FUNGI IN LOWLAND RIVER FLOODPLAIN ECOSYSTEMS Submitted by Janice Laraine Williams B. App. Sc. (Env. Anal.), B. Sc. (Hons) A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy Department of Environmental Management and Ecology School of Life Sciences Faculty of Science, Technology and Engineering La Trobe University Bundoora, Victoria 3086 Australia November 2010 Frontispiece: Sexual reproductive structures of an aquatic Oomycete growing on Eucalyptus camaldulensis leaves submerged in a laboratory mesocosm. ii Table of Contents Table of Contents iii List of Figures vi List of Tables xviii Abstract xix Statement of Authorship xx Acknowledgements xxi Chapter 1 1 General Introduction 1 1.1 Ecological Studies of Fungi in Australia 1 1.2 Floodplain Wetland Ecosystems 1 1.3 Carbon Cycles in Wetlands 2 1.4 Aim 2 1.5 Approach 2 1.6 Organisation of the Thesis 3 Chapter 2 5 Carbon Cycling in Lowland River Floodplain Wetlands 5 2.1 Introduction 5 2.2 The Breakdown and Decomposition of Plant Detritus 7 2.3 The Influence of Environmental Conditions 10 2.4 The Wetland Biota 21 2.5 Wetland Carbon Cycles 24 2.6 Plant detritus as a carbon resource 36 2.7 Fungal Ecology 38 2.8 Summary 52 Chapter 3 54 Pilot Study: Fungal Community Structure on a variety of substrates from within a floodplain wetland. 54 3.1 Introduction 54 3.2 Materials and Methods 56 3.3 Results 60 iii 3.4 Discussion 64 3.5 Conclusions 69 Fungal activity on Eucalyptus camaldulensis leaves under aquatic conditions is enhanced by terrestrial aging. 70 4.1 Introduction 70 4.2 Methods 72 4.3 Results 77 4.4 Discussion 99 4.5 Conclusions 108 Fungi in Drying Wetland Sediments 110 5.1 Introduction 110 5.2 Materials and Methods 112 5.3 Results 119 5.4 Discussion 127 5.5 Conclusions 131 Fungal biomass accumulation on E. camaldulensis leaves differs between rivers and wetlands 133 6.1 Introduction 133 6.2 Methods 136 6.3 Results 147 6.4 Discussion 166 6.5 Conclusions 177 Synchrotron FTIR micro-spectroscopy reveals chemical changes accompanying aquatic leaf decay. 179 7.1 Introduction 179 7.2 Materials and Methods 183 7.3 Results 191 7.4 Discussion 225 7.5 Conclusions 235 Chapter 8 237 General Discussion 237 8.1 Carbon Cycling in Australian Floodplain Wetlands 237 iv 8.2 The Importance of Fungi in Australian Floodplain Wetlands 240 8.3 Additional Observations 242 8.4 Knowledge Gaps 247 8.5 Future Directions 249 8.6 Concluding Remarks 250 Appendix 1: Optimising Noise Reduction 251 References 252 v List of Figures Figure 2.1: A stylised representation of the carbon compartments and fluxes found in most wetland systems. Based on Webster and Benfield, 1986. 26 Figure 3.1: A map of Normans Lagoon, showing the position of sample sites. The inset shows the location of Albury-Wodonga, situated on the Murray River, which forms the border between the states of New South Wales and Victoria. Normans Lagoon is found on the north side of the Murray River, south-east of Albury, NSW. 56 Figure 3.2: The western site at Normans Lagoon. The riparian vegetation at this site is dominated by Eucalyptus camaldulensis. Outside this zone, the vegetation is influenced by rural land use. 57 Figure 3.3: MDS plot of all T-RFLP data collected from Normans Lagoon. Sediment samples are shown in black, floc samples are shown in grey and plant detritus samples are shown in white. Symbols, as shown in the legend, have been used to differentiate between substrate types. 61 Figure 3.4: MDS plot of T-RFLP data from sediment and floc samples. Samples from the west lagoon site are shown in black, samples from the east lagoon site are shown in grey, and samples from the river at Doctors Point are shown in white. Symbols indicate sediment moisture class, i. e. dry (star), damp (diamond), water’s edge (squares), inundated (hexagons), deep (circles) and reed bed (triangles). Overlaid circles are intended to indicate site groups and have no statistical meaning. 62 Figure 3.5: MDS plot of T-RFLP data from sediment and floc samples from the Normans Lagoon west site. Symbols indicate sediment moisture class, i. e. dry (stars), damp (diamonds), water’s edge (squares), inundated (hexagons), deep (circles), and reed bed (triangles). Dry samples are shown in white, damp samples in grey and wet sediments in black. Overlaid circles illustrate samples are grouped by sediment moisture content, but have no statistical meaning. 63 Figure 3.6: MDS plot of T-RFLP data from plant detritus samples from Normans Lagoon. The west site is shown in black, while the east site is shown in grey. Overlaid circles indicate plant species groupings but have no statistical meaning. 64 Figure 4.1: A schematic diagram of mesocosm design. All mesocosms were comprised of fish tanks, with a 2cm layer of wetland sediment in the bottom. This was covered vi with nylon mesh, and leaf packs were placed upon the mesh. Leaf packs were covered with weighted wire. In wet treatments, filtered wetland water was added to a depth of 15cm. 73 Figure 4.2: Mass loss in the dry (i), moist (ii), and wet (iii) treatments over 140 days. Fresh leaves are indicated by closed symbols while aged leaves are indicated by open symbols. Mass loss, on the y-axis, is shown as percentage of the mass of the leaf pack prior to incubation. Error bars indicate the standard error of the data point. 80 Figure 4.3: Changes in organic matter content of leaf packs over 140 days are shown for the dry (i), moist (ii), and wet (iii) treatments. Fresh leaves are shown as closed symbols while aged leaves are shown as open symbols. Organic matter content (y- axis) is expressed as a percentage of the leaf dry mass. Error bars indicate the standard error of the data point. 81 Figure 4.4: Fungal biomass (mg.cm-2) on eucalypt leaves, as calculated from ergosterol concentration. Differences in fungal biomass between aged (open symbols) and fresh (closed symbols) leaves are shown for the dry (i), moist (ii) and wet (iii) treatments. Error bars indicate the standard error of the data point. 83 Figure 4.5: A three dimensional MDS plot based on a 2-stage resemblance matrix for oxidative and hydrolytic enzyme data for all times, treatments and leaf types. Fresh leaves are represented by solid symbols, while aged leaves are shown as open symbols. Treatments are symbolised by stars (dry treatment), diamonds (moist treatment) and circles (wet treatment). Arrows indicate that the direction of change in enzyme activity with different treatments differs between fresh leaves (solid arrow) and aged leaves (dashed arrow). Fresh and aged leaves in the wet treatment have similar enzyme activity. 86 Figure 4.6: The activity of cellobiohydrolase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 87 Figure 4.7: The activity of β-D-glucosidase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate vii (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 89 Figure 4.8: The activity of α-D-glucosidase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 90 Figure 4.9: The activity of β-D-xylosidase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 92 Figure 4.10: The activity of phenol oxidase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 94 Figure 4.11: The activity of peroxidase on aged (open symbols) and fresh (solid symbols) E. camaldulensis leaves in the dry (i), moist (ii) and wet (iii) treatments. Incubation time is indicated in days on the x-axis, while enzyme activity rate (µmol substrate converted per hour, per gram leaf dry weight) is shown on the y- axis. Error bars indicate the standard error of the data point. 95 Figure 4.12: An MDS plot of T-RFLP data from fresh (closed symbols) and aged (open symbols) E. camaldulensis leaves prior to incubation indicates that different fungal communities are present on the two leaf types (2D stress is 0.11).