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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.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance. / AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.

Thesis (MScAgric)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Characterizing the genetic structure of a pest population can provide an understanding of the factors influencing its evolution and assist in its ultimate control. The aim of the present study was to characterize the genetic structure of woolly apple aphid Eriosoma lanigerum (Hausmann) populations in the Western Cape Province in South Africa. Since this economically important apple pest has not previously been characterized at molecular level, it was necessary to evaluate methods for determining the genetic structure of E. lanigerum populations. Two different molecular techniques were evaluated viz. random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). This study represents the first application of the latter technique to members of the Aphididae. Aphids were sampled from four regions in the Western Cape in South Africa viz. Elgin, Ceres, Vyeboom and Villiersdorp. A spatially nested sampling design was used to establish the distribution of the genetic variance of aphids. A total of 192 individuals from 13 farms were analysed. Ten RAPD primers were chosen for analysis from an initial assay of 25 after fragment reproducibility had been confirmed. For AFLP analysis three different rare-cutting restriction enzymes were evaluated for AFLP analysis, viz. EcoRI, SseI and MluI. The latter yielded the best results in combination with the frequent-cutting enzyme MseI. Twenty-five AFLP selective primer pairs were evaluated, out of which five were chosen for analysis of the total population. Two hundred and fifty AFLP fragments and 47 RAPD fragments were scored for analysis. Both analyses indicated that a low level of genetic variation was apparent in E. lanigerum populations and that no differentiation resulted from geographic isolation. From RAPD analyses it was deduced that all variation could be attributed to differences between individuals. AFLP analysis indicated that, whereas genetic differences in E. lanigerum populations between orchards were negligible, a significant portion of genetic variation could be attributed to differences between farms and individuals within farms. Therefore, AFLP analysis allowed for finer discrimination of the genetic structure of E. lanigerum populations than RAPD analysis and is recommended for studies of other aphid species. The fact that most of the genetic variation present in E. lanigerum populations could be found on small spatial scales indicated that sampling individuals over a wide geographic area was an ineffective way of detecting the genetic diversity present in E. lanigerum populations. The low level of variation in populations is most likely due to the exclusive occurrence of parthenogenetic reproduction, founder effects (including distribution of infested plant material from a limited source) and selective factors such as the use of resistant rootstocks or pesticides. Furthermore, the low level of variation found indicated that the possibility of controlling E. lanigerum in the Western Cape using host plant resistance is favourable. Thus, plant breeders developing resistance to E. lanigerum can expect plant entries to be exposed to most of the genetic diversity present in Western Cape populations, regardless of location. / AFRIKAANSE OPSOMMING: Die bepaling van die genetiese struktuur van 'n landboukundige plaagpopulasie kan lei tot begrip van die faktore wat die populasie beïnvloed en kan uiteindelike beheer vergemaklik. Die doel van die huidige studie was om die genetiese struktuur van die appelbloedluis Eriosoma lanigerum (Hausmann) in die Wes-Kaap Provinsie van Suid-Afrika te bepaal. Aangesien hierdie belangrike appelplaag nie van tevore op molekulêre vlak bestudeer is nie, was dit nodig om metodes vir die bepaling van die genetiese struktuur van E. lanigerum populasies te evalueer. Twee molekulêre tegnieke is geëvalueer, nl. lukraak geamplifiseerde polimorfiese ONS (RAPD) en geamplifiseerde fragment-lengte polimorfismes (AFLP). Hierdie studie is die eerste om laasgenoemde tegniek te gebruik om lede van die Aphididae te bestudeer. Plantluise is verkry van vier verskillende gebiede in die Wes-Kaap Provinsie van Suid-Afrika nl. Elgin, Ceres, Vyeboom en Villiersdorp. 'n Hierargiese sisteem is gebruik om die verspreiding van die genetiese variasie van plantluise te bepaal. In totaal is 192 individue van 13 plase geanaliseer. Tien RAPD inleiers is gekies uit 'n analise van 25 verskillende inleiers nadat fragment reproduseerbaarheid bevestig is. Drie verskillende restriksie ensieme is geëvalueer vir AFLP analise nl. EcoRI, SseI en Mlul. Die beste resultate is verkry toe MluI saam met MseI gebruik is. Vyf-en-twintig AFLP selektiewe inleier pare is geëvalueer waarvan vyf gekies is vir analise van die totale populasie. Twee-honderd-en-vyftig AFLP fragmente en 47 RAPD fragmente is gedokumenteer vir analise. Beide RAPD en AFLP analises het getoon dat daar 'n lae vlak van genetiese variasie in E. lanigerum populasies is en dat geen differensiasie as gevolg van geografiese isolasie ontstaan het nie. Uit RAPD analise is daar afgelei dat al die variasie toegeskryf kon word aan verskille tussen individue. AFLP het aangetoon dat alhoewel verskille in E. lanigerum populasies tussen boorde laag was, kon 'n hoë persentasie van die variasie toegeskryf word aan verskille tussen plase en individue binne plase. AFLP analise het meer insig in die genetiese struktuur van E. lanigerum populasies verskaf, en word dus aanbeveel vir studies van ander plantluise. Omdat meeste van die genetiese variasie oor klein geografiese afstande verkry word, is steekproefueming oor groot gebiede 'n ondoeltreffende manier om die genetiese variasie binne 'n monster te meet. Die lae vlak van genetiese variasie is waarskynlik te wyte aan partenogenetiese vermeerdering, stigter gevolge (insluitend verspreiding van geïnfesteerde plantmateriaal vanaf 'n beperkte bron), sowel as selektiewe faktore soos die gebruik van bestande onderstokke en insekdoders. Verder dui die lae vlak van variasie aan dat die moontlikheid vir beheer deur gasheerplantbestandheid goed is in die Wes-Kaap. Planttelers kan verseker wees dat hulle plante blootgestel sal wees aan meeste van die genetiese variasie in die Wes-Kaap appelbloedluis populasies ongeag hulle ligging.