Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The Cape Floristic Region (CFR) is situated at the southern tip of Africa and possesses a flora that is unique amongst the floras of the rest of the world, both in terms of its incredibly high species richness, and its high levels of endemism. Proteaceae, the family to which Serruria belongs, is widely distributed amongst the landmasses of the southern hemisphere, with its centres of diversity occurring in Australia and southern Africa. Previous molecular and morphological analyses performed on the South African subfamily Proteoideae have shown Serruria, a CFR endemic, to form a well-supported monophyletic group. Based upon the strong monophyly of Serruria, DNA sequence data were collected for 53 of the 55 species from the plastid (rps16 intron, atpB-rbcL intergenic spacer, trnL-F region and psbA-trnH intergenic spacer) and nuclear (internal transcribed spacer region or ITS) genomes in order to investigate evolutionary relationships within the genus. Spatalla taxa were used as the outgroup. Both parsimony and Bayesian analyses were carried out on each of these data sets. The resulting trees were reasonably well resolved. All the Serruria taxa grouped together in a well-supported clade, except for S. f1ava, which emerged well within the Serruria clade in the analyses of the nuclear genome, but outside the clade in the plastid analyses. It was therefore proposed that this taxon represents a hybrid. Apart from this case, there was widespread agreement between the trees reconstructed using data from the two genomes. The plastid and nuclear data were therefore combined in order to analyse the data sets together. The molecular data does not support most of the groupings proposed by previous authors based on morphological data. Additionally, in some cases, multiple representatives of species do not group together. These specimens probably do not represent monophyletic taxa. Current ideas about relationships within Serruria are based predominantly on floral characters, and it is suggested that pollinator pressures have led to plasticity in the floral characters. Consequently, it is evident from this study that relationships within Serruria need to be re-examined in order to determine the patterns of evolution within the genus. / AFRIKAANSE OPSOMMING: Die Kaapse Floristiese Streek is aan die suiderpunt van Afrika geleë, en beskik oor 'n unieke flora relatief tot ander wêreldfloras, beide ten opsigte van die ongelooflike hoë spesie diversiteit en die hoë vlakke van endemisme. Proteaceae, die familie waaraan Serruria behoort, kom wydverspreid tussen die vastelande van die Suidelike Halfrond voor, en het diversiteitsentrums in Australië en suider Afrika. Vorige molekulêre sowel as morfologiese analises wat op die Suid-Afrikaanse subfamilie Proteoideae uitgevoer is, dui aan dat Serruria (wat endemies is tot die Kaapse Floristiese Streek) 'n goed ondersteunde monofiletiese groep is. Gebaseer op die sterk monofilie van Serruria, is DNA-volgorde-data vir 53 van die 55 spesies vanuit die plastied (rps16 intron, atpB-rbcL intergeniese spasie, trnL-F area en psbA-trnH intergeniese spasie) en kern (intern getranskribeerde spasie area, ook ITS genoem) ingewin om die evolusionêre verwantskappe binne die genus te ondersoek. Spatalla is as die buitegroep gebruik. Beide parsimonie en Bayesian analises is op elk van hierdie datastelle uitgevoer. Die resulterende bome het redelike hoë resolusie getroon. AI die Serruria-taxa het in 'n goed ondersteunde klade saam gegroepeer, behalwe vir S. f1ava, wat binne die Serruria klade val vir die kern genoom, maar buite die klade vir die plastied analise. Dit is dus voorgestel dat hierdie taxon as 'n hibried beskou mag word. Behalwe vir hierdie geval, was daar wydverspreide ooreenstemming tussen die bome wat verkry is vanaf data van die twee genome. Die plastied- en kern-data is derhalwe gekombineer om die datastelle saam te kan analiseer. Die molekulêre data ondersteun nie die meerderheid van morfologiese groeperings wat deur verskeie outeurs voorgestel is nie. Verder, in sommige gevalle, groepeer verskillende monsters van dieselfde spesies nie bymekaar nie. Dit is derhalwe voorgestel dat hierdia taxa nie monofileties is nie. Huidige idees omtrent die verwantskappe binne Serruria is grotendeels op blommorfologiese kenmerke gebaseer, en dit word voorgestel dat bestuiwing-druk gelei het tot plastisiteit van die blommorfologiese kenmerke. Verskille tussen die bome wat uit plastied- en kern-data gerekonstrueer is word aan vroeëre hibridisasie gebeure toegeskryf. Op grond van hierdie studie is dit duidelik dat die verhoudings binne Serruria verder ondersoek moet word om die patrone van evolusie binne die genus te bepaal.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Five new species of ophiostomatoid fungi, colon ising the infructescences of serotinous Protea species, have recently been discovered in South Africa. Prior to this, ophostomatoid fungi were thought to be restricted to the Northern Hemisphere. The discovery of these five species thus extensively expanded the known geographical range of these fungi, now also to include the Southern Hemisphere. Since this discovery, few studies have focused on the interesting ecology of this group, which is uniquely adapted for spore dispersal by arthropods. Studies focussed on ophiostomatoid fungi provide an unique opportunity to study inter-organism interactions between fungi, their host plants and the arthropods responsible for their spore dispersal. Very few similar studies have been undertaken, particularly in the Fynbos Biome, to which most of our economically important Protea species are confined. The current study provides insight into some ecological aspects of these complex interactions. The seasonal distribution of the ophiostomatoid fungi associated with the infructescences of members of the Proteaceae was investigated. Definite seasonal patterns were observed, with peak fungal colonisation occurring during the wetter winter months. While determining the host specificity of these fungi, a new Protea host for Ophiostoma splendens was identified, and a new species of Rhyncomeliola was discovered. All ophiostomatoid species native to Fynbos in the Stellenbosch region are exclusively associated with the infructescences of species in the plant genus Protea. By employing a multiplex Polymerase Chain Reaction (PCR), a set of group specific primers was developed for the identification of South African Ophiostoma and Gondwanamyces fungal DNA. This newly developed method was used to scan arthropods collected from the infructescences of P. repens for the presence of 0. splendens and G. proteae spores. By using these group-specific primers, three insects possibly responsible for long range dispersal of fungal spores were identified. Three further insect species, possibly contributing to the short-range dispersal of ophiostomatoid spores, were also implicated through this method. All arthropods associated with the fruiting structures of selected members of the Proteaceae were also investigated. A total of 62 pseudospecies (ca. 7500 individuals), belonging to 45 different arthropod families, were collected. Proteaceae species with larger fruiting structures housed more arthropod species and higher numbers of individuals than Proteaceae taxa with smaller fruiting structures. Some plant species housed similar arthropod communities, while others housed unique suites of arthropods. Seasonal patterns in arthropod numbers were observed, and it was found that, in most instances, arthropod numbers peaked during the autumn and winter months. Twenty-five fungal taxa were isolated from various Proteaceae arthropods, many of which are genera known to include Proteaceae pathogenic species. Ophiostomatoid fungi have a saprophytic relationship with their Protea hosts, and may deter some of these potentially harmful fungi from colon ising the infrutescences. Some fungi showed a high degree of specificity towards potential vectors, while others were found on a diverse range of arthropods. This study highlights the existence of complex inter-organismal interactions within the Fynbos Biome, a study area where the interactions between plants, fungi and insects have been grossly neglected. The few studies that have been conducted in this field have mostly focussed on a limited number of organisms, and no wide-scale attempts, such as presented here, have been published. It is important to obtain a holistic view in any ecological study that focuses on interactions between different suites of organisms. Ultimately this will aid in the development of better conservation strategies. This study thus provides a muchneeded start in studies on multi-organismal interactions in the Fynbos Biome. / AFRIKAANSE OPSOMMING: Vyf nuwe ophiostoma-agtige fungus spesies is onlangs in die vrug-liggame van Suid Afrikaanse Protea plante ontdek. Voor hierdie ontdekking, was die algemene opvatting dat ophiostoma-agtige fungi beperk is tot die Noordelike Halfrond. Die ontdekking van hierdie vyf spesies het dus die bekende geografiese verspreiding van hierdie tipe fungi vergroot om nou ook die Suidelike Halfrond in te sluit. Sederdien het min studies gefokus op die interesante ekologie van hierdie groep, wat aangepas is vir spoorverspreiding deur geleedpotiges. Studies wat fokus op die ophiostoma-agtige fungi voorsien 'n unieke geleentheid om die inter-organismiese-interaksies tussen fungi, hul gasheer plante en die geleedpotiges wat hul spore versprei, te bestudeer. Baie min soortgelyke studies is al voorheen in die Fynbos Bioom, waar die meeste van ons ekonomies-belangrike Protea spesies voorkom, onderneem. Die huidige studie verleen meer inligting oor sommige ekologiese aspekte van hierdie komplekse interaksies. Seisoenale verspreidingspatrone van ophiostoma-agtige fungi geassosieer met die vrug-liggame van lede van die Proteaceae, is ondersoek. Daar is defnitiewe seisoenale patrone in die aanwesigheid van fungi gevind, met 'n piek in fungi kolonisasie-getalle gedurende die vogtiger wintermaande. 'n Nuwe Protea gasheer vir Ophiostoma splendens en 'n nuwe fungus spesie (Rhyncomeliola sp.), is ontdek. Alle Fynbos ophiostoma-agtige spesies in die Stellenbosch area, is beperk tot Protea spesies. 'n Multipleks Polimerase Ketting Reaksie is ontwikkel om die spesifieke DNS van ophiostoma-agtige fungi te herken. Hierdie metode is gebruik om te toets vir die aanwesigheid van spore van hierdie fungi op die liggame van geleedpotiges (geassosieerd met P. repens). Hierdeur is drie insekte wat waarskynlik verantwoordelik is vir die langafstand verspreiding van spore geidentifiseer. Drie verdere insekte wat moontlik verantwoordelik is vir die kortafstand verspreiding van ophiostoma-agtige fungi is ook aangewys. Alle geleedpotiges geassosieer met die vrugstrukture van geselekteerde lede van die Proteaceae is ondersoek. 'n Totaal van 62 pseudo-spesies (omtrent 7500 individue), wat tot 45 families behoort, is versamel. Proteaceae spesies met groter vrugstrukture het meer geleedpotige spesies en individue gehuisves as taksa met kleiner vrugstrukture. Sommige plant spesies het soortgelyke geleedpotige gemeenskappe gehuisves, terwyl unieke groepe geleedpotiges in ander plante voorgekom het. Alhoewel die geleedpotiges seisonale variasies in getalle geopenbaar het, het die meeste groepe 'n piek in getalle getoon gedurende die herfs- en wintermaande. Vyf-en-twintig fungi taksa is geïsoleer vanaf verskeie Proteaceae geleedpotiges. Sommige van hierdie is genera wat Proteaceae patogeniese spesies insluit. Sommige fungi het 'n hoë graad van spesifisiteit teenoor potensiële vektore getoon, terwyl ander weer algemeen op verskeie geleedpotige spesies gevind is. Hierdie studie dui op die bestaan van komplekse inter-organismiese-interaksies in die Fynbos Bioom, in 'n area waar die studie van interaksies tussen plante, fungi en insekte nog baie min aandag geniet het. Die beperkte aantal studies wat tot dusver in hierdie veld onderneem is, fokus gewoonlik slegs op 'n paar organismes. Geen grootskaalse studies, soos die een hier voorgelê, is al gepubliseer nie. In enige ekologiese studie wat fokus op interaksies tussen verskeie organismes, is dit belangrik om 'n holistiese siening te probeer verkry. Uiteindelik sal dit bydra tot die ontwikkeling van beter bewarings-strategië. Hierdie studie verleen dus 'n noodsaaklike begin tot studies op multiorganismiese- interaksies.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Two genera of ophiostomatoid fungi occur in the seed-bearing structures of serotinous Protea species in the Cape Floristic Region. These fungi are dispersed by arthropods, including mites and beetles that visit the Protea host plants. Although the vectors of Proteaassociated ophiostomatoid fungi are known, their dispersal patterns remain unknown – especially the manner in which recently burnt fynbos vegetation is recolonized. Additionally, their reproduction strategy has not previously been investigated. The focus of this study was, therefore, to determine the extent of within- and between-plant dispersal of Proteaassociated ophiostomatoid fungi at the population level and to investigate their reproductive strategy. One Protea-associated ophiostomatoid fungus, Knoxdaviesia proteae, is found exclusively in the fruiting structures of P. repens and was the focus of this study. In order to interrogate natural populations of this fungus, 12 polymorphic microsatellite markers specific to K. proteae were developed with an ISSR-PCR enrichment strategy and pyrosequencing. These markers were amplified in two distantly separated populations of K. proteae. The genetic and genotypic diversities of both populations were exceptionally high and neither showed significant population differentiation. The lack of population structure in both populations implies that K. proteae individuals within a P. repens stand are in panmixia. As one of the sampling sites had burnt recently, the process whereby young fynbos is recolonized could be investigated. Compared to the adjacent, unburnt area, K. proteae individuals in the burnt area of this population had significantly less private alleles, suggestive of a young population that had experienced a genetic bottleneck. Knoxdaviesia proteae individuals that did not originate from the adjacent unburnt area were encountered within the burnt site and, additionally, isolation-by-distance could not be detected. The parsimony-based haplotype networks and the tests for linkage disequilibrium indicated that recombination is taking place within as well as between the two distantly separated populations. The observed panmixia in P. repens stands, widespread recolonization and the high genetic similarity and number of migrants between the two populations emphasizes long-distance dispersal and therefore the role of beetles in the movement of K. proteae. This cohesive genetic structure and connection across large distances is likely a result of multiple migration events facilitated by beetles carrying numerous phoretic mites. / AFRIKAANSE OPSOMMING: Twee genera ophiostomatoid swamme kom in die saad-draende strukture van bloeiende Protea spesies in the Kaapse Floristiese Streek voor. Hierdie Protea-verwante ophiostomatoid swamme word gekenmerk deur hul assosiasie met geleedpotige vektore – spesifiek die myt en kewer besoekers van die Protea gasheer plante. Alhoewel die geleedpotige vektore van Protea-verwante ophiostomatoid swamme bekend is, is die wyse waarop hierdie swamme versprei onbekend; veral die manier waarop onlangse gebrande fynbos geherkoloniseer word. Verder is die voortplantings-strategie van hierdie swamme nog nie voorheen ondersoek nie. Die fokus van hierdie studie was dus om die omvang van binne- en tussen-plant verspreiding van Protea-verwante ophiostomatoid swamme te bepaal op die populasie vlak en om hul voorplantings-strategie te ondersoek. Een Protea-verwante ophiostomatoid swam, Knoxdaviesia proteae, word uitsluitlik in die vrugdraende strukture van P. repens aangetref en was die fokus van hierdie studie. Om natuurlike populasies van hierdie swam te ondersoek is 12 mikrosatelliet-merkers spesifiek vir K. proteae ontwerp deur ‘n ISSR-PCR strategie en “pyro”-basisvolgorde bepaling te gebruik. Hierdie merkers is geamplifiseer in twee K. proteae populasies wat ver van mekaar geskei is. Die genetiese en genotipiese diversiteit van beide populasies was uitsonderlik hoog en nie een het beduidende populasie-differensiasie getoon nie. Die gebrek aan populasie struktuur in beide populasies veronderstel dat K. proteae individue binne ‘n P. repens stand in panmiksia is. Aangesien een van die steekproef terreine onlangs gebrand het, kon die herkolonisasie proses van jong fynbos ondersoek word. In vergelyking met die aangrensende, ongebrande area, het K. proteae individue in die gebrande area beduidend minder private allele gehad. Dit dui op ‘n jong populasie wat ‘n genetiese bottelnek beleef het. Knoxdaviesia proteae individue wat nie van die aangrensende, ongebrande area afkomstig is nie is ook binne die gebrande terrein aangetref. Verder is afsondering-deur-afstand nie aangetref nie. Die parsimonie-gebaseerde haplotiepe-netwerke en die toetse vir koppeling-onewewigtigheid het aangedui dat rekombinasie binne sowel as tussen die twee populasies plaasvind. Die panmiksia wat waargeneem is in P. repens populasies, wydverspreide herkolonisasie en die hoë genetiese ooreenkoms en hoeveelheid immigrante tussen die twee populasies beklemtoon lang afstand verspreiding en dus die rol van kewers in die beweging van K. proteae. Hierdie samehangende genetiese struktuur en die verband oor groot afstande is waarskynlik ‘n gevolg van verskeie migrasies gefasiliteer deur kewers wat talle foretiese myte dra.

Thesis (PhD) -- Stellenbosch University, 1989. / ENGLISH ABSTRACT: Arthropod communities of five proteaceous species viz. Protea repens. P. neriifolia. P.cynaroides, Leucospermum cordifolium and Leucadendron /aureolum were studied. A faunal list of the insects was compiled according to the feeding guilds of insects. Six phytophagous guilds were recognized: flower visitors, thrips, endophages, ectophages, sap-suckers, and ants. The guild composition, species packing and seasonal distribution for the free-IMng insects, spiders and other arthropods were investigated on the different plant species. The ants, flower visitors and sap-sucking species found on the plant species, were very similar, but the number of leaf chewing species common to all the plant species was low, suggesting that some species were monophagous. Insect abundance increased during the wet winter months when the plants were in flower. Of the total number of herbivores collected, flower visitors represented 69,5%, leaf feeders 14,5% and sap-suckers 16,1 %. The chewers constituted 60,1% of the total herbivore. biomass. Leaf damage increased with age ; young leaves were practically free of herbivore damage, while older leaves were stm acceptable as food. The degree of insect damage differed amongst the plant species varying from 2% to 15%. The following leaf characteristics were investigated to determine the factors involved in defence against insect herbivory : total phenolic content, protein precipitating ability, cyanogenesis, nitrogen content, leaf toughness, woodiness and fibre content lt appears that the plant species use different mechanisms to protect their leaves. The infructescences of Protea repens were exploited by the larvae of four Coleoptera and four Lepidoptera species. The community structure of these phytophagous insects was determined largely by negative interactions. Various strategies are followed (e.g. niche segregation in time and space) to lessen interspecific competition. / AFRIKAANSE OPSOMMING: 'n Studie van die arthropoocl gemeenskap op vyf proteasoorte, naamlik Protea repens. P. neriffolia. P. cynaroides. Leucospennum cordffolium en Leucadendron laureolum is gedoen. 'n Faunalys van insekte volgens hul voedingsgDdes is saamgestel. Die insekte is in ses fitofage gDdes verdeel naamlik blombesoekers, blaaspootjies, endovoeders, ektovoeders, sapsuiers en miere. Die samestelling van die gDdes, spesie-rangskikking en seisoenale verspreiding is bepaal vir die vrylewende insekte, spinnekoppe en ander arthropode. Die miere, blombesoekers en sapsuierspesies wat op die plantsoorte gevind is, is byna dieselfde, maar verslalle in die blaarvoederspesies is gevind wat 'n aanduiding is dat sommige spesies monofaag mag wees. Gedurende die nat wintermaande toon die insekgetalle 'n toename. Blombesoekers verteenwoordig 69,5%, blaarvoeders 14,5% en sapsuiers 16,1% van die aantal insekherbivore wat versamel is. Die kouers vorm 60,1% van die totale herbivoor biomassa Soos die blare verouder, neem die skade op die blare toe; jong blare toon byna geen skade nie, terwyl ouer blare steeds geskik is vir insekvoedsel. Die insekskade op die blare wissel by die verslallende plante tussen 2% en 15%. Om te bepaal watter blaareienskappe moontlik 'n rol kan speel in blaarverdediging teen insekte is die volgende ondersoek: totale fenoliese inhoud, die vermoe om proteiene te presipiteer, sianogenese. stikstofinhoud, blaartaaiheid, houtagtigheid en veselinhoud. Dit wil voorkom asof die plante verskillende meganismes gebruik om die blare te beskerm. Saadkoppe van Protea repens word deur vier Coleoptera en vier Lepidoptera spesies benut Die gemeenskapstruktuur van die boarders word tot 'n groat mate bepaal deur negatiewe interaksies. Verskeie strategiee word gevolg (bv. nissegregasie in ~ en ruimte) om kompetisie tussen spesies te verminder.

PhD / The waratah, Telopea speciosissma and its hybrids with other Telopea species, is an Australian native species grown for domestic and export cut flower markets. The showy floral bracts surrounding the inflorescence often suffer from bract browning, reducing the market value and export potential of the blooms. Prior to this project, the physiological cause of the disorder was not known, although bract browning had been attributed to water stress, heat stress, high light (particularly after frost), wind and mechanical damage. Bract browning was reportedly minimised when waratahs were grown in shaded conditions, although the reduction in browning by shade had not been quantified. The aim of this project was to examine the physiological cause of the bract browning disorder and investigate methods for control. The appearance, timing, and severity of the bract browning disorder was initially characterised by dissecting waratah buds from commercial growers throughout NSW. Bract browning became evident in the six to eight weeks prior to harvest, coinciding with rapid bract and flower expansion. A survey of commercial waratah growers, initiated by NSW Agriculture and the Waratah Industry Network and analysed by the author, corroborated these results. The survey showed that bract browning was observed in all years between 1999 and 2003, with relatively high severity (scores from three to five out of a possible five) in three of those years. Scores or counts of brown bracts were used to assess the severity of the disorder, the latter including the number of senesced floral bracts following browning as a measure of browning severity. The position and timing of browning suggest light damage or localised calcium deficiency could play a role in the development of browning. The bract browning disorder was studied in further experiments on potted red waratahs of cultivars ‘Fire and Brimstone’, ‘Olympic Flame’ and ‘Sunflare’ at the Mount Annan Botanic Garden; on commercially grown ‘Wirrimbirra White’ waratahs at Jervis Bay; and on natural populations in the Royal National Park. The effect of calcium nutrition on bract browning was studied at Mount Annan in 2001 and 2002, testing the hypothesis that browning may be caused by a localised calcium deficiency similar to lettuce tipburn or poinsettia bract necrosis. Waratah bracts had significantly less calcium in all fractions than leaves, with the procedure of Ferguson et al. (1980) used to separate physiologically active, oxalate associated and residual calcium. Calcium chloride sprays applied to developing bracts increased total bract calcium by about 25% in ‘Sunflare’ and ‘Olympic Flame’ cultivars, but not in ‘Fire and Brimstone’. However, application of calcium as a spray to the developing bracts, or as gypsum to the potting medium did not significantly reduce bract browning scores. These results and the development of bract browning in exposed, rather than enclosed tissue, suggest that factors other than calcium are involved in the development of bract browning. The light environment (full sun or 50% shade cloth) had a greater effect than irrigation frequency on bract browning of ‘Sunflare’ and ‘Olympic Flame’ waratahs in 2001. Waratahs grown under 50% shade cloth showed less bract browning at flower maturity than waratahs grown in full sun. This result was corroborated by subsequent experiments in 2002 and 2003. For example, in 2002, shade cloth reduced browning and bract loss by 30-60% at flower maturity, compared to waratahs grown in full sun. Shading waratahs from bud initiation in late summer (December-January) or bud opening in late winter (July-August) was equally effective in reducing browning. Shade cloth (50%) significantly reduced the light intensity experienced by waratah plants throughout the day, as well as reducing the daily maximum temperature and minimum relative humidity. Natural shade conditions at the Royal National Park effectively prevented browning of floral bracts, although the smaller basal bracts still turned brown and senesced. The development of bract browning as waratahs matured was linked to the development of chronic photoinhibition, measured as a decrease in predawn photosynthetic efficiency using chlorophyll fluorescence techniques. Waratah bracts were unable to maintain efficient photosynthesis in full sun conditions and reached saturation of non-photochemical quenching at lower light intensities than leaves. This suggests that bract tissue is adapted to a lower light environment than leaf tissue. Outer bracts had a significantly lower photosynthetic efficiency (Fv/Fm) than leaves early in flower development, as they were exposed to the environment for a prolonged period. Outer bracts also began to senesce towards flower maturity, particularly in full sun, increasing their susceptibility to damage. Inner waratah bracts were able to maintain a high photosynthetic efficiency prior to exposure, but photosynthetic efficiency decreased significantly at the intermediate stage of floral development, as inner bracts were no longer protected by outer bracts. Waratah leaves were more resilient than bracts, and did not suffer from chronic photoinhibition or browning during flower development. The increased susceptibility of bracts to photoinhibition and browning parallels results in other species, such as Dendrobium, where floral tissue experiences photoinhibition, bleaching and necrosis at lower light intensities than leaf tissue. Bracts on shaded waratahs maintained higher chlorophyll, carotenoid and anthocyanin concentrations than sun-exposed bracts, giving more intense flower colour and higher quality blooms. The significant decrease in bract pigmentation in the sun is likely to be a result of pigment destruction following photoinhibition, and has been noted in susceptible tissues of other species, such as Illicium (star anise) leaves. The presence of anthocyanins did not reduce bract browning in waratahs, with the concentration of UV-absorbing compounds showing a stronger positive correlation with protection from photoinhibition than the concentration of anthocyanins. However, anthocyanin concentrations were significantly lower in sun-exposed bracts, and brown compounds appeared to replace anthocyanins in the epidermal cells of brown bracts. Thus, it seems likely that browning in waratah bracts is the visible manifestation of oxidative damage to cell components, following chronic photoinhibition. Light-induced oxidative damage can lead to yellowing and pigment bleaching, lipid peroxidation, the development of necrotic lesions and senescence. However, lipid peroxidation as measured by the malionaldehyde assay gave no indication of oxidative damage to waratah bract tissue. This was probably due to the presence of anthocyanins and other flavonoids and sugars other than sucrose in bract tissue interfering with the colourimetric measurement of thiobarbituric acid reactive substances. The extensive planting of waratahs in NSW in the last five years suggests that the total value of lost production due to bract browning is likely to increase in the future. The browning disorder may also prevent the establishment of waratahs in other markets, as international cut-flower markets demand high quality blooms free from blemishes. The results of this study show that bract browning, photoinhibition and pigment loss are minimised by protecting waratahs from high light intensities from bud opening until harvest. However, the consequences of shading waratahs throughout the year require further investigation, as does the use of different percentages of shade cloth or other methods to reduce incident light.

PhD / The waratah, Telopea speciosissma and its hybrids with other Telopea species, is an Australian native species grown for domestic and export cut flower markets. The showy floral bracts surrounding the inflorescence often suffer from bract browning, reducing the market value and export potential of the blooms. Prior to this project, the physiological cause of the disorder was not known, although bract browning had been attributed to water stress, heat stress, high light (particularly after frost), wind and mechanical damage. Bract browning was reportedly minimised when waratahs were grown in shaded conditions, although the reduction in browning by shade had not been quantified. The aim of this project was to examine the physiological cause of the bract browning disorder and investigate methods for control. The appearance, timing, and severity of the bract browning disorder was initially characterised by dissecting waratah buds from commercial growers throughout NSW. Bract browning became evident in the six to eight weeks prior to harvest, coinciding with rapid bract and flower expansion. A survey of commercial waratah growers, initiated by NSW Agriculture and the Waratah Industry Network and analysed by the author, corroborated these results. The survey showed that bract browning was observed in all years between 1999 and 2003, with relatively high severity (scores from three to five out of a possible five) in three of those years. Scores or counts of brown bracts were used to assess the severity of the disorder, the latter including the number of senesced floral bracts following browning as a measure of browning severity. The position and timing of browning suggest light damage or localised calcium deficiency could play a role in the development of browning. The bract browning disorder was studied in further experiments on potted red waratahs of cultivars ‘Fire and Brimstone’, ‘Olympic Flame’ and ‘Sunflare’ at the Mount Annan Botanic Garden; on commercially grown ‘Wirrimbirra White’ waratahs at Jervis Bay; and on natural populations in the Royal National Park. The effect of calcium nutrition on bract browning was studied at Mount Annan in 2001 and 2002, testing the hypothesis that browning may be caused by a localised calcium deficiency similar to lettuce tipburn or poinsettia bract necrosis. Waratah bracts had significantly less calcium in all fractions than leaves, with the procedure of Ferguson et al. (1980) used to separate physiologically active, oxalate associated and residual calcium. Calcium chloride sprays applied to developing bracts increased total bract calcium by about 25% in ‘Sunflare’ and ‘Olympic Flame’ cultivars, but not in ‘Fire and Brimstone’. However, application of calcium as a spray to the developing bracts, or as gypsum to the potting medium did not significantly reduce bract browning scores. These results and the development of bract browning in exposed, rather than enclosed tissue, suggest that factors other than calcium are involved in the development of bract browning. The light environment (full sun or 50% shade cloth) had a greater effect than irrigation frequency on bract browning of ‘Sunflare’ and ‘Olympic Flame’ waratahs in 2001. Waratahs grown under 50% shade cloth showed less bract browning at flower maturity than waratahs grown in full sun. This result was corroborated by subsequent experiments in 2002 and 2003. For example, in 2002, shade cloth reduced browning and bract loss by 30-60% at flower maturity, compared to waratahs grown in full sun. Shading waratahs from bud initiation in late summer (December-January) or bud opening in late winter (July-August) was equally effective in reducing browning. Shade cloth (50%) significantly reduced the light intensity experienced by waratah plants throughout the day, as well as reducing the daily maximum temperature and minimum relative humidity. Natural shade conditions at the Royal National Park effectively prevented browning of floral bracts, although the smaller basal bracts still turned brown and senesced. The development of bract browning as waratahs matured was linked to the development of chronic photoinhibition, measured as a decrease in predawn photosynthetic efficiency using chlorophyll fluorescence techniques. Waratah bracts were unable to maintain efficient photosynthesis in full sun conditions and reached saturation of non-photochemical quenching at lower light intensities than leaves. This suggests that bract tissue is adapted to a lower light environment than leaf tissue. Outer bracts had a significantly lower photosynthetic efficiency (Fv/Fm) than leaves early in flower development, as they were exposed to the environment for a prolonged period. Outer bracts also began to senesce towards flower maturity, particularly in full sun, increasing their susceptibility to damage. Inner waratah bracts were able to maintain a high photosynthetic efficiency prior to exposure, but photosynthetic efficiency decreased significantly at the intermediate stage of floral development, as inner bracts were no longer protected by outer bracts. Waratah leaves were more resilient than bracts, and did not suffer from chronic photoinhibition or browning during flower development. The increased susceptibility of bracts to photoinhibition and browning parallels results in other species, such as Dendrobium, where floral tissue experiences photoinhibition, bleaching and necrosis at lower light intensities than leaf tissue. Bracts on shaded waratahs maintained higher chlorophyll, carotenoid and anthocyanin concentrations than sun-exposed bracts, giving more intense flower colour and higher quality blooms. The significant decrease in bract pigmentation in the sun is likely to be a result of pigment destruction following photoinhibition, and has been noted in susceptible tissues of other species, such as Illicium (star anise) leaves. The presence of anthocyanins did not reduce bract browning in waratahs, with the concentration of UV-absorbing compounds showing a stronger positive correlation with protection from photoinhibition than the concentration of anthocyanins. However, anthocyanin concentrations were significantly lower in sun-exposed bracts, and brown compounds appeared to replace anthocyanins in the epidermal cells of brown bracts. Thus, it seems likely that browning in waratah bracts is the visible manifestation of oxidative damage to cell components, following chronic photoinhibition. Light-induced oxidative damage can lead to yellowing and pigment bleaching, lipid peroxidation, the development of necrotic lesions and senescence. However, lipid peroxidation as measured by the malionaldehyde assay gave no indication of oxidative damage to waratah bract tissue. This was probably due to the presence of anthocyanins and other flavonoids and sugars other than sucrose in bract tissue interfering with the colourimetric measurement of thiobarbituric acid reactive substances. The extensive planting of waratahs in NSW in the last five years suggests that the total value of lost production due to bract browning is likely to increase in the future. The browning disorder may also prevent the establishment of waratahs in other markets, as international cut-flower markets demand high quality blooms free from blemishes. The results of this study show that bract browning, photoinhibition and pigment loss are minimised by protecting waratahs from high light intensities from bud opening until harvest. However, the consequences of shading waratahs throughout the year require further investigation, as does the use of different percentages of shade cloth or other methods to reduce incident light.

[Truncated abstract] Grevillea and Leucadendron belong to Proteaceae and both have economic importance to the floriculture industry. Grevillea is a highly diverse genus endemic to Australia and very attractive for landscaping. Leucadendron is a South African Proteaceae but is cultivated in Australia and is well known as a cut flower. This thesis focuses on the application of DNA-based molecular markers to these genera. Several groupings within Grevillea were suggested by previous researchers based on morphological characteristics. In this thesis the monophyly of the groupings among 12 Grevillea species from New South Wales was tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses. To test the robustness of the data, UPGMA using Jaccard similarity, Neighbor Joining using total character difference and Wagner parsimony analyses were undertaken. The relationship trees generated supported monophyly of the groupings. Chloroplast DNA (cpDNA) was used to develop phylogenetic relationships among Leucadendron species. Inheritance and variation of cpDNA were evaluated using PCR-RFLP. The study demonstrated that cpDNA was inherited maternally and a phylogenetic tree of Leucadendron species using parsimony analysis was constructed. ... A fingerprinting study conducted using ISSR, produced a dendrogram showing the relationships among 30 cultivars. From the results, i a fingerprinting key was developed. Three examples of synonymous cultivar pairs were identified. In Leucadendron the male and female flowers develop on separate plants, and sex identification is only possible at time of flowering. ISSR, suppression subtractive hybridisation (SSH), and SSH combined with mirror orientation selection (MOS) were used in attempts of identifying sex-dependent DNA fragments at earlier stages of plant development. Neither of these techniques was able to identify sex-specific markers in Leucadendron. Nevertheless, the results did indicate that cpDNA copy number may differentiate male and female plants. Also, it was demonstrated that the genomes of male and female plants are quite homologous, which increases the difficulty in identifying sex-specific sequences. This thesis highlights the potential of DNA-based markers to determine species relationships in Grevillea and Leucadendron, as well as to identify Leucadendron cultivars. The information produced during the research for this thesis provides a basis for Grevillea and Leucadendron variety development and may be used to assist the design of interspecific crosses, to identify cultivars and the parents of hybrids. In addition, the results offer insights into the likelihood, problems and strategies of finding sex-specific markers for genes controlling sex in Leucadendron. ii

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Leaf blackening, a postharvest disorder which is characterized by a dark brown to black discoloration, is found in most commercially important Protea cut flower species and cultivars. As this disorder is known to increase with storage time, it is a major concern to the South African industry as the use of sea freight is increasingly preferred due to lower transport costs and a more favourable carbon footprint. The cause of leaf blackening has been strongly linked to a carbohydrate stress exerted by the large inflorescence, thus requiring the utilization of sugar bound polyphenols in the foliage, which when removed, can oxidize enzymatically or non-enzymatically. A study where harvesting was done throughout the season as well as on selected days at 08:00, 12:00, 15:00 and 17:00, concluded that leaf blackening incidences in Protea cv. Sylvia stems varies significantly throughout the season, between years and even with the harvest time of day. Leaf blackening incidences increased from October onwards and remained high until February, before decreasing to acceptably lower levels towards March to May. Carbohydrate- and phenolic content together with water status of leaves at harvest was not able to accurately predict incidence of the associated leaf blackening. However, irrespective of the season of harvesting, leaf blackening was significantly lower when stems were harvested later in the day than compared to stems harvested in the morning. Low sucrose and high water content at these harvest times was positively correlated to high incidences of leaf blackening. In a next study where uptake dynamics of glucose pulsing was investigated, Protea cv. Sylvia was harvested at different times throughout the day, dehydrated to various levels and pulsed with an increasing range of glucose concentrations. Pulsing solution uptake per stem was found to be highly influenced by these factors, as dehydration of stems and a harvest time later during the day both decreased stem water potential, which then increased pulse-solution uptake within a certain time period. The daily harvest time influenced transpiration, whilst pulse-solution uptake decreased with an increase in glucose pulse concentration. When stems were pulsed pre-storage with an increasing range of glucose concentrations, not only did pulses of between 4.7 – 13.7% glucose significantly delayed the incidence of leaf blackening, but it also maintained a positive water balance longer in stems during vase life. Ethanol or acetaldehyde vapour did not provide a viable alternative for reducing leaf blackening incidence in Protea cv. Sylvia, although a synergistic effect was found when ethanol vapour or pulsing was used in combination with glucose. A commercial verification trial disclosed that Protea magnifica and Protea ‘Pink Ice’ reacted more beneficial to ethanol vapour than was observed in ‘Sylvia’. This study confirms that carbohydrate availability within the Protea cut stem remains a key factor in the control of leaf blackening. Factors which assist in maintaining high internal carbohydrate levels, such as enhanced glucose pulse uptake or effective vase solution utilization will contribute to providing an optimum control of leaf blackening during vase life following long-term cold storage. / AFRIKAANSE OPSOMMING: Loofblaarverbruining is ‘n na-oes defek wat gekarakteriseer word deur ‘n donker bruin na swart verkleuring wat voorkom in meeste kommersieël belangrike Protea snyblom spesies en kultivars. Hierdie defek is bekend daarvoor dat dit toeneem met stoortyd, dus is dit ‘n groot kommer vir die Suid-Afrikaanse industrie, met toenemende gebruik van seevrag as vervoer keuse wat laer vervoer kostes en meer gunstige ‘koolstof voetspoor’ bevoordeel. Die oorsaak van loofblaarverbruining word sterk gekoppel aan ‘n koolhidraat stres wat uitgeoefen word deur die groot bloeiwyse op die loofblare, waar suiker-gebonde polifenoliese verbindings ensiematies of nieensiematies geoksideer word met die verwydering van die suiker verbinding. 'n Studie waar geoes was regdeur die seisoen, sowel as op geselekteerde dae om 08:00, 12:00, 15:00 en 17:00, het bevind dat die voorkoms van loofblaarverbruining in stele van Protea kv. Sylvia aansienlik geskil regdeur die seisoen, tussen jare en selfs met die oes tyd gedurende die dag. Die voorkoms van loofblaarverbruining het toegeneem vanaf Oktober en het hoog gebly tot en met Februarie, voordat dit gedaal het tot aanvaarbare laer vlakke teen Maart, tot en met Mei. Koolhidraat-en fenoliese inhoud sowel as die water status van die blare by oes was onsuksesvol om die voorkoms van die gepaardgaande loofblaarverbruining akkuraat te voorspel. Loofblaarverbruining was egter aansienlik laer as stele geoes later in die dag teenoor stele geoes in die oggend, ongeag die seisoen van oes. Lae sukrose en 'n hoë water inhoud geassosieer met hierdie oes-tye was positief gekorreleerd met ‘n hoë voorkoms van loofblaarverbruining. In 'n volgende studie waar die opname dinamika van glukose pulsing ondersoek was, is Protea kv. Sylvia stele geoes op verskillende tye dwarsdeur die dag, gedehidreer tot verskillende vlakke en met 'n toenemende reeks van glukose konsentrasies gepuls. Pulsoplossing opname per steel is sterk beïnvloed deur hierdie faktore, aangesien dehidrasie van die stele asook stele geoes later gedurende die dag die afname van steel waterpotensiaal veroorsaak het, terwyl die puls-oplossing opname versnel het binne ‘n bepaalde tyd. Die tyd van oes beïnvloed ook transpirasie, terwyl vaas oplossing opname afgeneem met 'n toename in glukose puls konsentrasie. Wanneer ‘Sylvia’ stele gepuls was voor stoor met 'n reeks van toenemende glukose konsentrasies, het nie net die puls van tussen 4.7 – 13.7% glukose aansienlik die voorkoms van loofblaarverbruining vertraag nie, maar dit het ook ‘n positiewe water balans langer in stele gedurende die vaas lewe behou. Nie etanol of asetaldehied dampe is bevind as geskikte alternatief vir glukose pulsing om die voorkoms van loofblaarverbruining in Protea kv. Sylvia te verlaag nie, alhoewel ‘n sinergistiese effek waargeneem was wanneer etanol in kombinasie met glukose gebruik was. ‘n Kommersieële bevestigingstoetsing het bevind dat Protea magnifica en ‘Pink Ice’ meer voordeel uit ‘n ethanoldamp behandeling kon trek teenoor ‘Sylvia’. Hierdie studie het bevestig die belangrikheid van koolhidraat beskikbaarheid in die Protea snyblom, vir beheer van loofblaarverbruining. Faktore wat die handhawing van hoë interne koolhidrate vlakke, soos bevorderde glukose puls opname of effektiewe vaas oplossing benutting sal bydra tot ‘n optimal beheer van loofblaarverbruining tydens vaas lewe na langtermyn koueopberging. / National Research Fund (NRF) for their financial support in 2009; Protea Producers of South Africa (PPSA) and Productschap Tuinbouw (PT) as well as the Frank Batchelor Will Trust Grant for the financial support.

Thesis (MscAgric (Horticulture))--University of Stellenbosch, 2006. / Protea, Leucadendron and Leucospermum belong to the Proteaceae family. These three genera form an important part of the floriculture industry in South Africa and are commonly used as fresh cut flowers or dried flowers for both the local and international market. The distribution of macro and micro - nutrients was investigated in Protea ‘Cardinal’ using rooted cuttings grown from October 2001 to March 2002. The plants were divided into 1st flush leaf and stem, 2nd flush leaf and stem, basal leaf and basal stem, roots and the flower bud. These tissues were analysed to determine N, P, K, Ca, Mg, Fe, S, Na, B and Mn concentration. Results indicated that N decreased over time in all tissues and accumulated more in leaves than in stems. P in leaves and stems increased with time, while K increased in leaf tissues and remained stable in stem tissue. Ca did not change in young leaves but was high in old leaves. Mg in leaves increased but in basal stem, 1st flush stem and roots Mg decreased over time. Fe in leaves and roots increased with time and not a less significant change occurred in stems. In most tissues, B and Na decreased with time. S increased in leaves and decreased in stems with time. Mn was relatively stable in most tissues except in basal leaves where it increased. Nutrient concentration in tissues, especially in leaves, showed no distinct pattern in the distribution of nutrients. Eleven cultivars from the three genera, namely Protea, Leucadendron and Leucospermum, were used to develop deficiency symptoms of macro - nutrients by using different nutrient compositions. The plants were grown in 20cm pots from December 2002 to September 2003. The eleven cultivars were chosen because of their high market value. Visual symptoms were recorded in two stages with a five - month interval for each stage. The first stage was recorded from December 2002 to April 2003 and the second stage was recorded from May 2003 to September 2003. Observed symptoms indicated significant differences between the control and treatments in which specific nutrients were withheld. Some cultivars exhibited some symptoms that are commonly noticed under field conditions, e.g. in the N deficiency treatment the upper leaves of Protea ‘Sylvia’ were reddish and the lower leaves were chlorotic especially at the later stage. Protea ‘Red Rex’ in the P deficiency treatment had random red tints around the leaf including the petiole, these symptoms are also commonly noticed in Protea ‘Red ‘Rex’ in field conditions. Leucadendron ‘Rosette’ also showed some uncommon symptoms of accumulating “sugar” on leaf tips in Ca deficiency treatment that was not observed in any other cultivar. In some cultivars the symptoms were systematic e.g. Leucadendron ‘Chameleon’ while in other cultivars the deficiency affected a certain leaf age e.g. Leucospermum ‘High Gold’ in the Ca deficiency treatment. The buds in 'High Gold' died prematurely in the Ca deficiency treatment. Protea ‘Cardinal’ was used to determine the optimal N and P source and concentrations for optimal growth. Protea ‘Cardinal’ was grown in a temperaturecontrolled glasshouse for seven months using silica sand as growth medium. Different levels and sources of N and P were applied. The N was applied in a complete nutrient solution as NH4 +, NO3 -, NH4 +: NO3 - (1: 1, 1: 4 and 4: 1 ratios) and Urea, at different concentration levels: 5 mM, 1 mM and 0.1 mM. P was applied at 1 mM, 0.1 mM and 0.01 mM. The plants were fertigated manually with 1L nutrient solution on every second day of the week. P at 0.01 mM and 0.1 mM resulted in optimal plant growth. The 1 mM P resulted in marginal leaf scorching or dryness. When N was applied at 5 mM plant growth was more optimal than when N was applied at 1 mM and 0.1 mM. More dry weight was accumulated at 5 mM than at 1 mM or 0.1 mM N. The dry weight of leaves, stem and roots tissues at 5 mM were higher in the NO3 - treatment.

No description available.