Phosphorus uptake and utilization efficiency in cluster root and non-cluster root forming species of the Core Cape Subregion, South Africa Dunja Basic BSCDUN001 Thesis presented for the degree of Master of Science In the Department of Biological Sciences University of Cape Town February 2015 University of Cape Town Supervisors Dr Samson BM Chimphango, Assoc. Prof. Muthama A Muasya The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Podalyria calyptrata, seedlings grown in sand in the glasshouse (Photo: D Basic) Science: the creation of dilemmas by the solution of mysteries Brian Herbert and Kevin J. Anderson i Declaration I know the meaning of plagiarism and declare that all of the work in the document, save for that which is properly acknowledged, is my own. The thesis is submitted for the degree of Master of Science in the Department of Biological Sciences, University of Cape Town. It has not been submitted for any degree or examination at any other university. ______________________ Dunja Basic ii Abstract The Core Cape Subregion (CCR) is made up of a mosaic of highly weathered and nutrient leached soil substrates in the Western Cape. Plant available phosphorus (P) in these soils is very low, generally ranging from 0.4-3.7 µg P g-1 soil and as a result plants have evolved a number of traits to enhance P-acquisition, such as increased root surface area (SA) and specific root length (SRL), cluster root and root hair proliferation and exudation of organic acids and acid phosphatases (APase) from the roots. Crop yield is limited worldwide due to the unavailability of P and P-fertilization is showing limited success due to soil retention. Sustainable management of this would include exploiting plants with natural adaptations for enhanced P acquisition and utilization. The aim of this study was to discover whether cluster root forming species are more efficient at P acquisition than non-cluster root species. This was achieved by focusing on two objectives: (1) to characterize root traits for increased P acquisition in different soils of the CCR and (2) comparing P-uptake and utilization efficiencies of cluster root species to non-cluster root species under glasshouse and natural conditions. Plants from Fabaceae, Polygalaceae, Proteaceae, Cyperaceae, and Juncaceae were grown in two different glasshouse experiments and observed in a field study. In chapter one, a potted glasshouse experiment was conducted where selected species were grown in three different soil types for nine months before being analysed for root adaptations. In addition, a field study experiment was conducted where root samples were collected from two sites in the CCR with different soil types and analysed for organic acid exudation and phosphatase activity, and leaf samples were analyzed for N and P concentrations. The second chapter was another glasshouse experiment where plants were grown in a potted sand experiment under two P concentrations (10 and 25 mg.kg-1) for seven months before they were analysed for root adaptations. While cluster roots are a major adaptation for plants growing in P deficient conditions, they did not seem to give plants that produced them, an added advantage in P uptake or utilization. Polygala myrtifolia and Ficina trispicata were the most efficient species with regards to P uptake associated with their root morphology, while A. linearis and Pod. calyptrata were the most efficient species in P utilization most likely due to the addition of extra nitrogen they received through N2-fixation. It was shown that morphological traits such as total root length, total root SA, SRL, root diameter and root:shoot biomass ratios, along with carboxylate exudation are prime components in P acquisition in fynbos species. There is however, substantial genetic variation in all these traits and therefore selective breeding could be used to select for specific traits. iii Acknowledgements Firstly, a huge thank you to my supervisors, Dr Samson Chimphango and Assoc. Prof. Muthama Muasya, for providing the inspiration and necessary guidance for this project as well as opening my eyes to the wonders of plant physiology and sharing in their wisdom and knowledge. I would like to thank the technical staff at the Department of Biological Sciences, Des Barnes, Nazlie Davids, Gonzalo Aguilar, Dawood Hattas and the administration staff for all their assistance during my tenure. For assistance in the glasshouse as well as work on my samples, thanks to Saadiq Soeker as well as Edward Chirwa for doing a beautiful job grinding all my samples for further analysis. Thanks go to Pravin Mark Maistry for helping me understanding the methods and lab work needed for my study. For the use of his lab and equipment, while mine was under repair, as well as all his guidance throughout, I want to thank Simon Power. For the purchasing of my seeds and seedlings, thanks go to Rachel Saunders from Silverhill Seeds and Books, Cape Town; Andrea Durrheim from New Plant Nursery, George; Brenda and Lianne from Veld and Fynbos Propagation Nursery, Malmesbury and lastly Gael Gray from Good Hope Gardens Nursery, Kommetjie. Thanks go to Ian Newton from Archeology, UCT, for analysis of my samples for nitrogen. For running my organic acid samples quickly and efficiently, thanks go to Wernich Kühn and Meryl Patience from the LCMS Laboratory, Central Analytical Facility, Stellenbosch. For soil and plant analyses, thanks go to Maryna Kruger and Alta Visagie from the Department of Agriculture, Elsenburg and Mariëtte Rossouw from the analytical laboratory Bemlab (Pty) Ltd., Somerset West. For statistical advice, thanks go to Katja Mauff from the Department of Statistical Sciences, UCT. This project would not be possible without the financial support of the National Research Foundation (NRF) and the following bursaries: Harry Crossley Foundation Post Graduate Scholarship, Masters Research Scholarship and the Botany Post Graduate Scholarship. iv Many thanks go to Nkosinathi Dludlu who kept the Legume group in the department mentally stimulated with the biweekly discussion groups which took us out of our own field of study and into another’s. Many thanks go to my fellow colleagues Benny Lemaire, Kolisa Sinyanya and Caitlynne Francis, who are always available for stimulating conversations whether related to work or not. Huge thanks go to Wade Lane and Caitlynne Francis who patiently read through my work and provided incredibly useful advice and suggestions. Thanks go to Donald and Heather MacAlister for accepting me into their family and supporting my work. Finally, a special thank you goes to my grandparents, Vladimir and Dušanka, my parents, Ivan and Zlata and my brother, Bojan, without whom this would not have been possible. Whether it was helping with funds, driving me to campus every weekend to water my plants, inspiring my interest in the world and refusing to give up on me, I couldn’t ask for a more loving and supportive family. Last, but not least, a big thank you to my fiancé, John MacAlister, for all his support and for keeping me motivated, happy and feeling loved during all the work. v Table of Contents Declaration................................................................................................................................... ii Abstract ....................................................................................................................................... iii Acknowledgements .................................................................................................................... iv Chapter 1 – General Introduction ............................................................................................. 1 1.1 Soils of the CCR ......................................................................................................... 2 1.2 Phosphorus Availability .............................................................................................. 2 1.3 Adaptations to a low P lifestyle .................................................................................. 3 1.4 Mechanisms for phosphorus acquisition ..................................................................... 4 1.5 Problem Statement ...................................................................................................... 7 1.6 Hypothesis and thesis outline...................................................................................... 8 Chapter 2 – Morphological and physiological mechanisms for phosphorus acquisition of Fynbos species in different soils of the Western Cape ........................................................... 10 2.1 Introduction ............................................................................................................... 10 2.2 Materials and Methods .............................................................................................. 17 2.2.1 Species and growth conditions................................................................... 17 2.2.2 Fieldwork ................................................................................................... 18 2.2.3 Nutritional analysis of soil ........................................................................