M.Sc. (Botany and Plant Biotechnology) / Wheat (Triticum aestivum L.) is the one of the three most extensively cultivated cereal crops worldwide (Shewry, 2009). In South Africa, wheat is cultivated in both summer and winter rainfall regions as monocultures typical to modern industrialised agriculture. Monocultures provide uniform crop quality and allow processes such as planting and harvesting to be mechanised (Altieri et al., 2009). However, the genetically homogeneous nature of monocultures increases the vulnerability of the crop to both biotic and abiotic stresses (Faraji, 2011). Future food production is challenged by predicaments such as an increasing human population while the ratio of arable land to population is decreasing. Yield losses of wheat due to biotic factors alone were estimated as 29 % (2001-2003) (Oerke, 2006). The need to reduce the gap between attainable yield and actual yield is therefore crucial in order to maximise crop production for future food security (Duveiller et al., 2007). One of the most damaging pests to worldwide wheat production is the Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov) (Arzani et al., 2004). A native pest of central Asia, the RWA has spread to all cereal producing areas of the world with the exception of Australia (Burd et al., 2006). While feeding on susceptible hosts, the aphid injects an eliciting agent into the host, which causes the breakdown of the chloroplast and cellular membranes, leading to the appearance of symptoms typical of RWA feeding, including leaf rolling (Botha et al., 2005). Leaf rolling creates a sheltered environment for the aphid from insecticides and predators, and this together with their parthenogenic and viviparous reproductive nature makes their rapid increase in numbers extremely difficult to control (Goggin, 2007). Resistant wheat genotypes currently represent the most effective long term solution to control RWA infestations; however resistance breaking aphid biotypes are rapidly overcoming the incorporated resistance genes under field conditions (Burd et al., 2006; Jankielsohn, 2011). Understanding the molecular basis of plant resistance to the RWA is crucial in creating cultivars with durable resistance (Botha et al., 2005).

Please read the abstract in the section 00front of this document / Dissertation (MSc (Genetics))--University of Pretoria, 2002. / Genetics / unrestricted

Doctor of Philosophy / Department of Entomology / C. Michael Smith / The Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) (RWA), is an important pest in the U.S. Western Plains, causing hundreds of millions of dollars of losses to wheat and barley production through reduced yields and insecticide application costs. The objectives of this research were to evaluate the performance of two RWA-resistant barley varieties planted approximately one month earlier than normal in experimental fields at Fort Collins, Colorado; Tribune, Kansas; and Sidney, Nebraska during 2007, 2008, and 2009. The experimental design was a split-plot design with two main plot treatments (early and normal planting dates), and four split plot treatments (barley varieties) that were randomized within each main treatment plot. The varieties included two RWA-barley resistant varieties, Sidney and Stoneham, and the susceptible variety, Otis, under thiamethoxam-protected and unprotected regimes. Sampling of RWA, other cereal aphids, and natural enemy populations was conducted on four dates from mid May through early July. RWA populations collected from early-planted plots (first week of March) were significantly lower than normal-planted plots in 2007-2009 at the Fort Collins, Colorado and Tribune, Kansas sites. In samples collected from early planting date plots, RWA-resistant varieties yielded RWA populations similar to those found on the insecticide-treated susceptible variety at both Fort Collins and Tribune. At the Sidney, Nebraska site, very low RWA populations were present and there were no differences between either planting date or varietal treatments. The combined effect of early planting and RWA-resistant varieties reduced RWA populations at the Fort Collins, Colorado site in all three years. Results were similar at the Tribune, Kansas site in 2007, but differences due to planting date or variety were not observed in 2008 or 2009. The lowest RWA populations occurred at the Sidney, Nebraska site, were independent of planting date and varietal treatments. The RWA-resistant barley varieties had no negative impact on populations of other cereal aphids compared to those found on the susceptible variety, Otis at any of the three research sites. The only treatment effective in reducing other cereal aphids was the insecticide, thiamethoxam. There was also no clear response of populations of other cereal aphids to different planting date. Neither the RWA-resistant barley varieties nor the systemic, short residual action insecticide treatment had adverse affects on the abundance of natural enemies.

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 (MSc (Genetics))--University of Stellenbosch, 2005. / The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious insect pest of wheat and barley. It affects the quality and yield of grain by sucking plant sap from the newest growth whilst toxic substances are injected that destroy plant tissue. The Russian wheat aphid also acts as a vector of plant viruses. The cultivation of aphid resistant cultivars is the preferred control strategy and nine resistance genes, designated Dn1 to Dn9, have been identified. Another undesignated gene, Dnx, was found in the wheat accession PI220127. Mapping of the resistance genes relative to known markers will improve their use in breeding programs. The dominant RWA resistance gene, Dn5, was identified in the accession PI294994 and mapped to chromosome arm 7DL. However, recent reports have placed Dn5 on ...

M.Sc. (Botany) / Wheat is the second most important grain crop produced in South Africa. It is mostly used for human consumption (e.g. biscuits, rusks, breakfast cereals, and bread) with the remainder being used as seed and animal feed. Wheat is also used for production of alcohol for ethanol, absorbing agents for adhesives, disposable diapers and industrial uses as starch on coatings (Department of Agriculture, Forestry and Fisheries, 2011). The leading wheat producing areas in South Africa since 1994 are the Western Cape (38 %), Free State (26 %) and Northern Cape (17 %) (Department of Agriculture, Forestry and Fisheries, 2011). There are two main classes of cultivated wheat, these are durum wheat (Triticum durum) that contributes 5 % and common or bread wheat (Triticum aestivum L.), which contribute 95 % of the world wheat production (Kiplagat, 2005). One of the major threats to wheat production in South Africa and other countries is the Russian wheat aphid (RWA), (Kurdjumov), (Diuraphis noxia, Hemiptera: Aphididae) (Brooks et al., 1994). In South Africa, the Russian wheat aphid was reported as a pest of wheat in 1978 in the Eastern Free State (Walters, 1980). In the beginning of the 1979 season, it was detected only in the Eastern Free State, but quickly spread to the Western Free State and parts of Lesotho. Infestations were also isolated in some areas of KwaZulu-Natal, North West and Gauteng Province (Walters et al., 1980). Aphids are soft bodied insects and are the most economically important phloem feeders impacting on plants (Dixon, 1985; Klingler et al., 2009). Aphids damage plants in four ways. Firstly, they ingest vital plant metabolites including large volumes of translocated sugars from the phloem sap, diverting energy from the plant and reducing yield. Secondly, they secrete saliva into the plant tissues while probing the layers of leaf to find the phloem, in some instances the secreted saliva is thought to be phytotoxic (Dedryver et al., 2010). The physical damage done to the leaf along with the toxic attributes of the saliva leads to localized necrosis in some aphid plant interactions (Klingler et al., 2009). Thirdly, aphids act as virus vectors and they can be transmitted in a non-circulative or circulative manner (Chen et al., 2012).

This thesis investigates the effect of Russian wheat aphid (RWA; Diuraphis noxia) infestation on the defence responses of the bread wheat line, PI 137739, on a molecular level. PI 137739 is known to contain the RWA resistance gene, Dn1. The study was conducted by utilising and combining a vast array of molecular biological techniques. Chapter 1 introduces the reader to a summary of the resistance responses observed within infested plants. A detailed description of the Russian wheat aphid follows and the genes responsible for RWA resistance in wheat is discussed. A brief report of research performed on the bread wheat genome is given and the biochemical defence responses of plants against insect infestation are discussed. This is followed by a concise description of resistance (R) genes and resistance gene categories in plants. The last discussion concerns microarray technology, a molecular tool utilised during this study. Chapter 2 aims at identifying genes involved in resistance against RWA infestation; specifically, genes containing the conserved nucleotide binding site¬leucine rich repeat (NBS-LRR) motif. Genomic, as well as complementary DNA (cDNA), was utilised in order to compare functional gene expression in wheat infested with the RWA. This was executed by employing PCR-based methods, single-pass sequencing and basic local alignment search tool (BLAST) analyses. Chapter 3 introduces suppression subtractive hybridisation (SSH) as a tool to further identify NBS-LRR or other resistance-related sequences in RWA infested wheat plants. SSH allows the comparative analysis of differential gene expression in RWA infested and uninfested wheat in order to identify resistance-¬related genes expressed in the infested, resistant wheat plants. The effect of RWA infestation on wheat resistance responses was examined further in chapter 4 through microarray analysis. The aim was the introduction and establishment of the microarray technique and to test the feasibility of using microarrays for differential gene expression and regulation studies. Microarray slides were assembled in order to monitor the up- and down¬regulation of genes at different time intervals - day 2, day 5 and day 8 - of RWA infestation. Clones isolated throughout this study were assembled on microarray slides and probed with control and RWA infested RNA. Differential gene regulation was assessed and further confirmed through Northern blot analyses, as well as quantitative real-time PCR. The thesis concludes with a general summary of the results obtained in chapter 5 and future prospects are outlined. / Thesis (PhD(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

This study represents the first comprehensive analysis of gene regulation underlying the distinct categories of resistance afforded to wheat (Triticum aestivum, L.) by different Dn genes. Russian wheat aphid (Diuraphis noxia, Mordv.) feeding on susceptible wheat cultivars causes leaf rolling, chlorosis and the eventual death of the plant. Plants expressing Dn genes are resistant to D. noxia infestation, but different Dn genes afford phenotypically distinct modes of resistance: the Dn1 gene confers an antibiotic effect to lower aphid fecundity; Dn2 confers tolerance to high aphid pressure; and Dn5 confers antixenosis, and aphids do not prefer such plants as hosts. Little is known about the components involved in establishing a successful defence response against D. noxia attack and how these differ between the distinct resistance categories. It is assumed that the Dn genes function as classic R genes in plant defence, being receptors for elicitors in aphid saliva. Upon recognition, defence response signalling is initiated, but the exact mechanics of subsequent cellular events in aphid resistance have only recently come under investigation. Evidence from cDNA microarray and subtractive hybridization experiments indicated the involvement of kinase signalling cascades and photosynthetic proteins in the response against D. noxia. However, expression analysis describing how these processes differ between plants carrying different Dn genes and how these differences account for antibiosis, antixenosis or tolerance had not been conducted. We consequently investigated the downstream components involved in or affected by the generation of these resistance mechanisms by comparing the responses in transcript regulation of Tugela near-isogenic lines with different Dn genes to D. noxia infestation. cDNA-AFLP analysis was selected as an appropriate functional genomics tool, since it is semi-quantitative, does not require prior sequence information and allows for the discovery of novel genes. cDNA-AFLP analysis yielded 121 differentially regulated transcript-derived fragments (TDFs) grouped into eight expression clusters. We cloned and sequenced 49 representative TDFs, which were further classified into five broad functional categories based on inferred similarity to database sequences. Transcripts involved in such diverse processes as stress, signal transduction, photosynthesis, metabolism and gene regulation were found to be differentially regulated during D. noxia feeding. Many TDFs demonstrated homology to proteins with unknown function and several novel transcripts with no similarity to previously published sequences were also discovered. Detailed expression analysis using quantitative RT-PCR and RNA hybridization provided evidence that the time and intensity of induction of specific pathways is critical for the development of a particular mode of resistance. This includes: the generation of kinase signalling cascades and the induction of several ancillary processes such as ubiquitination, leading to a sustained oxidative burst and the hypersensitive response during antibiosis; tolerance as a passive resistance mechanism countering aphid-induced symptoms through the repair or de novo synthesis of photosystem proteins; and the possible involvement of ethylene-mediated wounding pathways in generating volatile organic compounds during antixenosis. This is the first report on the involvement of KCO1, a vacuolar K+ channel, in assisting cytosolic Ca2+-influx and preventing leaf rolling, as well as on the role of iron homeostasis as a gene regulatory mechanism for sustaining the oxidative burst during the antibiotic defence response. This study opens up several areas of investigation heretofore unexplored in cereal-aphid interaction research. Of particular interest is the induction of genes involved in photosynthetic compensation during Dn2 tolerance responses, since these constitute a novel, passive resistance mechanism exclusive to aphid defence as opposed to the active resistance triggered in the presence of the Dn1 gene in the form of a general hypersensitive response. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is one of the important insect pests of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and other grasses. To date, there are four RWA biotypes identified in South Africa. The virulent biotypes emerged, partly due to climate change and new genetic variations within populations of RWA; hence there is a need to improve host-plant resistance, as an effective control measure. Bread wheat (Triticum aestivum L.) accession Cereal Introduction (CItr) 2401 is known to be resistant to all RWA biotypes worldwide. The goal of this study was to use a backcrossed near-isogenic line (NIL) BC5F5 mapping population, developed from a cross between CItr 2401 and susceptible Kavkaz, to identify and validate single nucleotide polymorphism (SNP) markers linked to the resistance phenotype in CItr 2401. This was achieved by (i) conducting a preliminary study that evaluated the suitability of simple sequence repeat (SSR) markers previously reported in literature for discriminating stacked RWA resistance genes and, (ii) employing SNP markers for the first time in a RWA resistance study as a future alternative to the widely used SSR markers. None of the tested SSR markers showed potential use in marker-assisted selection (MAS). The mapping population was phenotypically evaluated for RWA resistance using the four South African biotypes, viz. RWASA1, RWASA2, RWASA3 and RWASA4. Analysis of variance (ANOVA) showed significant (P<0.001) differences of genotypes after confirming the normality of residuals and homogeneity of variance. The Illumina iSelect 9,000 wheat SNP platform was used to genotype the two crossing parents and a selection of 24 NIL genotypes from the mapping population. Eight SNP markers found to be linked to the phenotype were converted to breeder-friendly and high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) markers. The designed KASP markers were validated on the two crossing parents, the 24 NIL sent for SNP genotyping, on the mapping population and on the preliminary study genotypes for their effectiveness. The KASP assays developed in this study will be useful for stacking the RWA resistance from CItr 2401 with other Dn genes effective against the RWA. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Master of Science / Department of Entomology / C. Michael Smith / The Russian wheat aphid Diuraphis noxia (Kurdjumov) is a serious pest of world cereal grain crops, primarily barley and wheat. A phenotypic characteristic of D. noxia feeding, leaf rolling, creates a leaf pseudo gall which protects aphids, making it difficult to treat infested plants with insecticides or biological control agents. Therefore, the use of D. noxia-resistant crops is a desirable aphid management tactic. Because of the development of virulent D. noxia biotypes, the identification of new sources of barley and wheat resistance is necessary. Virus-induced gene silencing (VIGS) utilizes the plant defense system to silence viruses in inoculated plants. The accumulation of virus RNA in plants triggers the defense system to silence sequences homologous to the introduced virus and sequences of interest from a plant are inserted into the virus and silenced along with the virus. The VIGS method was tested to determine the ability of barley stripe mosaic virus (BSMV) to serve as a VIGS vector in wheat plants containing the Dnx gene for resistance to D. noxia. Dnx leaves with silenced BSMV virus yielded D. noxia populations that were significantly no different from populations produced on healthy Dnx leaves. Thus, BSMV silencing does not interfere with Dnx resistance. Several different methods were examined to determine how best to confine aphids to the silenced leaf, and a modified plastic straw cage was chosen as the optimum cage type. Microarray and gene expression data were analyzed to select two NBS-LRR type disease resistance protein genes - TaAffx.104814.1.S1_at and TaAffx.28897.1.S1 - (NBS-LRR1 and NBSLRR2), in order to assess their role in Dnx resistance. NBS-LRR1 and NBSLRR2 were silenced by inoculating leaves of Dnx plants with barley stripe mosaic virus (BSMV) containing sequences of each gene. Controls included Dnx and Dn0 plants inoculated with BSMV and non-BSMV inoculated plants. Aphids were allowed to feed on control and treatment plants to assess aphid population and mean weight of aphids surviving at the end of the experiment. There were no differences among treatments based on aphid population, but there were significant differences the mean weights of aphids reared on several different treatments.