White grub is the common name of most scarabaeid larvae and some are considered ! as grubs of economic importance to agricultural crops. Expansion of forestry into previous croplands has resulted in some pests of previous agricultural crops becoming important in forestry. Field trials planted over three seasons, determined the mortality factors influencing the establishment of commercial eucalypt and black wattle plantations in the Natal Midlands. White grubs had the highest pest status. I Characteristic raster pattern was used to identify larvae as a particular morphospecies with the aid of the dissecting microscope. Head I capsule width pattern was used to determine the larval instars responsible for the damage. High incidences of seedling damage was recorded from December to April. Several morphospecies were found I damaging seedlings simultaneously. Morphospecies 1, 2, 3, 4, 5 and 6 were responsible for the damage but morphospecies 1, 2, 4 and 5 were the most predominant. First, second and third instar larvae were found damaging seedlings and ! they sometimes occurred simultaneously. Second and third instar larvae were the predominant larval instars. / Dissertation (M Inst Agrar (Sustainable Insect Management))--University of Pretoria, 2006. / Zoology and Entomology / unrestricted

The fungal genus Botryosphaeria including its anamorphs has a cosmopolitan distribution and occurs on a wide range of monocotyledonous, dicotyledonous and gymnospermous hosts, including woody twigs and branches, herbaceous leaves, stems of grasses, and even lichen thalli. These fungi give rise to a wide variety of symptoms such as shoot blights, stem cankers, fruit rots, die-back and gummosis. They are also known as saprophytes on dead or dying stems, branches or leaves of plants. In Venezuela, the following species have been reported: Lasiodiplodia theobromae (the anamorph of Botryosphaeria rhodina), Diplodia pinea. D. mutila and Dothiorella spp. However, their characterization has been based only on morphological descriptions. The most common and well characterized species. L. theobromae, is associated with pines and other hosts. The focus of the studies presented in this thesis as to survey Eucalyptus and Acacia plantations in Venezuela for the presence and influence of Botryosphaeria spp., and to characterize these fungi using morphological characteristics and DNA sequence data. I also evaluated the pathogenicity and population biology of Botryosphaeria species present in the plantations. It was anticipated that the various studies would characterize a relatively large number of Botryosphaeria species, potentially recognize new species and provide some perspective of their relative importance to the Eucalyptus growing industry. The thesis has been divided into chapters that reflect discrete units prepared for future publication. The first chapter presents a review of the relevant literature. Two chapters treat the taxonomy of the Botryosphaeria spp. collected in this study. An additional two chapters consider the population biology of the most commonly encountered species and the remaining chapter deals with the pathogenicity of these fungi to Eucalyptus. In the literature review I treat the taxonomy of Botyosphaeria, which has been the subject of much uncertainty for many years. I also briefly review recent findings related to their molecular characterization. A focus is also placed on the pathogenicity of Botyosphaeria species on various host plants and their known importance in Venezuela. The focus concerns mainly Eucalyptus spp. Surveys that formed part of this study logically gave rise to a large collection of Botryosphaeria isolates. An important component of this thesis was to characterize these fungi. This was done based on morphological characteristics and also comparisons or DNA sequence data for various gene regions known to be informative for these fungi. Lasiodiplodia theobromae (anamorph of B.rhodina), B. ribis and B. parva were studied from a population biology perspective. These fungi were chosen for study because they were the most common species encountered on Eucalyptus in Venezuela. To study populations, I made use of simple sequence repeat markers (SS R). Three populations of each of L. theobromae (Venezuela, Mexico and South Africa) and B.ribis-B. parva complex (Venezuela, Colombia and Hawaii) were analysed and the data considered in terms of population differentiation, gene now, mode of reproduction, gene and genotype diversity. In the final chapter of this thesis, I evaluated the pathogenicity or the seven Botryosphaeria species identified from Venezuela. These seven species were thus inoculated on Eucalyptus urophylla x E. grandis hybrid clones. A second series of inoculations was then carried out with the two most pathogenic fungi to determine the relative tolerance or the most important clones to infection. This thesis was conducted over a period of four years. Work was undertaken both in Venezuela and South Africa and this necessitated long periods of time away from my home University and family. Surveys were conducted in many parts of Venezuela to collect the Botryospaeria spp. of interest for latter study in South Africa. The research chapters have been completed systematically and they evolved over time. Each represents a discrete unit implying that there is some overlap, at least in the references, between them. I would like to believe that the thesis will provide a firm foundation for further studies of Botryospaeria spp. and prove valuable to the small but important Eucalyptus growing industry in Venezuela. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Dwarf mistletoes are Parasitic flowering plants that infect conifer tree species. The effects of Arceuthobium americanum on the seed crop of Pinus contorta were examined over a two year period to evaluate the effects of altered host reproductive success on host population dynamics. Heavily infected trees produced significantly fewer cones than uninfected trees during the first season in which cones were collected. Cone length, number of seeds per cone, individual seed mass, and total calories per seed were significantly reduced in moderately and heavily infected trees. Trees with different levels of infection showed trends consistent with the above but no significant differences were observed during the second season. The number of seeds that were produced, disseminated, and that germinated per square meter of forest floor was significantly less in heavily infected plots than in lightly infected plots. Survival of one year old seedlings, however, was significantly higher in heavily infected plots. Significant increases in the abundance of woody litter due to increases in mortality of mature trees and witches brooms were positively correlated with increases in seedling survival in heavily infected plots. Survival of trees less than 45 years of age also increased as the level of infection in plots increased. This increases the fraction of total density that saplings represent in heavily infected stands. The total basal area of plots with different levels of infection were uniform, implying that all available environmental resources are being utilized. The differences in age distribution between lightly and heavily infected plots, however, indicate that resources are partitioned differently between saplings and mature trees in plots with different levels of infection. Dominant host trees experienced higher mortality in heavily infected plots than in lightly infected plots. This reduces the total biomass of all mature trees. Increased mortality of dominant host trees frees resources which reduces intraspecific competition thereby contributing to increased sapling survival in heavily infected plots. Consequently, stand density increases as the level of infection increases.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy in Science at the School of Molecular and Cell Biology, 2017 / Different host genes playing a role in replication, transcription and movement of geminiviruses have been identified, allowing a better understanding of host response during infection. The cytoskeletal protein myosin has been shown to associate with RNA viruses movement protein and mediate its movement, however no geminivirus association with myosin has been established. Arabidopsis thaliana nitric oxide associated protein 1 (AtNOA1), once thought to be an enzyme involved in a nitric oxide (NO) production, has been reported to be differentially regulated in response to biotic and abiotic stress. In this study we sought to identify the role that myosins and NOA1 play in the development of disease by south african cassava mosaic virus (SACMV). Using a bioinformatics approach, 24 myosin transcripts were identified in Nicotiana benthamiana, and phylogeny analysis revealed that seven were class VIII myosins and 17 class XI. Five myosins silencing constructs M15.1 (transcript Niben101Scf11288g00015.1), MYOSIN XI-F (M11.F), MYOSIN XI-K (M11.K), MYOSIN XI-2 (M11.2) and MYOSIN VIII.B were selected for silencing using a virus induced gene silencing (VIGS) approach with SACMV and TRV-VIGS vectors. At 14 days post inoculation (dpi), both SACMV and TRV-VIGS vectors successfully silenced myosins with SACMV-VIGS silencing all five and TRV-VIGS silencing all but M11. F. At 28 dpi, SACMV-VIGS induced silencing of myosin of only two myosins and TRV-VIGS three. TRV-VIGS was found to be more efficient at silencing as the suppression of myosin induced by TRV-VIGS was stronger than that of SACMV-VIGS. To assess the effect of myosin silencing on SACMV infectivity in a separate experiment, 7 dpi of silencing, N. benthamiana plants were challenged with SACMV and reduction of myosin expression was assessed as well as viral accumulation. TRV-VIGS did not induce any silencing of myosin at 14 dpi, and at 28 dpi, the expression of M11.K and M11.F were silenced. SACMV-VIGS induced silencing of M11.F at both 14 and 28 dpi. In TRV-VIGS silenced M11.K, viral load at 28 dpi was not lower than the control, however the fold increase in viral load at 28 dpi compared to 14 dpi was 3-fold (p value 0.03) for M11.K silenced TRV-VIGS plants and 86-fold for the control 6-fold for the M11.K suggesting that silencing of M11.K decreases the spread of SACMV. In TRV-VIGS silenced M11.K, viral load at 28 dpi was lower than the control (9-fold p value 0.03) and the increase in viral load at 28 dpi compared to 14 dpi was insignificant, suggesting that spreading of SACMV was also hampered. The reduction in myosin M11.F expression induced iv by SACMV-VIGS resulted in an increase in viral load compared to the control. We hypothesise that the increase in viral load observed in M11.F silenced plants induced by SACMV-VIGS is due to the perceived resistance of SACMV-VIGS control (SACMV-challenged no silencing construct) to SACMV-challenge, and therefore results from the SACMV-VIGS study were inconclusive. From the TRV-VIGS study however, we have identified two candidate myosins in N. benthamiana myosin XI-K and myosin XI-F as potential interactor of SACMV during infectivity. Further research into their role in the development of SACMV disease is warranted. Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation. To understand the role played by NOA1 in response to (SACMV) infection, a bioinformatics approach was used to identify NOA1 homologues in cassava T200. Using the cassava genome data on Phytozome, a putative NOA1 namely cassava 4.1_007735m, was identified. Based on its protein sequence, cassava4.1_007735m shared a 69.6% similarity to Arabidopsis NOA1 (AtNOA1). The expression of cassava4.1_007735.m (MeNOA1) and N. benthamiana NOA1 (NbNOA1) and the accumulation of NO in leaf samples was compared between SACMV-infected and non-infected at early infection stage (14 dpi for N. benthamiana and 28 dpi for cassava T200) and full systemic stage (28 dpi for N. benthamiana and 56 dpi for cassava T200). Real-time PCR was used to measure SACMV viral load which increased significantly by 2-fold (p value 0.05) from 14 to 28 dpi for N. benthamiana and 8-fold from 28 to 56 dpi in cassava T200 (p value 0.04) as chlorosis and symptom severity concomitantly progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p value 0.01 and 4 fold lower at 28, (p value 0.00) and the abundance of NO in infected N. benthamiana leaf tissue was 10% lower at 14 dpi and 40% lower at 28 dpi when compared to mock inoculated. In cassava T200, MeNOA1 expression was unchanged at 28 dpi and NO levels were decreased by 40% and at 56 dpi, MeNOA1 expression was 4-fold lower and NO accumulation was 37 % higher than that of mock inoculated leaf tissue. At 28 dpi for N. benthamiana and 56 for cassava T200, the decrease in NOA1 expression was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaf samples. Furthermore, the expression of v chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and) were found to be repressed in infected N. benthamiana and infected cassava T200 relative to mock inoculated plants. GC-MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection in N. benthamiana, decrease in photopigment and carbon stores, accompanied by an increase in glucose and decrease in fumarate, lead to a decline in NbNOA1 and NO levels. This is manifested by suppressed translation factors, and disruption of the chloroplast, resulting in chlorotic disease symptoms. In cassava T200 however, the link could not be established as the level of glucose was not significantly decreased and fumaric acid was not detected and although the concomitant decrease in the expression of MeNOA1 and chloroplast translation factors indicate dysfunction of the chloroplast, the link between MeNOA1 expression, carbon store, NO and chloroplast activity could not be established. / XL2017

A Thesis Submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the Degree of Doctor of Philosophy / The Epidemiology, occurrences and effect of BMV (Brome Mosaic virus) wheat (triticum aestivum) in the summer rainfall area. (Abbreviation abstract) / AC 2018

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree Master of Science Johannesburg, 2018 / Seed recalcitrance in avocado (Persea americana) has meant that avocado genetic material cannot be conserved in orthodox seed banks. Thus, biotechnological approaches have been considered for the long-term conservation of this species’ genetic material, through the cryopreservation of tissue culture-generated axillary buds. A study was conducted to develop a system for the proliferation of in vitro avocado cv. ‘Edranol’ axillary buds for the purpose of cryopreservation. Experiments were conducted to optimise avocado mother plant establishment and pretreatment. It was determined that potting soil mixes comprising of either 1:1:1 pine bark, perlite, river sand or 1:1:1 peat, perlite, river sand were suitable to culture healthy avocado mother plant seedlings. With these soil mixes approximately 2 shoots per plant developed after 11 weeks of transplanting and between 2.9 ± 0.31 and 3.37 ± 0.32 secondary shoots were produced after 5 months. Additionally, the mother plants produced well extended shoots (7.30 ± 1.29 cm; 8.77 ± 1.39 cm) with a sufficient number of axillary buds (7.75 ± 0.39; 6.33 ± 0.53), which were subsequently used as nodal explants. After surface decontamination, the establishment of an aseptic culture in vitro was successfully achieved. Six semi-solid tissue culture media were tested for the proliferation of in vitro axillary buds. Four media comprised of half (½) and full strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), with either 0.5 or 1mg/l 6-Benzylaminopurine (BAP). Two media were based on the P. indica medium as proposed by Nel et al. (1983), and comprised of half strength MS macronutrients, full strength MS micronutrients, 2mg/l BAP and 1mg/l GA3. All media were supplemented with 3g/l Gelrite and 30g/l sucrose at pH 5.6-5.8. Physiological measurements were taken six weeks after establishment, the first, the second and the third subculture. Tissue browning, death and contamination were observed in explants cultured on the media containing 0.5mg/l BAP, suggesting that this concentration of BAP was not suitable for cv. ‘Edranol’. Additionally, hyperhydricity appeared to be associated with the media containing ½ MS, which could be attributed to mineral deficiencies. Overall, there was no significant difference in the number of shoots and axillary buds developed across all the media tested, suggesting that endogenous auxin levels were higher than the concentration of cytokinin used in the media tested. In support of this, strong apical dominance and callus formation was observed. An increase in tissue browning, death and hyperhydricity on all the media tested, coupled with a decrease in shoot length, suggested a decline in the vigour of explants in vitro. 1MS + 1mg/l BAP was selected as the most appropriate medium for the initiation of cv. ‘Edranol’ cultures, producing between 3.2 ± 0.2 and 4.9 ± 0.5 axillary buds per explant. However, hyperhydricity, browning and death were observed in explants cultured on this medium. Overall, the in vitro axillary bud explants did not behave predictably or uniformly. Thus, the system was not optimised, indicating that further study is needed for the mass multiplication of axillary buds to be used for the cryo-conservation of avocado genetic material. It is recommended that future experiments will be needed to further test tissue culture media, with a focus on the optimisation of the nutrient and plant growth regulator concentrations. Additionally, the recalcitrance of explants to the in vitro environment may have been influenced by the physiological state of the mother plants, indicating that research should be focused on the effect which the mother plants may have on the endogenous responses of the in vitro explants. / MT 2018

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