Thesis (MSc Natural and Agricultural Sciences)--University of Pretoria, 2007. / Includes bibliographical references. Summary in English.

Thesis (Ph.D. (Botany)) - Rhodes University, 2008.

Penetration of sink as well as source leaves of wheat plants by the Russian wheat aphid, Diuraphis noxia (Mordvilko) was investigated using light, fluorescence and transmission electron techniques, to determine the feeding strategies adopted by the aphid in penetrating and successfully feeding from the phloem, and to assess the structural effects of the probing and feeding behaviour of D. noxia on the feeding sites. Examination of aphid-infested sink, as well as source leaf tissue, showed that D.noxia probed in cells of the vascular bundle more frequently than mesophyll cells. Within the vascular bundle, thin-walled sieve tubes were visited (probed) more than the other cells. In sink leaf material, 68 of 82 (83%) stylets and stylet tracks encountered during the examination of 1000 serial sections (from 5 different plants) terminated in thin-walled sieve tubes and only 14 (17%) in thick-walled sieve tubes. Thin-walled sieve tubes were visited more significantly than thick-walled sieve tubes. However, examination of the aphid.,.infested sink leaf on a per centimetre basis, from the tip of the leaf, revealed that thick-walled sieve tubes in the area closest to the tip (0-2cm from the tip) were as attractive to the aphid as were thin-walled sieve tubes, with no significant difference in the number of times thick- and thin-walled sieve tubes were probed in this area. Some 2-4cm from the tip however, thinwalled sieve tubes were significantly more probed and therefore more attractive than thick-walled sieve tubes. Examination of 2000 serial sections using aphid-infested source leaf tissue, showed that the thin-walled sieve tubes were significantly more probed than thickwalled sieve tubes, along the whole leaf, expressed as a total of all leaves, as well as on a per centimetre basis along the length of the leaf, with 212 (95%) of 222 terminations within the thin-walled sieve tubes and only 10 (5%) in thick-walledsieve tubes. The aphid probed the small vascular bundles (loading bundles) many more times than intermediate or large transport vascular bundles, in sink as well as source leaf. Of a total of 82 stylets and stylet tracks encountered in sink leaf tissue, 31 terminated in small vascular bundles ang the remaining 28 and 16 were located within large and intermediate vascular bundles respectively. In source leaf tissue 121 of 222 stylets and stylet tracks encountered were associated with small vascular bundles and only 58 tracks and 43 tracks with intermediate and large vascular bundles, respectively. The effect of sustained RWA feeding on the transport capacity was examined after the application of 5,6 carboxyfluoresceine diacetate (5,6-CFDA) in control (sink and source leaf tissue) and aphid-infested (source) wheat leaves, using fluorescence microscopy. After 3h acropetal longitudinal transport of 5,6-CF had occurred in sink leaves in longitudinal veins, as well as a lateral transfer via cross veins and subsequent unloading into mesophyll cells close to the tip of the leaf was observed. In control leaf tissue, the fluorescence front was detected up to about 5cm from the point of application and was only associated with the phloem and not unloaded. In contrast, aphid-infested leaf tissue showed very little 5,6-CF transport, being limited to 2cm or less from the point of application. Structural damage to the phloem in general and to the sieve tubes in particular within of control and infested wheat leaves was investigated using transmission electron microscopy (TEM). In addition, leaf strips were mounted in aniline blue to visualise callose deposition using the fluorescence microscopy. At the TEM level. infested leaf tissue showed various abnormalities, which included destruction of cell contents, membrane damage and subsequent loss of cell contents. TEM studies suggest severe osmotic shock resulted from the aphid's probing. Examination of leaf tissue using fluorescence microscopy showed that there was very little characteristic aniline blue-stained callose visible in control leaf tissue, other than the thin diffuse patches along the sieve plates and punctate spots associated with pore plasmodesmatal areas and plasmodesmatal aggregates. In contrast, the aphid-infested leaf tissue was heavily callosed, with callose deposited not only within the phloem tissue but also in neighbouring vascular parE:}nchyma cells as well. The data collectively suggest that D. noxia feeds preferentially within thin-walled sieve tubes, within the small longitudinal vascular bundles in sink , as well source leaf tissue. Based upon the data presented here the thin-walled sieve tubes in the wheat leaf appear to be more attractive to the aphid and that they are probably more functional in terms of transport system and unlo?lding in sink leaves. Aniline blue stained leaf material that had previously hosted large aphid colonies showed evidence of extensive callose deposits 24 to 36h after the aphids were removed, suggesting that the aphids caused severe mechanical damage to the vascular tissue and mesohyll cells as well. Damage (transient or more permanent) and the subsequent deposition of wound callose, disrupted phloem transport and hence the export of photoassimilate from the leaves.

The Russian wheat aphid (RWA) is a pest of cereals, such as wheat and barley. It feeds on these hosts by injecting saliva into the plants’ phloem tissue and consuming the mixture of saliva and photoassimilates. It has been proposed that the insect’s saliva contains elicitors or virulence factors, which cause the symptoms typically observed in susceptible wheat cultivars. These are leaf rolling, chlorotic streaking, a decrease in yield and death in cases of heavy infestation. In contrast, resistant plants display symptoms typical of defence responses, such as the formation of necrotic lesions and an increase in the expression of pathogenesis related proteins. But, most importantly, RWA feeding on these hosts does not result in their subsequent death. The objectives of the present study are thus to elucidate any putative virulence factors, present in insect saliva, that can result in the breakdown of resistance of cultivars and thus, lack of recognition and/or delayed onset of the plants’ defence responses. Thus, this thesis investigates the RWA on protein level to determine which components of these insects induce the different changes observed in the resistant and susceptible plants. Also, it examines whether or not the biotypes uniquely altered their elicitors in response to selective pressure. In Chapter 1 a brief introduction is presented on the Russian wheat aphid, its distribution and the effects of its feeding on resistant and susceptible wheat cultivars. In Chapter 2 a literature review provides insight on how the Russian wheat aphids feed and survive on wheat. It also outlines the control mechanisms which plants could employ to withstand attack from pests and pathogens. In Chapter 3 proteins were extracted from different parts of two Russian wheat aphid biotypes and separated on high pressure liquid chromatography (HPLC). Two biotypes were selected for the study to provide comparative information on the development of biotypes and/or their virulent elicitors. The presence of the potential elicitors was determined by examining the extent of leaf rolling chlorotic streaking/spots on injected plants’ leaves, determining the activity of defence related enzymes of the injected plants and visualizing the proteins extracted from these plants on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This was done in order to examine the plants on phenotypic, enzymatic and proteomic levels, which could confirm the results obtained on three different levels. It was found that resistant cultivars react similarly to the two biotypes, but that the RWA biotypes differ significantly on a protein level. Potential motivations for these variations are discussed. Results presented in this dissertation represent the outcomes of a study conducted from March 2005 to December 2006 in the Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, under the supervision of Prof. A-M Botha-Oberholster. Chapter 3 is being prepared to be submitted for review in Insect Biochemistry and Molecular Biology. / Dissertation (MSc (Plant Genetics))--University of Pretoria, 2008. / Genetics / unrestricted

Fifteen small grain fields, including eight wheat fields, two barley fields and five oat fields, throughout the grain growing areas of Graham, Greenlee and Cochise counties were surveyed weekly from the third week in March through the second week in June, to document the presence of Russian wheat aphid (RWA), other aphid, parasites and predators. RWA were found in all three counties and in all three grains. Presence of RWA caused chemical pest control applications in approximately 19%, 33% and 90% of the small grain fields in Graham, Greenlee and Cochise counties, respectively. Thirty three percent of the fields had beneficial parasites and predators were found 93% of the fields. Presence of parasites reduced the RWA populations in 80% of the cases and presence of predators had a dramatic effect on other aphids and was felt to be beneficial in controlling RWA.

During 1990 the emphasis on surveying was placed in the southeastern corner of the state, where damage was found to be most severe in previous years. Estimates of the incidence of and damage caused by the Russian Wheat Aphid (RWA) were made for the entire state. These estimates indicate that small grain producers in the state lost $212,000 due to this pest in 1990.

Thesis (MSc Natural and Agricultural Sciences (Genetics))--University of Pretoria, 2007. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (M.Sc. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2007.

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.

M.Sc. (Botany) / Cereals such as oats, rye, rice, barley, maize and wheat are a major source of food worldwide. Wheat (Triticum aestivum L.) is the largest winter cereal crop produced in South Africa (Crop Estimates Committee, 2011; South African Department of Agriculture, Forestry and Fisheries, www.daff.gov.za/crop estimates). Wheat production in South Africa includes both the summer and winter rainfall areas. Like other crops wheat is cultivated under monoculture conditions to increase yield per hectare. This increases the risk to pathogen exposure, as monocultures are genetically very similar or even identical, which makes them particularly vulnerable to both abiotic and biotic stresses (Haile, 2001). Both of these stressors negatively influence crop yield (Peterson and Higley, 2001; Wang et al., 2003).