), PS2-10 (Lane 10), positive control (very faint) (Lane 11), negative control (Lane 12).

L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 18 Amplification of PKS ketosynthase domain of PS6 endophytes using LC1/LC2c fungal specific primer set. L=ladder; PS6-1 (Lane 1), PS6-2 (Lane 2), PS6-3 (Lane 3), PS6-4 (Lane 4), PS6-5 (Lane 5), PS6-6 (Lane 6), PS6-7 (Lane 7), PS6-8 (Lane 8), PS6-9 (Lane 9), PS6-10 (Lane 10), PS6-11 (Lane 11), PS6-12 (Lane 12), positive control (Lane 13), (negative control below).

L 1 2 3 4 5 6

Figure 19 Amplification of PKS ketosynthase domain of PS6 endophytes using LC1/LC2c fungal specific primer set. L=ladder; PS6-13 (Lane 1), PS6-14 (Lane 2), PS6-15 (Lane 3), PS6-16 (Lane 4), positive control (Lane 5), negative control (Lane 6).

102 L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 20 Amplification of PKS ketosynthase domain of PS6 endophytes using LC1/LC2c fungal specific primer set. L=ladder; PS6-26 (Lane 1), PS6-27 (Lane 2), PS6-28 (Lane 3), PS6-29 (Lane 4), PS6-30 (Lane 5), PS6-31 (Lane 6), PS6-32 (Lane 7), PS6-33 (Lane 8), PS6-34 (Lane 9), PS6-35 (Lane 10), PS6-36 (Lane 11), PS6-37 (Lane 12), positive control (Lane 13), (negative control below).

L 1 2 3 4 5 6 7

Figure 21 Amplification of PKS ketosynthase domain of PS6 endophytes using LC1/LC2c fungal specific primer set. L=ladder, PS6-38 (Lane 1), PS6-39 (Lane 2), PS6-40 (Lane 3), PS6-41 (Lane 4), PS6-42 (Lane 5), positive control (Lane 6), negative control (Lane 7).

1 2 3 4 5 6 7 8 9 10

Figure 22 Amplification of PKS ketosynthase domain of upscaled endophytes using LC1/LC2c fungal specific primer set (Chapter 5). PS1-3 (Lane 1), PS2-2 (Lane 2), PS2-9 (Lane 3), PS2-19 (Lane 4), PS6-7 (Lane 5), PS6-7 (Lane 6), PS6-31 (Lane 7), PS6-31 (Lane 8), positive control (Lane 9), negative control (Lane 10). L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 23 Amplification of PKS ketosynthase domain of PS6 endophytes using LC3/LC5c fungal specific primer set. L=ladder; PS6-1 (Lane 1), PS6-2 (Lane 2), PS6-3 (Lane 3), PS6-4 (Lane 4), PS6-5 (Lane 5), PS6-6 (Lane 6), PS6-7 (Lane 7), PS6-8 (Lane 8), PS6-9 (Lane 9), PS6-10 (Lane 10), PS6-11 (Lane 11), PS6-12 (Lane 12), PS6-13 (Lane 13), positive control and negative control below).

103 L 1 2 3 4 5 6

Figure 24 Amplification of PKS ketosynthase domain of PS6 endophytes using LC3/LC5c fungal specific primer set. L=ladder; PS6-14 (Lane 1), PS6-15 (Lane 2), PS6-16 (Lane 3), PS6-16 (Lane 4), positive control (Lane 5), negative control (Lane 6).

L 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 25 Amplification of PKS ketosynthase domain of PS6 endophytes using LC3/LC5c fungal specific primer set. L=ladder; PS6-26 (Lane 1), PS6-27 (Lane 2), PS6-28 (Lane 3), PS6-29 (Lane 4), PS6-30 (Lane 5), PS6-31 (Lane 6), PS6-32 (Lane 7), PS6-33 (Lane 8), PS6-34 (Lane 9), PS6-35 (Lane 10), PS6-36 (Lane 11), PS6-37 (Lane 12), positive control (Lane 13), (negative control on another gel and was negative)

L 1 2 3 4 5 6 7 8 9 10

Figure 26 Amplification of PKS ketosynthase domain of upscaled endophytes (Chapter 5) using LC3/LC5c fungal specific primer set. L=ladder, PS1-3 (Lane 1), PS2-2 (Lane 2), PS2-9 (Lane 3), PS2-19 (Lane 4), PS6-7 (Lane 5), PS6-7 (Lane 6), PS6-7 (Lane 7), PS6-7 (Lane 8), positive control (Lane 9), negative control (Lane 10).

4.3.1.2 Screening for NRPS Encoding Genes

Screening all endophyte isolate DNAs revealed only 10 positive amplicons for

NRPS encoding genes using the MTF2/R2 and RJ016F/R primer stes. The majority of the NRPS screening positives using these primers were from the PS1 endophytes with

5/7 testing positive, with the only other positive amplifications from PS2-6 and PS7-2.

104 PCR using the fungal specific RJ016F/R only amplified faint bands when visualised on agarose gel for PS6-4, PS6-7 and PS6-8 (Table 12).

These screening results, coupled with those from the PKS screen, were used to eliminate the endophytes based on this indication of potential bioactivity and to continue with anti-microbial screening. Representitive gel photos of NRPS screening are presented below.

L 1 2 3 4 5 6 7 8 9

Figure 27Amplification of NRPS adenylation domain of PS1 endophytes using MTF2/MTR2 primer set. L=ladder; PS1-2 (Lane 1), PS1-3 (Lane 2), PS1-5 (Lane 3), PS1-6 (Lane 4), PS1-7 (Lane 5), PS1-8 (Lane 6), PS1-9 (Lane 7), PS1-R (Lane 8), positive control (Lane 9).

L 1 2 3 4 5 6 7 8 9 10 11

Figure 28 Amplification of NRPS adenylation domain of PS1 endophytes using MTF2/MTR2 primer set. L=ladder; PS1-11 (Lane 1), positive control (Lane 9 & 10), negative control (Lane 11).

105 L 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 29 Amplification of NRPS adenylation domain of PS6 endophytes using RJ016F/RJ016R fungal specific primer set. L=ladder; PS6-1 (Lane 1), PS6-2 (Lane 2), PS6-3 (Lane 3), PS6-4 (Lane 4), PS6-5 (Lane 5), PS6-6 (Lane 6), PS6-7 (Lane 7), PS6-8 (Lane 8), PS6-9 (Lane 9), PS6-10 (Lane 10), PS6-11 (Lane 11), PS6-12 (Lane 12), PS6-13 (Lane 13), positive control (Lane 14) (negative control shown on separate gel and was negative).

4.3.1.3 Endophytes that Possessed Both NRPS and PKS Genes

Endophytes PS1-3, PS1-6, PS2-6, PS6-4, PS6-7 and PS7-2 amplified both

NRPS and PKS encoding genes. This brought the total of identifiable endophyte isolates that have either a PKS or NRPS encoding gene to 31 out of 48 isolates.

Ten endophytes that were unidentifiable by 16S or 18S rRNA gene sequencing were also included in the bioassays based on their complex biosynthesis genes. PS2-10,

12, 13, 15 and 16 all showed positive PKS amplification along with PS1-2, PS6-8, 9 and 10, and PS6-32. The NRPS encoding gene was not amplified for any of these endophytes.

NRPS screening was initially carried out on the fungi, however, the focus shifted to PKS encoding genes as the majority of fungal natural products are PK based.

4.3.1.4 NRPS and PKS sequence analysis

Some of the genes were sequenced to check the right target was being amplified.

The majority of the returned sequences returned mixed sequence signals, low signal or both. Table 11 shows the results of the BLAST database comparison of the mixed signal sequence data. Sequences are included in the appendix.

106 Table 11 BLAST comparison of mixed sequence from NRPS and PKS screening Endophyte_ID Putative Primers used Sequence BLASTn match BLASTx match endophyte_ID (Table 10) quality (Similarity/coverage) (Similarity/coverage)

PS1-11 Bacillus sp. MTF2/MTR2 Good/ slightly Bacillus Conserved domains mixed thuringiensis serovar on [lcl|4736] kurstaki str. YBT- 1520, complete Peptide synthetase genome CP007607.1 [Bacillus cereus] (100/98) WP_000503027.1 (98/99) Bacillus sp. B49(2012) non- Peptide synthetase ribosomal peptide [Bacillus subtilis] synthetase gene, WP_025709609.1 partial cds (98/98) JQ585617.1 99/94 Non-ribosomal peptide synthetase [Bacillus sp. B49(2012)] AFH77871.1 (80/92)

PS1-3 Penicillium sp. DKF/DKR Mixed Penicillium N/A marneffei strain PM1 putative polyketide synthase PKS11 gene, complete cds HM070055.1 (16/74)

Penicillium marneffei ATCC 18224 polyketide synthase, putative, mRNA XM_002149701.1 (16/74)

PS2-2 Penicillium sp. LC1/2c Mixed Uncultured N/A endophytic fungus clone LJ1 putative polyketide synthase gene, partial cds JQ717038.1 (78/94)

Penicillium echinulatum isolate B1-4 polyketide synthase gene, partial cds KF986466.1 (78/86)

Phoma herbarum isolate K1-1 polyketide synthase gene, partial cds KF986451.1 (78/84)

Penicillium patulum gene encoding putative WA type ketosynthase, partial AJ132274.1 (77/81)

107 PS2-9 Penicillium sp. LC1/2c Good signal Uncultured Conserved domains endophytic fungus on [lcl|14826] clone LJ1 putative polyketide synthase Nonreducing gene, partial cds polyketide synthase JQ717038.1 (99/84) [Penicillium janthinellum], Penicillium ADY75762.1 99/97 brevissimum isolate H2-8-2 polyketide Nonreducing synthase gene, polyketide synthase partial cds [Penicillium raperi], KF986478.1 (99/82) ADY75767.1 99/97

Penicillium steckii Nonreducing isolate E1-10-2 polyketide synthase polyketide synthase [Penicillium gene, partial cds ochrochloron], KF986466.1 ADY75770.1 99/97 (100/82) Putative polyketide Penicillium synthase [uncultured janthinellum strain endophytic fungus] CBMAI 1031 AFP23847.1 99/97 nonreducing polyketide synthase gene, partial cds HM776143.1 (99/81) PS6-7 Botryospheria/ LC1/2c Mixed/ low Neofusicoccum N/A Neofusicoccum parvum UCRNP2 sp. putative polyketide synthase protein mRNA XM_007584296.1 (100/87)

Cladosporium sp. HDN13-337 isolate A2-1 polyketide synthase gene, partial cds KF986457.1 (99/78)

Scirrhia annulata ketosynthase-like gene, partial sequence DQ779557.1 (99/77)

Uncultured endophytic fungus clone TB1 putative polyketide synthase gene, partial cds JQ717040.1 (99/77)

PS6-31 Phomopsis sp. LC1/2c Low Dothiorella aegiceri Conserved domains putative polyketide on [lcl|29360] synthase gene, partial cds Nonreducing EU047579.1 polyketide synthase (100/93) [Diaporthe sp. CBMAI 1020], Diaporthe sp. ADY75774.1 CBMAI 1020 (98/99) nonreducing polyketide synthase Nonreducing

108 gene, partial cds polyketide synthase HM776154.1 (99/89) [Phomopsis sp. CBMAI 1019], Phomopsis sp. ADY75773.1 CBMAI 1019 (99/96) nonreducing polyketide synthase Polyketide synthase gene, partial cds [Diaporthe sp. HM776151.1 ASF2-3], (100/86) AIR77260.1 (99/96)

Phomopsis sp. Polyketide synthase FPSP-25 polyketide [fungal endophyte synthase (PKS) sp. CR38], gene, partial cds AAP68699.1 (99/95) JX839539.1 (78/91) PS2-6 Penicillium sp. LC1/2c Good Uncultured Conserved domains endophytic fungus on [lcl|17932] clone LJ1 putative polyketide synthase Polyketide synthase gene, partial cds [Penicillium steckii], JQ717038.1 (93/99) AHH02418.1 (99/94) Penicillium echinulatum isolate Polyketide synthase B1-4 polyketide [Penicillium synthase gene, brevissimum] partial cds AHH02433.1 KF986466.1 (93/83) (99/93)

Penicillium Polyketide synthase chrysogeum isolate [Penicillium K2-4 polyketide citrinum] synthase gene, AHI16969.1 (99/92) partial cds KF986452.1 (99/81) Nonreducing polyketide synthase Penicillium sp. ZJ01 [Penicillium putative WA type janthinellum] ketosynthase (KS) ADY75762.1 gene, partial cds (99/92) EF601570.1 (99/80) PS6-7 Botryospheria/ LC3/5c Mixed Neofusicoccum N/A Neofusicoccum parvum UCRNP2 sp. putative 6- methylsalicylic acid synthase protein mRNA XM_007585409.1 (100/76)

PS6-31 Phomopsis sp. LC3/5c Good/ slightly Phomopsis Conserved domains mixed liquidambaris strain on [lcl|5487] CBR polyketide synthase gene, Polyketide synthase partial cds [Phomopsis KM215685.1 (99/84) liquidambari] AIU47317.1 (38/97) Phaeosphaeria nodorum SN15 Putative MSAS-type hypothetical protein ketosynthase domain partial mRNA 2 [Phoma sp. C2932] XM_001791110.1 CAB44720.1 (38/89) (37/79) Putative 6- Phoma sp. C2932 methylsalicylic acid gene encoding synthase protein

109 putative [Neofusicoccum ketosynthase domain parvum UCRNP2] 2, partial XP_007585471.1 AJ132278.1 (37/75) (37/91)

Beta-ketoacyl synthase [Macrophomina phaseolina MS6] EKG12695.1 (37/86) PS2-19 Aspergillus sp. LC1/2c Good/slightly Aspergillus sydowii Conserved domains mixed ketoacyl synthase on [lcl|20198] gene, partial cds KF880427.2 Polyketide synthase (100/100) [Aspergillus nidulans] Aspergillus nidulans AHH02415.1 isolate C2-1 (100/100) polyketide synthase gene, partial cds Ketoacyl synthase KF986460.1 [Aspergillus sydowii] (100/100) AHL29081.1 (100/100) Aspergillus versicolor isolate Polyketide synthase C1-8 polyketide [Aspergillus synthase gene, versicolor] partial cds AHH02414.1 KF986459.1 100/100 (100/100)

4.3.2 Preliminary Crude Extract Anti-Microbial Assays

Endophyte broth cultures were extracted using ethyl acetate as the primary organic solvent. The dry extract weights varied between isolates. The variation of the extract weights varied (Table 12) from 3 mg for PS6-8 to 72.7 mg for PS2-2 over the same incubation time and conditions.

A total of 31 endophytes that possessed either a NRPS and/or PKS amplified gene were grown in liquid culture for testing their extracts for anti-microbial activity.

Anti-microbial activity assays revealed that almost all endophyte extracts that were tested showed some activity against at least one of the test indicator organisms.

However, 12 endophytes were not screened due to their very slow growth rates and hence low biomass production when grown in liquid culture, low to nil extract being produced, contamination of the extract, or some endophytes not growing in liquid broth at all. Therefore, a total of nineteen of the 31 identifiable endophytes that possessed

110 either a NRPS or PKS encoding gene were included in the crude extract screening along with 10 of the unidentifiable endophytes. A single endophyte, PS2-15, showed no activity although it had a amplified PKS-encoding gene.

The anti-microbial activity on the disc diffusion assay ranged from slight activity to strong activity for the crude extracts. Of the extracts tested against the test organisms, 19 showed activity against E. faecalis with PS2-19 being the most active, 21 extracts showed activity against E. coli, 12 extracts had activity against P. aeruginosa,

PS3-17 was the most active of 14 extracts active against S. aureus, 25 showed activity against B. subtilis, 13 showed activity against C. diphtheriae and 12 showed activity against Cryptococcus albidus. However, only 50% of the extracts were tested against C. albidus or C. diphtheriae due to repeated non-growth of C. albidus at the time of testing and the C. diphtheriae strain was not available from the culture collection during second round of testing.

Some endophytes (PS2-18, PS2-19, PS6-4, PS6-7, PS6-11, PS6-12 and PS6-31) showed activity against all test organisms. PS1-3, PS2-2, PS2-12, PS3-17, PS6-10, PS6-

32 and PS6-33 had activity against all but one of the test organisms. PS3-17 showed the highest level of activity against both S. aureus and B. subtilis, while PS2-2 and PS2-19 were the most active over the whole range of test organisms.

Table 12 summarises the endophytes that showed bioactivity against one or more test strains, in addition to possessing either a NRPS and/or a PKS gene. Following the table is a series of representitive electrophoresis gel photographs demonstrating the amplification of NRPS and PKS encoding genes.

111 Table 12 Endophyte NRPS and PKS Encoding Gene Screening and Bioactivity Results 5 Extract Test organisms Positive Endophyte1 Closest match2 weight3 NRPS4 PKS4 A B C D E F G control6

PS1-2^ n/a 37 - + c - - - - ++ + + ++++

PS1-3 Penicillium sp. 34 + a + c, d, e + + - + ++ + ++ ++++

PS1-6 Burkholderia sp. 23 + a + c - + - - ++ n/t n/t ++++

PS1-10 Bacillus sp. 6.7 + a - - + + - ++ - + ++++

PS1-11 Bacillus sp. 5.3 + a - + + - + ++ - + ++++

PS2-2 Penicillium sp. 72.7 - + c, d, e ++ ++ - ++ ++ n/t n/t ++++

PS2-9 Penicillium sp. 59.5 - + c, d, e - + - ++ +++ n/t n/t ++++

PS2-10^ n/a 4.8 - +c - - - - + n/t n/t ++++

PS2-12^ n/a 10.6 - +c + ++ ++ - ++ n/t n/t ++++

PS2-13^ n/a 3.3 - +c - - - - + n/t n/t ++++

PS2-15^ n/a 8.2 - +c - - - - - n/t n/t ++++

PS2-16^ n/a 11.5 - +c + - - - - n/t n/t ++++

PS2-17 Cladosporium/

Davidella sp. 7.3 - + c - - - - - + n/t ++++

PS2-18 Chrysoporthe sp. 10.6 - + c ++ + + + + n/t n/t ++++

PS2-19 Aspergillus sp. 24.6 - + c ++ ++ ++ ++ ++ n/t n/t ++++

PS3-17 Penicillium sp. 7.3 - +c, d, e + ++ - +++ +++ n/t n/t ++++

PS6-4 Botryosphaeria sp. 12.5 + b + c + + + ++ ++ n/t n/t ++++

PS6-6 Phomopsis sp. 14 - + d,e + + ++ - ++ ++ - ++++

PS6-7 Botryosphaeria sp. 15 + b + d + + + + ++ ++ + ++++

PS6-8^ n/a 3 + b + c, d, e + + - - ++ ++ + ++++

PS6-9^ n/a 4 - + d, e + + - - + ++ + ++++

PS6-10^ n/a 7 - + c, d, e + + + - ++ ++ + ++++

PS6-11 Colletotrichum sp. 7 - + c + + + + ++ ++ ++ ++++

PS6-12 Colletotrichum sp. 6 - + c + + ++ + ++ ++ + ++++

PS6-26 Phomopsis sp. 6 - + c, d, e - + - - + - - ++++

PS6-31 Phomopsis sp. 15 - + c, d, e + + + + ++ ++ ++ ++++

PS6-32^ n/a 9 - + c, d + + - + ++ ++ + ++++

112 PS6-33 Graphium sp. 4.9 - + d + - ++ ++ ++ n/t n/t ++++

PS7-2 Agrobacterium/

Rhizobium sp. 5 + a + c - - - - - + n/t ++++

1Endophyte identification (e.g. PS1-3 = Plant species 1 number 3) ^ indicates that no 18S rRNA gene sequence data was obtained due to no/low amplification but bioassays were conducted; 2Closest matched organism from BLAST search; 3Weight of extract in mg; 4NRPS and PKS primers that had successful amplification. Primers: a=MTF2/MTR2; b=RJ016F/RJ016R; c=DKF/DKR; d=LC1/LC2c; e=LC3/LC5c; 5Bioassay results (-) = No Activity, (+) = 0-4mm, (++) = 4-6mm, (+++) = 6-8mm, (++++) = >8mm; (All measurements taken from centre of disk to the outermost edge of the clearing zone); Test organisms A. E. faecalis, B. E. coli, C. P. aeruginosa, D. S. aureus, E. B. subtilis, F. C .diphtheriae, G. Cryptococcus albidus. n/t= Not tested. Negative controls (DMSO) were all negative.6 Ampicillin (100 µg/mL) and cycloheximide (100 µg/mL) were used as a positive control for the bacterial and fungal assay, respectively.

From the results of the crude extract bioactivity assays, PS1-3, PS2-2, PS2-19,

PS6-7 and PS6-31 were chosen as candidates for further compound isolation and

chemical characterisation (Chapter 5). These endophytes were chosen due to the

presences of PKS or NRPS encoding genes, had broad range of activity against most to

all test organisms and were readily culturable in liquid broth.

4.4 DISCUSSION

4.4.1 Screening for NRPS and PKS Encoding Genes

The use of ethnobotanical information coupled with the directed genetic

screening for potential bioactive compounds from plants or endophytes is becoming a

more frequently reported topic of research in the search for novel pharmaceutically

important compounds [203, 240, 273].

NRPS/PKS genes were chosen as an indictor for bioactivity and to de-replicate

and narrow the search for the candidate endophytes that may produce bioactive

compounds. Sequence data for NRPS and PKS screening the majority of the time

returned a mixed and or low signal data. Although mixed, BLAST database

comparisons using the BLASTn tool were still carried out. Sequences that returned no

significant matches, due to mixed or low signal, were noted for ongoin work but

113 assumed positive for further compound selection. However, some mixed sequences returned database matches to NRPS and PKS genes ranging from 16/74% low similarity/query coverage respectively, (PS1-3 PKS) to 100% query similarity and query coverage (PS2-19 PKS).

The fungal NRPS and PKS PCR screening positive control was PS2-6. The fungal endophyte was identified as a Penicillium sp. and amplified correct band sizes for both NRPS and PKS encoding genes for all primers used during PCR screening.

However, sequencing always returned a mixed/low signal and was unable to get a positive BLAST ID for the sequence. NRPS and PKS amplification may amplify multiple, sometimes 20-30 or more sequences at a time giving mixed sequences. The signal returned from the sequencing was so low and mixed gave very low coverage and similarity to anything and thus BLAST results for all sequenced amplicons were not included.

This study revealed that 31of the 48 identified endophytes amplified either a

NRPS or PKS encoding gene. Of these, six endophytes possessed both NRPS and PKS encoding genes. In addition, ten endophytes that were not identified in Chapter 3 also amplified a NRPS or PKS encoding genes.

It is widely reported that NRPS and PKS are responsible for the production of many bioactive compounds used today [274]. Using NRPS and PKS screening as a basis for bioactivity was demonstrated in the research of 8 traditional Chinese medicine

(TCM) plants from Yunnan Province, China [203]. It was found that 74 bacteria and 36 fungal isolates were cultured and screened for NRPS/PKS encoding genes as well as the crude extract bioactivity. Seventeen endophytes with an NRPS and/or PKS positive amplicons were tested against a range of bacterial, fungal and myeloma cell line RPMI-

8226. Although nearly all extracts showed activity in some form, endophytes F53 and

114 F48 were identified as potential anti-cancer compound producing candidates, with isolate B42 having very potent anti-bacterial properties.

Another study that searched for bioactive compounds using NRPS and PKS gene screening as a guide, targeted endophytic actinomycetes from the rainforest of

Yunnan province, China. All bar one of the 41 tested endophytes showed activity against at least one of the cytotoxicity test cell lines and all endophyte extracts showed anti-microbial activity against at least one of the five test strains [273].

In this study, a PKS fragment was amplified in PS2-15; however, the extract showed no activity against the test organisms. Similarly, Zhao et al. (2011), used these methods to study the endophytes of the TCM plants in Panxi Plateau, China [241]. This study found 56 out of 60 endophytes had at least one NRPS or PKS encoding gene present, and 4 endophytes that showed anti-microbial activity did not possess

NRPS/PKS encoding genes. Similarly to PS2-15, the isolated endophyte SAUK6023 possessed both NPRS and PKS encoding genes but showed no activity against the tested strains.

Qin et al. (2009) reported the isolation, diversity and anti-microbial activity of endophytes isolated from TCM found in the rainforests of Xishuangbanna, China [275].

Forty-six actinomycete strains were selected and screened for NRPS and PKS encoding genes. It was found that just over half of the selected endophytes showed activity against at least one of the test organisms in the bioactivity assays, only 26.1% had amplified a NRPS gene and 10.9% and 15.2% amplifiying a PKS-I and II, respectively, showing no direct correlation between genetic make-up and activity in that study.

The endophytes found to posses a NRPS or PKS gene but produce extracts with no bioactivity could be due to a number of reasons, such as the gene codes for a pigment, the active compound becoming unstable during the extraction or storage

115 conditions or the NRPS or PKS encoding genes being down-regulated or non functional in the particular conditions used for the laboratory culturing of the isolates. It may also be that the endophyte may rely on the host plant for intermediate substrates or otherwise participate in a step of metabolite production. As reported by Kasuri et al. (2011), an isolated endophyte Fusarium solani, capable of producing camptothecin (CPT), a monoterpenoid indole alkaloid, has decreased production after repeated subculturing. It was shown that the endophyte relies on the plant to complete the biosynthesis of CPT by providing a crucial enzymatic step in the reaction [276].

4.4.1.1 Other Biosynthetic Pathways

This study prioritised the screening of NRPS and PKS encoding genes as indicators for bioactivity, as most antibiotics are produced via NRPS, PKS or hybrids of these biosynthetic pathways (Chapter 1, section 1.9). Endophytic bacteria and fungi have indeed been shown to produce different classes of natural products, as reviewed in

Zhang, et al, 2006 [277] and Keller et al, 2005 [278], independent of NRPS and PKS biosynthetic pathways. These include alkaloids such as indoles, pyrrolizidines, quinazolines ergot and quinolones, mono-, sesqui- and diterpene and derivatives, fatty acid and triacylglycerols, isocoumarin derivatives, phenylpropanoids and lignans, and also including ribosomal peptides such as bacteriocins [279, 280], that are all synthesised via a myriad of biosynthetic pathways.

There have been over 55 000 terpene compounds reported in the literature from all kingdoms of life [281]. Terpenes are produced via two main pathways: mevalonate pathway in all terpene producing organisms and the mevalonate independent pathway in only bacteria, plants, algae and protozoa (reviewed by Eisenreich etal, 2000 [282]).

Generally, fungal terpenes are synthesised via the mevalonate pathway producing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as the starting

116 molecules [283]. As reviewed in Wang and Ohnuma, 2001 [284], the IPP is attached to the DMAPP and is followed by additions of isoprene molecules, by specific isoprenyl diphosphate synthases to produce linear pyrophosphate molecules that are the building blocks for terpenes, such as geranyl diphosphate (GPP; monoterpenes C10), farnesyl diphosphate (FPP; sesquiterpene C15), geranylgeranyl diphosphate (GGPP; diterpene

C20) and geranylfarnesyl diphosphate (GFPP; sesterterpene C25). The linear pyrophosphate molecules are subjected to further cyclisation reactions by terpene synthases as well as further modifications such as oxidation, methylation, protein/amino acid addition or oxygen substitution, to produce a diverse range of terpene or terpenoid compounds [285, 286]. Examples of terpene compounds produced by fungi include diterpene taxol [81, 82, 287], bisabolane-type sesquiterpenoids sydonic acid and derivatives [288-290], diterpene gibberellins [291] and the indole diterpene paxilline

[292]. Despite their abundance, the genetics underpinning the biosynthesis is less understood compared with NRPS and PKS clusters.

As reported in chapter 5, some examples of the natural product classes mentioned above were isolated from the endophytes in this study. These classes of compounds were isolated and show activity against the test organisms (Table 12). This indicated that even though NRPS and PKS encoding genes are targeted in order to identify potentially bioactive producers, active compounds from other classes or compounds can still be identified.

4.4.2 Taxonomy of Bioactive Endophytes

In this study, the endophyte was chosen for upscaled fermentations (for Chapter

5) based on 1. the presence of targeted biosynthetic genes, 2. being the most active strain over a broad range of test organisms and 3. being capable of growing and producing enough extract in liquid culture for further tests. The most active extracts

117 tested were from PS1-3, PS2-2, PS2-19, PS6-7 and PS6-31, and these were taxonomically closest matched to two Penicillium spp., one Aspergillus sp., one

Botryosphaeria sp. and a Phomopsis sp., respectively.

4.4.3 Screening of Endophytic Penicillium Species

Endophytes PS1-3 and PS2-2 phylogenetically grouped with Penicillium sp.

PS1-3 amplified both PKS and NRPS encoding genes and showed activity against all test organisms but P. aeruginosa, with greatest activity against B. subtilis and C. albidus. PS2-2 had PKS encoding genes amplified and showed activity against E. faecalis, E. coli, S. aureus and B. subtilis. Historically, Penicillium sp. are known to produce a number of important compounds including the first pure-isolated and mass- produced antibiotic, penicillin. Endophytic Penicillium spp. have been isolated from numerous plants and have been the source of a number of bioactive and industrially relevant compounds. These include the unique quinazoline alkaloid extracted from a

Penicillium endophyte of Crocus sativus, displaying cytotoxic and anti-fungal properties [228], and an endophytic Penicillium glandicola isolated from Opuntia ficus- indica Mill that showed strong pectinase active with potential for biotechnological applications [293]. Another study reported that two Penicillium sp., from the Brazilian plant Alibertia macrophylla, produced three novel compounds each. The compounds, identified as orcinal and 4-hydroxymellein, displayed anti-fungal activity while a separate compound, Cyclo (L-Pro-L-Val), showed acetylcholinesterase (AChE) inhibitory activity that has the potential for Alzheimer’s therapy [294]. Lastly, the endophyte Penicillium expansum 091006 isolated from Excoecaria agallocha

(Euphorbiaceae) in Wenchang, Hainan, China, produces 4 polypenolic compounds with

Exansol A and B both showing cytotoxic activity against HL-60 cell line with Exansol

B also showing proliferation of A549 [295].

118

4.4.4 Screening of Endophytic Botryosphaeria and Neofusicoccum Species

Endophyte PS6-7 phylogenetically grouped to Neofusicoccum and

Botryosphaeria sp. which are both in the Botryosphaeriaceae [296]. They are commonly associated with plant diseases; however their natural products also possess bioactive properties. PS6-7 was one of four endophytes that amplified a NRPS encoding gene using the fungal specific primers. It also amplified a PKS encoding gene and had broad range activity against both bacterial and fungal test oragnisms showing greatest activity against B. subtilis and C. diphtheriae.

An endophytic Botryosphaeria isolated from Vitus quinquangularis produced

Resveratrol (3,5,4’-trihydroxystilbene), a compound known for slowing and preventing numerous diseases [297]. The crude extracts from Botryosphaeria rhodina isolated from the Indian medicinal plant Tinospora cordifolia exhibited strong anti-bacterial activity comparable to the drug ciprofloxacin [298], while Botryosphaeria sp. M76 isolated from the medicinal plant Garcinia mangostana in southern Thailand showed anti-bacterial activity against S. aureus and MRSA S. aureus, and anti-fungal activity against Microsporum gypseum [299].

4.4.5 Screening of Endophytic Phomopsis Species

Endophyte PS6-31 was was identified as a Phomopsis sp. Phomopsis are common endophytic fungi with numerous reports of isolation and bioactivity from different plant sources. PS6-31 had amplified PKS encoding genes and showed broad activity against all bacterial and fungal test organisms. Other examples of bioactive compounds isolated from endophytic Phomopsis include the novel phytotoxic nonenolide, (6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone, from endophytic

Phomopsis sp. HCCB03520 [300]. Isolation of pinoresinol diglucoside (PDG), a stable

119 microbial anti-hypertensive compound, was also achieved from an endophytic

Phomopsis sp. from the TCM plant Eucommia ulmoides Oliv. [301]. A mixture of volatile organic compounds (VOCs) was detected from an endophytic Phomopsis sp. isolate from Odontoglossum sp. in a cloud forest in Northern Ecuador. These VOCs showed a range of anti-fungal activity against plant pathogen organisms as well as being a source for fuel production [301]. Four new compounds, named pyrenocines J–M, have been isolated from a Phomopsis sp. within the plant Cistus salviifolius. Pyrenocines J,

K and M showed strong anti-fungal, anti-bacterial and algicidal properties with pyrenocines L exhibiting anti-bacterial and algicidal activity [302]. Lastly, the isolation of a new slightly cytotoxic polyketide (2-(7′-hydroxyoxooctyl)-3-hydroxy-5- methoxybenzeneacetic acid ethyl ester) was reported from an endophytic Phomopsis sp. isolated from the mango tree, Excoecaria agallocha, from Dong Zai, Hainan, China

[303].

4.4.6 Screening of Endophytic Aspergillus Species

Endophyte PS2-19 was identified as Aspergillus sp. PS2-19 had an amplified

PKS encoding gene and had the most active broad range anti-bacterial activity against all test organisms. Examples of Aspergillus sp. as endophytes producing bioactive compounds include thirty-nine compounds that were produced from one endophytic

Aspergillus fumigatus LN-4, isolated from the bark of Melia azedarach. Eight indole diketopiperazine alkaloids, five fumiquinazolines, tryptoquivaline O, a 4,8-dihydroxy-1- tetralone and the nordammarane triterpenoid helvolic acid from LN-4 all showed anti- fungal activity against a range of plant pathogens. Other isolated compounds from this endophyte also showed activity in the brine shrimp toxicity assay and anti-feeding activity against army worms [304]. An endophytic Aspergillus fumigatus isolated from

Juniperus communis L. Horstmann produced deoxypodophyllotoxin which showed

120 activity against S. aureus subsp. aureus, K. pneumoniae subsp. ozaenae and P. aeruginosa [259]. Aspergillus niger isolated from Roccella montagnei produced chitosan, and exhibited anti-bacterial activity against V. cholera, S. aureus and E. coli

[305].

4.4.7 Rationale for Selection of Endophytes for Up-scaled Fermentation

Five endophytes were chosen for up-scale fermentation, described in Chapter 5 section 5.2.1. In addition to amplification of a NRPS or PKS encoding gene, PS2-19,

PS6-7 and PS6-31 were chosen because they exhibit activity against all test organisms.

PS6-7 also amplified both fungal-specific NRPS and a PKS encoding genes. PS1-3 amplified both NRPS and PKS encoding genes and along with PS2-2, was selected because they also showed activity on all but one of the test organisms. The literature presented above strongly suggested and supported the decision for up-scaling and further compound isolation of the selected endophyte isolates in this study since they have all been shown to produce bioactive or biotechnologically important compounds when isolated as endophytes. It also indicates that endophytes isolated in this study have the potential to produce natural products with activity in addition to anti-bacterial and anti-fungal activity and is a viable option for future investigations.

Endophytes PS2-12, PS2-18, PS3-17, PS6-4, PS6-10, PS6-11, PS6-12, PS6-32 and PS6-33, which produced bioactive extract, were not pursued further because of low biomass or slow growth rates in the 100 mL cultures, difficulty growing in the larger volume or no growth when up-scaled cultures were seeded. This included PS3-17, which showed the highest activity of all extracts against S. aureus and B. subtilis; however, upscale liquid culturing conditions did not suit biomass growth. The selected strains took priority due to time constraints and the limited number of strains that could be analysed. These endophytes showed potential for bioactive compound production

121 and will be studied further, possibly using heterologous expression of their biosynthetic gene clusters. Another six endophytes, which were excluded from the results table, also showed slight activity against test organisms; however, these did not test positive for

PKS or NRPS encoding genes. This may indicate production of the bioactive compounds via other biosynthetic pathways. These endophytes were not within the scope of the NRPS and PKS screening aims of this study and were not examined further.

4.5 Conclusion

In summary, amplification of a NRPS and PKS encoding gene was successful in thirty-one of the forty-eight identifiable endophytes. Endophyte DNA from strains PS1-

3, PS2-2 and PS6-31 were amplified for the PKS encoding genes using all three PKS primer pairs. This indicates that these endophytes contain a both reducing and nonreducing PKS-encoding genes that has the potential to produce a number of bioactive compounds.

The crude extracts of all but one of the endophyte extracts tested showed activity against at least 1 test organism. PS1-3, PS2-2, PS2-19, PS6-7 and PS6-31 were chosen for up-scaled growth, further bioactivity testing and compound isolation and identification.

The five endophytes chosen for up-scaled growth for compound isolation, purification and identification were done so based on the amplification of PKS or NRPS encoding genes being present and the extract being biological activity against a range of test organisms.

These five endophytes grouped phylogenetically with Penicillium, Aspergillus,

Botryosphaeria and Phomopsis. Literature reveals that endophytes in these genera can

122 produce bioactive, biotechnologically, or pharmaceutically valuable compounds and are therefore prime candidates for further investigation.

In the next chapter the five nominated endophytes extracts will be examined using silica column fractionation, MPLC, HPLC, TLC, NMR and mass spectrometry in an effort to isolate and structurally characterise the bioactive constituents of these endophyte extracts.

123 5 Chapter 5 Investigation into the Bioactivity of Natural Products

Isolated from Endophytic Penicillium, Phomopsis,

Neofusicoccum/Botryospheria and Aspergillus species

5.1 Introduction

Fungi are the most frequently isolated plant endophytes [177]. It is estimated that every plant species on Earth has at least one endophytic microorganism residing within its tissue. Since 1991, it has been accepted that there are approximately 1.5 million fungal species on earth [306]; however recent estimations have been presented at approximately 5.1 million species [307]. Fungal derived natural products include alkaloids such as the potent anti-neoplastic, pentacyclic quinoline alkaloid camptothecin from the endophyte Camptotheca acuminata [308]; the anti-phytopathogenic fungal alkaloid, 12β-hydroxy-13α-methoxyverruculogen TR-2, isolated from endophytic

Aspergillus fumigatus [304] and the cytotoxic compounds camptothecine, 9-methoxy

CPT (9-MeO-CPT) and 10-hydroxy CPT (10- OH-CPT) produced by the three endophytes, Fomitopsis sp. P. Karst, Alternaria alternata (Fr.) Keissl and Phomopsis sp. (SACC.) [309]. Other compounds produced by fungi, classed as terpenoids include sesquiterpenes from endophytic Trichoderma atroviride (anti-inflammatory) [310],

Biscogniauxia nummularia (anti-germinating) [311], Myrothecium roridum (cytotoxic)

[312], and Xylaria sp. BL321 (α-glucosidase inhibitor) [313]; diterpenoids from endophytic Fusarium oxysporum (potent antagonist effects on platelet activating factors) [314], Aspergillus sp. YXf3 (neuraminidase inhibitory activity) [315],and various taxol-producing endophytes (anti cancer) [81, 316-319]; triterpenoids from endophytic Pichia guilliermondii Ppf9 (anti-bacterial and anti-germinative) [320],

Eupenicillium parvum (anti-feeding on insects and anti-malarial) [90] and Fusarium sp.,

124 Aspergillus sp. (anti-bacterial and anti-phytopathogenic fungi) [200]; and phenolic compounds isolated from Botryosphaeria obtusa (phytotoxin) [321].

Two other pharmacologically important classes of fungal natural products are non-ribosomal peptides (NRPs) and polyketides (PKs). Fungal natural products that are

NRPs include the anti-bacterial β-lactam antibiotics penicillin and cephalosporin [251], the anti-tumour, anti-malarial and anti-insecticidal efrapeptins [322] and the immunosuppressive compound cyclosporine A [323]. PKs are the most abundant class of fungal secondary metabolites produced [278]. Fungal PK compounds include the anti-cholesterol compound lovastatin [145] , antimicrobial and cytotoxic Paeciloside A

[324], 1,8 dihydroxynaphthalene (DHN)-melanin-type compound [325], and the anti- bacterial and cytotoxic hybrid PKs and NRPs compound, chaetoglobosin A [326].

Endophytic Penicillium, Aspergillus, Neofusicoccum sp. and Botryosphaeria sp. are frequently isolated from a variety of plants. Examples of natural products that they produce as endophytes are the unique cytotoxic and anti-fungal quinazoline alkaloid, (-

)-(1R,4R)-1,4-(2,3)-indolmethane-1-methyl-2,4-dihydro-1H-pyrazino-[2,1-b]- quinazoline-3,6-dione, two new enzyme inhibitory p-terphenyls and a cytotoxic compound citriquinochroman, all isolated from various endophytic Penicillium sp. [228,

327, 328]. The anti-bacterial compounds fumigaclavine C and pseurotin A and cytotoxic furandiones, named asperterone B and C isolated from Aspergillus, [329, 330] and a phytotoxic cyclobotryoxide have all been extracted from an endophytic

Neofusicoccum australe [331].

Discussed here are the results of a chemistry based investigation. This is the first ever examination of natural products produced by endophytic Penicillium,

Neofusicoccum/Botryospheria and Aspergillius isolated from specific medicinal plants of Botany Bay. Positive genetic screening for PKS and NRPS encoding genes and

125 testing of crude extracts of the selected endophytes have shown activity in bioassays against at least one test organism (Chapter 4).

Figure has been removed due to Copyright restrictions.

Figure 30 Examples of natural products produced by fungi. (1) Penicillin and cephalosporin [332]; (2) Cyclosporine A [333]; (3) Lovastatin [334]; (4) a. Camptothecin, b.10-hydroxycamptothecin, c. 9-methoxycamptothecin and d. 10- methoxycamptothecin [335]; (5) Taxol [336].

126 5.2 Materials and Methods

5.2.1 Culturing and Extract Isolation

Cores of agar containing actively growing biomass were removed from plates and placed into 3 x 2L flasks containing 400mL of potato dextrose broth (BD Difco).

The flasks were incubated at room temperature with occasional agitation approximately every 12 h for a minimum of 21 days. Biomass was streaked onto PDA plates to confirm that the fungi were still growing in pure culture. The fungal biomass and broth were extracted with an equal volume of ethyl acetate and shaken overnight. The biomass was filtered off and the organic layer separated from the aqueous layer. The organic layer was treated with anhydrous sodium sulphate to remove any residual moisture. The solvent was removed under vacuum reduced pressure.

5.2.2 Thin Layer Chromatography

The extracts were analysed by Thin Layer Chromatography (TLC) on silica 60

WF245s (Merek) using a mobile phase of dichloromethane:methanol 95:5 (v/v).

Separation on TLC plate was visualised under UV light at both long and short wavelengths and were further developed by charring in cerium sulphate, vanillin or p- anisaldhyde/sulphuric acid.

5.2.3 Fractionation and Compound Isolation from Endophytic Penicillium (PS1-

The crude extract recovered from an endophytic Penicillium from plant species

1, named PS1-3, was first fractionated on Medium Pressure Liquid Chromatography

(MPLC). The sample was dissolved in dichloromethane:methanol 95:5 (v/v) and loaded onto the RevelerisTM flash chromatography system (Grace). A Reveleris® silica 4g column was equilibrated prior to loading of the sample. A 2 mL injection containing the

127 extract was loaded and separated using a step-wise gradient elution from dichloromethane (DCM) to methanol at a flow rate of 18 mL/min. The solvent gradient used was as follows; initially 1% methanol in DCM then ramped up to 6% over 2.8 min, then held for 2.1 min, then ramped up to 100% as a wash step. Fractions were collected based on UV absorbance peaks and time intervals (Figure 31).

5.2.4 Fractionation and Compound Isolation of PS1-3 Fractions

Fractions eluting at 36, 42 and 54 s were further fractionated on a silica gel column. The SPE silica gel column was packed in ethyl acetate, and the column was equilibrated with 100% hexane and eluted under gravity. The sample was added to the top of the column and the solvent gradient run through the column using the ratios of

100% hexane, 90% hexane: 10% ethyl acetate, 80% hexane: 20% ethyl acetate, 50% hexane: 50% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate: 50% methanol and

100% methanol (Figure 31).

5.2.5 Fractionation and Compound Isolation from Penicillium (PS2-2),

Aspergillus (PS2-19), Phomopsis (PS6-7) and Neofusicoccum/Botryospheria

(PS6-31)

All other large-scale extracts were initially fractionated on LH20 sephadex column. The LH20 column was prepared by immersing the LH20 into analytical grade methanol, the column was packed and then equilibrated with a methanol and DCM mixture at a ratio of 4:1 respectively. The sample was loaded to the top of the column and was gravity fed through the column using the same mixture and ratio of methanol and DCM as the mobile phase. Any separation into “visible bands” on the column were collected into test tubes and later transferred to pre-weighed scintillation vials (Figure

32, Figure 33 and Figure 34).

128

5.2.6 High Pressure Liquid Chromatography (HPLC)

All analytical and semi preparative HPLC was performed using a HP 1100 series equipped with a 1100 series diode array detector and a 1046A fluorescence detector (Hewlett Packard, USA) to detect UV fluorescence at 254 and 280nm. The solvent system was run at 5% acetonitrile :95 % water with 0.5% trifluoroacetic acid

(TFA) for 10 min, from 5% acetonitrile :95 % water with 0.5% TFA to 95% acetonitrile:5% water with 0.5% TFA for 30 min and then 100% acetonitrile for 5 min.

To optimise the solvent system conditions, the analytical HPLC was run first using a

Zorbax SB-C18 (4.6 x 250 mm, 5µm) reversed phase analytical column (Agilent

Technologies) for separation of the compounds at a flow rate of 1mL/min.

Under optimised conditions, the semi-preparative run was carried out using a

Discovery BIO Wide Pore C18, (10 x 250 mm, 5 µm; Supelco), reversed phase column at a flow rate of 3.5mL/min with the same solvent conditions (5% acetonitrile :95 % water with 0.5% TFA for 10 min, from 5% acetonitrile :95 % water with 0.5% TFA to

95% acetonitrile:5% water with 0.5% TFA for 30 min and then 100% acetonitrile for 5 min). UV detection was at 254 and 280nm.

5.2.7 NMR and MS Analysis

Proton-nuclear magnetic resonance (1H-NMR), carbon nuclear magnetic resonance (13C-NMR), and two-dimensional NMR correlation spectra (1H−1H COSY, and 1H−13C HSQC and HMBC) were recorded on a Bruker Tesla 600 MHz instrument

(operating at 600 MHz for 1H and 150 MHz for 13C) in either deuterated chloroform

(CDCl3), deuterated methanol (CD3OD) or deuterated dimethylsulfoxide (DMSO-d6).

Spectra were referenced to all solvents that were used in the sample preparation. The mass spectrometry data were obtained using a Thermo Scientific LTQ FT ULTRA

129 instrument, operating in negative and positive electrospray ionisation modes. This was carried out at the Bioanalytical Mass Spectrometry Facility (BMSF) at the Mark

Wainwright Analytical Centre, UNSW. NMR data was analysised and compound structures determined, by Dr John Kalaitzis, a natural product chemist within the lab, as well as by Ingrey under the supervision and assistance of Dr Kalaitzis.

5.2.8 Anti-bacterial and Anti-fungal Activity Bioassay

Bioassays were conducted using seven commonly used test microorganisms to determine the anti-bacterial and anti-fungal activities of the selected endophyte extracts from Chapter 4. The extracts were tested against the Gram-negative bacteria,

Escherichia coli NCTC 10418 and Pseudomonas aeruginosa NCTC 10490; the Gram- positive bacteria, Staphylococcus aureus NCTC 6571, Bacillus subtilis NCTC 10418 and Corynebacterium diphtheriae (UNSW# 046300); and the fungal strain Candida albicans ATCC 10231. The bacterial test strains were aseptically transferred to 10 mL of nutrient broth (Sigma) and incubated at 30°C (C. diphtheriae and B. subtilis) or 37°C

(E. coli, S. aureus and P. aeruginosa). The fungal test strain was grown in Malt Extract

Broth (Bacto Laboratories) at 30°C. The cultures were incubated until exponential growth phase was reached at an optical density between 0.100-0.600nm @ 625 nm. The concentration of cells was adjusted using nutrient broth to an OD625nm between 0.080 and 0.100 nm. Fifty microliters of the prepared cells were seeded into a sterile flat- bottomed 96-well microtiter plate as well as an equal volume of the prepared endophyte extract. The extracts were prepared to give a concentration of 200 µg/mL and concentration of 20 µg/mL to give final concentrations in the wells of 100 µg/ml and 10

µg/ml respectively. Bacterial microtiter plates were incubated for 24 h and fungal plates for 48 h, at the appropriate temperature. The optical density was then read at 625 nm using a SpectraMax 340 plate reader (Molecular Devices). The controls included in the

130 bioassay were media only and either ciprofloxacin (20 µg/ml; Sigma-Aldrich), or nystatin (50 µg/ml; Invitrogen) as a positive control in bacterial or fungal assays, respectively. Each experiment was performed in triplicate with controls on each microtiter plate.

The calculation of the cell growth inhibition was as follows

%CGI=[1-(A600test/A600cont)]x100

Cell growth inhibition as a percent absorbance of test sample divided by the absorbance of control sample at 600 nm. Standard error of the mean (SEM) was calculated with n=3.

5.3 Results

5.3.1 Isolation of Compounds from Endophytic Penicillium (PS1-3)

Penicillium (PS1-3) ethyl acetate extract (609 mg) was fractionated using

Medium Pressure Liquid Chromatography (MPLC) yielding nine fractions.

Visualisation of the fractions by TLC gave a total of seven fractions after combining identical fractions. Fraction 3, 7 and 8 showed activity against all preliminary bacterial test organisms (Chapter 4 section 4.2.4).

Fraction 3 was fractionated on a silica column and twenty-three fractions were collected. One fraction was collected from the mobile phase of 95% hexane:5%ethyl acetate, one fraction from 90% hexane:10% ethyl acetate, seven from 80% hexane: 20% ethyl acetate (SDI-04, SDI-74 to 78 SDI-05), 6 from 50% hexane:50%ethyl acetate

(SDI-79, SDI-04 and SDI-03), one from 100% ethyl acetate (fractioned by HPLC), five from 50% ethyl acetate:50% methanol (SDI-80 to 84) and one fraction from the 100% methanol wash. This fractionation is summarised in Figure 31. 1H NMR was acquired for all fractions to determine purity and as a basis for preliminary compound structure elucidation and prioritisation for further work. SDI-81 was subjected to HPLC for

131 further purification and fourteen peaks were collected. Five of these were selected to run 1H NMR and two compounds (SDI-139 and SDI-141) were judged to be sufficiently pure for further analysis. Mass spectrometry and 2D NMR data were collected for these, together with SDI-05 for structure elucidation.

The 100% ethyl acetate fraction was purified by HPLC to give five further compounds (SDI-3 to 7). 1H NMR was carried out on all compounds; however insufficient material was recovered for 2D NMR experimentation and bioassay studies.

3;<=5%>)?;(;?% ?@;'()*+,% !"#$%!"#$% !"#$% !"#$%&'()*+,(*+,%-.%/'()*+,01% &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-.9. &'()*+,-. &'()*+,-.#/0. &'()*+,-.%/1. &'()*+,-.6/7. &'()*+,-.9. &'()*+,-.8/2. #/0. %/1. 6/7. &'()*+,-.9. 8/2. A#$BC4+(00(=% #/0. %/1. 6/7. 8/2. D64E?E%0?5?)*+,%

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

&'()*+,-. &'()*+,-. Figure&'()*+,-.2. 2 Isolation and purification of extracts obtained from PS1-3! #7. "34$#%2.#8. "34$#1#. Figure 31 Isolation and purification of extracts obtained from PS1-3 ! ! &'()*+,-. "#$%&'()! "34$56. ! &'()*+,-.9. &'()*+,-#5. "#$%&'()!*!#$6. 5.3.2Isolation Isolation of !compounds of Compounds from endophytic from an EAspergillusndophytic ( PS2Aspergillus-19) (PS2-19) ! +, ! AspergillusAspergillus (PS2 (PS2-19)-19) ethylethyl"34$#%2. acetate extractsextracts"34$#1#. (172 (172 mg) mg) was was extracted extracted from from three three 2 !

L flasks.2 L flasks The. The extracts extracts recovered recovered from from PS2 PS2-19-19 were were fractionated fractionated on aa sizesize exclusion exclusion LH-

20LH column,-20 column resulting, resulting in twelve in twelve fractions. fractions. Fractions Fractions were were combined combined on on the the basis basis of of TLC visualisationTLC visualisation to give to a givetotal a of total eight of eightfractions fractions (SDI (SDI-63 to-63 67, to 67,SDI SDI-58,-58, 69 69and and 72). 72). SDI - SDI-63, SDI-66 and SDI-69 were all determined to be sufficiently pure, by 1H NMR, 63, SDI-66 and SDI-69 were all determined to be sufficiently pure, by 1H NMR, for for further mass spectrometry and 2D NMR analysis. further mass spectrometry and 2D NMR analysis.

132

!"#$%& 5678(%9):6*6:% :;60*)1+.%

'()*+,-./ !"#$%&'(')*%)+(,-.%/0*)1+.*1+.%%'()*+,-./ '()*+,-./%/ '()*+,-./5/!"#$%& '()*+,-./7/ '()*+,-./8/ #43/ 23%/0*)1+.&4%642/ '()*+,-./ '()*+,-./ '()*+,-./%/ '()*+,-./5/ '()*+,-./7/ '()*+,-./8/ ,'?:?% "01$23/ #43/ "01$22/ 642/ "01$2&/ &:(:)1+.%

"01$23/ "01$22/ "01$2&/ @"%ABC%>,'?:?% &:(:)1+.%

! "#! 3;<=5%>)?;(;?% ?@;'()*+,% !"#$%!"#$% !"#$% !"#$%&'()*+,(*+,%-.%/'()*+,01% &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-.9. &'()*+,-. &'()*+,-.#/0. &'()*+,-.%/1. &'()*+,-.6/7. &'()*+,-.9. &'()*+,-.8/2. #/0. %/1. 6/7. &'()*+,-.9. 8/2. A#$BC4+(00(=% #/0. %/1. 6/7. 8/2. D64E?E%0?5?)*+,%

&'()*+,-. &'()*+,-. Figure&'()*+,-.2. 2 Isolation and purification of extracts obtained from PS1-3! #7. "34$#%2.#8. "34$#1#. ! ! &'()*+,-. "#$%&'()! "34$56. ! &'()*+,-.9. &'()*+,-#5. "#$%&'()!*!#$6. Isolation of !compounds from endophytic Aspergillus (PS2-19) ! +, ! Aspergillus (PS2-19) ethyl"34$#%2. acetate extracts"34$#1#. (172 mg) was extracted from three !

2 L flasks. The extracts recovered from PS2-19 were fractionated on a size exclusion

LH-20 column, resulting in twelve fractions. Fractions were combined on the basis of

TLC visualisation to give a total of eight fractions (SDI-63 to 67, SDI-58, 69 and 72).

SDI-63, SDI-66 and SDI-69 were all determined to be sufficiently pure, by 1H NMR,

for further mass spectrometry and 2D NMR analysis.

!"#$%& 5678(%9):6*6:% :;60*)1+.%

"01$23/ "01$22/ "01$2&/ @"%ABC%>,'?:?% &:(:)1+.%

Figure 32 Isolation and purification of extracts from PS2-19 ! "#!

5.3.3 Isolation of Compounds from an Endophytic Phomopsis (PS6-7)

Phomopsis (PS6-7) ethyl acetate extract was obtained in two separate collections from two 2 L flasks yielding 63 and 167 mg. The extracts recovered from PS6-7 were fractionated on a size exclusion LH-20 column and resulted in six fractions. Fractions were combined on the basis of TLC visualisation to give a final total of five fractions

(SDI-41 to 44 and SDI-47). SDI-42 and SDI-47 were subjected to HPLC with eight peaks collected for each. Fraction 7 of SDI-47 was further purified by HPLC and yielded SDI-100 as a white precipitate. All fractions collected from SDI-42 were recovered at yields <1mg. These fractions were not considered for further NMR determination at this time.

133 Figure 3 Isolation and purification of extracts from PS2-19

Isolation of compounds from endophyte Phomopsis (PS6-7)

Phomopsis (PS6-7) ethyl acetate extract was obtained in two separate

collections from two 2 L flasks yielding 63 and 167 mg. The extracts recovered from

PS6-7 were fractionated on a size exclusion LH-20 column and resulted in six

fractions. Fractions were combined on the basis of TLC visualisation to give a final

total of five fractions (SDI-41 to 44 and SDI-47). SDI-42 and SDI-47 were subjected

to HPLC with eight peaks collected for each. Fraction 7 of SDI-47 was further

purified by HPLC and yielded SDI-100 as a white precipitate. All fractions collected

from SDI-42 were recovered at yields <1mg. These fractions were no longer

considered for further NMR determination at this time.

6789(%:);7*7;% !"#$% ;<70*)1+.%

!"#$%&'(')*%)+(,-.%/0*)1+.*1+.%%!"#$% &'()*+,-./01. 23%/0*)1+.&4% &'()*+,-.8. &'()*+,-.5. &'()*+,-.90#. 2"34$5%6. &'()*+,-./01. &'()*+,-.8. &'()*+,-.5. &'()*+,-.90#. =!>%?,'@;@% 2"34$5%6. 2"34$516. &;(;)1+.% "34$/77. !"#$% "E!>%> )+(,-.%/0*)1+.*1+.%!"#$% "34$/77.A5% 25%/0*)1+.&4% 25%/0*)1+.&4%

&'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-./01. &'()*+,-. A &'()*+,-.9. &'()*+,-.8. &'()*+,-.5. &'()*+,-.90#. "%BCD%?,'@;@% #/0. %/1. 6/7. 2"34$5%6. 8/2. &;(;)1+.% F.&,G)';.7% &'()*+,-. &'()*+,-. -*7;0'*(%% &'()*+,-.2. #7. #8. "34$/77.

&'()*+,-. "34$56. &'()*+,-.9. &'()*+,-#5.Figure 4 Isolation and purification of extracts from PS6-7 #$6. Figure 33 Isolation and purification of extracts from PS6-7

"34$#%2. 5.3.4"34$#1#. Isolation of Compounds from Endophyte Neofusicoccum/Botryosphaeria Isolation of compounds from endophyte Neofusicoccum/Botryoshperia!(PS6-31)

(PS6-31) Neofusicoccum/Botryoshperia!(PS6-31) ethyl acetate extracts (337 mg) were Neofusicoccum/Botryosphaeria (PS6-31) ethyl acetate extracts (337 mg) were extracted from three 2 L flasks. The extracts recovered from PS6-31 were fractionated extracted from three 2 L flasks. The extracts recovered from PS6-31 were fractionated

on a !LH-20 column and resulted in seven fractions. Fractions were combined on the ""!

basis of TLC visualisation for the final total of six fractions (SDI- 24 to 29). SDI-25

was determined to be sufficiently pure, by 1H NMR and was subjected to HPLC and

resulted in a further 27 fractions collected. These compounds, however, will be a part of

ongoing investigation outside this study.

134 !"#$%

&'()*+,-. &'()*+,-. &'()*+,-.!"#$% &'()*+,-. &'()*+,-.9. #/0. %/1. 6/7. 8/2. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-. &'()*+,-.9. &'()*+,-.2.#/0. %/1. 6/7. 8/2. on a LH#7.-20 column and resulted#8. in seven fractions. Fractions were combined on the &'()*+,-. &'()*+,-. &'()*+,-.2. &'()*+,-. "34$56. basis of#7. TLC visualisation&'()*+,-.9. for #8.the&'()*+,-#5. final total of six fractions (SDI- 24 to 29). SDI-25 #$6. 1 was &'()*+,-.determined to be sufficiently pure, by H NMR and was subjected to HPLC and "34$56. &'()*+,-.9. &'()*+,-#5. #$6. "34$#%2. "34$#1#. resulted in a further 27 fractions collected.

"34$#%2. "34$#1#. !"#$% 5678(%9):6*6:% "#$%&'! :;60*)1+.%

&'()*+,-. &'()*+,-.!"#$%&'(')*%)+(,-.%/0*)1+.*1+.%%&'()*+,-. &'()*+,-. #()%*+! #()%*,! #()%*$!!"#$% #()%*-!&'()*+,-.9.#()%*.! #()%*/! #/0. %/1. 6/7.23%/0*)1+.&4%"#$%&'! 8/2.

&'()*+,-.#()%*+!&'()*+,-.&'()*+,-.#()%*,! #()%*$!&'()*+,-.&'()*+,-.#()%*-! #()%*.! &'()*+,-.#()%*/! ,'?:?% &'()*+,-.2. &'()*+,-.9. #/0. #7.%/1. 6/7.#8. 8/2. &:(:)1+.% ! ! &'()*+,-. &'()*+,-. @ &'()*+,-.2. &'()*+,-. "%ABC%>,'?:?% "34$56. ! #7. &'()*+,-.9.#8.&'()*+,-#5. ! #$6. &:(:)1+.% ! ! Figure! 34 Isolation&'()*+,-. and purification of extracts from PS6-31 "34$56.Figure! 5 Isolation and purification&'()*+,-.9. of extracts&'()*+,-#5. from PS6-31 #$6. "34$#%2. "34$#1#. ! ! ! ! 5.3.5NMR NMR and structure and structure elucidation elucidation "34$#%2. "34$#1#.

5.3.5.1 NMR and Structure Elucidation of C! ompounds from PS1-3 NMR and structure elucidation of compounds from PS1-3

5.3.5.1.1SDI-139 SDI -139 RP-HPLC purified SDI-139 was obtained as a reddish brown solid. The positive RP-HPLC purified SDI-139 was obtained as a reddish brown solid. The mode HR-ESI-MS of SDI-139 displayed a sodiated molecular ion peak at m/z 313.0678 positive mode HR-ESI-MS of SDI-139 displayed a sodiated molecular ion peak at + corresponding to [M+Na] consistent with +the formula C15H14O6Na requiring 9 rings m/z at 313.0678 corresponding to [M+Na] consistent with the formula C15H14O6Na 13 andrequiring double 9bonds rings equivalentsand double bonds(RDBE). equivalents The C (RDBE). NMR spectrum The 13C revealedNMR spectrum 14 carbons

andrevealed a further 14 carboncarbons resonance and a further 107.3 carbon8ppm resonance were observed 107.38ppm in the was HMBC. observed in the

1 HMBC.The H NMR spectra in CD3OD-d-4 displayed a methyl singlet (3H, s) at δ1.93,

1 a methoxyThe methyl H NMR (CH spectra3-O) at inδ3.83 CD3 andOD- fourd-4 displayed aromatic aproton methyl peaks singlet at (δ3H,6.19 s) (d,at 2.5Hz),

δ6.45!1.93, (d, a 2.5Hz), methoxy δ 6.50methyl (1H, (CH s)3 -andO) atδ6.61 !3.83 (1H, and s).four Signals aromatic at δproton6.19 and peaks δ6.45 at ! were6.19

determined(d, 2.5Hz), by !6.45 1H NMR (d, 2.5Hz), and HMBC !6.50 (1H, to be s) meta and !-coupled.6.61 (1H, s). Signals at !6.19 and

1 !6.45The were 1 Hdetermined-13C connectivities by H NMR were and determined HMBC to bethrough meta- coupled.2D-HSQC experiments that

showed the protons at chemical shift δ6.61, δ6.50, δ6.45, δ6.19, δ3.83 and δ1.93 were

bound to carbons at chemical shift 117.49, 116.79, 100.71, 111.58, 56.06 and 19.43 ! "#! ppm, respectively. Key HMBC showed three-bond correlations from the protons at

δ1.93 ppm (3H, s) to the quaternary carbon at 135.53 and the protonated carbon at

117.49 ppm together with a two-bond correlation to a carbon at 127.48 ppm. The proton

bonded to the carbon at 117.49 ppm shows a shift of δ6.61 ppm (1H, s) indicative of an

135 aromatic proton. This proton shows a three-bond correlation to a methyl carbon at

19.43ppm, and quaternary carbons at 135.53 and 143.47 ppm. The only other proton that showed correlation with the already mentioned is another aromatic proton at δ 6.50 ppm that shows a three-bond correlation to carbons at 127.48 and 145.12 ppm. Using the 1H and HMBC’s allowed for the assignment of partial structure A Figure 35.

A database search of Scifinder (https://scifinder.cas.org/scifinder) was conducted using the molecular formula and returned 898 hits. A refined search to include the partial structure A resulted in 3 significant matches. One result referenced a natural product virtual target molecule for inhibitors of HIV-1 integrase [337], one result had no references and the most probable structure of SDI-139 had 44 references.

Using this as a template and incorporating the remaining HMBC data for constructing the remaining sections of partial structure B seen in Figure 36. The remaining HMBC correlations were used to satisfy the molecular formula and complete the compound NMR assignment. The protons at δ 6.19 (1H, d 2.5Hz) and δ 6.45 (1H, d

2.5Hz) are meta-coupled protons as well as being in the shift range of protons attached to Sp2 hybridized (ring) carbons. This was confirmed by HMBC through the three-bond correlation of δ 6.19 with the carbon at 100.71 (HSQC H-C correlated to proton δ

6.45ppm) and 107.38 ppm and the three-bond correlation of δ 6.45 to carbons at 111.58

(HSQC H-C correlated to proton δ 6.19ppm) and a two-bond correlation to a quaternary carbon at 165.92ppm. The meta-coupled 1H at δ 6.19 and δ 6.45 show a common three- bond correlation. The shift of this quaternary carbon suggests that it is attached to the carboxylic carbon at 107.38. The protons at δ3.83 are attached to a carbon at 56.06 ppm indicative of an O-methyl group and show a three-bond correlation to a carbon at

165.15 ppm. The only remaining unassigned carbon at 174.45ppm is typical of a

136 carboxylic acid functional group. As the NMR data were collected in CD3OD, no OH correlations were observed due to the OH being exchanged.

HO 2' 3' 1' 4' 6' 5' HO CH3 7' H

Figure 35 Partial structure A of SDI-139 determined from HSQC and HMBC correlations. Arrows indicate HMBC correaltions.

O OH C 2-COOH

OH 2 1 3 6 4 H 5 H

CH3 5-OCH3

Figure 36 Partial structure B of SDI-139 determined from HSQC and HMBC correlations. Arrows indicate HMBC correaltions.

137 It was concluded that the partial structure B fitted the requirements of the molecular formula and the searched molecule in Scifinder database. With all the C, H and O’s assigned, it was logical to join fragments A and B through a single carbon to carbon bond at 135.53 ppm (fragment A) and 165.92 ppm (fragment B). The compound

[1,1`-Biphenyl]-2-carboxylic acid, 3,4`,5`-trihydroxy-5-methoxy-2`-methyl-, commonly known as altenusin, satisfied the required 9 RDBE. SDI 139 1H NMR data presented in the appendix.

HO H 3' O OH 4' 2' C 2-COOH 5' 1' 6' OH H 2 1 3 7' CH3 6 4 5 H H

O 5-OCH3 CH3

Figure 37 Final structure of SDI-139 Table 13 NMR data for SDI-139 collected at 600Hz (1H) & 150 MHz (13C) in methanol-d-4 2 Position δH (multi. J in Hz) δC, multi. gHMBC ( JCH and

3 JCH)

1 165.92

2 107.38a

3 148.23

4 6.19 (d, 2.5Hz) 111.58 C2, C6

5 165.15

6 6.45 (d, 2.5Hz) 100.71 C1, C2, C4

1` 135.53

138 2` 6.50 (s) 116.79 C4`, C3`, C6`

3` 143.47

4` 145.12

5` 6.61 (s) 117.49 C1`, C3`, C7`

6` 127.48

7` 1.93 (s) 19.43 C1`, C5`, C6`

2-COOH 174.45

5-OMe 3.83 (s) 56.06 C5 a =determined by HMBC

5.3.5.1.2 SDI-05 Silica gel column purified SDI-05 was obtained as a white precipitate. HR-MS suggests that the most likely mass of [SDI-05+Na]+ is at 449.36 m/z and corresponds to

1 the molecular formula C30H57O2Na. H NMR spectra show methyl protons at δ 0.90, overlapping peaks at δ 1.25−1.39 representing CH2 envelope, a multiplet at δ 1.64, three multiplet peaks at δ 2.06, δ 2.36 and δ 2.78 indicating CH2 protons adjacent to double bonds and a broad multiple peak at δ 5.3-5.42 indicating protons typical of double bonds. HR-MS suggests that the most likely mass of [SDI-05+Na]+ at 449.36 m/z and

1 corresponds to the molecular formula C30H57O2Na. The H NMR is very similar to that of a triacylglycerol (TAG); however the absence of α-chain indicator 1H NMR signals at δ4-4.5 [338] (Figure 39) and carbons between 60 and 70 ppm and a single carboxylic acid (Figure 38) suggests that SDI-05 is a polyunsaturated fatty acid (PUFA). SDI 05

1H NMR data presented in the appendix.

5.3.5.1.3 SDI-80 Silica gel column purified SDI-80 was obtained as light reddish precipitate. The 1H

NMR suggests that a polyketide is present with aromatic signals around δ 6, phenolic

139 signals at ~ δ 5, methoxy protons at ~ δ 4 and methyl attached to rings indicated at ~

δ 2. This also indicates that there are more than 2 compounds present in the sample.

This NMR data suggest that the fraction was not pure enough for further 2D NMR and due to low yield of fraction SDI-80 further purification by HPLC was not pursued at this time. SDI 80 1H NMR data presented in the appendix.

130719-ROC-SDI05.005.esp

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 Chemical Shift (ppm)

130612-ROC-SDI63-NEWER.005.esp #" O b O a a O b b O O a O

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 Chemical Shift (ppm) Figure 38 Comparison of 13C NMR of fatty acid SDI-05 (1) and TAG SDI-63 (2). (a) represents 3 carboxyl carbons and (b) represents carbons of the TAG backbone. (a) represents one carboxyl in fatty acids and (b) is absent in fatty

140 acids 130719-ROC-SDI05.001.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm)

130612-ROC-SDI63-NEWER.001.esp

O #" b O a a O b b O O a O

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) Figure 39 Comparison of 1H NMR spectra of fatty acid SDI-05 (1) and TAG SDI-63 (2). (b) refers to proton shifts observed for TAG backbone. These are absent in fatty acid SDI-05 (1)

5.3.5.2 NMR and Structure Elucidation of Compounds from PS2-19

5.3.5.2.1 SDI-63 LH-20 silica column purified SDI-63 was isolated as a reddish brown oily substance. 1H NMR spectra suggested that SDI-63 is sufficiently pure for 2D NMR.

Literature reviews of known 1H NMR spectra led to comparisons between the 1H NMR spectrum of SDI-63 was compared to NMR spectra of known triacylglycerol (TAG) which led to suggestions that SDI-63 could possibly be a TAG. The HR-MS spectrum of SDI-63 was also compared to HR-MS spectra that also showed similarities to known

TAGs. The HR-MS of SDI-63 revealed [M+Na]+ ion at 855.7398 m/z and [M+Na]+ ions at 577.5163 and 551.5007 m/z suggesting that SDI-63 contains a TAG with two palmitic acid and one oleic acid side chain (POP). Zhang et al, 2012, reported that a

TAG standard [POP+Na]+ ion at 855.72 m/z and fragments ions [M+Na-PCOONa]+ at

141 577.5 m/z and [M+Na-OCOONa]+ at 551.5 m/z corresponding to sodiated palmitic and oleic acid (POP) fatty acid ions [339].

The HR-MS of SDI-63 also shows other signals suggesting minor TAG in the fraction. Ions representing [M2+Na]+ at 881.7549 m/z and signal at 603.5317 m/z corresponding to [M2+Na-PCOONa]+ were observed on the spectra. M2 corresponds to the mass of the 2nd TAG present and M2+Na-PCOONa is the mass of the 2nd TAG minus a palmitic acid side chain. These two ions are related differing from POP ions by

26 m/z. Overlapping signals of the 1H NMR and 2D NMR spectra make it difficult to interpret exactly what TAG’s are present. SDI 63 1H NMR data is presented in the appendix.

O O

Figure 40 Structure of 1,3-dipalmitoyl-2-oleoylglycerol (POP)

5.3.5.2.2 SDI-66 LH-20 silica column purified SDI-66 was isolated as a reddish precipitate and a red oily substance. SDI-66 was analysed by HR-MS and gave two positive ions at m/z

305.1356 and at m/z 287.1251. A molecular formula of C15H22O5Na and C15H20O4Na

142 was predicted from these data. 1H and 13C NMR data analysis gave a clear indication of a partial structure that is common to a number of bisabolane sesquiterpenoids. A database search of Scifinder (https://scifinder.cas.org/scifinder) was conducted using the partial structure and two compounds were predicted that satisfied all NMR and HR-MS data. SDI-66 was thought to be a pure compound; however closer 2D NMR analysis revealed that it was a mix of two compounds. The data collected correspond to literature where the predicted compounds have been reported as (7S,11S)-(+)-12-hydroxysydonic acid [340] and a dehydrated analogue of this compound. SDI 66 1H NMR data is presented in the appendix.

SDI_66_Positive_Full #1-18 RT: 0.02-0.49 AV: 18 NL: 1.46E7 T: FTMS + p NSI Full ms [100.00-2000.00] 305.1355 100

O Na+ 90 O HO C CH3 80 HO C CH3 CH3

70 OH O Na+ OH OH 60 CH3 OH 50

40 RelativeAbundance 30

251.0466 347.1459 20 307.1417

344.8981 10 287.1250 294.1753 327.1174 335.1459 349.1524 257.1508 273.1457 283.1049 303.1202 319.1511 0 250 260 270 280 290 300 310 320 330 340 350 m/z Figure 41 HR-ESI-MS positive mode spectra with predicted structures of the mixture SDI-66

5.3.5.2.3 SDI-69 LH-20 silica column purified SDI-69 was isolated as a reddish brown substance.

SDI-69 was analysed by HRESIMS and giving a positive ion at m/z 547.1569 and negative ion at m/z 523.1591. Using this data the molecular formula C28H28O10 was

1 13 proposed with SDI-69 having 15 RDBEs. The H and C NMR suggests one CH2, three

CH3, at least 8 protons attached to double bonds most likely attached to rings and at

143 least six phenolic protons. Two partial structures were tentatively identified and both determined to be 1,3-dihydroxybenzene moieties, common in most fungal PKS compounds (Figure 42). A database search of Scifinder

(https://scifinder.cas.org/scifinder), using the mass and formula determined by HR-MS analysis, revealed that there was no structure that will satisfy the NMR data. The compound seems to be highly unusual and based on preliminary NMR and mass spectrometry data, SDI-69 is a novel polyketide. The complete structure of SDI-69 is not yet known and will require further expert analysis as it is out of the area of expertise of SDI. Despite this, screening of this novel compound for activity was pursued. SDI 69

1H NMR data is presented in the appendix.

~~~~

~~~~ HO CH3 HO

OH OH

Figure 42 Two partial structures of SDI-69.

5.3.5.3 NMR and Structure Elucidation of Compounds from PS6-7

5.3.5.3.1 SDI-100 RP-HPLC purified SDI-100 was obtained as a white precipitate. The 1H NMR spectra showed low signal to noise, however, indicated that it is maybe a polyketide.

The 1H NMR displayed a methyl singlet (3H, s) at δ 1.93, a singlet at δ 3.83 (3H, s) typical of a CH3 attached to a ring system, protons at δ 6.19 and δ 6.46 (both 2.6 H/z) are meta-coupled protons as well as being in the shift range of protons attached to Sp2 hybridised (ring) carbons and two singlets at δ 6.50 and δ 6.60 indicating aromatic

144 protons. However SDI-100 was no longer pursued, as the final yield of material was too low for further analysis. SDI-100 1H NMR data is presented in the appendix.

5.3.6 Biological Activity Assay Investigation

A total of six endophytic, semi-purified fractions were included in the anti- bacterial and anti-fungal bioassay tests. Fractions SDI-80 was tested for bioactivity on the basis of interesting 1H NMR however, could not be purified due to low yield for

HPLC. SDI-146 and SDI-147 amounted to the wash fractions or materials between peaks that were collected in bulk. SDI-05, SDI-100 and SDI-139 were not tested for bioactivity because insufficient amount of material was recovered.

Activity was shown by at least one of the extracts against both the Gram- positive and Gram-negative test microorganisms. The fractions tested against the gram positive bacteria (Figure 43, Figure 44 and Figure 47), SDI-69 was shown to be the most active fraction showing activity at 100 µg/mL against S. aureus and C. diphtheriae as well as being active against B. subtilis with 76%, 74% and 68% growth inhibition, respectively (Table 14). SDI- 66 showed greatest activity against B. subtilis with a growth inhibition of 97%. All of the extracts showed growth inhibition activity against

C. diphtheriae at both 100 µg/mL and 10 µg/mL and against S. aureus at 10 µg/mL. On a number of occasions increased cell growth inhibition was observed at 10 µg/mL and increased cell growth at 100 µg/mL. SDI-63, 66, 80, 146 and 147 all followed this trend when tested against S. aureus. SDI 80 and SDI-146 also showed the same trend against

B. subtilis.

Endophyte extracts were also tested against Gram-negative bacteria E.coli and

P.aeruginosa (Figure 45 and Figure 46). Endophyte extracts SDI-69, 146 and 147 were the most active against E. coli at 100 µg/mL with inhibition of 35%, 29% and 34%

145 respectively, with SDI-146 being more active at 10 µg/mL with 41% growth inhibition.

All extracts showed some inhibition of growth of P. aeruginosa at 100 µg/mL with

SDI-147 showing 35% inhibition (Table 14). Only extracts SDI-66 and 147, showed weak inhibition at 10 µg/mL (Table 15).

Anti-fungal activity was measured against C. ablicans (Figure 48). Extracts

SDI-66, 80 and 146, showed slight inhibition (all <20% inhibition) at 100 µg/mL, 10

µg/mL and both 10 µg/mL and 100 µg/mL, respectively. SDI-63, 69 and 147 all showed increased growth at both concentrations.

Table 14 Growth inhibition of test organisms by endophyte fractions at 100 µg/mL (%+SEM) Endophyte Endophyte Genus % Cell growth inhibition of bacterial and fungal test strains at 100µg/mL extract S. aureus C. diptheriae E. coli P. aeruginosa B. subtilis C. albicans

SDI-63 PS2-19 Aspergillus -53.48+ 21.02+ 13.64+ 3.62+ 0.00 -29.43+ -3.78 0.68 0.40 0.13 -0.97

SDI-66 PS2-19 Aspergillus -44.9+ 28.68+ -1.45+ 23.98+ 93.67+ 13.78+ -2.61 1.11 -0.21 0.83 10.33 0.88

SDI-69 PS2-19 Aspergillus 76.14+ 74.66+ 35.27+ 10.86+ 67.81+ -4.88+ 0.63 4.05 1.84 1.34 2.94 -0.23

SDI-80 PS1-3 Penicillium -80.91+ 29.47+ -3.27+ 22.93+ -3.43+ -20.39+ -2.29 1.16 -0.32 0.37 -0.45 -0.56

SDI-146 PS1-3 Penicillium -18.49+ 18.27+ 28.91+ 13.27+ -8.44+ 8.04+ -2.48 0.48 5.65 0.52 -0.86 0.41

SDI-147 PS1-3 Penicillium -18.78+ nt 33.52+ 35.17+ nt -35.82+ -0.25 3.44 0.84 -0.43

Table 15 Growth inhibition of test organisms by endophyte fractions at 10 µg/mL (%+SEM) Endophyte Endophyte Genus % Cell growth inhibition of bacterial and fungal test strains at 10µg/mL extract S. aureus C. diptheriae E. coli P. aeruginosa B. subtilis C. albicans

SDI-63 PS2-19 Aspergillus 49.50+ 16.70+ 13.64+ -14.63+ -20.05+ -28.72+ 2.70 3.01 0.26 -0.41 -0.88 -0.22

SDI-66 PS2-19 Aspergillus 29.22+ 25.15+ 13.27+ 13.57+ 30.61+ -3.88+ 1.91 1.32 0.29 1.63 4.66 -0.32

SDI-69 PS2-19 Aspergillus 5.57+ 33.20+ -16.73+ -8.45+ -3.96+ -19.60+ 0.38 3.06 -3.51 -1.41 -0.42 -1.31

SDI-80 PS1-3 Penicillium 10.54+ 26.33+ 7.27+ -3.92+ 32.72+ 19.24+ 1.05 3.02 0.51 -0.27 5.67 1.10

SDI-146 PS1-3 Penicillium 49.70+ 14.93+ 41.45+ -6.64+ 15.30+ 12.99+ 2.39 1.05 11.30 -0.21 0.96 0.12

146 SDI-147 PS1-3 Penicillium 23.80+ nt 19.71+ 20.27+ nt -10.41+ 0.85 1.77 5.34 -0.44

5.4 Discussion

Traditional medicine plants play a major role in the survival and continuation of

Aboriginal culture. Plants such as Eucalyptus sp., Acacia sp. and Eremophila sp. have been well documented as possessing medicinal properties. However, further research into endophytes of the plants may reveal that the medicinal properties could be a result of the natural products produced by the microbes.

S. aureus 100μg/mL 75 10μg/mL

-5

-25

-45 Proliferation Inhibition (%) Proliferation -65

-85

Figure 43 Cell growth inhibition of Staphylococcus aureus at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is ciprofloxacin (50 µg/mL). All data are mean +/- SEM n=3.

147 B

C. diphtheriae

90 100μg/mL 80 10μg/mL 70 60 50 40 30 20

Proliferation Inhibition (%) Proliferation 10 0 -10

Figure 44 Cell growth inhibition of Corynebacterium diptheriae at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is ciprofloxacin (50 µg/mL). All data are mean +/- SEM n=3.

E. coli

80 100μg/mL 70 10μg/mL 60 50 40 30 20 10 0

Proliferation Inhibition (%) Proliferation -10 -20 -30

Figure 45 Cell growth inhibition of Escherichia coli at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is ciprofloxacin (50 µg/mL). All data are mean +/- SEM n=3.

148 D

P. aeruginosa

100

100μg/mL 80 10μg/mL

20 Proliferation Inhibition (%) Proliferation 0

-20

Figure 46Cell growth inhibition of Pseudomonas aeruginosa at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is ciprofloxacin (50 µg/mL). All data is mean +/- SEM n=3. E

B. subtilis

120 100μg/mL 100 10μg/mL 80

0 Proliferation Inhibition (%) Proliferation -20

-40

Figure 47 Cell growth inhibition of Bacillus subtilis at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is ciprofloxacin (50 µg/mL). All data are mean +/- SEM n=3.

149 F

C. albicans

100 100μg/mL 80 10μg/mL

-20 Proliferation Inhibition (%) Proliferation -40

-60

Figure 48 Cell growth inhibition of Candida albicans at extract concentration of 10 µg/mL and 100 µg/mL. Positive control is nystatin (50 µg/mL). All data are mean +/- SEM n=3.

In this study, endophyte PS1-3, with closest match to a Penicillium sp., was isolated from a medicinal plant in the Smilacaceae, that is used traditionally to treat skin infections and sore throats and as a tonic for general well being. PS1-3 was found to produce the compound altenusin, isolated as SDI-139, together with SDI-05, a polyunsaturated fatty acid and SDI-80 suggested to be a mix of compounds including a polyketide. The structure of altenusin was first reported and isolated by Rosett et al in

1957, from the funus Alternaria [341]. It has since been isolated from other fungi, including endophytes from the genera Alternaria, Penicillium, Talaromyces and

Phomopsis. Altenusin isolated from Alternaria sp. exhibited broad anti-microbial activity against a range of test organisms including MRSA [342], inhibits the enzyme trypanothione reductase from Trypanosoma cruzi [343], and showed cytotoxic activity against L5178Y mouse lymphoma cells as well as protein kinase inhibition activity

[344]. Reports of Penicillium species that produce altenusin and structurally similar

150 compounds show myosin light chain kinase inhibition [345] and neutral sphingomyelinase inhibition [346]. Talaromyces flavus-isolated altenusin showed slight inhibition of anti-HIV-1 integrase [347]. Although SDI-139 was not included in the bioassay tests as the activity of altenusin produced by fungi has been shown in literature mentioned above and thus is likely to be a bioactive constituent possessing anti- infective properties in the plant.

Endophyte PS2-19,with a closest match to an Aspergillus sp., was isolated from a medicinal plant in the Myrtaceae. This plant is used traditionally to treat infection of the skin, and to help burn healing, and toothache and to relieve joint pain. Three fractions isolated from PS2-19, SDI-63, SDI-66 and SDI-69 were further investigated and purified.

In this study, fraction SDI-63 was identified as a mixture of triacylglycerol

(TAG) by NMR and mass spectrometry data. TAG’s are the main components of natural oils and are produced by plants and seeds [338, 348-350], fungi [351, 352], bacteria [353-355] and algae [356]. Organisms usually accumulate and store fatty acids in the form of TAGs for times of metabolic stress or seed germination. TAG’s are now being targeted as a source of biofuels such as biodiesel [357-359]. Aspergillus have previously been reported to produce fatty acids and TAGs [360]. Using fungal TAG’s instead of other sources of TAG such as utilising old cooking oil, allows for production of a more consistent quality of biofuel [360]. Fungal production of TAGs allow for readily available and a continual renewable source of consistent quality TAGs for production of biofuels. PS2-19 may be a good candidate for further investigation for biofuel production.

Fraction SDI-66 was identified as (7S,11S)-(+)-12-hydroxysydonic acid and a dehydrated analogue that belongs to the phenolic bisabolane sesquiterpenoids. In this

151 study, SDI-66 showed activity against all bioassay test organisms at either 10 µg/mL or

100 µg/mL. Greatest activity was shown at 100 µg/mL against Bacillus subtilis. In the literature, (7S,11S)-(+)-12-hydroxysydonic acid, obtained from Aspergillus sydowii isolated from marine sediment, showed slight anti diabetic and anti-inflammatory activities [340]. In the same study, a structural analogue, synodol, showed the most anti diabetic potential and also exhibited significant anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by fMLP/CB-induced human neutrophils. A related compound, (-)-sydonic acid, isolated from a sponge associated Aspergillus sp., showed broad anti-bacterial activity with the strongest inhibition against Bacillus subtilis, Sarcina lutea, E. coli, Micrococcus tetragenus and two marine bacteria Vibrio parahaemolyticus and Vibrio anguillarum [290]. Sydonic acid was first isolated and reported by Hamasaki et al, 1978, from Aspergillus sydowii

[288]. Sydonic acid has also been isolated from other fungi including Glonium sp., endophytic Penicillium sp. and marine sponge-derived Aspergillus sp. [289, 290, 295,

361]. Sydonic acid exists in both the S and R forms and has a number of derivatives depending on the functional group that may be attached. In a separate study another sample from a marine derived Aspergillus sp. exhibited weak anti-bacterial activity against S. aureus [362].

Fraction SDI-69 was suggested to be a novel polyketide based on NMR and mass spectrometry data, with the structure not yet fully identified. Aspergillus spp. are known producers of bioactive PKS derived compounds. Active PK compounds include the anti-cholesterol lovastatin [145], the pathogenic and carcinogenic toxins aflatoxin and sterigmatocystin [363], anti-cancer compound asperfuranone [364], cytotoxic curvularins and 10,11-dehydrocurvularin [365, 366]. In this study, SDI-69 showed activity against all bacterial test organisms at least one of the tested concentrations. The

152 two partial structures obtained by NMR and mass spectrometry data are common structures in fungal metabolites. Similar types of structures have been reported to be biosynthesised by type III PKS from Aspergillus oryzae [367]. The novel PKS compound, SDI-69, may need to be crystallised and the structure determined using x- ray crystallography for completion of compound identification. The novelty and activity of this purified PKS compound satisfied the proposed outcome from the project aims.

Potential active compounds present that are not detected by UV or are produced at low levels may not be collected from the HPLC. This was demonstrated in this study by fractions SDI-146 and SDI-147, which were included in the bioassay testing as they represented the remaining fractions not collected as peaks from HPLC. SDI-146 showed activity against all test organisms at least one of the tested concentrations while SDI-

147 was active against three of the four test organisms. This would indicate that active fractions are present that have not been detected by the HPLC machine.

Observed in this study, there was both cell proliferation and cell inhibition of the test organism at either higher concentration, lower concentration or both. This could be explained by a number of factors, for example, if there was no cell proliferation inhibition, the concentration of the test compound may be below its MIC. Kjer et al,

2009, shows the MIC of altenusin when tested against C.albicans is 125µg/mL [342].

Compared to this study that shows cell proliferation of C.albicans at both 100µg/mL and 10 µg/mL (Table 13 and 14). Working on the same principle, the activity at lower concentration but not higher concentration, may indicate that the MIC of the compound is between the upper and lower concentrations tested. Cellular inhibition at higher concentrations but at lower concentration may indicate that the active compound may be in small amounts and requires a higher concentration to be cytotoxic on the test organism.

153 As the majority of the test fractions are mixtures or semi pure compounds, the activity, or lack of, may be resultant from synergistic effects to enhance anti- proliferation or antagonism effects that may inhibit activity and cause the test organisms to grow [368]. Activity within the mixture such as SDI 80 that had a potential PKS identified from 1H NMR, SDI 146 and SDI 147, both collected wash from the HPLC, will remain a high priority for on going and future works.

5.5 Conclusion

In this chapter, seven natural products collected from endophytic fungi were completely or partially purified and identified. PS1-3 was most closely matched with a

Penicillium sp. produces altenusin (SDI-139), a PUFA (SDI-05) and a mixture of compounds that may contain a PKS (SDI-80). An Aspergillus, PS2-19, produced a TAG

(SDI-63), a mixture of known compounds as (7S,11S)-(+)-12-hydroxysydonic acid and dehydrated analouge (SDI-66), and an unknown novel PKS (SDI-69). PS6-7 also produced a compound that may be a PKS based on 1H NMR data. These endophytes were selected on the basis of amplifying NRPS or PKS encoding genes and with crude extract anti-microbial activity. The reported biological activities of the known compounds, altenusin and the bisabolane analogues were discussed and showed various anti-microbial, cytotoxic and anti-inflammatory activities. Anti-microbial bioassays conducted in this study also revealed the activities of SDI-63, SDI-66, SDI-69 and SDI-

80. The two plants that PS1-3 and PS2-19 were isolated from are traditionally used for thier anti-bacterial, anti-inflammatory, and anti-fungal properties. The activity shown in this study and reported activities of the known compounds coincide with the traditional use of the plant species that their respective endophytes were isolated from. This suggests that the endophytes isolated from the medicinal plants may play a role in providing the medicinal properties of the plant and that NRPS and PKS genetic

154 screening is a suitable method for detecting endophytes that produce bioactive compounds.

155 6 General Discussion

This PhD was conducted in collaboration with the Dharawal elders group and members of the La Perouse Aboriginal community on medicinal plants used by the

Dharawal people of Botany Bay, Sydney, Australia. The objective of this thesis was to utilise the ethnobotanical information of medicinal plants to study the diversity of endophyte communities, and in turn, genetically screen the isolates for natural product biosynthesis genes and isolate bioactive compounds of these endophytes.

The Dharawal people have used these plants for many generations to manage and cure a range of ailments. Some have been documented and researched, and had compounds isolated on a commercial scale. Examples include tea tree oil from

Melaleuca alternifolia [26], active ingredients of buscopan from Dubsobia sp. [51] and

Eucalyptus oil from various Eucalyptus sp.

Respected Dharawal elders, who had continually practiced their culture and had credible traditional knowledge, granted permission to collect and study medicinal plant species from Botany Bay. This was an extremely important step in the initiation of the project. Interviewing elders, in this case the Dharawal elders, who have an unbroken and continual practice of their culture brings credibility, accuracy and authenticity to the knowledge being presented. Documented use of traditional medicine plants in Australia have been documented by doctors, anthropologist, and scientist since the arrival of first

British ships. The information that was collected may have problems of inaccuracy and not reflect or include the proper use of the medicine plant. This may be due to translational boundaries, bias of the British (superiority views), and observation by the

British without explanation or Aboriginal people keeping the plant use secret. In any case, when accessing traditional knowledge for ethnobotanical studies elders from that area should first be consulted. A discussion point in these initial interviews was the

156 access rights and permission to use this information once it is shared. The elders had shared some of their information with trust and confidence that the information will be handled sensitively based on their requests. This is the reason that, in this thesis, the plants used are represented by the plant family classification rather than the plant species. The request and instructions of the knowledge holders should always be followed and respected. Another point to consider is the great importance of ethnobotanical information and associated modern-day studies. Four of the seven women who were engaged in the interview process passed away during my work. The traditional knowledge and cultural experience that these elders held was priceless and is no longer directly accessible to their following generations. This reiterates the importance of how this knowledge is endangered to being lost forever and ethnobotanical studies and documentation of the knowledge held by the elders and elderly people of the community should remain a high priority.

Collection of the plants used in this study were from areas in close proximity to known pre-European contact, Dharawal occupation sites, determined by traditional knowledge and archaeological information, within the Kamay/Botany Bay National

Park from La Perouse and Kurnell. The protection of the plant species within the

National Park together with their close proximity to known occupation sites enhanced the possibility to use plants that are growing under the same or similar condition to pre-

British contact. This was also aimed at giving a fair representation of the host plant- microbe interaction and isolating the diverse endophyte communities as close as possible to this time in history.

Endophytes exist as a highly diverse range of microorganisms that are present in every plant species on earth. It has been increasingly accepted that endophytes have the ability to produce bioactive natural products that are important industrial and

157 pharmaceutical compounds, this being the scientific and technical basis for this thesis.

In this thesis, medicinal plants were chosen for endophytes isolation as it is hypothesised that this will increase the likelihood of isolating endophytes that produce bioactive compounds. Although plant species are documented and used for their medicinal properties, it can be postulated that the endophytic microbes actually produce the bioactive compounds or are partly involved in the steps of plant metabolites. This is demonstrated in the production of taxol by various endophytic fungi [81, 299, 356], endophytic fungal production of camptothecin [291] and in the case of a study conducted on an Australian medicinal plant snakevine (Kennedia nigriscans), the production of novel antibiotics munumbicins [95, 117]. In this study, 48 culturable endophytic microorganisms were isolated and identified from five medicinal plants of

Botany Bay, Sydney. The isolated endophytes from this study, together, are a unique data set and are comparable to several phylogenetic analyses, by order, of the fungal communities in several different endophytic and symbiotic environments. [218-222].

The fungal endophytes were grouped into 10 orders with the genera identified from these orders; all being isolated in previous studies regarding endophyte diversity [182,

200, 201, 203, 223, 276, 279, 302]. Three divisions of bacteria were also isolated that phylogenetically grouped into α-proteobacteria, β−proteobacteria and Firmicutes.

Previous studies have also reported genera belonging to these bacterial orders isolated from plant sources [192, 203, 230-233]. Furthermore extracts from these previously isolated fungal and bacterial endophytes have also been reported to have produce bioactive compounds [200, 203, 302, 357].

Although, culturable endophytes were isolated from the medicinal plants in this study, this is by no means all of culturable endophytes present from the plants examined. The harsh surface sterilisation process, particularly on the more fragile plant

158 tissues, the endophyte isolation media used, and the growth conditions may have selected endophytes that survive and thrive under these particular conditions. A more accurate determination of total endophyte population that also includes non-culturable endophytes will require more genetic-based methods and techniques such as metagenomics and environmental PCR. Other approaches for characterising the total endophyte communities maybe include a wider range of isolation media and culturing conditions in order to draw comparisons of endophytes from the same species of plants at different times of year, different biomes from within Australia and possibly comparisons between the same species from different countries.

In the search for bioactive compounds PCR amplification of genes that encode enzymes involved in the biosynthesis of such compounds proved successful. Primer sets targeting NRPS and PKS encoding genes were used to indicate if the gene encoding the respective enzyme complex was present or not. It is acknowledged that NRPS and PKS enzyme complexes may not always produce bioactive compounds, for example, the production of pigments that are polyketide-based. A positive result for the presence of these genes was used as an indication that the endophyte may produce bioactive NRPS- or PKS-derived compounds and therefore was considered a candidate for further crude chemical extraction and bioactivity tests. The bioactivity of the crude extracts then guided further purification and compound analysis of the most active compounds. This method resulted in the isolation of a novel PKS-based compound (SDI-69), several minor NRPS and PKS compounds, as well as other non-NRPS and PKS compounds.

The NRPS and PKS PCRs may amplify multiple, sometimes 20 or more, sequences at a time, leading to mixed DNA sequences. For this reason not all of the PCR amplicons were successfully sequenced. The sequences obtained from the PCR screening (Chapter

4 table 11) showed significant homology to the targeted NRPS or PKS encoding gene.

159 There is therefore certainty that the gene amplified was either a NRPS or PKS encoding gene, however, the exact identity of the amplified NRPS or PKS gene was not identified. This indicates that the derived gene sequences and probably their enzyme products are novel. Clone libraries could also be constructed and sequenced to confirm that number and type of genes that have been amplified. Alternatively, this could be clarified in the future by sequencing the entire genome of these endophytes or by analysing the metagenome of total plant DNA.

It is also acknowledged that bioactive compounds can be produced via alternate pathways from NRPS and PKS but was not the key focus of this study. Non-ribosomal peptide synthetases (NRPS) and polyketide synthase (PKS) encoding genes were targeted, and in this study, the targeted genes were amplified from nineteen identifiable endophytes and ten unidentified endophytes. The NRPS/PKS PCR was used as a means for selecting candidates for further compound isolation. Some of the genes were sequenced to check the right target was being amplified with the majority of the NRPS and PKS sequence data coming back as mixed sequence signals. A search of the NCBI database using blastn tool compared these mixed sequences to known sequences and some of the sequences matches returned the intended gene target to verify that these genes were being amplified. While NRPS screening was initially carried out on the fungi, the focus shifted to PKS encoding genes, as the majority of fungal natural products are PK based.

In this thesis, preliminary crude extract bioassay testing was only conducted on endophytes that had amplified a NRPS or PKS encoding gene by PCR. Endophyte PS2-

15 was the exception as no NRPS or PKS was amplified but was included and was shown to have slight bioactivity that suggests that bioactive compounds produced via other pathways e.g. terpenes, were still detected by the initial bioactivity screening

160 process. A study of endophytes from the traditional Chinese medicine (TCM) plants in

Panxi Plateau, China found that the endophyte community of the plants studied is very diverse [241]. Fifty-six out of sixty endophytes isolated were reported to possess at least one NRPS or PKS encoding gene. Four of the endophytes DNA did not amplify either

NRPS or PKS genes, but still showed anti-microbial activity. Similarly, Qin et al.

(2009) reported forty-six actinomycete strains, isolated from a TCM found in the rainforests of Xishuangbanna, China. They were selected and screened for NRPS and

PKS encoding genes. [274]. Half of the selected endophytes showed activity against at least one of the test organisms in the bioactivity assay, but only 26.1% of the total endophytes isolated had an amplifiable NRPS gene with 10.9% and 15.2% amplifying a

PKS-I and II, respectively, which led to the conclusions that there was no direct correlation between biosynthetic gene and target activity.

Five endophytic fungi were chosen for scale up growth based on their bioactivity and NRPS or PKS gene content. Based on compound recovery and purity, the main compounds discussed in Chapter 5 were produced by endophytic, Penicillium

(PS1-3) and Aspergillus (PS2-19). An endophytic Aspergillus, PS2-19, was isolated from a medicinal plant that is traditionally used to treat infection of the skin, help burn healing, toothache and relieve joint pain. This possessed a PKS gene and also produced a novel anti-bacterial polyketide compound, SDI-69. The compound SDI-69 showed activity against all bacterial test organisms (figure 43-48). PS2-19 also produced an anti-microbial triacylglycerides (SDI-63) and the bisabolane sesquiterpenoids, (7S,11S)-

(+)-12-hydroxysydonic acid and its dehydrated analogue (SDI-66) (Section 5.3.5.2.2).

The compound SDI-63 was most active against S. aureus, E. coli and C. diphtheriae and SDI-66 showed activity against all test organisms. Literature shows (7S,11S)-(+)-

12-hydroxysydonic acid (contained in mixture SDI-66) and its analogue sydonic acid

161 have anti-microbial, anti-diabetic, anti-inflammatory and cytotoxic properties [324,

349].

Endophyte PS1-3 was isolated from a plant that is used traditionally to treat skin, throat and eye infections as well as stomach complaints and as a “good blood” medicine, PS1-3 produced the compound SDI-139, which was determined to be a known biphenyl, altenusin. The altenusin-producing Penicillium sp. coincides with reported activities in the literature that altenusin, is produced by a number of fungi, and possesses anti-microbial, cytotoxic and anti-viral activities [327, 329, 331].

With the example of PS2-19, genetic screening of NRPS and PKS was targeted and possessed a PKS gene and produced a novel PKS compound. PS2-19 also produced other classes of compounds that are bioactive. This shows that if specific genes are targeted, other classes of bioactive compounds can still be isolated via bioactive screening. The bioactivity of the isolated compounds from PS1-3 and PS2-19 correlated with the traditional use of their respective plants, suggesting that endophytes may have a contributing role in the medicinal properties of the plant. An example of endophytic contribution is the production of the anti-cancer precursor compound, podophyllotoxin, obtained from the rhizomes from the endangered medicinal plant Podophyllum hexandrum [358]. An endophytic Phialocephala fortinii isolated from the rhizomes of

Podophyllum peltatum was shown to produce podophyllotoxin at concentrations of 0.5 to 189 µg/L. Although this is low-level production, it can be sustainably produced independent of the plant [359]. This also supports the premise of this work that isolation of a productive endophyte will lead to a ready supply of a required bioactive compound.

Traditional techniques of natural product extraction require the removal of massive amounts of plant material for little compound in return. In contrast, techniques used to isolate endophytes only require small amounts of plant material (section 3.2.3)

162 to isolate a number of endophytes (section 3.3) that produce multiple bioactive compounds (chapter 4 and 5). The isolated endophytes can then be grown in large cultures and produce the targeted compound making it readily available and produced sustainably. Endophyte-targeted research, as described here, also play a part in helping to conserve and allow a continuation to practice the culture of Indigenous peoples with full access to plants and areas of significance. This can also be achieved without the rules and restrictions that may be imposed by companies that obtain exclusive licences and access over plants for pharmaceutical investigations.

Discovering new information at cellular and molecular levels with the introduction of the latest technology and methods, and the increased participation of Indigenous researchers, it is now possible to classify traditional knowledge at a more technical and in depth level. Discoveries of these molecular and cellular applications in regards to traditional knowledge of medicinal plants can also be viewed as evolving traditional knowledge. With increased involvement of Indigenous communities at all levels from initial interviews right through to research, publishing and to the market place if applicable, will instil a greater level of confidence in Indigenous people for the use of their traditional knowledge in future scientific research. Positions in higher education and training opportunities of Indigenous people in areas that utilise traditional knowledge is also an issue that needs to be addressed and encouraged so that this type of work can be carried out by the knowledge holders themselves and not as by-standing participants. This will also create awareness in the Indigenous communities of current research that may lead to increased empowerment, alertness, safeguarding and protection of the knowledge that has the potential to uncover sources of novel and economically valuable chemical compounds.

163 Appendix 1

Fungal endophyte 18S rRNA gene sequences. Release date 07-02-2015

PS1-3 KM659040 PS3-17 KM659052 PS6-15 KM659064 PS2-2 KM659041 PS6-1 KM659053 PS6-16 KM659065 PS2-6 KM659042 PS6-2 KM659054 PS6-29 KM659066 PS2-9 KM659043 PS6-3 KM659055 PS6-31 KM659067 PS2-17 KM659044 PS6-4 KM659056 PS6-34 KM659068 PS2-18 KM659045 PS6-5 KM659057 PS6-39 KM659069 PS2-19 KM659046 PS6-6 KM659058 PS6-40 KM659070 PS3-3 KM659047 PS6-7 KM659059 PS6-41 KM659071 PS3-5 KM659048 PS6-11 KM659060 PS7-14B KM659072 PS3-7 KM659049 PS6-12 KM659061 PS7-14WF KM659073 PS3-14 KM659050 PS6-13 KM659062 PS7-15 KM659074 PS3-16 KM659051 PS6-14 KM659063 PS7-17 KM659075

>PS1-3 [organism=Penicillium sp.] Smilacaceae stem isolated endophytic Penicillium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAAAGGTGAAATT CTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGAT GTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGGGGTTTCTATGATGACCCGCTCGGCACCTTACGAGAAATCAAA GTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGAT TGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGGGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGGATAACG AACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCGTTTGCGGGCCG CTGGCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTACGTG GCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGC GCTACACTGACAGGGCCAGCGAGTACAT

>PS2-2 [organism=Penicillium sp.] Myrtaceae branch/stem isolated endophytic Penicillium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTTTGGGGGCGTCAGTATTCAGCTGTCAGAGGAGAAATTCT TGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGATGTTT TCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCG TCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGGGATTCT ATGATGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTGGGTTCTG GGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGC ACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAAC TCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTT GATCTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT GTCTGCTTAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCG GTCCGCATTTGCGGGCCGCTGGCTTCTTAGGGGGACTATCGGCTCAAGC CGATGGAAGTGCGCGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCT GGGCCGCACGCGCGCTACACTGACAGGGCCAGCGAGTACATC

164 >PS2-6 [organism=Penicillium sp.] Myrtaceae branch/stem isolated endophytic Penicillium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAATT CTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGAT GTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG ACGGGATTCTATGATGACCCGTTCGGCACCTTACGAGAAATCAAAG TTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGATT GACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTCGGCCCTTAAATAGCCCGGTCCGCATTTGCGGGCCGCTG GCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGGGCCAGCGAGTACATCA

>PS2-9 [organism=Penicillium sp.] Myrtaceae branch/stem isolated endophytic Penicillium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTTTGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAAT TCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGA TGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATC AGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCG GACGGGATTCTATGATGACCCGTTCGGCACCTTACGAGAAATCAAA GTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGAT TGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGA ACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCATTTGCGGGCCGCT GGCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGGC AATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGGGCCAGCGAGTACATC

>PS2-17 [organism=Cladosporium sp.] Myrtaceae branch/stem isolated endophytic Cladosporium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATTCTT GGATTGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTT CATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGT CGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGGTGTTAGT ATTTTGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTGGGTTCTG GGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGC ACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAAC TCACCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTT GATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTT GTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCA GGCCCGCTTTGGCGGGTCGCCGGCTTCTTAGAGGGACTATCGGCTCAAG CCGATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTC TGGGCCGCACGCGCGCTACACTGACAGAGCCAACGAGTTCAT

165 >PS2-18 [organism=Chrysoporthe sp.] Myrtaceae branch/stem isolated endophytic Chrysoporthe sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGATGTTTTTCTTTGACTCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGCGCCGCTCAGGCGGCACGCTG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACGGAGCCAGCGAGTTCCTCC

>PS2-19 [organism=Aspergillus sp.] Myrtaceae branch/stem isolated endophytic Aspergillus sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAATT CTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGAT GTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGGCGTTTCTATGATGACCCGCTCGGCACCTTACGAGAAATCAAA GTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGAT TGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGA ACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCGTCCGCGGGCCGC TGGCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGG CAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCG CTACACTGACAGGGCCAGCGAGTACATC

>PS3-3 [organism=Acremonium sp.] Myrtaceae branch/stem isolated endophytic Acremonium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAACTGTCCGAGGTGAAATTCTT GGACCAGTTGAAGACTACCTACTGCGAAAGCATTTGCCAAGGATGTTTT CATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTC GTAGTCTTAACCATAAACTATGCCGACTAGGGATCCGGCGGTGTTATTT ATTGACCCGCTGGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGG GGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCAC CACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTC ACCAGGTCCAGACACAATGAGGATTGACAGATTGAGAGCTCTTTCTTGA TTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACGAGACGTCCGCCTGCTAACTAGACTGC ATTGCTTCGGCAGCGCACTGTCTTCTTAGAGGGACTATCGGCTCAAGCC GATGGAAGTTGGACTCAATAACAGGTCTGTGATGCCCTTAGATGTTCTG GGCCGCACGCGCGCTACACTGACGGTGGAAACGAGTTCTTCC

166 >PS3-5 [organism=Guignardia sp.] Myrtaceae leaf isolated endophytic Guignardia sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGG GCGATGTTATTATTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGATT GACAGATTGAGAGCTCTTTCTTGATTCTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCCGGCCCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTGATGCCCTTAGATGTCCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTTAT

>PS3-7 [organism=Rhodotorula sp.] Myrtaceae branch isolated endophytic Rhodotorula sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTTGGGGGCATTTGTATTCCGTCGTCAGAGGTGAAATTC TTGGATTGCCGGAAGACAAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTGATCAAGAACGAAGGAAGGGGGATCGAAAACGATTAG ATACCGTTGTAGTCTCTTCTGTAAACTATGCCAATTGGGGATCGGTA CAGGATTTTTAATGACTGTATCGGCACCCGAAGAGAAATCTTTAAAT GAGGTTCGGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAAT TGACGGAAGGGCACCACCAGGTGTGGAGCCTGCGGCTTAATTTGAC TCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATTGACA GATTGATAGCTCTTTCTTGATCTTGTGGTTGGTGGTGCATGGCCGTTC TTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGATAACGAACGAGA CCTTAACCTGCTAAATAGACCAGCCGGCTTTGGCTAGCTGCTGTCTT CTTAGAGGGACTATCAGCGTTTAGCTGATGGAAGTTTGAGGCAATA ACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACA CTGACAGAGCCAGCGAGTCTACCA

>PS3-14 [organism=Acremonium sp.] Myrtaceae branch isolated endophytic Acremonium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAACTGTCCGAGGTGAAATT CTTGGACCAGTTGAAGACTACCTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCCGG CGGTGTTATTTATTGACCCGCTGGGCACCTTACGAGAAATCAAAGTG CTTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACGTCCGCCTGCTAACTAGACTGCATTGCTTCGGCAGCGCACTGT CTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTGGACTCAAT AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTAC ACTGACGGTGGAAACGAGTTCTTCC

167 >PS3-16 [organism=Acremonium sp.] Myrtaceae branch isolated endophytic Acremonium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAACTGTCAGAGGTGAAATT CTTGGACCAGTTGAAGACTACCTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCCGG CGGTGTTATTTATTGACCCGCTGGGCACCTTACGAGAAATCAAAGTG CTTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACGTCCGCCTGCTAACTAGACTGCATTGCTTCGGCAGCGCACTGT CTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTGGACTCAAT AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTAC ACTGACGGTGGAAACGAGTTCTTCC

>PS3-17 [organism=Penicillium sp.] Myrtaceae branch isolated endophytic Penicillium sp. 18S ribosomal RNA gene, partial sequence.

AAGGGATAGTCGGGGGCGTCAGTATTCCGCTGTCAGAGGTGAAATT CTTGGATTTGCGGAAGACTAACTACTGCGAAAGCATTCGCCAAGGA TGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGATC AGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCG GACGGGATTCTATGATGACCCGTTCGGCACCTTACGAGAAATCAAA GTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACAAAATAAGGAT TGACAGATTGAGAGCTCTTTCTTGATCTTTTGGATGGTGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGA ACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCATTTGCGGGCCGCT GGCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGGC AATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGGGCCAGCGAGTACATCA

>PS6-1 [organism=Phomopsis sp.] Sapindaceae stem isolated endophytic Phomopsis sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGGTGTTATTTCTTGACCCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGTGCCGCCTAGGCGGCACGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCAGCGAGTTCCTCC

168 >PS6-2 [organism=Neofusicoccum sp.] Sapindaceae stem isolated endophytic Neofusicoccum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCCGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGG GCGATGTTATTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCCCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTTATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTTAC

>PS6-3 [organism=Phomopsis sp.] Sapindaceae stem isolated endophytic Phomopsis sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGGTGTTATTTCTTGACCCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGTGCCGCCTAGGCGGCACGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCAGCGAGTTCCTTC

>PS6-4 [organism=Neofusicoccum sp.] Sapindaceae stem isolated endophytic Neofusicoccum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGAGTGAAAT TCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATC AGATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCG GGCGATGTTATTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAA GTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGAT TGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGA ACGAGACCTTAACCTGCTAAATAGCCAGGCCCGCTTTGGCGGGTCG CCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAG GCAATAACAGGTCTGTTATGCCCTTAGATGTTCTGGGCCGCACGCGC GCTACACTGACAGAGCCAACGAGTTTA

169 >PS6-5 [organism=Neofusicoccum sp.] Sapindaceae stem isolated endophytic Neofusicoccum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGG GCGATGTTATTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCCCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTTATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTTAC

>PS6-6 [organism=Phomopsis sp.] Sapindaceae stem isolated endophytic Phomopsis sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGGTGTTATTTCTTGACCCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGTGCCGCCTAGGCGGCACGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCAGCGAGTTCCTCC

>PS6-7 [organism=Neofusicoccum sp.] Sapindaceae stem isolated endophytic Neofusicoccum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGG GCGATGTTATTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCCCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTTATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTTAC

170 >PS6-11 [organism=Colletotrichum sp.] Sapindaceae leaf isolated endophytic Colletotrichum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTTATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAG ATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGAC GATGTTATTTTTTGACTCGTTCGGCACCTTACGAGAAATCAAAGTGC TTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAA TTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGA CTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGAC AGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGA GACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCAGTACGCTGGC TTCTTAAAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAAT AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGTTAC ACTGACAGAGCCAGCGAGTACTTCC

>PS6-12 [organism=Colletotrichum sp.] Sapindaceae leaf isolated endophytic Colletotrichum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTTATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAG ATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGAC GATGTTATTTTTTGACTCGTTCGGCACCTTACGAGAAATCAAAGTGC TTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAA TTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGA CTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGAC AGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGA GACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCAGTACGCTGGC TTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAAT AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGTTAC ACTGACAGAGCCAGCGAGTACTTCC

>PS6-13 [organism=Colletotrichum sp.] Sapindaceae leaf isolated endophytic Colletotrichum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATTGTAGAGGAGAAATTC TTGGATTTATTGAAGACTAACTACTGCGAAAGAAATGCCAAGGATG TTTTCATTTATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGA TACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACG ATGTTATTTTTTGACTCGTTCGGCACCTTACGAGAAATCAAAGTGCT TGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAAT TGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGAC TCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACA GATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTT CTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAG ACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCAGTACGCTGGCT TCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATA ACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGTTACA CTGACAGAGCCAGCGAGTACTTCCTT

171 >PS6-14 [organism=Colletotrichum sp.] Sapindaceae leaf isolated endophytic Colletotrichum sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATTGTAGAGGAGAAATTC TTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATG TTTTCATTTATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGA TACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACG ATGTTATTTTTTGACTCGTTCGGCACCTTACGAGAAATCAAAGTGCT TGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAAT TGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGAC TCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACA GATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTT CTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAG ACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCAGTACGCTGGCT TCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATA ACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGTTACA CTGACAGAGCCAGCGAGTACTTCCT

>PS6-15 [organism=Helicoon sp.] Sapindaceae root isolated endophytic Helicoon sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATTGTAGAGGTGAAATTC TTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATG TTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCAG ATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGC GGTGTTTTAATAATGACTCGCTCGGCACCTTACGAGAAATCAAAGTG TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGATGAAATAAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTTCAGGTGGTGGTGCATGGCCG TTCTTAGTTCGTGGGGTGACTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCCGG CTTCTTAGAGGGACTATCGGCTCAAGCCGAGGGAAGTTTGAGGCAA TAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTA CACTGACAGAGCCAACGAGTTCTTT

>PS6-16 [organism=Graphium sp.] Sapindaceae root isolated endophytic Graphium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCGTCAGTATTCAATTGTAGAGGAGAAATTC TTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATG TTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCAG ATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGC GATGTTATCTTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAGTC TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCCGG CTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAA TAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTA CACTGACAGAGCCAACGAGTTCATC

172 >PS6-29 [organism=Phomopsis sp.] Sapindaceae root isolated endophytic Phomopsis sp. 18S ribosomal RNA gene, partial sequence.

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCATAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGGTGTTATTTCTTGACCCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGTGCCGCCTAGGCGGCACGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCAGCGAGTTCCTCC

>PS6-31 [organism=Phomopsis sp.] Sapindaceae root isolated endophytic Phomopsis sp. 18S ribosomal RNA gene, partial sequence

TAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAGGTGAAATT CTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGA TGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAAACGATCA GATACCGTTGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGG CGGTGTTATTTCTTGACCCGCTCGGCACCTTACACGAAAGTAAAGTT TTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAA ATTGACGGAAGGGCACCACAAGGGGTGGAGCCTGCGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCCAGACACAACTAGGATTGA CAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCG TTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGTGCCGCCTAGGCGGCACGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCAGCGAGTTCCTCC

>PS6-34 [organism=Collophora sp.] Sapindaceae root isolated endophytic Collophora sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGAGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGATGTTATCTTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCTCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACACTGCCAACGAGTTTCT

173 >PS6-39 [organism=Graphium sp.] Sapindaceae root isolated endophytic Graphium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGAGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGATGTTATCTTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTATTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAAC GAGACCTTAACCTGCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCCG GCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCT ACACTGACAGAGCCTACGAGTTCATC

>PS6-40 [organism= Graphium sp.] Sapindaceae root isolated endophytic Graphium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGATGTTATCTTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTCAT

>PS6-41 [organism= Graphium sp.] Sapindaceae root isolated endophytic Graphium sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG GCGATGTTATCTTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTCAT

174 >PS7-14B [organism=Phyllosticta sp.] Myrtaceae leaf isolated endophytic Phyllosticta sp. 18S ribosomal RNA gene, partial sequence.

>PS7-14WF [organism=Phyllosticta sp.] Myrtaceae leaf isolated endophytic Phyllosticta sp. 18S ribosomal RNA gene, partial sequence.

>PS7-15 [organism=Phyllosticta sp.] Myrtaceae leaf isolated endophytic Phyllosticta sp. 18S ribosomal RNA gene, partial sequence.

TAGGGATAGTCGGGGGCGTCAGTATTCAATTGTCAGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGAGATCGG GCGGTGTTTCTTTTTATGACCCGCTCGGCATCTTACGAGAAATCAAA GTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAA GAAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAAT TTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGAT TGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGG CCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGTTAACGA ACGAGACCTTGACCTGCTAAATAGCCAGGTTCACTTTGGTGGACCGC AGGCTTCTTAGAGGGACTTTTGGCTCAAGCCAATGGAAGTACGAGG CAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCG CTACACTGACGGAGCCAGCGAGTACA

175 >PS7-17 [organism=Neofusicoccum sp.] Myrtaceae leaf isolated endophytic Neofusicoccum sp. 18S ribosomal RNA gene, partial sequence.

TGGGGATAGTCGGGGGCATCAGTATTCAATTGTCCGAGGTGAAATT CTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGAT GTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGG GCGATGTTATTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAG TCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG AAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATT TGACTCAACACGGGGAAACTCACCAGGTCCAGACACAGTAAGGATT GACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAA CGAGACCTTAACCTGCTAAATAGCCAGGCCCGCTTTGGCGGGTCGCC GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGC AATAACAGGTCTGTTATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCAACGAGTTTAC

Bacterial endophyte 16S rRNA gene sequences. Release date 07-02-2015

PS1-5 KM659076 PS6-18 KM659082 PS1-6 KM659077 PS6-22 KM659083 PS1-7 KM659078 PS6-24 KM659084 PS1-8 KM659079 PS6-25 KM659085 PS1-10 KM659080 PS7-2 KM659086 PS1-11 KM659081 PS7-11 KM659087

>PS1-5 [organism=Bacillus sp.] Smilacaceae root isolated endophytic Bacillus sp. 16S ribosomal RNA gene, partial sequence.

GCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGT AGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAA CGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAAC AAGTGCTAGTTAAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTAT TGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTC AACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGTGGA ATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTGGCGAAG GCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGT TTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGC CGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAGT

>PS1-6 [organism=Burkholderia sp.] Smilacaceae root isolated endophytic Burkholderia sp. 16S ribosomal RNA gene, partial sequence.

GCCGATGGCGGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCCGT AGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAA TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAA TCCTTTTCGATAATACCGGAAGGGGATGACGGTACCGGAAGAATAAGCACCGGCTA

176 ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACT GGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACCGATGTGAAATCCCCGGGCTTA ACCTGGGAACTGCATTGGTGACTGCAAGGCTTGAGTATGGCAGAGGGGGGTAGAA TTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGG CAGCCCCCTGGGTCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGG ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTCGGGTCTTCA TTGACTTGGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACCGGTCGC AGATTAAAACTCAAGGAATTGACGGGGACCCGCACAAGCGGT

>PS1-7 [organism=Bacillus sp.] Smilacaceae root isolated endophytic Bacillus sp. 16S ribosomal RNA gene, partial sequence.

ACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGT AGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAA CGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAAC AAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTAT TGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTC AACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGA ATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAG GCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGT TTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGC CGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG

>PS1-8 [organism=Bacillus sp.] Smilacaceae root isolated endophytic Bacillus sp. 16S ribosomal RNA gene, partial sequence.

>PS1-10 [organism=Bacillus sp.] Smilacaceae stem isolated endophytic Bacillus sp. 16S ribosomal RNA gene, partial sequence.

177 TGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTC AACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGA ATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAG GCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGT TTTCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCC GCAGGCTGAACCTCAAAGGAATTGACGGGGGCCCGCACAAGC

>PS1-11 [organism=Bacillus sp.] Smilacaceae stem isolated endophytic Bacillus sp. 16S ribosomal RNA gene, partial sequence.

>PS6-18 [organism= Burkholderia sp.] Sapindaceae root isolated endophytic Burkholderia sp. 16S ribosomal RNA gene, partial sequence.

GCCGATGGCAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGT AGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAA TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAA ACCTCCTGGTTAATACCTGGGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTA ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACT GGGCGTAAAGCGTGCGCAGGCGGTTCGCTAAGACAGATGTGAAATCCCCGGGCTTA ACCTGGGAACTGCATTTGTGACTGGCGGGCTAGAGTATGGCAGAGGGGGGTAGAA TTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGG CAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGG ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTCGGGTCTTCA TTGACTTGGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCA AGATTAAAACTCAAGGAATTGACGGGGACCCGCACAAGCGGT

>PS6-22 [organism= Burkholderia sp.] Sapindaceae root isolated endophytic Burkholderia sp. 16S ribosomal RNA gene, partial sequence.

178 ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTCGGGTCTTCA TTGACTTGGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCA AGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGG

>PS6-24 [organism= Burkholderia sp.] Sapindaceae root isolated endophytic Burkholderia sp. 16S ribosomal RNA gene, partial sequence.

>PS6-25 [organism= Burkholderia sp.] Sapindaceae root isolated endophytic Burkholderia sp. 16S ribosomal RNA gene, partial sequence.

>PS7-2 [organism= Agrobacterium sp.] Myrtaceae branch isolated endophytic Agrobacterium sp. 16S ribosomal RNA gene, partial sequence.

GCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCAT AGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCT ACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCAT GCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGATGAAGATAA TGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATA CGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGAT ATTTAAGTCAGGGGTGAAATCCCGCAGCTCAACTGCGGAACTGCCTTTGATACTGG GTATCTTGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTA GATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTG AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGT AAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCAT TAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGG GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGC

179 >PS7-11 [organism=Agrobacterium sp.] Myrtaceae branch isolated endophytic Agrobacterium sp. 16S ribosomal RNA gene, partial sequence.

GCCCGCGTTGGATTACGTAGTTGGTGGGGTAAAGGCCTACCATGGGGACGATCCAT ATCTGGTCTGAGAGGATGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCT ACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCAT GCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGATGAAGATAA TGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATA CGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGAT ATTTAAGTCAGGGGTGAAATCCCGCAGCTCAACTGCGGAACTGCCTTTGATACTGG GTATCTTGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTA GATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGGTCCATTACTGACGCT GAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGAATGTTAGCCGTCTGGCAGTATACTGTTCGGTGGCGCAGCTAACGCA TTAAACATTCCGCCTGGGGGAGTACGGTCGCCAGATTAAAACTCAAAGGAATTGAC GGGGGGCCCGCACAAGCGGTGGGAGCATGTGGTTTAATTCT

NRPS and PKS sequences (BLAST Results Table 11 pg. 106)

PS1-11 NRPS CAGGAAGTTACTACGGTATGCACGAGTGAAGTGACGGGCGATAACTTAGCCTATAT TATCTATACTTCAGGTTCCACTGGAAATCCAAAGGGAGTAATGATTGAGCACAGGA ATACAGTAACAATGATTCATTGGGCGTATCACACGTATTCTCGAAAGGAATTAGCG GGAGTATTACCTTCAACTTCACTATCTTTTGATTTATCCGTTTTTGAAGTATTTGTTC CCTTAACGATGGGTGGTAAGGTGATTGTCGCTGAAAATGCTCTACATCTAGACAAG TTGTCTACCAAAGGTGTGACTTTGGTTAATACTGTGCCATCGGCAGCAAAGGAACT GGTTCGAGCAAAAACAAATTCCTTCTTCAGTAAAGGTAATAAAATTGGCTGGAGAA CCGTTACCATATCCGCTTGTTCAAGATTTATACGAGAGAAAGTACGA

PS1-3 PKS AGCAAGACCGTTTCGATTGGTGTGGCTGCCGAGGAGCGAATCGAATATTGTTCCCA TCGCTGACTGCTTGAGCATCGACTTTCTTTTGCCGTGAGGGCTCCTCCCCTTGCCTA ACCGTACCCATCTCGTCATGTGGGTTGGTGTGGCCTGTGTTGCTCGGCGAGGTACGC TGACATTGACTGTAATCCTGTGTTGGTCGATTGACGTGATGATGTTGACGCCACCGC CCTCGGCCATTCCCCAGACTGAGGAGCTTTGCGCGCGTGATGGACTGCTTCTAACG AGGACGAACGAGCAGGGTCCATTATTTATTACGGCACTATGGCCCCGAAATGCAAA AATATTTCCCCACTTAGGAATGATTATTCGCTTACCGTAGCCGAATGCTCGGTAAGT GTATCAAAAGTGATGTTCCTTGAGTGTCCTCTGTTGGTTGTCCCCATGGAGCAACCC ACATTGCACGACACTGTTGCTCCCTGAGTCCTGATCCATAGTTACACCTGCAGAGTC AAGAACCTCATCTCAAACTTAAAACGTCT

PS2-2 PKS CCAGTGACGATTACCGTGAGATCAACAGCGGCCAAGACATTGATACCTATTTCATT CCCGGTGGTATCCGTGCCTTTTCCCCCGGTCGCATTATTTTTTATTTCAAATTCACGG TCCTACTTATTTCATACGGTTGTTCTTTCGTTTGCTG

PS2-9 PKS TGACTAGCGACGATTACCGTGAGATCAACAGTGGCCAGGACATTGATACTTACTTT ATTCCCGGTGGTAACCGAGCGTTCACCCCTGGCCGAATCAACTATTACTTCAAATTC AGCGGTCCCAGTGTGAGCGTCGACACGGCTTGCTCTTCTAGTCTTGCAGCTATCCAC GTGGCCTGCAACTCTCTTTGGAGGAATGACTGCGACTCTGCGGTAGCGGGTGGTGT GAACATTCTGACTAACCCTGACAACCACGCTGGTCTTGACCGCGGCCATTTCCTTTC TCGGACAGGAAACTGTAATACTTTCGATGATGGTGCCGATGGTTACTGTCGAGCTG ATGGAATTGGCTCAGTGGTCCTTAAGCGACTTGAAGATGCTCAAGCAGACAATGAC

180 CCTATTTACGGTATCATCAACGGCGCTTACACAAATCACTCTGCCGAGGCTGTTTCT A

PS6-7 PKS ACTTCGGACGACTGGCGAGAAATCAACGCGGCCGAGAACGTGTACACCTACTTCAT CACGGGCGTTCCTTAAAGCCTTCTCTCCCGGCCGCATCATCTACTATTTCAAGTTCT CCGGCCCGTCCTACTCGATCGATACCGCTGGCTTGTCCTGTCTTGCTGTCATACAGT TGGCCTGCACCTCAATTGAGTGCCGGTGATTGATACACAGCGTGCGCTGGTGGC

PS6-31 PKS CGGTCAGACCTCGGACGACTGGCGCGAGATCAACGCCGCCCAAGAGGTCGACACC TATTACATCACGGGTGGTGTCCGAGCCTTTGGTCCTGGTAGAATCAACTACCACTTC GGCTTCAGCGGGCCCAGTCTCAACATCGATACTGCATGCTCCTCCAGCGCTGCCGCT ATGAATGTCGCCTGCTCTTCGCTTTGGGCTCGCGACTGCGATACGGCCATCGTGGGA GGTCTCTCTTGTATGACCAACTCTGACATCTTTTCCGGTTTGAGTAGAGGACAGTTC CTCTCCAAGAAAGGGCCGTGCGCTACTTTCGATAACGATGCAGATGGATACTGCAG AGGCGATGGCTGTGCGTCGGTGATTGTCAAGCGCTTGGAGGATGCTGAAGCCGACC AAGACCGCGTCCTTGCCGTCATTCTCGGCACTGCTACCAACCACTCTGCTGACGCCA TCTCTATTACGCATCCTCATGG

PS2-6 PKS TGCTTACTGCTTACGAGGCATTTGAGATGGCTGGTTTCATCCCGGACAGCACCCCAT CCACCCAGAAGAACCGAGTTGGTGTGTTCTATGGTATGACCAGTGACGATTACCGT GAGATCAACAGCGGCCAAGACATTGATACCTATTTCATTCCCGGTGGTAACCGTGC CTTTACCCCCGGTCGCATTAACTACTATTTCAAATTCAGCGGTCCTAGCGTGAGCGT CGATACGGCTTGCTCTTCCAGTCTTGCTGCGATTCATGTCGCCTGCAACTCGCTCTG GAAGAATGATTGTGACTCTGCCGTGGCCGGTGGTGTCAACGTTTTGACCAACCCTG ACAACCACGCTGGACTTGACCGAGGTCATTTCCTCTCGCGAGGCGGAAACTGCAAC ACCTTCGACGACGGTGCCGATGGTTACTGCCGTGCTGATGGTATCGGCTCGGTCGTT TTGAAGAGACTTGAAGATGCTCAAGCAGACAATGATCCCATCTACGGTATCATCAA CGGCGCATACACGAACCACTCCGCCGAGTCTGTCTCCATTACTCGTCCTCTTGCCGG AGCACAGTCTTTCATCTTTGACAAGCTTCTCAATGAAGCTAATGTCAGCCCCAAGG ATGTCAGCTACATTGAGATG

PS6-7 PKS TCCCCCTCATCGAGGCCTGGATGGGCATCGGCTTACGGCGCACGTCATCCCGAACC GGATATCCTATCTTCTCGACCTCTTGGGCCCCAGTTCGGCGGTAGATGCATCATGTG CGTCGTCGCTGGTGGCCGTCCTCTTGTGCCCGCCAGGCGATTGCAATTTGCAAGTCC CATGTCCCCTTCTTTTTGTTTTTTGAACGTGCTGTGCTCTCCTGCATTGTTCTGCATG CTGGGTATTGCCGGAGTGCTG

PS6-31 PKS AGATATTCCGAATATCGAGGCGTGGATGGGGATCGGTTCAACACCACATGGAATCC CAAACAGGATTTCTTACCACCTGGATCTCATGGGCCCTAGCGCTGCAGTAAGTATA CCTCCTTTCAAAAGTGAGACCTCTTCGTTTCTTGTTTGTTTCGAGTTTCGAGGTACCC TATGTATGATGTTCATAAGCTCACGGTACTTTCTCTCACCTTCGAAGAAAACCGCTG ACTCTCGAGCCCGTATAGGTGGACGCACCATGTGCTTCATCTGGTGTGGCGG

PS2-19 PKS GGAATGACAAGCGACGACTATCGCGAAATCAACAGTGGTCAAGACATTGATACAT ACTTCATTCCAGGTGGCAACCGCGCGTTCACCCCCGGGCGCATCAATTATTACTTCA AGTTCAGCGGACCTAGTGTCAGCGTTGATACGGCATGTTCTTCCAGTCTTGCTGCGA TCCACTTGGCTTGCAATTCCATCTGGAGGAACGACTGCGACACAGCCATTTCTGGTG GCGTGAACATCTTGACCAACCCAGACAACCATGCTGGCTTGGACCGCGGCCATTTC CTCTCCAGAACCGGGAACTGCAATACATTCGATGACGGCGCGGACGGCTACTGCAG

181 AGCAGACGGGGTTGGAACAGTTATCCTTAAACGGCTTGAGGACGCTGAAGCAGAT AATGATCCGATTATTGGTGTCATCAACGGCGCGTATACAAATCATTCAGCGGAAGC GGTTTCAATTACTCGCCCGCAT

Appendix 2

1H NMR spectra

SDI 63 1.0 130612-ROC-SDI63-NEWER.001.001.1R.ESP

0.9

0.8

0.7

0.6

0.5

0.4 Normalized Intensity Normalized

0.3

0.2

0.1

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

SDI 66 1.0 6-66.001.1R.esp

0.9

0.8

0.7

0.6

0.5

0.4 Normalized Intensity Normalized

0.3

0.2

0.1

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

182

SDI-69 0.40 130524-ROC-SDI-69.001.001.1R REFERENCED DDMSO.ESP

0.35

0.30

0.25

0.20

0.15 Normalized Intensity Normalized

0.10

0.05

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 Chemical Shift (ppm)

SDI 05 1.0 130719-ROC-SDI05.001.001.1R.esp

0.9

0.8

0.7

0.6

0.5

0.4 Normalized Intensity Normalized

0.3

0.2

0.1

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

183

SDI 80

SDI 100 0.055 130820-ROC-SDI-139-141-100.009.001.1R.esp

0.050

0.045

0.040

0.035

0.030

0.025

Normalized Intensity Normalized 0.020

0.015

0.010

0.005

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

184 SDI 139 130820-ROC-SDI-139-141-100.001.001.1R.esp

0.15

0.10 Normalized Intensity Normalized

0.05

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

185 Reference

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