Browsing by Author "Fisher-Smith, Nadia"

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    An investigation into the wheat (Triticum aestivum L.) host response to Russian wheat aphid (Diuraphis noxia Kurd.) feeding
    (Stellenbosch : Stellenbosch University, 2023-12) Fisher-Smith, Nadia; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjomov), is a major pest of wheat, causing damage and high yield losses worldwide. One of the undesirable effects of aphid feeding is leaf rolling, which serves as a shelter for the aphids protecting them from their natural predators and from insecticide spraying. Leaf rolling reduces the plant’s ability to photosynthesise and grow effectively, in addition, leaf rolling can also increase the aphid fitness, as it provides an ideal environment for growth. Therefore, identifying natural sources of resistance and introducing them into susceptible cultivars seems to be the most efficient strategy against RWA feeding. The use of genetic resistance is an efficient and environmentally safe method for controlling the RWA. This study aimed to ascertain if enhanced tolerance to biotic stress can be achieved by modifying plants either through genetic manipulation or chemical mutagenesis. Various studies have been done at a transcriptome level, allowing the identification of genes likely involved in RWA resistance. Utilising proteomics data in this study, allowed for the identification of differentially expressed peptides between resistant and susceptible wheat lines. Among the identified proteins were: glutathione-S-transferase (GST) and peroxidase. Literature suggests that GST forms part of the detoxification system in plants against biotic stress. This protein was uniquely expressed in the W1320-W1278 line that contains the Dn5 resistance gene. Peroxidase is associated with the oxidative burst, usually in response to stress, was identified in this study in the Gamtoos-S (Dn0) susceptible cultivar. Utilising genetic manipulation, a partial gene fragment of glutathione-S-transferase F6 (GSTF6b) was isolated from wheat and sequenced to confirm its identity. The gene fragment was cloned into a plant expression vector in the antisense orientation and bombarded into four- to six-day-old wheat immature embryos. Resulting in a putative transgenic plant, namely Gamtoos-S (Dn0)-pUBI-510:GSTF6b. Quantitative reverse- transcriptase-linked polymerase chain reaction (RT-qPCRs) were conducted to quantify the expression of the GSTF6b gene with/without RWA infestation. A reduction of nearly 50% was observed in GSTF6b expression in the respective transgenic plants when compared with the control. The T₁ was successfully hardened off, and allowed to seed and a T₂ generation was generated, which was functionally analysed through phenotypic screening, aphid fecundity, enzymatic responses and measuring oxidative burst. A decrease GST transcript level was observed post-infestation in the transgenic plants suggesting that plant susceptibility can probably be linked to a decrease in GST transcript promoting aphid growth and increasing the rate of reproduction. The last part of the study involved chemical mutagenesis, whereby drought-tolerant mutagenic M6 lines were screened for aphid resistance. A phenotyping assessment was performed on available mutant lines infested with South African (SA) biotype 1. A total of 33 mutant lines selected for drought tolerance, consisting of 21 ethyl methanesulfonate (EMS) and 12 Sodium azide (NaN₃) mutants, showed variation in aphid tolerance. Furthermore, drought-tolerant mutants were found to be more susceptible to aphid infestation, excluding the M12 (RYNOB8.012) line, shown to be intermediate to aphid feeding. The anti-oxidative enzyme GSTF6b expression was found to be significantly up-regulated in the mutagenic lines before infestation, therefore, contributing to the notion that GSTF6b is present at the basal level. A positive correlation was observed between GSTF6b gene expression and the intrinsic rate of increase (rm) in 25 mutagenic lines.