Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression. The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv "Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system. In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a "recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups. This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine. / AFRIKAANSE OPSOMMING: Fitoplasma siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan die onlangse ontdekking van fitoplasma in Suid-Afrikaanse wingerde baie nadelige gevolge vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat in amper al organismes as deel van hulle nie-spesifieke verdedegingsstelsel tot uitdruk kom. Hierdie peptiede kan beskerming aanbied teen ʼn wye verskeidenheid van bakteriële en swampatogene in plante. As gevolg van die feit dat fitoplasmas geen buitenste membraan en selwand het nie, word AMPe oorweeg as middle om weerstand te verleen teen hierdie fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die wingerdstok patogeen aster geel fitoplasma (AYp) deur middle van Agrobakteriumbemiddelde tydelike uitdrukkingssiteme, te ondersoek. Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer van aartappel en wingerd plante) is gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-viruse 35S uitdrukkingsvektore met vier verskillende AMP-kodering rye, is dus ontwikkel. As ʼn aternatiewe method om die moontlike effek van Vv-AMP1 op AYp in planta in ag te neem, is oorplantings van die Vv-AMP1 transgeniese Vitis vinifera cv "Sultana‟ plantmateriaal gebruik. Om voorsiening te maak vir AMPe se doeltreffenheid in hierdie tydelike uitdrukkingsvektore, is pogings aangewend om die ruimlike verspreiding en patogeen konsentrasie van AYp in V. vinifera cv "Chardonnay‟ te beskryf. Addisioneel is transmissie eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana te besmet met AYp deur die insekvektor, Mgenia fuscovaria. Plantmateriaal is getoets vir AYp deur van ʼn PCR protokol gebruik te maak met universele inleiers (grondlae) soos beskyf deur Grundersen en Lee (1996). Vir kwantifiseering van die AYp infeksie, is n semi-kwantitatiewe qPCR protokol geoptimaliseer, met hulp van die SYBR Groen-gebaseerde stelsel. In geheel is 86 V. vinifera cv "Chardonnay‟ planties getoets vir AYp infeksie – twee-, drie-, vier-, sewe- en elf weke na die bekendstelling aan die in vitro voorwaardes. Geen AYp infeksie kon egter opgespoor word en die plante het “herstel fenotipe‟ vertoon. Om die verspreiding van AYp in stingelknope van ʼn besemtte V. vinifera cv "Cardonnay‟ plant, blaar en ooreenstemmende stingelknope uit vyf stingels te ondersoek, is hulle getoets deur ʼn PCR protokol. Daar kon afgelei word dat AYp hoofsaaklik in die stingelknop in vergelyking met die blaarmaterial laat in die season, gevind word. Dit is hoogs oonwaarskynlik om fitoplasma infeksies in blaarmaterial te vind, as in die ooreenstemmende stingelknop daar geen AYp oopgespoor kon word nie. As gevolge daarvan dat die AYp-geinfekteerde wingerdmateriaal nie in vitro gegroei kon word nie, kon die effek van VvAMP-1 transgeniese wingerd teen AYp nie getoets word nie. Infeksies van C. roseus en N. benthamiana plante met AYp is suksesvol bereik deur transmissie eksperiemente. met ʼn insekvektor. Tydellike uitdrukkingvektore toetse is uitgevoer op die AYp besmette N. benthamiana material. Kwantifisering van fitoplasma in hierdie material het die afname van AYp in altwee, die AMP behandelings groepe en die kontrole groepe getoon. Hierdie studie het ʼn qPCR geoptimaliseer om besmette plantmaterial met AYp op te spoor en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in hierdie studie gebruik is, was nie vertroubaar genoeg, want geen beduidelike effek van die AMPe op AYp konsentrasie kon waargeneen word nie. Hierdie studie het bewys dat AYp nie vasgestel is en in stand gehou kan word deur in vitro aankweeking van V. vinifera cv "Chardonnay‟ material nie, en weefselkulture kan dus ʼn manier wees om AYp in hierdie kultivar uit te roei. Tot kennis, is hierdie studie die eerste studie om die ruimtelike verspreiding van AYp in stingelknope van ʼn besmette V. vinifera cv "Chardonnay‟ wingerstok te rapporteur. / Winetech and DAAD

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The species Haliotis midae is of great commercial value to the South African abalone industry and is mainly exported to Asian markets, specifically China. Up to 50% is sold as canned products with H. midae registering an average canning yield of approximately 35%. The species is presently genetically undomesticated and breeding programmes are being introduced to improve a range of production traits of which growth and yield is of primary importance. The objective of the study was to determine genetic parameters such as heritability, genotypic and phenotypic correlations of yield-related traits to assess the potential genetic improvement through selective breeding. A series of yield-related parameters were identified that is of relevance to the standard abalone canning procedure. Low to moderate heritabilities where recorded for most traits, including pre-shuck/live weight (0.20 ± 0.06), post-shuck weight (0.15 ± 0.05), post-gut weight (0.15 ± 0.05), post-brine weight (0.19 ± 0.06), pre-canning weight (0.19 ± 0.06), post-canning weight (0.21 ± 0.06), shell weight (0.16 ± 0.05), canning yield percentage (0.08 ± 0.03) and shell weight to post-gut weight ratio (SW: PGW) (0.09 ± 0.04). Weight related parameters are phenotypically highly correlated (0.86 ≤ r ≤ 0.99) but show negative correlation with canning yield percentage (-0.38 ≤ r ≤ 0.04). The nett yield of abalone shows a relatively strong positive correlation with the live weight (r = 0.66). Shell length is highly heritable (h2 ≈ 0.48) and show a strong positive correlation with live weight (r = 0.94). Shell weight is also highly correlated with live weight (r = 0.80) and the SW: PGW ratio does not show a significant correlate with live weight (r = 0.03). Weight-related traits show heritability values ranging from 0.15 to 0.20 that could allow a positive genetic response. Shell length (as a linear growth parameter) shows a high heritability (h2 ≈ 0.48) and a strong positive correlation with live weight (r = 0.94) which also makes it suitable for use as a selection criterion in breeding programmes for improved growth rate. Direct selection for canning yield is compromised by the destructive nature of measurement and the low heritability (h2 < 0.10). The negative correlations between yield as a percentage and growth traits (-0.38 ≤ r ≤ 0.04) further complicate its use as a direct breeding objective. Although the canning yield as a percentage shows a decrease with an increase in live weight, the nett canning yield increases (r = 0.66) with the live weight. It is therefore recommended to use shell length as a criterion for selection for increased growth rate and nett yield, thereby optimising profitability. / AFRIKAANSE OPSOMMING: Die spesie Haliotis midae is van groot kommersiёle waarde tot die Suid-Afrikaanse perlemoenindustrie en word meestal uitgevoer na markte in Asiё, spesifiek China. Tot 50% van die perlemoen wat in Suid-Afrika geproduseer en uitgevoer word, word verblik en huidiglik is die verblikkingsopbrengspersentasie ongeveer 35%. Haliotis midae is tans geneties onderontwikkeld en die gebruik van teelprogramme word nou geimplementeer met die doel om 'n verskeidenheid eienskappe te verbeter, waarvan groei en opbrengs van primêre belang is. Die doelwit van die studie was om genetiese parameters soos oorerflikheid en ook die genotipiese en fenotipiese korrelasies van obrengsverwante eienskappe te bepaal om sodoende die potensiёle genetiese verbetering as gevolg van selektiewe teeling te assesseer. 'n Reeks obrengsverwante eienskappe is geidentifiseer wat relevant is binne bestaande en standaard kommersiёle perlemoenverblikkingsprotokolle. Lae tot matige oorerflikheidswaardes is waargeneem en sluit in lewende, of voor-ontskulpingsgewig (0.20 ± 0.06), na-ontskulpingsgewig (0.15 ± 0.05), na-oopvlekkingsgewig (0.15 ± 0.05), na-pekelgewig (0.19 ± 0.06), voor-verblikkingsgewig (0.19 ± 0.06), na-verblikkingsgewig (0.21 ± 0.06), skulpgewig (0.16 ± 0.05), verblikkingsopbrengspersentasie (0.08 ± 0.03) en 'n skulpgewig tot na-oopvlekkingsgewig verhouding (SW: PGW) (0.09 ± 0.04). Gewigsverwante parameters is fenotipies hoogs gekorreleerd met mekaar (0.86 ≤ r ≤ 0.99) maar toon 'n negatiewe korrelasie met die verblikkingsopbrengspersentasie (-0.38 ≤ r ≤ 0.04). Die netto opbrengs van perlemoen dui op 'n relatiewe sterk positiewe korrelasie met lewende gewig (r = 0.66). Skulplengte is hoogs oorerflik (h2 ≈ 0.48) en toon 'n sterk positiewe korrelasie met lewende gewig (r = 0.94). Skulpgewig is ook hoogs gekorreleerd met lewende gewig (r = 0.80) en die SW: PGW verhouding toon geen beduidende korrelasie met lewende gewig nie (r = 0.03). Gewigsverwante eienskappe toon oorerflikheidswaardes wat varieer tussen 0.15 en 0.20 en kan 'n moontlike genetiese respons lewer. Skulplengte (as 'n liniêre groeiparameter) toon 'n hoё oorerflikheid (h2 ≈ 0.48) en 'n sterk positiewe korrelasie met lewende gewig (r = 0.94) wat dit gepas maak vir gebruik as 'n seleksiekriterium in 'n teelprogram met verbeterde groeitempo as doel. Direkte seleksie in terme van verblikkingsopbrengs word ingeboet danksy die destruktiewe natuur van die metingsmetodiek asook 'n lae oorerflikheid (h2 < 0.10). Die negatiewe korrelasies tussen verblikkingsopbrengs (uitgedruk as 'n persentasie) en groeieienskappe (-0.38 ≤ r ≤ 0.04) dien as 'n verdere komplikasie in die gebruik van dié eienskap as direkte teeldoelwit. Alhoewel die verblikkingopbrengs 'n afname toon soos lewende gewig toeneem, is daar steeds 'n positiewe korrelasie tussen die netto verblikkingsopbrengs en die lewende gewig (r = 0.66). Dit word dus aangeraai om skulplengte as seleksiekriterium vir verbeterde groeitempo en netto opbrengs te gebruik om sodoende wins te maksimaliseer.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Wheat rust, caused by the Puccinia spp., is a global biotic cause of wheat yield losses. This disease can effectively be combatted by implementing rust resistant wheat cultivars. The release of new resistant wheat cultivars is however prolonged due to the time needed to fix resistance genes in a good quality background and develop pure breeding wheat lines. The aim of this study was the pyramiding of novel species derived leaf and stripe rust resistance genes in bread wheat lines through the utilization of high throughput marker assisted selection and microspore derived doubled haploid technology. / AFRIKAANSE OPSOMMING: Koringroes het wêreldwyd verliese in koringopbrengste tot gevolg. Dit word veroorsaak deur die Puccinia fungi. Hierdie siekte kan effektief beveg word deur die verbouing van roesbestande kultivars. Die vrystel van nuwe weerstandbiedende kultivars is egter ‘n langdurige proses weens die tyd verbonde daaraan om weerstandsgene te fikseer in ‘n genetiese agtergrond met ‘n goeie kwaliteit en om dan suiwertelende lyne te ontwikkel. Die doelwit van hierdie studie was om nuwe spesie-verhaalde blaar- en streeproes weestandsgene in koringlyne te stapel met behulp van merker bemiddelde seleksie en mikrospoor geassosieerde verdubbelde haploïede tegnologie.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aim of this study was to critically assess the implementation of the internal reference method within the most recent 173 full-sibling growth trial of the Innovation Fund Abalone Breeding Project. The trial was conducted over two locations for a period of five years, with minimal replication for the majority of test families and a single full-sibling family was entered into each experimental unit (basket) as an internal reference group. The primary focus was firstly, to validate the performance of the internal reference group as a control for comparisons and correction of environmental variation in test family performances. Secondly, to identify areas of weakness and either make recommendations to remedy areas of weakness or justify devoting resources to alternative methods of reducing extraneous environmental variance with limitations on replication. The efficiency and statistical power associated with utilising internal reference information as a covariate and for manual correction respectively were examined for the 6 full-sibling test families that were replicated. This study reports on the evaluation of factors which are potential sources of bias in the internal reference method, the first of which, tag loss, was found to be significant after 6- 12 months. However, it was not found to bias internal reference group performances as the factors which contribute to tag loss were found to act randomly. Variability in size ratio of internal reference to test family at co-stocking proved a significant source of bias, as reference groups smaller than their test family counterparts had reduced performances. Testing for genotype by environment interactions was precluded due to the inherent lack of replication and the subsequent confounding of genotype effects with inter-rearing structure effects at one of the locations. However, significant differences were detected for both traits of interest of the internal reference group over the two locations. Significant antagonistic interactions were detected and identified as a source of bias for average daily weight gain of replicate test families. The evaluation of average daily length gain for the efficiency of adjustment when the internal reference is a covariate and the change in statistical power when the internal reference is used for a manual correction, yielded conflicting results. The latter shows a decrease in statistical power and the former shows an increase in efficiency, both resulting in poor goodness of fit in the respective models. There was however evidence that when no antagonistic interactions occurred “between replicate variance” decreased and therefore the internal reference method has statistical merit provided all critical success factors are satisfied. Recommendations were made for future implementation of the internal reference method to facilitate adequate statistical testing for sources of bias and the prevention thereof. Additionally, an alternative method which may have merit in decreasing environmental variance and the need for replication, is discussed. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die gebruik van ʼn interne verwysingsgroep te ontleed, soos toegepas tydens die evaluering van 173 volsib families as deel van die Innovasiefonds Perlemoen Teelprogram. Die evaluering is gedoen op twee lokaliteite oor 'n tydperk van vyf jaar, met minimale replikasie van die toets families en die gebruik van ‘n enkele volsib familie as 'n interne verwysingsgroep in elke eksperimentele eenheid (mandjie). Die primêre fokus was eerstens om die gebruik van die interne verwysingsgroep vir die korreksie van omgewingsvariasie in die toets familie optredes te evalueer. Tweedens, om spesifieke gebreke te identifiseer ten opsigte van die gebruik van die interne verwysingsgroep en aanbevelings maak dit reg te stel en om die meriete van alternatiewe metodes te oorweeg. Die doeltreffendheid en statistiese onderskeidingsvermoë van die gebruik van interne verwysingsgroep as 'n kovariaat is ondersoek met betrekking tot die 6 volsib groepe wat oor voldoende replikasies beskik het. Die studie doen voorts verslag oor die evaluering van potensiële oorsake van sydigheid ten opsigte van die gebruik van die interne verwysingsgroep, insluitend die beduidende verlies van identifikasie vanaf 6 tot 12 maande. Geen aanduiding van sydigheid is egter gevind en die aanleidende oorsake van verlies van identifikasie blyk van ʼn ewekansige aard te wees. Verskille in die grootte tussen die interne verwysingsgroep en toets-families met aanvang van evaluering blyk 'n belangrike bron van sydigheid te wees, waar die kleiner groepering aan verminderde prestasie gekoppel word. Bepaling van genotipe-omgewing-interaksies kon nie uitgevoer word nie as gevolg van die inherente gebrek van replisering oor lokaliteite. Beduidende verskille is egter waargeneem tussen interne verwysingsgroepe oor die twee lokasies ten opsigte van die beide groei eienskappe. Beduidende antagonistiese interaksies is waargeneem en geïdentifiseer as 'n bron van sydigheid ten opsigte van die gemiddelde daaglikse gewigstoename van replikaat toetsfamilies. Die evaluering van gemiddelde daaglikse lengtetoename met die interne verwysingsgroep as is 'n kovariaat en die verandering in statistiese ontledingsvermoë tydens die gebruik van die interne verwysingsgroep het teenstrydige resultate opgelewer. Laasgenoemde toon 'n afname in statistiese ontledingsvermoë en die eersgenoemde toon 'n toename in doeltreffendheid, met beide swak passing op die onderskeie modelle. In die afwesigheid van antagonistiese interaksies tussen replikasies het variansie afgeneem en beskik die interne verwysingsgroep oor die nodige statistiese meriete indien daar aan al die kritiese vereistes voldoen word. Aanbevelings is gemaak ten opsigte van die toekomstige implementering van die interne verwysingsmetode met verwysing na voldoende statistiese toetsing vir bronne van sydigheid en die voorkoming daarvan. 'n Verdere metode wat oor die nodige meriete beskik om die omgewingsvariasie en die noodsaaklikheid vir replikasie te verminder, word bespreek.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Most terrestrial plants make use of beneficial symbiotic associations to obtain nutrients (eg. nitrogen (N) and phosphorous (P)) from fungi in exchange for photoautotrophic carbon. However, plant parasitism (defined here as the ability of certain plants to parasitize other living material) has evolved in the plant kingdom and such plants obtain some, or all, of their nutritional needs from a host, which is severely negatively impacted by the parasite. While the physiological adaptations are well studied, the underlying molecular and biochemical mechanisms of plant parasitism remain largely unknown. As a first approach, a biochemical blueprint of primary metabolites present within parasitic plant species was constructed. The metabolomes of nineteen parasitic plants, ranging from hemi- and holoparasitism to mycoheterotrophism, were profiled via gas chromatography mass spectrometry (GC MS) based technology and targeted spectrophotometric assays. Based on these analyses, three important observations were made. First, parasitic plants were severely carbon deprived, despite being successful in colonizing and exploiting their hosts. Second, the levels of organic acids participating in mitochondrial respiration decreased and certain amino acids and soluble protein content increased. This suggests that parasitic plants utilize alternative respiratory substrates to compensate for a limitation in carbon supply. Third, although characterized by reduced carbohydrate pools, minor sugars normally not associated with plant metabolism, dominated the soluble sugar pool. The presence and significance of one of these sugars, namely turanose (α-D-glucopyranosyl-(1→3)-α-D-fructofuranose), was further investigated. Turanose biosynthetic reactions could be demonstrated in Orobanche minor extracts. Protein purification and mass spectrometry identification suggested that turanose biosynthesis occurred uniquely in parasitic plants. Future work will elucidate the functional significance of turanose metabolism in plant parasitism. Taken together, this study significantly contributes to our understanding of plant parasitism through development of metabolic signatures associated with distinct parasitic classes. These biochemical profiles highlighted several important strategies and alternative metabolic pathways that are either expressed or constitutively activated during parasitism. This knowledge broadens the scope of using parasitic plants in several biotechnological applications or as a novel research tool to address fundamental questions in plant science. / AFRIKAANSE OPSOMMING: Meeste landelike plante maak gebruik van voordelige simbiotiese assosiasies met swamme om voedinsgtowwe (bv. stikstof (N) en fosfor (P)) van hulle te verkry in ruil vir koolstof geproduseer deur die plant. Plant parasitisme (gedefinieer hier as die vermoë van sekere plante om ander lewende materiaal te parasiteer) het ontwikkel in die planteryk waar hulle sommige, of al hul voedings stowwe van 'n gasheer plant ontvang, wat erg negatief geraak word deur die parasiet. Terwyl die fisiologiese aanpassings goed gebestudeer is, is die onderliggende molekulêre en biochemiese meganismes van plant parasitisme steeds grootliks onbekend. As 'n eerste benadering, was hierdie projek geïnisieer om 'n biochemiese bloudruk op te bou van primêre metaboliete teenwoordig in parasitiese plante. Die metabolome van negentien parasitiese spesies, wat wissel van hemi - en holoparasiete tot mikoheterotrofiese plante, is ondersoek deur gas chromatografie – massa spektrometrie (GC MS) gebaseerde tegnologie en geteikende spektrofotometriese toetse. Gebaseer op hierdie ontledings was drie belangrike waarnemings gemaak. Eerstens, parasitiese plante was erg koolstof arm, ten spyte daarvan dat hulle suksesvol is in die aanhegting en ontginning van voedingstowwe vanaf gasheer plante. Tweedens, die vlakke van organiese sure wat deelneem aan mitochondriale respirasie het afgeneem, terwyl sekere aminosure en oplosbare proteïen inhoude toegeneem het. Dit dui daarop dat parasitiese plante gebruik maak van alternatiewe respiratoriese substrate om te vergoed vir 'n beperking in koolstof aanbod. Derde, alhoewel parasitiese plante gekenmerk word deur verminderde koolhidraat inhoude, het skaarse suikers wat normaalweg nie verband hou met plant metabolisme nie, hulle oplosbare suiker inhoud oorheers. Die teenwoordigheid en betekenis van een van hierdie suikers, naamlik turanose (α -D -glucopyranosyl-(1→3)-α-D-fructofuranose), was verder ondersoek. Die sintese reaksie van turanose kan gedemonstreer word in Orobanche hederae uittreksels. Proteïen suiwering en massa spektrometrie identifikasie het voorgestel dat turanose biosintese uniek plaasvind in parasitiese plante. Toekomstige werk sal aandui wat die betekenis is van turanose metabolisme in plant parasitisme. Saamgevat het hierdie studie aansienlik bygedra tot ons begrip van plant parasitisme deur ontwikkeling van metaboliese handtekeninge wat verband hou met onderskeie parasitiese klasse. Hierdie biochemiese profiele beklemtoon verskeie belangrike strategieë en alternatiewe metaboliese paaie wat óf uitgedruk of konstitutief geaktiveer word tydens parasitisme. Hierdie kennis verbreed die omvang van die gebruik van parasitiese plante in verskeie biotegnologiese toepassings of as 'n nuwe navorsings instrument om fundamentele vrae in plant wetenskap aan te spreek.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) is an epidemic disease characterised by wet, persistent coughing, weight loss, fever, fatigue, and blood in the sputum. It has been reported that one in every three individuals is currently infected with Mycobacterium tuberculosis, the causative agent of TB, and that 10% of them will develop the active disease. The high prevalence and low penetrance of this disease has resulted in increased research performed to ascertain what factors play a role in susceptibility to M. tuberculosis infection. Some factors known to play a role in a minority of cases may include: HIV infection, diabetes, alcohol abuse and old age, but racial differences in susceptibility have also been observed. However, the influence of genetic factors is gaining popularity in current research. M. tuberculosis requires iron to proliferate, which it must appropriate from its host. For this reason the genes involved in the metabolism of iron in the human host are of particular interest when considering susceptibility to M. tuberculosis infection. In order to determine whether the expression of these genes may influence disease susceptibility, the promoter region of the CYBRD1, HAMP, HFE, LTF and SLC40A1 genes have been targeted for investigation. The aim of this study was to determine whether DNA variation in the promoter region of these genes involved in iron metabolism is associated with M. tuberculosis susceptibility. The study cohort consisted of 49 TB patients and 51 healthy, unrelated, population-matched controls, all of whom were Black, Xhosa-speaking individuals. Initially, 15 patient samples were randomly selected for exploratory screening, utilising semi-automated bi-directional sequencing analysis. In this manner, 40 variants were identified of which 30 were previously described. The novel variants included CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; and LTF: -1377 T/G, -457 T/C, -437 C/G. A number of loci demonstrated a statistically significant difference in allele and genotype frequencies, and in iron parameter levels when comparing the patient and control groups and for each variant separately. In silico analyses revealed the creation and/or abolishment of several transcription factor binding sites (TFBSs) due to the presence or absence of certain identified variants. The change in the composition of TFBSs in the promoter region may lead to differential expression of the gene. This study served as a pilot investigation to identify promoter region variants in the candidate genes involved in iron metabolism, in TB patients. The results presented here indicate that the identified variants (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) could possibly contribute to the increased absorption of iron in the TB patient group, which could subsequently increase the occurrence of pathogenic infection. The findings of this study could further aid in the elucidation of the exact mechanism(s) by which iron, its metabolism, and the genes involved affect disease susceptibility, more specifically, M. tuberculosis susceptibility. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is „n epidemiese siekte gekarakteriseer deur nat, aanhoudende hoes, gewigsverlies, moegheid, en bloed in die speeksel. Dit is gerapporteer dat een in elke drie individue tans geïnfekteer is met Mycobacterium tuberculosis, die veroorsakende agent van TB, en dat 10% van dié individue die aktiewe vorm van die siekte sal ontwikkel. Die hoë voorkoms en lae effek van hierdie siekte het daartoe gelei dat meer navorsing mettertyd gedoen is om die faktore wat „n rol mag speel in M. tuberculosis infeksie, te bepaal. Sommige faktore bekend vir hul rol in „n minderheid van gevalle sluit in: MIV infeksie, diabetes, alkoholmisbruik en bejaardheid, maar etniese verskille in vatbaarheid vir die siekte is ook al waargeneem. Die waarskynlikheid van genetiese invloed op die ontwikkeling van TB word ook meer deur navorsers ondersoek. M. tuberculosis benodig yster om te vermeerder, wat dit moet bekom vanaf die gasheer. Vir hierdie rede is die gene betrokke by yster metabolisme in die menslike gasheer veral van belang vir die oorweging van vatbaarheid vir M. tuberculosis. Om te bepaal of die uitdrukking van hierdie gene moontlik „n invloed het op vatbaarheid vir die siekte, was die promoter areas van die CYBRD1, HAMP, HFE, LTF en SLC40A1 gene geteiken. Die doel van hierdie studie was om te bepaal of DNS variasie in die promoter area van hierdie gene betrokke in yster metabolisme moontlik verband kan hou met vatbaarheid vir M. tuberculosis. Die studie kohort het uit 49 TB pasiënte en 51 gesonde, onverwante, populasie-gepaarde kontroles, waarvan almal Swart, Xhosa-sprekende individue was, bestaan. Aanvanklik was 15 pasiënt monsters lukraak gekies vir ondersoekende sifting, deur die gebruik van semi-outomatiese twee-rigting volgordebepalings. Op hierdie manier is 40 variante geïdentifiseer waarvan 30 voorheen beskryf is. Die nuwe variante sluit in CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; en LTF: -1377 T/G, -457 T/C, -437 C/G. „n Aantal loci het statisties betekenisvolle verskille getoon in alleel en genotipe frekwensies, en in yster parameter vlakke met die vergelyking van die pasiënt groep met die kontrole groep. In silico analise het verder die skepping en/of afskaffing van verskeie transkripsiefaktor bindingsetels (TFBSs), as gevolg van die teenwoordigheid of afwesigheid van sekere variante, getoon. Die verandering in die samestelling van TFBSs in die promoter area kan lei tot differensiële uitdrukking van die geen. Dié studie het gedien as „n voorlopige ondersoek om te bepaal of promoter area variante, geïdentifiseer in kandidaat gene betrokke by yster metabolisme, „n invloed het in die ontwikkeling van TB. Die resultate wat hier gewys word dui aan dat die geïdentifiseerde variante (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) moontlik die verhoogde opname van yster kan veroorsaak, wat later die toename van die patogeniese infeksie kan veroorsaak. Die bevindinge van hierdie studie kan moontlik bydra tot die toeligting van die presiese meganisme(s) waardeur yster, yster metabolisme, en die betrokke gene vatbaarheid vir siekte, meer spesifiek M. tuberculosis vatbaarheid, beïnvloed.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: South Africa is ranked eighth in the world as far as international wine production is concerned and in terms of area under bearing vines South Africa is ranked 12th. In 2011 the wine industry contributed R4 204.4 million to the South African economy in state revenue from wine products. The importance of viticulture to the economy of South Africa forces the industry to limit the effect of all disease causing pathogens in order to keep their competitive edge. Aster yellows (AY) phytoplasma 16SrI-B subgroup was reported for the first time in grapevine (Vitis vinifera L. (Vitaceae)) in South Africa in 2006. Worldwide phytoplasma diseases of grapevine cause serious damage ranging from lower yields to the death of vines. The lack of knowledge about the epidemiology of AY disease makes it difficult to determine the impact of the disease on the South African wine industry. The aim of this study was to conduct surveys in disease-affected vineyards in the Vredendal region to determine the incidence and spatial distribution of the disease in a variety of cultivars. The field surveys based on visual symptoms of AY disease were confirmed by polymerase chain reaction (PCR). A survey was also conducted in and around AY-infected vineyards in search of possible alternative host plants of the phytoplasma. Spatial distribution of AY-affected vines were analysed using the PATCHY spatial analysis package. A rapid decline of AY-affected Chardonnay eventually leading to the death of vines was observed, confirming the sensitivity of Chardonnay towards grapevine yellows infections. Symptomless AY infections occurred and AY could not be detected in all symptomatic vines, which indicate uneven distribution of AY in individual vines. Molecular analyses using PCR-RFLP showed that all vines sampled in the Vredendal vicinity contained AY phytoplasma only. No phytoplasmas were present in any weeds or other possible host plants tested. Although the mean yearly disease incidences of Chardonnay (29.95%) and Chenin blanc (16.64%) were higher than Pinotage (5.80%) over the four-year survey period, there was no statistically significant difference between the disease incidences of these three cultivars. The mean yearly disease incidence showed a trend over time and the disease incidence of the first year was significantly lower than that of the other years. Chardonnay showed a cumulative disease incidence of 37.77% at the end of the 4-year study which means that Chardonnay vineyards can be 100% AY infected in ten years’ time. Spatial distribution patterns of AY-infected vines were mostly non-random with clustering of disease affected vines along and across vine rows. With the exception of one vineyard, aggregation of AY-affected vines mostly occurred on the edge of vineyards adjacent to infected vineyards. This epidemiological study gives an indication of the sensitivity of the different cultivars towards AY, the tempo of spreading and the future impact of the disease on the South African wine industry. It also contributes valuable information towards the development of a management strategy for grapevine yellows disease in South African vineyards. / AFRIKAANSE OPSOMMING: Suid- Afrika is op agtste op die wêreld ranglys wat internasionale produksie van wyn aan betref, en in terme van oppervlakte onder wingerd, is Suid-Afrika 12de. In 2011 het die wynbedryf R4 204.4 miljoen tot die Suid-Afrikaanse ekonomie bygedra in staats inkomste uit wyn produkte. Die belangrikheid van wingerd tot die ekonomie van Suid-Afrika dwing die bedryf om die effek van alle siekteveroorsakende patogene te beperk, om sodoende hul kompeterende voordeel te behou. Aster vergeling (AY) fitoplasma 16SrI-B subgroep is vir die eerste keer in 2006 in wingerd (Vitis vinifera L. (Vitaceae)) in Suid-Afrika waargeneem. Fitoplasma siektes van wingerd veroorsaak wêreldwyd ernstige skade wat wissel van laer opbrengste tot die afsterf van wingerdstokke. Die gebrek aan kennis oor die epidemiologie van astervergeling siekte maak dit moeilik om die impak van die siekte op die Suid-Afrikaanse wynbedryf te bepaal. Die doel van hierdie studie was om ‘n opname te maak in siekte geaffekteerde wingerde in die Vredendal omgewing om sodoende siekte voorkoms en verspreidingspatrone van die siekte in 'n verskeidenheid van kultivars te bepaal. Die veld opnames, gebaseer op visuele simptome van aster vergeling siekte, was bevestig deur polimerase kettingreaksie (PKR). ‘n Opname is ook in en om aster vergeling geaffekteerde wingerde uitgevoer, op soek na moontlike alternatiewe gasheer plante van die fitoplasma. Verspreidingspatrone van astervergeling geaffekteerde wingerde is ontleed met behulp van die PATCHY ruimtelike analise pakket. 'n Vinnige agteruitgang van AY geaffekteerde Chardonnay, wat uiteindelik gelei het tot die afsterf van wingerde, is waargeneem, wat die sensitiwiteit van Chardonnay teenoor wingerdvergeling infeksie bevestig. Simptoomlose astervergeling fitoplasma infeksies kom voor en astervergeling fitoplasma kon nie opgespoor word in alle simptomatiese wingerdstokke nie, wat op oneweredige verspreiding van AY fitoplasma in individuele wingerdstokke dui. Molekulêre ontledings met behulp van PKR-RFLP het getoon dat alle wingerdstokke, wat in die Vredendal omgewing getoets is, slegs astervergeling fitoplasma bevat. Geen fitoplasmas was teenwoordig in enige onkruide of ander moontlike gasheer plante. Hoewel die gemiddelde jaarlikse siekte voorkoms van Chardonnay (29,95%) en Chenin Blanc (16,64%) oor die vier-jaar opname periode hoër was as dié van Pinotage (5,80%), was daar geen statisties beduidende verskil tussen die siekte voorkoms van hierdie drie kultivars nie. Die gemiddelde jaarlikse siekte voorkoms het 'n tendens oor tyd getoon, en die siekte voorkoms van die eerste jaar was betekenisvol laer as dié van die ander jare. Chardonnay het ‘n kumulatiewe siekte voorkoms van 37.77% aan die einde van die 4-jaar studie getoon, wat beteken dat Chardonnay wingerde binne 10 jaar 100% besmet kan wees met AY. Verspreidingspatrone van AY geaffekteerde wingerdstokke was meestal nie-ewekansig met bondeling van geaffekteerde wingerdstokke in en oor wingerd rye. Bondeling van AY geaffekteerde wingerdstokke het, met die uitsondering van een wingerd, meestal op die kant van wingerde aanliggend aan besmette wingerde, voorgekom. Die epidemiologiese studie gee 'n aanduiding van die sensitiwiteit van die verskillende kultivars ten opsigte van AY, die tempo van die verspreiding en die toekomstige impak van die siekte op die Suid-Afrikaanse wynbedryf. Dit dra ook waardevolle inligting by tot die ontwikkeling van 'n strategie vir die bestuur van wingerdvergeling siekte in Suid-Afrikaanse wingerde.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Efavirenz is an antiretroviral drug used in the treatment of HIV-positive patients as part of first line triple-highly active antiretroviral therapy. Treatment response varies among individuals and adverse drug reactions tend to occur, as a result of the variation in the rate of efavirenz metabolism among individuals. This is partly caused by genetic variation; therefore the study of genes involved in the metabolism of efavirenz, such as CYP2B6, could potentially enhance treatment success. The effect of CYP2B6 SNP 516G>T (part of the CYP2B6*6 allele) is particularly important, as individuals homozygous for the minor allele of this SNP have significantly increased efavirenz levels. Furthermore, nuclear receptors, specifically constitutive androstane receptor, encoded by NR1I3, and pregnane X receptor, encoded by NR1I2, are involved in the regulation of the genes responsible for efavirenz metabolism and could therefore indirectly influence the pharmacokinetics of efavirenz. The current study identified variants in the NR1I3 and NR1I2 genes through in silico analysis, bi-directional sequencing and literature searches. A total of nine NR1I3 and ten NR1I2 target variants were subsequently genotyped in 132 HIV-positive female patients from the Xhosa and Cape Mixed Ancestry populations. The resulting genotype and allele frequencies were statistically analysed to search for correlations between genetic variations and available efavirenz levels in hair samples, treatment outcome as measured by viral load, and the occurrence of adverse drug reactions. The minor allele of a NR1I2 5’-upstream SNP, rs1523128 (6334A>G), was significantly associated with decreased efavirenz levels. From analysis of the effect of composite SNPs, NR1I3 5’-upstream SNP rs55802895 (258G>A) in conjunction with CYP2B6*6, was significantly associated with efavirenz-levels. It was found that the minor allele of rs55802895 inhibited the effect of CYP2B6*6, resulting in normal efavirenz levels for individuals homozygous for the minor allele of both SNPs. Additionally, when the target NR1I3 and NR1I2 variants were analysed in conjunction with six SNPs from CYP1A2, CYP2A6, CYP3A4 and CYP3A5, 11 compound genotypes were shown to be statistically associated with mean EFV plasma levels. The study emphasises the complexity of efavirenz metabolism, and the importance of transcriptional regulation in xenobiotic metabolism. / AFRIKAANSE OPSOMMING: Efavirenz is ‘n antiretrovirale middel wat gebruik word in die behandeling van HIV-positiewe pasiënte as deel van drievoudige hoogs-aktiewe antiretrovirale terapie. Reaksie op behandeling verskil tussen individue en nadelige newe-effekte, wat veroorsaak word deur die verskil in tempo waarteen efavirenz gemetaboliseer word, neig om voor te kom. Hierdie verskille word gedeeltelik veroorsaak deur genetiese variasie; dus kan die studie van gene betrokke by die metabolisme van efavirenz, soos CYP2B6, moontlik die sukses van behandeling verhoog. Die effek van CYP2B6 SNP 516G>T (deel van die CYP2B6*6-alleel) is veral belangrik, want individue wat homosigoties is vir die minderheids-alleel het betekenisvol hoë efavirenz-vlakke. Nukleêre reseptore, spesifiek konstitutiewe androstane reseptor, deur NR1I3 gekodeer, en pregnane X reseptor, deur NR1I2 gekodeer, is betrokke by die regulering van die gene verantwoordelik vir efavirenz-metabolisme en kan dus die farmakokinetika van efavirenz beïnvloed. Die huidige studie het variante in NR1I3 en NR1I2 identifiseer deur in silico-analise, bi-direksionele volgordebepaling en ’n literatuurstudie. Nege NR1I3 en tien NR1I2-variante in totaal is vervolglik gegenotipeer in 132 HIV-positiewe vroulike pasiënte van Xhosa en Kaapse Gemengde Afkoms populasies. Die gevolglike genotipe- en alleelfrekwensies is statisties geanaliseer om vir korrelasies tussen genetiese variasies en beskikbare efavirenz-vlakke in haarmonsters, uitkoms van behandeling gemeet in virale lading en die voorkoms van nadelige newe-effekte te soek. Daar is gevind dat die minderheids-alleel van ’n NR1I2 5’-stroomop SNP, rs1523128 (6334A>G), betekenisvol geassosieer is met ’n daling in efavirenz-vlakke. Vanuit die saamgestelde SNPs, is die NR1I3 5’-stroomop SNP rs55802895 (258G>A), tesame met CYP2B6*6, betekenisvol geassosieer met efavirenz-vlakke. Daar is gevind dat die minderheids-alleel van rs55802895 die effek van CYP2B6*6 demp, en gevolglik normale efavirenz-vlakke in individue homosigoties vir die minderheids-allele van albei SNPs veroorsaak. Addisioneel is die teiken NR1I3 en NR1I2 variante gemeenskaplik met ses SNPs van CYP1A2, CYP2A6, CYP3A4 en CYP3A5 geanaliseer en 11 gekombineerde genotipes is statisties geassosieer met gemiddelde EFV plasma vlakke. Hierdie studie beklemtoon die kompleksiteit van efavirenz-metabolisme en die belangrikheid van transkripsionele regulering in xenobiotiese metabolisme. / National Research Foundation (NRF)

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis-like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation. / AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) algemeen bekend as die Russiese koringluis (RWA), is ‘n klein floëem-voedende pes van koring (Triticum aestivum L). Virulente D. noxia biotipes wat instaat is om op voorheen bestande koring kultivars te voed gaan ontwikkel voortdurend, en daarom is die identifisering van faktore wat kan bydrae tot virulensie so belangrik. Omdat energie-metabolisme ‘n sleutelrol in die oorlewing van organismes speel, kan gene en prosesse wat by die produksie en regulering van energie betrokke is belangrike bydraers tot virulensie lewer: soos onder andere mitokondria en die NAD+/NADH-verhouding wat die gesondheid en metaboliese aktiwiteit van ‘n sel reflekteer. Die betrokkenheid van karotenoïede in die produksie van energie deur 'n fotosintese-verwante proses kan 'n belangrike faktor bydraend tot luis fiksheid wees, asook die bydra daarvan tot plantluis-immuniteit deur bemiddeling van oksidatiewe stres. Die volledige mitochondriale genoom van globale Diuraphis noxia populasies is met behulp van volgende generasie DNA volgordebepaling gekarrakteriseer, en daar is bevind dat dit 15 721 bp in grootte is en uit 38 gene bestaan wat tipies binne insekte voorkom. Enkelnukleotied- polimorfisme (SNP) ontleding van die genome van nege populasies het onthul dat daar 125 SNPs in die proteïen-koderende gene voorkom, met die meerderheid van die SNPs in die ND-gene, en die minste in die ND4L-geen. Lae SNP-frekwensies is gevind vir die atp6- en atp8- gene, wat verskil van verslae oor ander Hemiptera. Veranderlike ND5-uitdrukkingsvlakke onder die biotipes is waargeneem, alhoewel geen korrelasie duidelik was tussen ND5-uitdrukking en die virulensie geassosieer met elke biotipe nie. Die transkripsie van atp6 was hoër in die hoogs virulente biotipe (SAM) onder normale en stresvolle toestande in vergelyking met die minste virulente biotipe (SA1). ‘n Aansienlike hoër NAD+/NADH-verhouding is ook waargeneem vir die SAM-biotipe onder spanningsvolle omstandighede in vergelyking met die minder virulente biotipes. UPLC-MS-analise het geen likopeen of β-karoteen geïdentifiseer nie as gevolg van lae verbinding konsentrasies in die onttrekte monsters, maar verskeie hidrofobiese verbindings was in verskillende konsentrasies tussen die biotipes teenwoordig. Die karoteen desaturase-uitdrukkingsprofiel het aangetoon dat SA1 die laagste relatiewe uitdrukking van gene betrokke by karotenoïed produksie het, terwyl SAM die hoogste relatiewe uitdrukking onder normale en spanningsvolle omstandighede het. Die resultate van die studie dui daarop dat die volgorde bewaring in mitochondriale gene verband hou met die sleutel energie prosesse om 'n toestand van homeostase onder wisselende omstandighede te handhaaf en dat die produksie van energie 'n bydraende faktor tot die ontwikkeling van virulensie in D. noxia is. Die resultate toon ook aan dat karotenoïede moontlik kan bydra tot fiksheid van D. noxia deur reaktiewe suurstofspesies te aas of deur die produksie van addisionele energie, maar verdere ondersoeke word benodig ter bevestiging.