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THE EFFECT of SILVER NANOPARTICLES on Trichoderma Harzianum, Rhizoctonia Spp., and FUNGAL SOIL COMMUNITIES

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THE EFFECT of SILVER NANOPARTICLES on Trichoderma Harzianum, Rhizoctonia Spp., and FUNGAL SOIL COMMUNITIES THE EFFECT OF SILVER NANOPARTICLES ON Trichoderma harzianum, Rhizoctonia spp., AND FUNGAL SOIL COMMUNITIES Hartati Oktarina Doctor of Philosophy School of Biology Newcastle University June 2017 Declaration I declare that this thesis is the result of my own investigations and that no part of it has been submitted for any degree other than Doctor of Philosophy at the Newcastle University. Where other sources of information have been used, they have been dully acknowledged. Hartati Oktarina ii Abstract Silver nanoparticles (AgNPs) have known anti-microbial properties and are applied in many industrial applications ranging from medicine to fabric preservation. Recently, researchers have proposed the use of AgNPs in agriculture to control plant pathogenic fungi. The use of AgNPs to control plant pathogen fungi does raise concerns as they may potentially affect the activity of beneficial soil microorganisms. In this work the effects of AgNPs on the plant pathogenic fungus, Rhizoctonia spp., and their biocontrol agent, Trichoderma harzianum, and fungal soil communities were investigated. The results showed that T. harzianum mycelium growth was very tolerant to high levels of AgNPs (up to 600 mg L-1) while Rhizoctonia spp. were more sensitive (mycelial growth was affected at 20 mg L-1). Nevertheless, AgNPs effect on reproductive stage of T. harzianum, e.g. spore production, was not clear as it only showed on one concentration. Despite the decrease in spore production of T. harzianum after AgNPs exposure, the spores successfully germinated when cultivated on fresh growth medium (more than 60%). Following up these findings, T. harzianum and AgNPs were combined to examine the synergistic potential of these chemical and biological controls on growth of Rhizoctonia spp. Interestingly, the combination of AgNPs and Trichoderma did not appear to act synergistically to reduce Rhizoctonia growth in vitro. In subsequent work the effect of AgNPs contamination on soil fungal communities was assessed by Illumina MiSeq Next Generation Sequencing (NGS) and processed using the UPARSE pipeline run with USEARCH. The soil contamination experiments were carried out over a period of 2 years as previous studies have only examined effects of AgNPs contamination over a few months. Before analysing the metabarcoding data from the Illumina sequencer, a method was developed to find a suitable technique to process the data. It was found that single forward read sequences produced more operational taxonomic unit (OTU) than single reverse and paired end sequences. Therefore, single forward read sequences were used to investigate the effect of AgNPs on soil fungal communities in this study. Soil contamination by AgNPs reduced fungal species richness, evenness, and changed the community structure. For example, species such as Cryptococcus terreus was the most abundant in controls but these were replaced by other species including Trichocomaceae sp. in AgNPs contaminated soil. Tolerant species, such as T. spirale were identified in highly contaminated soil (660 mg kg-1 of AgNPs) and this species has been found in previous studies examining metal contamination. Overall the findings from this thesis suggest that more intensive study will be required when considering AgNPs as an alternative to synthetic fungicides to control plant pathogenic fungi as they have a negative impact on the fungal community in soil even at lower levels e.g. 3 mg kg-1 of AgNPs. iii Acknowledgments All praise be to Allah the Almighty. He is the one who gave me courage to gain knowledge and made it possible to accomplish my study. All respects are for His prophet Muhammad (Peace be Upon Him). I offer my gratitude to Prof. Ian Singleton for supervising and guiding me through my study time. Also to my second supervisor Prof. Anne Borland. I have received support from people within School of Biology. For that I would like to thank to Dr. Matthew Peake, Dr. Kirsten Wyness, Ros Brown for their help with laboratory matters, and to my colleagues for their friendships. I owe my appreciation to Dr. James Woodhall for providing isolates, Dr. Lidija Siller for nanoparticles analysing, Michael Botha for collecting soil sample from Cockle Park Farm, and to Fiona Maclachlan for soil analysing. My acknowledgment also goes to DIKTI (Ministry of Research, Technology and Higher Education of the Republic of Indonesia) for their financial support, to Syiah Kuala University for their administration support and to Indonesia Community in Newcastle Upon Tyne for making me feel like home. Last but not least I would like to express my deepest gratitude to my parents and my family for their support, affection, and patience in accompany me through my PhD life. I dedicate this achievement to them. iv Table of Contents Declaration ................................................................................................................ ii Abstract ..................................................................................................................... iii Acknowledgements ................................................................................................... iv Table of Contents ...................................................................................................... v List of Figures ........................................................................................................... ix List of Tables ............................................................................................................. xiv Nomenclature ............................................................................................................ xvi Chapter I. General Introduction 1.1 Introduction ....................................................................................................... 2 1.2 The Application of Nanoparticles ..................................................................... 3 1.3 Classification of Nanoparticles ......................................................................... 4 1.4 Synthesis of Nanoparticles ................................................................................ 6 1.5 Silver Nanoparticles (AgNPs) ........................................................................... 8 1.6 Release of AgNPs into the Environment .......................................................... 8 1.7 Effect of AgNPs on Soil Microorganisms and Other Soil-associated Organisms 9 1.8 Effect of AgNPs on Plants ................................................................................ 10 1.9 Anti-microbial Mechanism of AgNPs .............................................................. 10 1.10 Trichoderma Species Roles ............................................................................... 11 1.11 Biological Control Mechanisms of Trichoderma ............................................. 12 1.11.1 Competition through rhizosphere competence ....................................... 12 1.11.2 Mycoparasitism and antibiotic (toxin) production ................................. 13 1.11.3 Enzyme production ................................................................................ 14 1.12 Factors Affecting the Success of Trichoderma spp. as a Biological Control Agent ................................................................................................................. 14 1.14 Project Aims ...................................................................................................... 16 Chapter II. The Effect of Silver Nanoparticles on Trichoderma harzianum Growth in Vitro 2.1 Introduction ....................................................................................................... 18 2.2 Materials and Methods ...................................................................................... 19 2.2.1 Fungal isolation ........................................................................................ 19 2.2.2 Morphological identification .................................................................... 19 2.2.3 Molecular identification ........................................................................... 19 A. Deoxyribonucleic acid (DNA) extraction ............................................. 19 v B. DNA quality check ................................................................................ 21 C. Polymerase chain reaction (PCR) amplification ................................... 21 D. DNA agarose gel electrophoresis .......................................................... 22 2.2.4 Characterisation of silver nanoparticle (AgNPs) ..................................... 22 2.2.5 The impact of AgNPs on T. harzianum colony diameter ......................... 23 2.2.6 The impact of AgNPs on T. harzianum spore production ....................... 23 2.2.7 The impact of AgNPs on T. harzianum spore viability ........................... 23 2.2.8 Statistical analysis .................................................................................... 24 2.3 Results ............................................................................................................... 24 2.3.1 Identification of fungal isolates ................................................................ 24 2.3.2 AgNPs analysis ........................................................................................ 26 2.3.3 The impact of AgNPs on T. harzianum colony diameter ......................... 28 2.3.4 The impact of AgNPs on T. harzianum spore production ....................... 30 2.3.5 The impact of AgNPs on T. harzianum
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