The Russian wheat aphid (Diuraphis noxia, RWA), is a serious pest in most wheat producing countries around the world. Infestation of wheat fields by this pest has a severe economic impact, as a result of heavy losses in crop yield. Because of the importance of wheat as a food source and its ever growing supply demand, the study of wheat-Russian wheat aphid interactions on the molecular level are integral to the development of management strategies. This is highlighted by the fact that new RWA biotypes that overcome resistance in a number of wheat varieties, continually emerge. Therefore, this study aims to contribute to this endeavour, by elucidating the molecular mechanisms by which the RWA resistance gene Dn7 confers resistance to three different RWA biotypes (one from SA, and two from the USA). Firstly, suppression subtractive hybridization (SSH) was applied in order to isolate transcripts differentially expressed in the RWA resistant wheat line, 94M370, carrying the Dn7 gene. There are two main advantages to this technique. One is that the relative representation of rare transcripts is increased in the subsequent cDNA population, and it is these low abundance transcripts that are arguably the ones of particular interest. Secondly, this method allows for the isolation of unknown transcripts, without the need for existing sequence information. Experiments with this method however, failed, leading to an investigation as to probable causes. The various steps involved in the SSH procedure were individually assessed in an attempt to identify and correct the problem. Various adjustments were made to PCR procedures, template enzyme digestions and ligation reactions, without success. After creating a basic cDNA-AFLP fingerprint from the existing cDNA template, in order to confirm that the template is not responsible for experimental difficulties – it was decided to apply a different strategy in order to meet research objectives. Consequently, the study on Dn7 mediated defence responses was continued with cDNA-AFLP. In addition to studying the response by Dn7 to South African biotype RWA infestation, its responses to infestation by two United States RWA biotypes was also explored. This allowed us to gain a greater comprehension of the methods by which Dn7 activates defences against different aphid eliciting agents. Findings suggest that this gene activates responses that are unique to each of the different aphid interactions. Although the interactions between Dn7 and the two US biotypes were very similar, this can possibly be explained by the fact that the differences between these two biotypes on molecular level are minuscule. Dn7 responds to the South African biotype of the RWA in a completely different manner, as judged by the very dissimilar expression patterns obtained during cDNA-AFLP analysis. Reasons for this phenomenon could include molecular differences between the South African and US RWA biotypes, differences in response generating elicitor molecules (which has indeed been shown to be the case between South African and US aphid biotypes), or a combination of both. The sequencing of fragments displaying differential expression patterns during cDNA-AFLP fingerprinting, provides us with additional information as to the exact mechanisms potentially involved. As expected, various compounds related to plant defence were identified, such as a number of Leucine rich repeat (LRR) domain containing proteins, genes related to cell signalling and genes involved in protein processing (proteases, peptidases). Finally, these results are consistent with theories that Dn7 may recognise and interact with its distinct aphid elicitors either directly, by the presence of multiple bindings sites on the same protein, or indirectly. In that case, in accordance with the guard hypothesis, Dn7 may simply monitor interactions between aphid elicitors and other recognition factors- after which a response cascade is activated. Useful potential research would focus on Dn7 itself, including mapping, isolation as well as structural and functional characterization. / Dissertation (MSc)--University of Pretoria, 2007. / Genetics / unrestricted

Please read the abstract in the section 07chapter7. / Dissertation (MSc (Genetics))--University of Pretoria, 2002. / Genetics / unrestricted

The Russian wheat aphid, Diuraphis noxia is one of the most destructive exotic invaders of South Africa, capable of reducing individual wheat plant yields by up to 90%. Entomopathogenic fungi are important natural mortality factors associated with this aphid in its Eurasian endemic habitats as well as in the United States and Canada; their impact often exceeding that of predators and parasitoids. The principal objectives of this study included (1) baseline characterization of the aphid-pathogenic flora associated with aphids from South Africa, with special reference to six common cereal aphids, (2) quantification of the comparative impact of the different fungal species on the cereal-aphid complex in three different wheat producing regions of South Africa, (3) field evaluation of the Hyphomycete Beauveria bassiana against D. noxia on resistant wheat, (4) screening of six fungicides for their potential usage in managing entomophthoralean epizootics within greenhouse rearings of the Russian wheat aphid, and (5) development and evaluation of a novel bioassay protocol for screening entomopathogenic Hyphomycetes against D. noxia. A total of nine species of fungi known to infect and kill aphid hosts were collected, including the six entomophthoraleans, Pandora neoaphidis, Conidiobolus thromboides, Conidiobolus obscurus, Entomophthora planchoniana, Conidiobolus coronatus and Neozygites fresenii, and three Hyphomycetes, Beauveria bassiana, Verticillium lecanii, and Paecilomyces farinosus. The former four entomophthoraleans are considered first reports from this country. For the first time, morphological characteristics of these nine South African-collected species are visually depicted and techniques for their isolation and in vitro culture discussed. Seven species of fungi were recorded from D. noxia, of which P. neoaphidis was the most important, causing up to 50% mortality during the late season under dryland conditions in the summer-rainfall region. Mycoses at epizootic levels, together with the large diversity of fungal species recorded from this host, indicated a high level of susceptibility to fungal infection. In contrast, infection of the oat aphid Rhopalosiphum padi remained < 5% despite favourable numbers of hosts and apparently suitable environmental conditions. This phenomenon strongly suggests some level of low susceptibility to fungal infection in this species. Under irrigated conditions m KwaZulu-Natal, the rose-gram aphid Metopolophium dirhodum was the predominant aphid but remained below economical injury levels. Field surveys revealed that this aphid was effectively targeted by P. neoaphidis and C. obscurus, and findings suggest that in some areas of South Africa entomophthoralean fungi effectively suppress this aphid, negating the need for insecticide applications. On average, ca. 61% control of D. noxia on resistant wheat was observed following an application of B. bassiana (5 x 10¹³ conidia per hectare) during the early flag-leaf stage of the wheat. Efficacy of B. bassiana applications on younger plants appeared to be influenced by the level of aphid activity, possibly explained by secondary pick-up of inoculum by D. noxia. These findings accentuate the importance of understanding the tritrophic relationship between host plant, pest and pathogen. The fungicides copper oxychloride, mancozeb + oxadixyl, captab + metalaxyl, bittertanol, iprodione, and mancozeb at a rate of 0.1% active ingredient moderately to strongly inhibited C. thromboides vegetative growth (mean inhibition 81.1 %). Mancozeb at concentrations of 10.0, 2.0, 1.25, 0.5, 0.08, and 0.016% was further evaluated in vitro. The fungus growth cut-off point, midway between 1.25 and 2.0% mancozeb, was calculated and a rate of 1.625% active ingredient per litre of water was used to decontaminate the fungus-infected D. noxia cultures. A novel bioassay protocol was developed, employing live host plants for rearing aphids post inoculation. Using this design against D. noxia, an average LC₅₀ estimate of 85 conidia per mm² for B. bassiana strain GHA was calculated. Control mortality was restricted to levels below 4%. The data indicated high precision due to an average coefficient of variation for slope of less than 20%, and an average chi-squared value of 5.46 ± 2.74 (n = 10 assays). The design will accommodate the use of cereal aphid species other than D. noxia, while live host plants will facilitate tritrophic studies on the effect of host-plant resistance on fungus-induced mortality of D. noxia. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

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.

Genetic analysis of 433 samples of serious crop pest aphid Diuraphis noxia was conducted with the use of 8 microsatellites loci. Statistical analysis revealed sexual reproduction of D. noxia in temperate regions. The linkage disequilibrium was detected because of the excess of heterozygotes. These results support the theory of RNDr. Starý about the invasion of D. noxia to American continent via states of North Africa, Spain and France.

Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.

The infestation of the cultivated grain crops by phloem feeding aphids has generated a great deal of interest over the years, due to the serious damage they cause to the crops and yield losses that result. The mechanism of the interaction between aphids and host plants remains largely unknown in spite of efforts to understand the basis of aphid feeding on grain crops. Greater efforts are required to explain the mechanism(s) of this interaction in order to achieve sustainable agriculture. This thesis focused on an investigation of the mechanism of feeding by the Russian wheat aphid, Diuraphis noxia Mordvilko (RWA) and the bird cherry-oat aphid, Rhopalosiphum padi L. (BCA) on barley and wheat cultivars. These two aphids co-occur naturally, but they inflict very different feeding effects on host plants. Structural and functional approaches were employed to investigate their feeding habits and these were then related to the observed differences in their host plants. Transmission electron microscopy (TEM) techniques were used to study the ultrastructural damage, while fluorescence microscopy techniques – using aniline blue fluorochrome (a specific stain for callose) and 5, 6-CFDA (a phloem-mobile fluorophore) – were employed to investigate the functional response to damage via wound callose formation and phloem transport capacity respectively. RT-PCR and quantitative real-time RT-PCR techniques were used to investigate the regulation of the genes involved in callose synthesis and degradation at the transcriptional level. Morphological observation of the damage caused by the aphids show that infestation by RWA results in extensive leaf chlorosis, necrosis and rolling, while infestation by BCA does not lead to any observable symptoms within the same period. Interestingly, the population study shows that BCA breeds faster than RWA within the two-week experimental period. The ultrastructural study of feeding damage caused by the two aphids on the vascular bundles of susceptible barley cv Clipper, shows a different patterns of damage. Probing the vascular bundles results in the puncturing of vascular parenchyma by both aphids, but severe damage occurs in sieve tubes-companion cell complex during sustained feeding by RWA. In contrast, less damage occurs when BCA feeds on the phloem. Drinking from the xylem by RWA results in deposition of a large quantity of electron-dense watery saliva, which apparently seals the xylem vessels completely, by blocking all the pit membrane fields between the xylem vessels and associated parenchyma cells. In contrast, drinking from xylem by BCA results in deposition of a dense, granular saliva into the xylem vessels only, which does not appear to totally occlude the pit membrane fields. This is the first known report in which ultrastructural evidence of aphids’ drinking in xylem is provided. The comparative effects of RWA feeding on a susceptible Betta and resistant Betta-Dn1 wheat cultivars showed that after two weeks, the Betta cultivar expressed damage symptoms such as chlorosis, necrosis and leaf roll, while few chlorotic patches and necrotic spots occur in resistant Betta-Dn1 cultivars. An ultrastructural investigation of the feeding damage caused to all leaf tissues revealed, for the first time, that RWA is capable of both intra- and inter-cellular probing within mesophyll cells. Probing in the mesophyll cells induces a more severe damage in susceptible Betta than in the resistant Betta-Dn1 counterpart. Similar differences in damage occurred during feeding in the thin-walled sieve tubes of the phloem, with the sieve tubes of the Betta showing more damage than that of the resistant Betta-Dn1. However, drinking from xylem resulted in the characteristic occlusion of metaxylem vessels by copious deposition of saliva by RWA in both Betta and Betta-Dn1 cultivars. In all cases of probing, feeding, and drinking by RWA in both cultivars, all probed cells with evidence of salivary material deposit and those cells adjacent to salivary material deposit, exhibit significant damage in susceptible Betta cultivar, whereas similar cells in Betta-Dn1 cultivars do not show as damage as severe. Investigation of the functional response of the plants to feeding by aphids through the deposition of wound-induced callose shows that formation and deposition of wound callose occurs in both longitudinal and cross veins within 24h of feeding by RWA. This deposition increases through short-term feeding (72h) and prolonged feeding (14d). This is in sharp contrast to the observations with BCA feeding,where little or no callose formation occurs within the same time frame. Callose formation and deposition occurs only when a higher population of BCA feeds on barley leaves. This is the first report of aphid-induced wound callose by BCA. In all cases of callose deposition, aphid stylet tracks were associated with callose and the deposition of callose appears to be a permanent feature, because wound callose remained in the leaf tissues even after 120h of the aphids’ removal. Wound callose signals (defence and anti-defence) are discovered to be transported in the phloem tissues and are dependent on the direction of assimilate flow. Examination of the possible regulation of wound callose genes at the transcriptional level shows that the two expressed glucan synthase gene sequences (GSL – genes involved in callose formation) analysed did not show any significant increase or regulation upon aphid infestation. Contrary to expectation, all three aphid-induced β-1, 3-glucanases (genes which are thought to be involved in callose degradation) showed higher expression in RWA-infested tissue than in BCA-infested tissue. The results of the feeding damage on the transport capacity of the phloem shows that BCA infestation does not lead to a significant reduction in the phloem transport capacity during short-term feeding (72h), while RWA-infested leaves showed considerable reduction in the transport capacity of the phloem within the same period. However, prolonged feeding (14d) by BCA induces a considerable reduction on the transport capacity of the phloem on the infested tissues. In contrast, a marked reduction in the transport capacity of the phloem occurs in RWA-infested leaves and in most cases, complete cessation of transport ensues. In conclusion, these data collectively suggest that RWA is a serious and most destructive phloem feeder in comparison to the BCA. RWA causes severe damage to all cellular tissues of the host plants, which result in apoplasmic and symplasmic isolation of xylem and phloem tissues, while BCA infestation does not result in such isolation within the same time and population levels. Resistance genes appear to function by conferring resistance to cell damage on the resistant cultivars during aphid feeding. Responses by plants to aphid infestation via wound callose deposition are again shown to be species-specific. A quick response results when RWA feeds, even at a very low population level, while a response occurs only at a higher infestation level by BCA, and this response was shown as not regulated at the transcriptional level. Differences in the damage to leaf tissues and wound callose deposition eventually lead to varying degrees of damage to the transport capacity of the phloem. These differences in the damage signatures are hereby suggested to be the cause of the diversity in the observed damage symptoms and the yield losses upon infestation by the two aphid species.

The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is a major pest of cultivated small grains. It is particularly devastating because of is high reproductive rate which results in the growth of large populations which become damaging to its host plants. Development of resistant barley (Hordeum vulgare L.) cultivars is complicated as resistance is polygenic. As a result, the industry remains at risk now that the RWA has spread throughout South Africa. It has, as recently as, 2013, been identified in the SW Cape, which was previously geographically isolated. This is South Africa‟s principle barley growing region. Now a potentially huge problem exists. Therefore, it is imperative that an alternative to pesticide use is found. Testing potential innate resistance in barley cultivars is thus, critical. In this thesis, I present data on four barely cultivars where I have examined their resistance/ lack of resistance to three known RWA biotypes, RWASA1, RWASA2 and RWASA3. The barley varieties used were two economically important South African malt barley cultivars (S5 and SSG 564) along with two potentially RWA resistant Afghan accessions (CIho 4125 and CIho 4159). The RWA biotype population growth rates on each of the plants were determined over a 14 day period. The aim was to establish baseline data of the effects of RWA population growth on the host plants under ambient CO2 (380 – 400 ppm) conditions. The extent of RWA feeding damage was investigated at the cell level by examining saliva deposition and cell disruption using Transmission Electron Microscopy; at the tissue/vascular level using fluorescence microscopy, to determine the extent of callose formation; at a whole leaf level by recording percent chlorosis and leaf roll; and finally, at a whole plant level by measuring biomass loss.The experiments were repeated under elevated CO2 (450 ppm) to model any changes in RWA/plant interaction with respect to future climate change. The effects of an elevated CO2 environment and RWA feeding on host plant foliar N and C:N ratio were compared to ambient CO2 conditions, to provide a clearer picture of the potential nutrient drain that a feeding RWA colony exacts on its host. Of the varieties tested, the CIho accessions performed better than the two SA barley cultivars as the CIho accessions appeared to express a mild antibiosis resistance response as RWA populations, particularly those of RWASA1, were smaller than those observed on either S5 or SSG 564. In addition, less damage was evident in the two CIho accessions due to RWA feeding. II RWASA2 was the most virulent of the three RWA biotypes tested, followed by RWASA3 while RWASA1 was the least virulent. Under elevated CO2 conditions, RWA feeding damage was exacerbated but the trend of biotype virulence remained the same. Higher aphid population sizes were recorded under elevated CO2, meant that even the more resistant CIho accessions were overcome by the increased demand made by the larger aphid colonies on the host plants. The % foliar N data showed that under elevated CO2 aphid-free control plants had increased N levels in their leaves. Increased “food” supply (as shown by the increased N levels) therefore allowed significantly larger aphid populations to develop on the plants exposed to elevated CO2, due to improved nutrient status of the phloem sap taken up by RWA. The knock-on effect of a higher aphid population was increased cell disruption as a result of extensive probing, extensive formations of wound callose, with the result that phloem damage impeded nutrient flow through the vascular tissues which contributed to chlorosis and (eventually plant) death. The major conclusion from this study is that even a mild CO2 elevation resulted in an increase aphid population which may pose a severe and very real threat to a barley crop. Therefore, without effort to identify and deploy resistant barley cultivars, it could well be possible that future barley cultivation in South Africa may no longer be viable.

Cereal crop productivity is hampered when these plants are infested by phloem feeding aphids. A great deal of research has been carried out with the direct aim of a clearer understanding of the mechanism involved in the interaction between aphids and their host plants. Research has directly or indirectly been geared towards enhanced plant productivity and achieving sustainable agriculture. Barley (Hordeum vulgare L.) is an important small grain crop in South Africa, whose crop performance is negatively affected by fluctuations in weather patterns as well as by agricultural pests. One of the insect pests infesting barley is the Russian wheat aphid, Diuraphis noxia Kurdjumov (RWA), of which the two South African biotypes, codenamed RWASA1 and RWASA2, were studied in this thesis. During dry spells, RWA infestation becomes a more serious threat to barley productivity. Resistant plants have been used to combat RWA infestation of small grains. In South Africa, 27 RWA-resistant wheat cultivars are currently used in commercial cultivation, but no resistant barley lines have, unfortunately, been developed, in spite of this grain’s significant economic importance. This informed the study in this thesis, and this interest particularly focussed on three RWA-resistant lines developed by the USDA, testing their performance against South African RWA biotypes, for possible adaptation to South Africa. The aim was thus to examine the plant-aphid interactions, aphid breeding rates, plant damage and sustainability, evidence of resistance or tolerance and finally potential performance under elevated CO2 (a very real climate change threat). Two major avenues of research were undertaken. The first aspect involved examination of structural and functional damage caused by RWASA1 and RWASA2 on the three resistant and a non-resistant line. Aphid population growth and damage symptoms (chlorosis and leaf roll) of infestation by these aphid biotypes were evaluated. This was followed by a structural and functional approach in which the effects of feeding on the transport systems (phloem and xylem) of barley were investigated. Fluorescence microscopy techniques (using aniline blue fluorochrome, a specific stain for callose and 5,6-CFDA, a phloem-mobile probe) were applied to investigate the feeding-related damage caused by the aphids, through an examination of wound callose formation and related to this, the resultant reduction in phloem transport capacity. Transmission electron microscopy (TEM) techniques provided evidence of the extent of the feeding-related cell damage. The second aspect involved a study of the effect of changing CO2 concentrations ([CO2]) on the resistant and susceptible barley cultivars to feeding by the two RWA biotypes. Leaves of plants grown at ambient and two elevated levels of [CO2] were analysed to investigate the effect of changing [CO2] on biomass, leaf nitrogen content and C:N ratio of control (uninfested) and infested plants. The population growth studies showed that the populations of the two RWA biotypes as well as bird cherry-oat aphid (BCA, Rhopalosiphum padi L.) increased substantially on the four barley lines. BCA was included here, as it had been the subject of several previous studies. RWASA2 bred faster than RWASA1 on all lines. The breeding rates of the two RWA biotypes were both suppressed and at near-equivalent levels on the three resistant lines, compared to the non-resistant PUMA. This suggests that the resistant lines possessed an antibiosis resistance mechanism against the feeding aphids. Feeding by the aphids manifested in morphological damage symptoms of chlorosis and leaf roll. The two biotypes inflicted severe chlorosis and leaf roll on the non-resistant PUMA. In the resistant plants, leaf rolling was more severe because of RWASA2 feeding compared to RWASA1 feeding. In contrast, chlorosis symptoms were more severe during RWASA1 feeding than was the case with RWASA2 feeding. Investigation of the effect of aphid feeding on the plants showed that callose was deposited within 24h and that this increased with longer feeding exposure. Wound callose distribution is more extensive in the non-resistant PUMA than in the resistant plants. RWASA2 feeding on the resistant lines caused deposition of more callose than was evident with RWASA1 feeding. During long-term feeding, it was evident that variation in the intensity and amount of wound callose was visible in the longitudinal and transverse veins of the resistant plants. Of the three STARS plants, STARS-9301B had the least callose. Interestingly, wound callose occurred in both resistant and non-resistant plants, in sharp contrast to what has been reported on resistant wheat cultivars that were developed in South Africa. The relative reduction in the wound callose deposited in the resistant line, when compared to the non-resistant lines, suggests the presence of a mechanism in the resistant lines, which may prevent excessive callose formation. Alternatively, the mechanism may stimulate callose breakdown. RWASA2 feeding on the resistant lines deposited more wound callose than RWASA1 feeding. This evidence supports the hypothesis that RWASA2 is a resistance breaking and more aggressive feeder than RWASA1 is; and further underscores the urgent need for development of RWA-resistant barley cultivars. The ultrastructural investigation of the feeding damage showed that the two biotypes caused extensive vascular damage in non-resistant plants. There was extensive and severe cell disruption and often obliteration of cell structure of the vascular parenchyma, xylem and phloem elements. In sharp contrast, among the resistant plants, feeding-related cell damage appeared to be substantially reduced compared to the non-resistant PUMA. Low frequency of damaged cells indicated that majority of the cell components of the vascular tissues were intact and presumed functional. There was evidence of salivary material lining the secondary walls of the vascular tissue, which resulted in severe damage. Within xylem vessels, saliva material impregnated half-bordered pit pairs between the vessels and adjacent xylem parenchyma. This is believed to prevent solute exchange through this interface, thereby inducing leaf stress and vi leaf roll. A notable finding is that RWASA2 effectively induced more cell damage to vascular tissues in the resistant lines than did RWASA1. In general the experimental evidence (see Chapter 5) suggests that the resistant lines are possibly more tolerant (or able to cope with) to RWA feeding. Evidence for this is the reduction of wound callose and at the TEM level, a comparatively less obvious cell damage in the resistant lines, which suggests that they possess antibiosis and tolerance capacity. The apparent reduction of feeding-related cell damage from the TEM study confirmed the disruptive action of the feeding aphids in experiments using the phloem-mobile probe, 5,6-CF. Results showed that feeding by RWASA1 and RWASA2 reducedthe transport functionality of the phloem in all cases, but that RWASA2 feeding caused a more obvious reduction in the rate and distance that 5,6-carboxyfluorescein was transported, than did RWASA1. Investigation of the effect of changing [CO2] on the barley cultivars showed that in the absence of aphids and under elevated CO2 conditions, the plants grew more vigorously. In this series of experiments, the infested plants suffered significant reduction in biomass under ambient (as was expected) and under the two elevated CO2 regimes. Biomass loss was greater at elevated CO2 than under ambient [CO2]. The infested nonresistant PUMA plants showed a more significant biomass loss than did the resistant cultivars. Clearly, the benefits derived from elevated CO2 enrichment was thus redirected to the now-advantaged aphids. Uninfested vii plants showed an increase in leaf nitrogen under the experimental conditions. However, feeding aphids depleted leaf nitrogen content and this was more apparent on plants exposed to RWASA2 than was the case with RWASA1. The end result of this was that C:N ratio of infested plants were higher than uninfested plants. Clearly, the faster breeding rates of the aphids at elevated CO2 caused depletion of N and a resultant deficiency exacerbated chlorosis as well as leaf rolling due to the higher aphid population density under elevated CO2 than at ambient. By 28 days after infestation (DAI), majority of the plants exposed to enriched CO2 treatments had died. A major finding here was thus that although this study demonstrated that elevated CO2 resulted in an increase in biomass, this was detrimentally offset in plants infested by the aphids, with a decline in biomass and loss of functionality leading to plant death at 28DAI. The overriding conclusion from this study is a clear signal that the twin effects of CO2 enrichment (a feature of current climate change) and aphid infestations may precipitate potential grain shortages. A disastrous food security threat looms.

M.Sc. / Wheat is one of the most extensively grown small grain crops in Southern Africa. It is a staple food source used by humans, animals and other living organisms around the globe (Feldman, 2001). One of the major threats of wheat production in South Africa is the Russian wheat aphid (RWA), (Kurdjumov), (Diuraphis noxia, Homoptera: Aphididae) (Brooks et al., 1994; Du Toit & Walters, 1984). The RWA was first described as a pest in South Africa in 1978 and in the US in 1986. Thereafter it was rapidly reported as becoming a major pest of cereals in these countries (Brooks et al., 1994; Du Toit & Walters 1984). A short life span, asexual reproduction, the development of resistance towards insecticides and rapid colonisation of the host plant are all factors that have made the management of the RWA a difficult task (Dogimont et al., 2010; Hein et al., 1998). As a result of complications such as these, host plant resistance is the most viable option to counter the RWA (Dogimont et al., 2010). The method of feeding of the RWA is detrimental to the plant as they insert their stylets into, and feed primarily from, the phloem sieve elements. This damages the plants through nutrient drainage (Dixon, 1985; Klingler et al., 2009) and results in a variety of symptoms, the most common being chlorosis, necrosis, wilting, stunting, curling of the leaves (which provides the pest with a sheltered environment protected from predators and pesticides), misshapen or nonappearance of new growth, and localised cell death at the site of aphid feeding. The RWA elicits an increase in essential amino acids in the phloem sap, by triggering the breakdown of proteins in infested wheat leaves (Burd & Burton, 1992; Du Toit, 1986; Haley et al., 2004; Ma et al., 1998; Miller et al., 2001; Walters et al., 1980). It also reduces transport of labelled tracers (amino acids) from the feeding site to the roots and other sinks in the plant. This increases the nutrient concentration at the site of aphid feeding by increasing the import of resources from other sites in the plant, mobilising local resources and blocking their export to other organs. The damage of the foliar tissue that occurs as a result of the RWA feeding is thought to play a role in the pest’s ability to increase the nutritional quality of the host plant (Botha et al., 2006; Goggin, 2007; Shea et al., 2000).