1 Genetic analysis and substrate utilization of fungal isolates from the standing dead material of the moss Schistidium apocarpum from a High Arctic site A thesis submitted to the University of Manchester for the degree of MPhil in the Faculty of Engineering and Physical Sciences 2011 Garwai Leung School of Earth, Atmospheric and Environmental Sciences 2 Contents List of figures and tables Page 7 Abstract Page 9 Declaration Page 10 Copyright statement Page 11 Acknowledgements Page 12 Chapter 1: Introduction Page 13 1.1 Arctic environment Page 13 1.2 What has been found? Page 14 1.2.1 Fungal taxa Page 14 1.2.2 Function Page 14 1.2.2.1 Endophyte Page 15 1.2.2.2 Saprotrophic Page 15 1.2.3 Habitat Page 16 1.3 Which techniques to use for fungal identification and to determine breakdown of carbon substrates? Page 16 1.3.1 Traditional techniques Page 17 1.3.2 BIOLOG Page 17 1.3.3 Genetic analysis Page 17 3 1.4 Substrate utilization Page 18 1.4.1 Casein Page 19 1.4.2 Cellulose Page 19 1.4.3 Chitin Page 20 1.4.4 Lignin Page 20 1.4.5 Starch Page 21 1.4.6 Tannic acid Page 22 1.4.7 Pectin Page 22 1.4.8 Xylan Page 23 1.5 Gaps in knowledge Page 24 1.6 Aims and hypothesis Page 25 Chapter 2: Methods Page 26 2.1 DNA Extraction Page 26 2.2 DNA Amplification Page 27 2.3 Sequencing Page 28 2.4 Subculturing of fungal isolates Page 29 2.5 Mycelia extension rate and carbon substrate tests Page 30 2.5.1 Mycelial extension rate test Page 31 2.5.2 Casein medium Page 31 2.5.3 Cellulose medium Page 32 2.5.4 Chitin medium Page 32 4 2.5.5 Lignin medium Page 33 2.5.6 Pectin medium Page 33 2.5.7 Starch medium Page 34 2.5.8 Tannic acid medium Page 34 2.5.9 Xylan medium Page 34 Chapter 3: Results Page 35 3.1 Identifications Page 35 3.2 Phylogenetic trees Page 37 3.3.1 Cumulative mycelia extension Page 42 3.3.2 Mean Extension Rate Page 45 3.4 Carbon substrate tests Page 48 3.4.1 Casein Page 49 3.4.2 Cellulose Page 49 3.4.3 Chitin Page 50 3.4.4 Lignin Page 50 3.4.5 Pectin Page 50 3.4.6 Starch Page 51 3.4.7 Tannic acid Page 52 3.4.8 Xylan Page 53 Chapter 4: Discussion Page 54 4.1 Isolate identities Page 54 5 4.1.1 Cadophora luteo-olivacea Page 54 4.1.2 Debaryomyces hansenii Page 54 4.1.3 Fimetariella rabenhorstii Page 55 4.1.4 Hypocrea viridescens Page 55 4.1.5 Monodictys arctica Page 55 4.1.6 Penicillium camemberti Page 56 4.1.7 Phoma sclerotioides Page 56 4.1.8 Phoma herbarum Page 57 4.2 Relationship between temperature and extension rate Page 57 4.3 Inference of function from substrate utilization Page 58 4.4 Temperature relation to substrate utilization Page 59 4.5 Future work Page 59 Chapter 5: Conclusion Page 60 References Page 62 Appendices Page 68 Appendix 1 Page 68 Appendix 2 Page 69 Appendix 3.1 Page 70 Appendix 3.2 Page 71 6 Appendix 4 Page 74 Appendix 5 Page 79 Appendix 6.1 Page 85 Appendix 6.2 Page 104 Appendix 6.3 Page 121 Final word count: 17,985 7 List of Figures and Tables Chapter 1: Introduction Figure 1.1- Map of Svalbard Page 13 Figure 1.2- ITS section of fungal DNA Page 18 Figure 1.3- Cellulose structure Page 19 Figure 1.4- Chitin structure Page 20 Figure 1.5- Lignin structure Page 21 Figure 1.6- Starch structure Page 21 Figure 1.7- Tannic acid structure Page 22 Figure 1.8- Pectin structure Page 23 Figure 1.9- Xylan structure Page 24 Chapter 2: Methods Table 2.1- List of isolates grouped by morphology from Schistidium apocarpum and Dryas octopela and isolates subcultured in this study Page 27 Table 2.3- List of components for a PCR reaction Page 28 Chapter 3: Results Table 3.1- Identities of subcultured isolates and percentages to Page 36 Figure 3.1.1- Complete phylogenetic tree of isolates Page 39 Figure 3.1.2- Phoma sclerotioides and Monodictys arctica tree section of Figure 3.1.1 Page 40 Figure 3.1.3- Phoma herbarum and Penicillium tree section of Figure 3.1.1 Page 41 Figure 3.1.4- Remaining tree section of figure 3.1.1 Page 42 Figure 3.2.1- Mean cumulative mycelial extension, in millimetres, at 4oC Page 43 Figure 3.2.2- Mean cumulative mycelial extension, in millimetres, at 10oC 8 Page 44 Figure 3.2.3- Mean cumulative mycelial extension, in millimetres, at 25oC Page 45 Figure 3.3.1- Mean extension rate of Phoma sclerotioides isolates at all temperatures Page 46 Figure 3.3.2- Mean extension rate of Penicillium taxa at all temperatures Page 46 Figure 3.3.3- Mean extension rate of mesophilic taxa at all temperatures Page 47 Figure 3.3.4- Mean extension rate of Phoma herbarum and Monodictys arctica at all temperatures Page 47 Table 3.2- Average reaction strength to carbon substrates in semi-defined solid media Page 48 Figure 3.4.1- Picture of negative to strongly positive reactions of casein test Page 49 Figure 3.4.2- Picture of negative to strongly positive reactions of cellulose test Page 50 Figure 3.4.5- Picture of negative to strongly positive reactions of pectin test Page 51 Figure 3.4.6- Picture of negative to strongly positive reactions of starch test Page 52 Figure 3.4.7- Picture of negative to strongly positive reactions of tannic acid test Page 53 Appendix Table A1- Original isolate sequencing data Page 79 Figure A1- MP phylogenetic tree of Phoma sclerotioides ITS sequences Page 81 Figure A2- ML phylogenetic tree of Phoma sclerotioides ITS sequences Page 82 Figure A3- MP phylogenetic tree of ITS, G3P, HIS loci of Ph. Sclerotioides Page 83 Figure A4- MP phylogenetic tree of ITS, G3P, HIS loci of Ph. Sclerotioides Page 84 9 Abstract The University of Manchester Garwai Leung MPhil Genetic analysis and substrate utilization of fungal isolates from the standing dead material of the moss Schistidium apocarpum from a High Arctic site 2011 Fungi isolated from the litter of the moss Schistidium apocarpum, from a site in Svalbard, Norway (78°56 N, 11°50 E), were placed into 12 different groups using culture morphology. Representative isolates from each group were then subcultured and identified by sequencing the Internal Transcribed Spacer region (ITS1-5.8S-ITS2) of the ribosomal DNA. Sequences were compared to species from the BLASTn database and were aligned using CLUSTALW. A phylogenetic tree was constructed using MEGA 5 and bootstrapped 1000 times. Subcultured isolates were tested to see if they could degrade several pure carbon sources (casein, cellulose, lignin, pectin, starch, tannic acid, and xylan) as analogues of carbon substrate, found in plant litter, at 6oC. Isolates were also grown at three different temperatures (4, 10 and 25oC) and mycelia extension rates were measured. The majority of isolates were identified as Phoma sclerotioides, a known cause of brown root rot in alfalfa in temperate regions with harsh winters. Other isolates identified included Debaryomyces hansenii, Fimetariella rabenhorstii, Hypocrea viridescens, Monodictys arctica, Penicillium camemberti and Phoma herbarum. Isolate identities and enzyme activity were similar to mid-late stage of decomposition although isolates were from standing-dead material and it is possible to be at the start of decomposition. This study shows a variety of fungi have the potential to utilize carbon sources from Schistidium apocarpum litter at low temperatures, therefore they are likely to be contributing to the mineralization of carbon in the arctic environment. 10 Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. 11 Copyright statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and he has given The University of Manchester certain rights to use such copyright including for administrative purposes. ii. 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Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses 12 Acknowledgements I would like to take this opportunity to show my gratitude to all those who supported me throughout this thesis.