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Endophytes of Pseudowintera Colorata (Horopito)

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Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Endophytes of Pseudowintera colorata (horopito) ________________________________________ A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Microbiology and Microbial Ecology at Lincoln University by Neeraj Purushotham Balraj 2017 Lincoln University i Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy Endophytes of Pseudowintera colorata (horopito) By Neeraj Purushotham Balraj Pseudowintera colorata (horopito) commonly known as New Zealand pepper tree is a native medicinal plant, known for its antimicrobial properties. International studies have demonstrated that endophytes of medicinal plants play key roles in maintaining plant health, tolerance to biotic and abiotic stresses and production of secondary metabolites. However, there have been no studies on the endophytes of P. colorata. The objectives of this study were to i) investigate the community structure of the endophytic bacteria, Actinobacteria and fungi in the tissues of P. colorata and identify members of the core endomicrobiome, ii) investigate the bioactive potential of the culturable endophytic bacteria, Actinobacteria and fungi, iii) investigate the influence of selected members on the growth and chemistry of P. colorata in vitro and in vivo. The endophytic communities in P. colorata were characterized by denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq. Plants from ten sites across New Zealand were analysed by DGGE and it was revealed that tissue type was the main factor influencing the endophytic communities in P. colorata (PERMANOVA, P=0.001). Richness of Actinobacteria, Betaproteobacteria and fungi was higher in stems compared to leaves and roots. For a subset of three sites, the interaction of plant location with maturity influenced the microbial communities across all groups analysed except for Alphaproteobacteria and total fungi (PERMANOVA, P=0.226 and P=0.164 respectively). Using Illumina MiSeq for analysing the bacterial communities from ten sites, it was confirmed that tissue type affected the bacterial communities in P. colorata. Illumina data revealed that Gammaproteobacteria was the most abundant class (89.1%) followed by Alphaproteobacteria (10%). In addition, two OTUs belonging to Pseudomonas were identified as members of P. colorata core endomicrobiome. A total of 350 endophytic bacteria, 200 endophytic fungi and nine endophytic Actinobacteria were recovered from P. colorata plants from ten sites across New Zealand. The majority of ii endophytic bacteria were isolated from the stem (57.1%, n=200), followed by roots (37.1%, n=130) and leaves (5.7%, n=20). Eleven endophytic bacteria showed strong antagonistic activity against four phytopathogenic fungi Neofusicoccum luteum, N. parvum, Neonectria ditissima, Ilyonectria liriodendri and four endophytic bacteria were active against bacterial pathogens Pectobacterium atrosepticum, P. brasiliensis, Staphylococcus aureus, Escherichia coli. In addition, endophytic bacteria also produced siderophores on chrom-azurol S agar (CAS). Based on 16S rRNA gene, the endophytic bacteria were identified as members of genera Bacillus, Pseudomonas, and Pantoea. The endophytic fungi Pezicula sp. PRY2BA2, Metarhizium sp. PR1SB1 were active against all the phytopathogenic fungi tested. Six endophytic fungi showed high activity against Candida albicans in vitro. A total of nine endophytic Actinobacteria were recovered onto selective agar. Sequencing the 16S rRNA gene revealed that the culturable members belonged to genera Streptomyces, Micromonospora, Nocardia, Nakamurella and Microlunatus. Major bands (n=20) from DGGE gels were sequenced and were identified as uncultured bacteria, Streptomyces sp. and Angustibacter peucedani. Nocardia sp. TP1BA1B and Streptomyces sp. UKCW/B solubilized phosphate in tricalcium phosphate agar (TCP) and secreted siderophores. This is the first study to identify Actinobacteria communities in P. colorata and to examine the functional traits of cultured representatives. The effect of endophytes displaying in vitro activity was examined by reintroduction as a soil drench. Bacillus sp. TP1LA1B and Nocardia sp. TP1BA1B in addition to increasing the shoot height, also significantly increased the shoot, root biomass and the number of internodes of P. colorata seedlings compared to the control (P=0.016, P <0.001, P=0.007 and P <0.001 respectively). This is the first study to investigate the influence of endophytes on the growth of P. colorata. Overall, this study revealed the community structure of endophytic bacteria, Actinobacteria and fungi in P. colorata for the first time and is one of the only two studies on native plants in New Zealand. Members of the endomicrobiome displayed in vitro activity as measured by antimicrobial and nutrient mobilisation assays. The endophytes were able to influence host plant growth when applied as soil drenches and some were able to recolonize the host endophytically demonstrating a route from soil to root. This study indicated that P. colorata harbours unique endophytes which have a key role in the ecology of the plant. iii Keywords: Endophytes, Microbial Ecology, Medicinal Plant, Pseudowintera colorata, horopito, biological control, DGGE, NGS, Illumina MiSeq, Plant growth promotion, Endophytic Actinobacteria, biotransformation. iv Acknowledgements First and foremost, my sincere thanks to my lord, my saviour Shiridi Sai Baba for bestowing me with this opportunity to pursue my dream and for giving me strength, guidance and courage to complete my Ph.D. I would like to thank my supervisors Assoc. Prof. Hayley Ridgway, Assoc. Prof. Eirian Jones and my advisor Dr Jana Monk for their expert supervision, guidance and timely support throughout my entire study. My sincere thanks to Dr Nigel Perry and Dr John Van Klink at Plant and Food Research for their invaluable inputs and help with the NMR analysis in my study. I thank the New Zealand Ministry of Foreign Affairs and Trade (MFAT) for the Commonwealth scholarship, which has made this study possible. I extend my thanks to Lincoln University for funding this study and am also thankful for Lincoln University faculty research writing scholarship. Special thanks to ICPP 2003 inc. for the ICPP international travel grant to attend conferences in Canada and Germany. I would like to thank the lab managers of the plant pathology group Candice Hume and Sandy Hammond for their help with equipment and consumables. I extend my thanks to Stu Larsen for my helping me with my experimental work in Biotron, Neil Smith for helping me with freeze drying my samples, Brent Richards for providing me with space and equipment for my pot experiments in the glasshouse. I would like to thank Estelle, Thanh Le, Dr Celine Blond and John Ramana for helping me with my pot trial set up and harvest. I thank my colleagues at Lincoln University Dr Shanika Tennekoon, Anish Shah, Kritarth Seth, Dr Kathryn Wigely and Dr Wisnu Wicaksono for their advise on different aspects of my study and help during my Ph.D. My heartfelt thanks to my mom and dad for their blessings and support which has seen me through this study. My greatest thanks to my wife Neelima for bearing with me during my most stressful times and for all the unconditional love and support given to me to complete my study. A massive thank you to my son, my dearest Karthikey for being my reason, my solace and my motivation to find strength during the toughest of times and for me to strive and focus on completing my study. This thesis is dedicated to the most important people in my life.. my family. v Table of Contents Abstract……………………………………………………………………………………………………………………………ii Acknowledgements………………………………………………………………………………………………………….v Table of contents…………………………………………………………………………………………………………….vi List of Tables…………………………………………………...................................................................xi List of Figures………………………………………………………………………………….................................xii 1 Literature Review….………………………………………………………………………………………………………1 1.1. Endophytes ……………………………………………………………………………………………………………..1 1.1.1. Endophytic fungi……………………………………………………………………………………………1 1.1.2. Endophytic bacteria……………………………………………………………………………………….3 1.1.3. Endophytic Actinobacteria…………………………………………………………………………….4 1.2 The function of endophytes……………………………………………………………………………………….5 1.2.1 Nutrient uptake……………………………………………………………………………………………..6 1.2.2 Protection against phytopathogens……………………………………………………………....7 1.3 Commercial interest in endophytes……………………………………………………………………….....7 1.4 Endomicrobiome and functionality analysis………………………………………………………………8 1.4.1 Culture independent methods……………………………………………………………………….9 1.4.1.1 Denaturing gradient gel electrophoresis (DGGE)………………………………………9 1.4.1.2 DNA metabarcoding using next generation sequencing technologies……..10 1.4.2 Culture dependent techniques…………………………………………………………………….11 1.5 Endophytes of
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  • Pseudowintera Colorata) Leaves

    Pseudowintera Colorata) Leaves

    Korean J. Chem. Eng., 36(2), 272-280 (2019) pISSN: 0256-1115 DOI: 10.1007/s11814-018-0191-9 eISSN: 1975-7220 INVITED REVIEW PAPER INVITED REVIEW PAPER The potential use of pulsed electric field to assist in polygodial extraction from Horopito (Pseudowintera colorata) leaves Joanna Nadia, Marliya Ismail, Kaveh Shahbaz, and Mohammed Farid† Department of Chemical and Materials Engineering, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand (Received 30 July 2018 • accepted 14 November 2018) AbstractHoropito (Pseudowintera colorata) contains polygodial as an active compound that has many health benefi- cial properties. The potential of applying a continuous pulsed electric field (PEF) as a pretreatment step prior to sol- vent extraction of polygodial from Horopito leaves was studied. Horopito leaves suspended in water were subjected to PEF at electric field intensity ranging from 5 to 25 kV/cm and pulse frequencies from 200 to 800 Hz. The interaction between electric field intensity and pulse frequency was found to have a significant role in extraction. Both electro-per- meabilization and temperature increase from treatment caused some polygodial leaching from the leaves prior to sol- vent extraction. The study revealed that PEF at low electric field intensity and high frequency is the most effective way to achieve higher solvent extraction yield while minimizing the effect of leaching. The maximum improvement was obtained when PEF at 5 kV/cm and 800 Hz for 348 s were applied, giving a polygodial extraction yield of about 16.6% higher than that of non-PEF treated leaves. Keywords: Extraction, Horopito (Pseudowintera colorata), Polygodial, Pulsed Electric Field INTRODUCTION organic solvent [5,17], hydrodistillation [18], and steam distillation [19].