Thesis (PhD (Microbiology))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy in Microbiology at the University of Stellenbosch. / ENGLISH ABSTRACT: The interaction between a soil yeast, Cryptococcus laurentii and a medicinal plant, Agathosma betulina (Berg.) Pillans (Rutaceae), was studied. Cryptococcus laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina and liquid chromatographytandem mass spectrometry analysis revealed that the yeast was capable of producing polyamines such as cadaverine and spermine. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when A. betulina seedlings grown under axenic and low nutrient conditions were inoculated with C. laurentii CAB 578 and cultivated for five months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. Furthermore, fluoromicroscopy, in combination with the fluorescent probes Calcofluor White and Fungolight revealed the presence of metabolic active yeast colonies on the rhizoplane. The first part of the study thus provided evidence for a symbiosis between A. betulina and C. laurentii CAB 578. During the second part of the investigation, the effect of this symbiosis on quantitative elemental distribution in A. betulina roots grown under axenic, nutrient-poor conditions was assessed using micro-particle-induced x-ray emission spectrometry. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers and endophytic C. laurentii CAB 578 in root tissues were located using fluorescence microscopy and transmission electron microscopy, respectively. The results showed that the average concentrations of iron, manganese and phosphorus were significantly (P < 0.05) higher within roots of yeast-inoculated plants, compared to control plants. It was shown that the yeast was not a root endophyte and that elemental enrichment in the epi/exodermal-outer cortical tissues correlated with the presence of Casparian bands in the exodermal cells of both treatments. This was the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont. In the final part of the investigation, the effect of C. laurentii CAB 578 on the photosynthetic nitrogen, phosphorus and water-use efficiencies, as well as the carbon economy of A. betulina was studied. Agathosma betulina plants inoculated with C. laurentii CAB 578, as well as controls, were grown under axenic conditions and the following parameters measured: Apparent photon yield, foliar nitrogen and phosphorus concentrations, leaf dark respiration, maximum photosynthetic rate, photosynthetic nitrogen-use efficiency, photosynthetic phosphorus-use efficiency, photosynthetic wateruse efficiency, root construction cost, stomatal conductance, substomatal CO2 and transpiration rate. The data showed that the higher photosynthetic resource-use efficiencies in yeast-inoculated plants were a consequence of higher maximum rates of CO2 assimilation, which was not related to foliar nitrogen and phosphorus content. We hypothesize that photosynthetic stimulation in yeast-inoculated plants was a result of the increased demand for photosynthates of the yeast-root symbiosis. In summary, the study revealed that a symbiosis exists between A. betulina and the soil yeast C. laurentii CAB 578. This interaction has a significant effect on the size of the yeast population as well as on the physiology of the plant. / AFRIKAANSE OPSOMMING: Die interaksie tussen ‘n grondgis, Cryptococcus laurentii, en ‘n medisinale plant, Agathosma betulina (Berg.) Pillans, is ondersoek. Cryptococcus laurentii CAB 578 is vanuit die risosfeer van A. betulina in sy natuurlike omgewing geisoleer en vloeistof chromatografie tandem massa spektrofotometriese analise het bewys dat die gis poliamiene insluitend kadaverien en spermien produseer. Dit is bevind dat die eksogene aanwending van poli-amiene wortelgroei bevorder. Hierdie bevinding staaf die waargenome 51% verhoging in wortelgroei van mikroob-vrye A. betulina saailinge geinokuleer met C. laurentii CAB 578 en gekweek vir vyf maande onder lae nutriënt kondisies in ‘n glashuis. Met gebruik van die grond verdunningsplaat-metode, is dit verder bewys dat gisgetalle hoër was in die teenwoordigheid van wortels as in die omliggende grond. Dit is ook bewys met die gebruik van die fluoressente peilers Calcofluor White en Fungolight, in kombinasie met fluoressensie-mikroskopie, dat metabolies aktiewe giste die wortels se oppervlak gekoloniseer het. Die eerste deel van die studie het dus bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Tydens die tweede deel van die ondersoek is die effek van C. laurentii CAB 578 op die konsentrasie en verspreiding van elemente binne A. betulina wortels, gekweek onder lae-nutriënt, mikroob-vrye kondisies, bepaal met behulp van mikro-partikel geinduseerde X-straal emissie spektrofotometrie. Om die interpretasie van heterogene verspreidingspatrone van die onderskeie elemente te ondersteun, is daar met behulp van fluoressensie en transmissie-elektron-mikroskopie vir apoplastiese versperrings en endofitiese C. laurentii CAB 578 in die wortelweefsel getoets. Dit is bevind dat die gemiddelde konsentrasie van fosfaat, mangaan en yster beduidend (P < 0.05) hoër was in gis-geinokuleerde plante, as in kontrole plante. Die gis was nie ‘n wortel endofiet nie en elementale verryking in die epi/eksodermale-buitenste korteks weefsels het gekorreleer met Casparian bande in die eksodermale selle van beide behandelings. Hierdie was die eerste verslag wat die rol van ‘n grondgis as ‘n nutriënt-bekommende mikrosimbiont vir plante beskryf het. In die laaste gedeelte van hierdie ondersoek is die effek van C. laurentii CAB 578 op die fotosintetiese fosfaat, stikstof en water-verbruiksdoeltreffendheid, asook die koolstof ekonomie in mikroob-vrye Agathosma betulina plante geinokuleer met C. laurentii CAB 578 asook kontrole plante bestudeer. Die volgende parameters is getoets: Blaar donker respirasie, blaar fosfaat en stikstof konsentrasies, fotosintetiese fosfaatverbruiksdoeltreffendheid, fotosintetiese stikstof-verbruiksdoeltreffendheid, fotosintetiese water-verbruiksdoeltreffendheid, huidmond konduktansie, huidmond CO2 konsentrasie, klaarblyklike foton opbrengs, maksimum fotosintetiese spoed, wortel konstruksie-koste, en transpirasie spoed. Die resultate het getoon dat die hoër maksimum fotosintestiese spoed in gis-geinokuleerde plante gelei het tot ‘n hoër fotosintetiese verbruiksdoeltreffendheid van fosfaat, stikstof en water en dat dit nie verband gehou het met blaar fosfaat en stikstof konsentrasies nie. Dit word voorgestel dat die stimulasie van fotosintese in gisgeinokuleerde plante ‘n gevolg is van die verhoogde aanvraag na fotosintaat deur die giswortel simbiose. Om op te som, die bevindings van hierdie studie het bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Hierdie simbiose het ‘n beduidende effek op die populasie grootte van die gis sowel as die fisiologie van die plant.