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Needle Fungi and Tree Health of Pinus Radiata in Tasmania

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Needle Fungi and Tree Health of Pinus Radiata in Tasmania Needle fungi and tree health of Pinus radiata in Tasmania Istiana Prihatini M. Sc. (Gadjah Mada University, Indonesia) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of Tasmania October 2014 i DECLARATION OF ORIGINALITY This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief, contain no copy of material previously published or written by another person, except where due reference is made in the text of the thesis. This thesis does not contain any material that infringes copyright. AUTHORITY OF ACCESS This thesis may be made available for loan. Copying of any part of this thesis is prohibited for two years from the date this statement was signed; after that time limited copying and communication is permitted in accordance with the Copyright Act 1968. STATEMENT ON PUBLISHED WORK The publishers of the papers comprising Chapter 3, 4, 5 and 6 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. October, 2014 ii STATEMENT OF CO-AUTHORSHIP The following people and institutions contributed to the publication of work undertaken as part of this thesis: Istiana Prihatini, School of Land and Food, University of Tasmania (Candidate) Caroline Mohammed, School of Land and Food, University of Tasmania (Supervisor) Morag Glen, School of Land and Food, University of Tasmania (Co-supervisor) David Ratkowsky, School of Land and Food, University of Tasmania Tim Wardlaw, Forestry Tasmania Author details and their roles: Paper 1: Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania Located in Chapter 3 Istiana Prihatini (60%) designed and conducted the experiment, analysed the data and wrote the manuscript. Morag Glen (25%) and Caroline Mohammed (10%) contributed to experimental design, analysed the data, and edited the manuscript. As the industry partner, Tim Wardlaw (5%) advised on experimental design. Paper 2: Multigene phylogenetic study of Cyclaneusma species Located in Chapter 4 Istiana Prihatini (60%) designed and conducted the experiment, analysed the data and wrote the manuscript. Morag Glen (25%) and Caroline Mohammed (10%) contributed to experimental design, analysed the data, and edited the manuscript. As the industry partner, Tim Wardlaw (5%) advised on experimental design. iii Paper 3: Survey of needle fungi in Pinus radiata trees with varying levels of resistance to spring needle cast (SNC) Located in Chapter 5 Istiana Prihatini (60%) designed and conducted the experiment, analysed the data and wrote the manuscript. Morag Glen (25%) and Caroline Mohammed (10%) contributed to experimental design, analysed the data, and edited the manuscript. As the industry partner, Tim Wardlaw (5%) assisted with fieldwork and advised on experimental design. Paper 4: Needle fungi in Tasmanian Pinus radiata plantations in relation to elevation and rainfall. Located in Chapter 6 Istiana Prihatini (60%) designed and conducted the experiment, and wrote the manuscript. Morag Glen (20%) and Caroline Mohammed (10%) contributed to experimental design, and edited the manuscript. As the industry partner, Tim Wardlaw (5%) advised on experimental design. David Ratkowsky (5%) analysed the data. We the undersigned agree with the above stated “proportion of work undertaken” for each of the above published or submitted peer-reviewed manuscripts contributing to this thesis Istiana Prihatini, School of Land and Food, University of Tasmania (Candidate) Oct 2014 Assoc. Prof. Caroline Mohammed, School of Land and Food, University of Tasmania (Supervisor) Oct 2014 iv Dr Morag Glen, School of Land and Food, University of Tasmania (Co-supervisor) Oct 2014 Dr David Ratkowsky, School of Land and Food, University of Tasmania Oct 2014 Dr Tim Wardlaw, Forestry Tasmania Oct 2014 Prof. Holger Meinke, Head of School, School of Land and Food, University of Tasmania Oct 2014 v PUBLICATIONS AND COMMUNICATIONS Journal articles: 1. Prihatini I, Glen M, Wardlaw T.J. and Mohammed C. 2014. Multigene phylogenetic study of Cyclaneusma species. Forest Pathology Volume 44, Issue 4, 299–309. 2. Prihatini I, Glen M, Wardlaw T.J. and Mohammed C. 2014. Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania. Southern Forests (accepted subject to revision, currently under revision). 3. Prihatini I, Glen M. Wardlaw T.J. and Mohammed C. 2014. Survey of needle fungi in Pinus radiata trees with varying levels of resistance to spring needle cast (SNC). Forest Pathology (accepted subject to revision, currently under revision). 4. Prihatini I, Glen M, Wardlaw T.J. Ratkowsky D.A. and Mohammed C. 2014. Needle fungi in Tasmanian Pinus radiata plantations in relation to elevation and rainfall. New Zealand Journal of Forest Science (submitted, currently under review). Conference proceedings: 1. I. Prihatini, A.H. Smith, J.M. Gonnet, T.J. Wardlaw, C. Beadle, M. Glen and C.L. Mohammed, 2007. Linking environmental stress in pine plantation to fungal attack, 16th Biennial Australasian Plant Pathology Society Conference, 24 - 27 September 2007, Adelaide, Australia, poster presentation. 2. I. Prihatini, M. Glen, A.H. Smith, T.J. Wardlaw and C.L. Mohammed, 2009. Survey of the needle fungi associated with Spring Needle Cast in Pinus radiata, 17th Biennial Australia Plant Pathology Society Conference, 29 September - 1 October 2009, Newcastle, Australia, poster presentation. 3. M. Glen, I. Prihatini, A. Smith, C. Mohammed and T. Wardlaw, 2011. Spring needle cast in Tasmania—fungal communities and environmental factors, Inaugural joint 4th Asian Conference on Plant Pathology and the 18th Biennial Australasian Plant Pathology Society Conference, 26–29 April 2011, Darwin, Australia, oral presentation. vi ABSTRACT Pinus radiata is one of the most important species of forestry plantation worldwide and many pests and diseases have followed the expansion of this species around the globe. Tasmanian P. radiata plantations suffer from a needle cast disease referred to as spring needle cast (SNC) which has similar symptoms to Cyclaneusma needle cast, a serious disease in New Zealand. Stands with moderate or severe SNC can be expected to suffer potential losses in clear-fall volume. The aetiology of SNC is poorly understood and has long been a point of debate between Tasmania and New Zealand foresters with the New Zealanders attributing SNC to Cyclaneusma minus. Tasmanian research in the 90s proposed that SNC is caused by one or a suite of fungi that are triggered by an unknown environmental stress. Three species of fungi have been putatively associated with SNC, i.e. Lophodermium pinastri, Strasseria geniculata and C. minus. Understanding endophyte diversity is crucial for determining their role in forest health. Endophytes are increasingly recognized for having a role in plant-pathogen interactions leading to disease. In particular, they may play an important role in determining the presence of a disease such as SNC, which does not appear to have a single pathogenic causal agent and which may be associated with environmental stress. The main objective of this study was therefore to identify needle fungal communities which are associated with both healthy and SNC-affected trees in P. radiata plantations in Tasmania. Needle fungi have been extensively studied in conifers but rarely in P. radiata. Previous studies in P. radiata have mainly been based on fungal isolation. In this study, fungal communities were characterized by various detection methods including the isolation of pure cultures, morphotype grouping of cultures supported by the DNA sequencing, direct DNA analysis from needle tissue and DNA sequencing of cloned PCR products. A total of vii 544 isolates were obtained from 180 needle samples. The cultures were grouped according to macro-morphological characters, resulting in 26 morphological groups that each consisted of at least three cultures and 14 singletons. After phylogenetic analysis, the appropriate identity of each Operational Taxonomic Unit (OTU) was confirmed which resulted in 48 OTUs, including five basidiomycetous OTUs. The starting point of 98% sequence similarity for grouping of Molecular Operational Taxonomic Units (MOTUs) produced 130 MOTUs from 1303 sequences derived from DNA of fungal isolates and DNA extracted from P. radiata needles. After further analyses, a total of 154 MOTUs were recognised from isolates and DNA fragments amplified from pine needles. Only 10 of the 154 MOTUs were detected by direct DNA amplification and fungal isolation, 35 by isolation only, and the remaining 129 were detected by DNA only. The most frequently detected group of fungi detected from P. radiata needles in Tasmania was Dothideomycetes. Leotiomycetes was the second most frequently detected group, by culturing as well as by DNA amplification. Sordariomycetes was the third most frequently detected group, but the most highly represented by isolation. Most of the commonly isolated fungal species and many of the commonly detected MOTUs were consistent with previously known fungal associates of P. radiata or other pine or conifer species, e.g. species of Lophodermium, Cyclaneusma and Coniochaeta. PCR
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