The role of phytotoxic and antimicrobial compounds of Euphorbia gummifera in the cause and maintenance of the fairy circles of Namibia by Nicole Galt Submitted in partial fulfillment of the requirements for the degree Magister Scientiae Department of Plant and Soil Sciences Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria Supervisor: Prof. J.J.M. Meyer March 2018 i The role of phytotoxic and antimicrobial compounds of Euphorbia gummifera in the cause and maintenance of the fairy circles of Namibia by Nicole Galt Department of Plant and Soil Sciences Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria Supervisor: Prof. J.J.M. Meyer Degree: MSc Medicinal Plant Science Abstract Fairy circles (FC) are unexplained botanical phenomena of the pro-Namib desert and parts of the West Coast of South Africa. They are defined as circular to oval shaped anomalies of varying sizes that are left bereft of vegetation. Even though there are several distinctly different hypotheses that have aimed to explain the origin of fairy circles, none have done so to satisfaction of the scientific community. The aim of this study was to determine if phytotoxic and antibacterial properties of a co-occurring Euphorbia species, E. gummifera plays a role in the creation of fairy circles. Representative soil samples (from inside-, outside fairy circles and underneath dead E. gummifera plants) and plant samples (aerial ii parts of E. gummifera and intact grasses, Stipagrostis uniplumis) were collected from the area. The collected samples were used for a several biological assays. A soil bed bio-assay was done using the three collected soil types. A germination inhibition study was done using a methanolic E. gummifera extract. A soil-agar bio-assay was done with collected soil as well as with rhizosphere soil from grass roots. All data was analyzed statistically. The E. gummifera methanolic extract was used to test its antibacterial activity against several bacterial species. Among the tested bacteria were two isolates from the rhizosphere soil that were identified through 16S rRNA sequencing. Several compounds with biological activity of interest were identified through silica column chromatography and gas chromatography mass spectrometry in the E. gummifera methanolic extract. The results from the germination inhibition assay indicated that E. gummifera does possess phytotoxic properties in terms of significant germination inhibition (P-value <0.05) at concentrations between 40mg/ml to 10mg/ml extract if the seeds were water stressed. The results of the soil bed bio-assay further corroborated the synergistic theory. The phytotoxic soil from underneath E. gummifera prevented germinated seeds to transition to mature seedlings when water stressed. The soil-agar bio-assay indicated that rhizosphere soil had a stimulatory effect on germination while the dead plant soil and soil from inside FCs inhibited germination significantly. The methanolic extract exhibited antibacterial activity against the two identified rhizosphere isolates, Kocuria polaris and Pseudomonas paravulva, as well as other bacterial species tested in the study. Several compounds that have previously been found in other studies to have antibacterial and phytotoxic activity were identified. In conclusion, due to E. gummifera’s phytotoxicity (possibly allelopathy) and antibacterial activity, especially under water stressed conditions, a possible cause for the creation of fairy circles has been identified in the southern parts of Namibia where fairy circles co- occur with E. gummifera. iii Table of contents Chapter 1: Introduction 1.1 Introduction ........................................................................................................................ 5 1.2 Literature review: Fairy circle theories ............................................................................ 7 1.2.1 Insect related theories .................................................................................................... 7 1.2.1.1 Shortcomings and discrepancies of the insect related theories .................................. 20 1.2.2 Vegetation patterning ....................................................................................................... 25 1.2.2.1 Shortcomings and discrepancies of the vegetation patterning theories ............... 30 1.2.3 Hydrocarbon microseepage and other gas related theories ......................................... 33 1.2.3.1 Shortcomings and discrepancies of the gas-related theory .................................. 40 1.2.4 Microbial related theories ............................................................................................ 41 1.2.5 Allelopathy theory ....................................................................................................... 49 1.3 Aims and objectives .......................................................................................................... 51 1.4 References .......................................................................................................................... 54 Chapter 2: Allelopathic properties of Euphorbia gummifera 2.1 Introduction ........................................................................................................................... 66 2.1.1 History of plant allelopathy .............................................................................................. 66 2.1.2 Important aspects of the allelopathic process in the environment .................................... 67 2.1.2.1 Release of allelochemicals into the environment ...................................................... 67 ............................................................................................................................................... 68 2.1.2.2 Fate of allelochemicals in the environment ............................................................... 69 2.1.2.3 Phytotoxic effects/mode of action of allelochemicals on target plant ....................... 70 2.1.3 The Euphorbia genus and allelopathy .............................................................................. 75 2.2 Aims and objectives ............................................................................................................... 77 2.3 Materials and methods .......................................................................................................... 77 2.3.1 Field collection ................................................................................................................. 77 2.3.1.1 Soil collection ............................................................................................................ 77 2.3.1.2 Plant collection .......................................................................................................... 78 iv 2.3.2 Extract preparation ........................................................................................................... 78 2.3.3 Germination inhibition assay ............................................................................................ 79 2.3.4 Soil-bed bioassay .............................................................................................................. 80 2.3.5 Soil-agar bioassay ............................................................................................................. 80 2.4 Results ..................................................................................................................................... 81 2.4.1 Extract preparation ........................................................................................................... 81 2.4.2 Germination inhibition assays .......................................................................................... 82 2.4.2.1 Germination inhibition assay- Not water stressed ..................................................... 82 2.4.2.2 Germination inhibition assay- Water stressed ........................................................... 82 2.4.2.3 Germination inhibition assay using S. uniplumis extract .......................................... 84 2.4.2.4 Germination inhibition assay with the separated E. gummifera extract .................... 85 2.4.3 Soil-bed bioassay .............................................................................................................. 87 2.4.4 Soil-agar bioassay ............................................................................................................. 89 2.4.4.1 Soil-agar bioassay using nutrient agar ....................................................................... 89 2.4.4.2 Soil-agar bioassay using pure agar ............................................................................ 90 2.5 Discussion ............................................................................................................................... 91 2.5.1 Germination inhibition assays .......................................................................................... 91 2.5.1.1 Germination inhibition assay- Not water stressed ..................................................... 91 2.5.1.2 Germination inhibition assay- Water stressed ........................................................... 92 2.5.1.3 Germination inhibition assay using S. uniplumis extract .......................................... 93 2.5.1.4 Germination inhibition assays with the two parts of the E. gummifera extract ......... 93 2.5.2 Soil-bed bioassay .............................................................................................................