Bemisia tabaci is the vector of cassava mosaic begomoviruses and cassava brown streak viruses which are main production constraints to cassava in sub-Saharan Africa. Current vector dynamics involved in the spread of both viruses in the region was established through comparison of the mitochondria cytochrome oxidase I DNA. Two distinct species were obtained: sub-Saharan Africa clade 1 (SSA1), comprising of two sub-clades (I & II), and a South West Indian Ocean Islands (SWIO) species. SSA1 sub-clade I whiteflies were widely distributed in East Africa. SSA1 sub-clade II whiteflies predominated the coast regions of Kenya, southern & coast regions of Tanzania and widespread in Uganda. SWIO whiteflies occurred in the coastal region of Kenya. This study also revealed that SSA1 sub-clade I haplotypes performed significantly better than SSA1 sub-clade II haplotypes with respect to mean number of eggs laid, developing instars and hatched adults on healthy, African cassava mosaic virus-[Tanzania:2001 ] and East African cassava mosaic Kenya virus-infected plants. There was no boost in whitefly numbers by the CMB-infected plants. The fecundity and development differences observed between SSA1 sub-clade I and II haplotypes have major epidemiology implications on the CMGs in the region

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2013. / Unable to load abstract.

Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. Johannesburg, 2015. / With rising global food prices, growing populations, climate change and future demand for tuber crops for feed and potential energy source, cassava is well positioned to meet the needs of many countries in the SADC region, including South Africa. However a major constraint to cassava cultivation is cassava infecting begomoviruses (CBVs), including African cassava mosaic virus (ACMV) and South African cassava mosaic virus (SACMV). ACMV and SACMV belong to the family Geminiviridae, comprising of circular single-stranded bipartite. Symptoms associated with CBVs infection include yellow and/or green mosaic, leaf deformation, leaf curling and stunted plant growth. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava to express hairpin RNA (hpRNA) silencing constructs against CBV. However cassava is recalcitrant and difficult to transform and regenerate. The aim of this study was to produce hpRNA/inverted repeat (IR) hpRNA constructs targeting ACMV AC1/4:AC2/3 open reading frames (ORF) and hpRNA targeting SACMV BC1 ORF to engineer hpRNA expressing transgenic cassava resistant to ACMV and SACMV. Furthermore, the approach was to stack two ACMV contiguous overlapping reading frames (AC1/4) and (AC2/3) in an attempt to improve resistance to CBV. However IR sequences are prone to unfavourable tight secondary structure formation known as cruciform structures. To circumvent this, one set of constructs (mutated sense-arm: mismatch constructs) were designed to contain sodium bisulfite deamination-induced mutations in the hairpin sense-arm making it less complementary to the antisense arm and therefore enhancing IR stability and cruciform junction formation. MM2hp (mismatch construct targeting ACMV AC1/4:AC2/3) and MM4hp (mismatch construct targeting SACMV BC1) were generated. The second construct set, non-mismatch: gateway, was designed based on the most currently used Gateway construct system. Gateway constructs contained an intron positioned between the IR fragments. MM6hp (non-mismatch construct targeting ACMV AC1/4:AC2/3) and MM6hp (non-mismatch construct targeting SACMV BC1) were generated. Similar to the deamination-induced mutations, the intron assisted with IR stability. ACMV- or SACMV-derived hpRNA constructs were transformed into model cassava cultivar cv.60444. Additionally, since few farmer-preferred cultivars or landraces have been transformed for resistance, South African high starch landrace T200 was also transformed with the hpRNA constructs. Agrobacterium-mediated transformation of friable embryogenic callus (FEC) was used and plants regenerated. Several transgenic cv.60444 and T200 lines were regenerated. Cassava landraces are generally less amenable to transformation however were able to report 79 % and 76 % for model cv.60444 and landrace T200, respectively. T200 transformation efficiency reported in this study is 43% higher than previously reported. This is also the first report of South African cassava landrace T200 transformation with ACMV and SACMV-derived hpRNA constructs. Transgenic lines were selected and infected with ACMV and SACMV infectious virus clones. Lines were then monitored at 12, 32 and 67 days post infection (dpi) for symptom development, plant growth and SACMV and ACMV viral load. At 67 dpi, a more significant difference between transgenic lines and untransformed infected cv.60444 was observed. At 67 dpi, 69 % and 75% of ACMV AC1/4:AC2/3 and SACMV BC1 transgenic lines, respectively, showed lower symptoms and reduced viral load compared to control susceptible wild-type cv.60444, but comparable to virus-challenged non-transgenic tolerant landrace control TME3. Notably, a lack of correlation between viral load and symptoms was not always observed. Plant to plant variation was observed between individual transgenic lines generated from each construct (MM2hp; MM4hp; MM6hp and MM8hp) transformation events (A-MM2, A-MM4, C-MM6 and C-MM8). However, overall a positive correlation between symptoms and viral load was observed for virus challenge trials of transgenic lines generated from A-MM4, C-MM6 and C-MM8 transformation events, this overall positive correlation was observed at all 3 dpi (12, 32 and 67 dpi). A number of ACMV and SACMV tolerant transgenic lines were obtained for both mismatch and non-mismatch hpRNA expressing transgenic lines, where virus replication persisted, but symptoms were lower at 67 dpi compared to non-transgenic plants. CBV tolerance levels observed in transgenic lines expressing mismatch technology hpRNA was not significantly different to CBV tolerance levels observed in transgenic lines expressing non-mismatch hpRNA. Expression of ACMV and SACMV- derived constructs generated tolerant cassava lines, where tolerance is defined as plants displaying virus replication but lower to no symptoms. In addition to this, a recovery phenotype was observed in five MM2hp (ACMV AC1/4:AC2/4)- derived hp expressing transgenic lines at 365 dpi, where recovery is defined as no to mild symptoms after an initial period of symptoms, and a reduction in or no viral load. In five MM4hp (SACMV BC1)-derived hpRNA expressing transgenic lines, complete recovery was observed at 365 dpi; no symptoms and no detectable virus. From this study we propose that expression of CBV- derived hpRNA targeting ACMV AC1/4:AC2/4 and SACMV BC1 in CBV susceptible cv.60444 enhances cv.60444 ACMV and SACMV tolerance. Mismatch (mutated sense-arm) construct technology offered tolerance levels comparable to the more conventional and more expensive non-mismatch (Gateway) technology. We therefore also propose that the use of mismatch hpRNA technology in cassava genetic engineering can be used as an alternative approach to transgenic crop production. Promising transgenic lines, showing moderate SACMV and ACMV resistance, were identified and these will be used in further trials as they could be considered favourable to farmers.

Cassava Manihot esculenta Crantz (Euphorbiaceae), is a source of food for more than 700 million people in developing countries and is cultivated in estimated global area of 18.6 million hectares with total annual production of 238 million tonnes. Diseases however, take a substantial toll of yield, with CMD being the most important disease and major constraint for cassava production in Tanzania and Africa. The disease causes an estimated loss of over US$ 14 million per annum. A study was undertaken in 2006/2007 to investigate the influence of satellite DNA molecules on severity of cassava begomoviruses and the breakdown of resistance to cassava mosaic disease (CMD) in Tanzania. The goal was to appraise the nature of resistance to CMD in indigenous and improved cassava cultivars in the presence of resistance-breaking satellites. Three specific aims were earmarked: to identify and characterize cassava mosaic virus isolates and satellite DNA molecules in major cassava growing areas of Tanzania; to screen cassava cultivars for resistance to begomoviruses in presence and absence of the satellite DNA molecules; and to determine the nature of interaction between begomovirus DNAs and Satellite DNA molecules in Nicotiana benthamiana. To achieve these aims, a survey was done in the major cassava growing areas of Tanzania to investigate occurrence of cassava mosaic begomoviruses and associated satellites namely, SatDNA-II and SatDNA-III. Stems from plants showing CMD symptoms were collected from field. The stems were re-planted in screenhouse to study more about the symptoms. Symptomatic leaves from sprouting cuttings were collected for DNA extraction to be used in two downstream assays - amplification of EACMV, ACMV, SatDNA-II and SatDNA-III by polymerase chain reaction (PCR) and sequencing. In another experiment to evaluate cassava cultivars for resistance to CMD in presence of satellites, stem cuttings of the classical CMD-resistant cultivars were planted in greenhouse. Infectious clones of EACMV-TZ and EACMV-UG2 comprising both DNA-A and DNA-B components of bipartite begomoviruses (EACMV-TZ and EACMV-UG2) as well as infectious clones of SatDNA-II and SatDNA-III were bombarded onto the greenhouse cassava plants using a gene gun. Emerging disease symptoms on inoculated plants were scored using standard procedure. Total nucleic acid extraction from the inoculated plants was done and PCR was performed to amplify AC1 and βC1 genes as well as full length SatDNA-II and SatDNA-III. Southern blot analysis was performed to determine the presence of AC1, βC1, SatDNA-II and SatDNA-III on the DNA. In order to study interaction between cassava mosaic begomovirus (EACMV-TZ) and satellites, infectious clones of EACMV-TZ (DNA-A and DNA-B) and that of SatDNA-II and SatDNA-III were used. The clones (DNAs) were used to infect Nicotiana benthamiana by abrasion. Inoculated plants were covered with a plastic dome and placed in insect-free growth chamber for symptom development, which were scored on a standard scale of 1 to 5. Total DNA was extracted from the N. benthamiana leaves and used for Southern blot analysis. Results from the field survey showed that disease incidences varied from 60 to 90% in the Lake Victoria Zone and from 10 to 90% in the Eastern Zone. Cultivar Lyongo had the highest disease symptom severity in the Lake Victoria Zone while in the Eastern zone plants with high severity levels were from cvs Maiza and Tabora. In the screenhouse, some sprouted cuttings remained healthy up to 16 days after planting (DAP) and others recovered from the disease. Reversion was also observed in some cultivars. Using PCR, East African cassava mosaic Tanzania virus (EACMV-TZ) was amplified from 72.8% of tested samples while African cassava mosaic virus (ACMV) was amplified from 4.3%. Five percent of plants had dual infection of the two viruses. While ACMV was detected in samples collected from Lake Victoria, EACMV-TZ was mostly found on samples from the Eastern zone. Sequencing showed the presence of two new virus isolates: EACMV-TZ [TZ113] and EACMV-TZ [TZ108]. Seventy five percent of plants, which showed reversion of symptoms, contained SatDNA-II. It was found that full length SatDNA-II occurred in both zones, while SatDNA-III was exclusive to the Lake Zone. Multiple DNA bands were noted in PCR agarose gels, more so in SatDNA-II than SatDNA-III. For SatDNA-II, the multiple bands were more evident for samples collected from Eastern zone than for those from the Lake Zone. Using primers based on expressed sequence tags (EST-primers) for SatDNA-II (895 bp) and SatDNA-III (306 bp), genome integrated forms of the satellites were amplified from 68% and 71.17% of samples, respectively. Thirty percent of the samples showed co-infection of the satellites. While EST-primers for detection of the integrated forms of SatDNA-III produced single bands on gels, those of SatDNA-II still produced additional bands, most noteworthy being the closely spaced „double bands‟. Upon sequencing, the satellite DNA isolates showed similarity with sequences deposited in the genebank and bearing accession numbers AY836366 and AY836367 for SatDNA-II and SatDNA-III isolates, respectively. Alignment reports (Clustal W) revealed presence of GC-rich regions, TATA protein binding motifs (TATAAAT) and CAAT boxes as well as poly (A) signal. GC-rich regions in SatDNA-II were mostly trinucleotides (CGC) and hexanucleotides (CCGCCG) while in SatDNA-III the regions were trinucleotides (CGC) and pentanucleotides (CCGCC). Following biolistic inoculation, five-week scoring for the symptoms showed that plants from cvs AR37-92, CR27-24 and AR16-3 remained symptomless while plants from cv T200 were symptomatic. PCR amplification of βC1 gene five weeks post inoculation (wpi) gave PCR products in 19.6% of the samples while AC1 was amplified from only two plants. Full-length SatDNA-II was amplified from 70% of DNA samples, mostly from plants in which a begomovirus was co-inoculated with SatDNA-II. Amplification of full-length SatDNA-III from bombarded plants was unsuccessful. Amplification of integrated fragments of SatDNA-II from bombarded plants using EST-primers gave a PCR product in 93.7% of the samples. PCR amplification of the fragments from DNAs extracted from plants of cvs AR17-5 and CR27-24 previously inoculated with EACMV-TZ + SatDNA-II and EACMV-UG2 + SatDNA-III, respectively, gave closely spaced bands on 13% of the DNA samples. Amplification of integrated forms of SatDNA-III gave bands in 52.4% of samples. Probing for full-length SatDNA-II, SatDNA-III and AC1 from DNAs extracted from plants pre-inoculated with these DNAs using DIG- labeled probes gave hybridization signals in 60%, 83% and 68% of the samples, respectively. Further analysis of the signals in the context of screening suggested that cvs AR37-92 and AR37-96 were highly resistant to CMD while cv AR40-10 was susceptible. In the interaction experiment, Nicotiana benthamiana plants inoculated with an infectious clone of EACMV-TZ developed moderate CMD symptoms 7 days post inoculation (dpi) with symptoms consisting of leaf distortion and moderate stunting of plants. There were also plants which recovered from the symptoms by 35 dpi. Plants inoculated with EACMV-TZ + SatDNA-II produced similar symptoms with N. benthamiana plants developing symptoms 7 dpi that became severe by 14 dpi and without recovery even after 35 dpi. Very severe symptoms were also observed when N. benthamiana plants were inoculated with EACMV-TZ + SatDNA-II + SatDNA-III. Plants inoculated with SatDNA-II or SatDNA-III alone remained asymptomatic even after 35 dpi. Southern blot analysis showed clear increase in DNA accumulation when EACMV-TZ was inoculated together with both SatDNA-II and SatDNA-III as compared to when EACMV-TZ was inoculated alone or with SatDNA-II only and probed with EACMV-TZ. In conclusion, symptom recovery and reversion of symptoms in screenhouse plants is associated with virus resistance. There is a wide occurrence of satellites (SatDNA-II and SatDNA-III) across the sampled regions consistent with distribution of their helper cassava begomoviruses. The satellites are of a wider occurrence and diversity in Eastern zone than elsewhere in the country. The occurrence of SatDNA-III was not confined to the Lake zone as previously thought. There is evidence for satellite sequence integration into host plant genome, a further indication that the satellites are wider spread in cassava germplasm than earlier conceptualized. In few instances, both SatDNA-II and SatDNA-III isolates co-existed in the same plant though its effect on symptom enhancement could not be immediately established. The observed recovery in screening studies is thought to result from resistance introduced in the plant materials involved. Since labeled probes for satellites that were used in hybridization had been prepared from satellite sequences considered to be integrated, the hybridization signals did not depend on whether the leaf samples were picked from symptomatic or asymptomatic plants. From the study, three observations clearly suggest that SatDNA-II and SatDNA-III are biologically functional and that their effects on host plants are distinctly different. The study has demonstrated enhanced cassava begomovirus symptoms in N. benthamiana in the presence of satellite DNA molecules. This is the first detailed study undertaken to highlight the occurrence and role played by satellite DNA molecules in breaking the resistance to CMD of cassava cultivars grown in Tanzania. Keywords: Cassava mosaic disease, Cassava mosaic begomoviruses, Satellite DNA molecules, Tanzania.

Com a crescente preocupação por práticas agrícolas mais sustentáveis, o uso de rizobactérias promotoras de crescimento de plantas (PGPR) tem recebido cada vez mais atenção por sua capacidade em melhorar a performance das plantas assim como aumentar sua resistência contra insetos e patógenos. Neste trabalho foi demonstrado que a inoculação de plantas de trigo com a rizobacteria Azospirillum brasilense reduziu alguns sintomas característicos de plantas infectadas pelo vírus do nanismo amarelo da cevada (BYDV), bem como a população do afídeo vetor, Rhopalosiphum padi, nessas plantas. A redução de alguns sintomas relacionados a doença foi associada à resistência sistêmica induzida, desencadeada pelo aumento nos níveis de ácido jasmônico, fitohormônio amplamente conhecido na indução de resistência por bactérias benéficas contra patógenos. Entretanto, não foi possível elucidar as vias de defesa que auxiliaram na diminuição da população do vetor. Adicionalmente, verificou- se que a inoculação bacteriana alterou os padrões de preferência hospedeira por ápteros não- virulíferos e virulíferos e alados de R. padi. Os dados apresentados nessa tese sugerem o grande potencial que a inoculação com A. brasilense pode ter no manejo do BYDV, influenciando não apenas o vírus, mas todo seu patossistema, uma vez que o crescimento populacional e o comportamento de seu vetor, R. padi, foram afetados pela interação BYDV-trigo-PGPR. / With the increasing concern for agricultural practices more sustainable, the use of plant growth promoting rhizobacteria (PGPR) has been received increasing attention for its ability to improve plant performance and increase plant resistance against insects and pathogens. In this work, it was demonstrated that the inoculation of wheat plants with the rhizobacteria Azospirillum brasilense, reduced some characteristic symptoms of plants infected by the barley yellow dwarf virus (BYDV), as well as the performance of its aphid vector, Rhopalosiphum padi. The reduction in disease severity was related to induced systemic resistance, triggered by the increase in the levels of jasmonic acid, phytohormone widely known to induce resistance by beneficial bacteria against pathogens. However, it was not possible to elucidate the pathways of defense involved in decrease the vector population. In addition, bacterial inoculation was found to affect the host preference of R. padi aptera non-viruliferous and viruliferous and alates. The data presented here suggest the great potential that the inoculation with A. brasilense can be in the management of BYDV, influencing not only the virus but all its pathosystem, considering that the population growth and the behaviour of its vector, R. padi, were affected by BYDV-wheat-PGPR interaction.

Bibliography: leaves 116-117.

Viruses or viruslike particles of eukaryotic algae are ubiquitous in aquatic habitats, however, suprisingly little is known about them. The research presented here focused on one such virus which infects a multicellular filamentous brown alga of the genus Feldmannia. Although preliminary studies had been performed on the genome structure of the Feldmannia sp. Virus (FsV), little was known. The purpose of this study was to analyze the structure of the FsV genome in detail. During the experiments aimed at mapping the FsV genome, cross-hybridization was observed among five BamHI-fragments of the digested FsV DNA. Sequence analysis of one of those fragments revealed the presence of 173 by direct repeats. There are two FsV genomes of different size-classes (158 and 178 kbp). The 173 by repeats in the cross-hybridizing BamHI-fragments were confined to a small region of each virus genome. The number of these repeats in the 178 kbp genome was estimated to be about 109 and in the 158 kbp genome to be about 41. in the 178 kbp genome, the repeats are contained within a 22 kbp region and in the 1.58 kbp genome, the repeats are contained within a 10 kbp region. These viruses are actively replicated in sporophyte plants. A family of related 173 by direct repeats was discovered in an encrypted FsV genome. The family of repeats estimated to be greater than 50 kbp in length were found inserted into a protein kinase gene encoded within the 3.6 kbp viral BamHI-fragment Z. Southern analysis indicates that these repeats in the encrypted FsV genome are distinct from the previously characterized repeats in the amplified FsV genome. The translated protein kinase shares highest homologies to the SNF1 subfamily of serine/threonine protein kinases and contains a potential autophosphorylation site in a region unique to this protein kinase. A DNA polymerase gene was identified in the FsV genome. The predicted peptide sequence of the FsV DNA polymerase gene contains all of the conserved motifs found in B-family (a-like) DNA polymerases. A TTTTTNT sequence motif shown to be a transcription termination signal for Vaccinia virus early genes is found at the 3' end of the DNA polymerase gene. Phylogenetic analysis of the FsV DNA polymerase gene and other viral DNA polymerase genes indicates that FsV belongs to a group of algal viruses recently defined as Phycodnaviridae. / Graduation date: 1998

Graduation date: 1996

Bibliography: leaves 116-117 / viii, 131, [82] leaves, [20] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1983