A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell Biology. Johannesburg 2017 / With growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. 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 with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct. / MT2017

Bemisia tabaci (Genn.) is the vector of cassava mosaic geminiviruses (CMGs), which are the main production constraint to cassava, both in Uganda and elsewhere in Africa. A severe form of cassava mosaic disease (CMD) was responsible for the devastation of cassava in Uganda beginning in the late 1980s. In subsequent years the severe CMD epidemic spread throughout Uganda, and to neighbouring countries, causing devastating effects to cassava production, and its geographical range continues to expand with the pandemic. To further understand the virus-vector dynamics involved in the spread of CMD in the post epidemic zone in Uganda, we investigated the current distribution of B. tabaci genotypes in selected cassava-growing regions. Additionally, the relationship between the vector genotypes and distribution of CMGs in the post-epidemic zone was examined also. CMD-affected cassava leaves were collected from 3 to 5 month-old cassava plants, and B. tabaci adults and fourth instar nymphs were collected from cassava and twenty-two other plant species occurring adjacent to the sampled cassava fields. The mitochondrial cytochrome oxidase I (mtCOI) sequence was used to establish the genotype of B. tabaci adults and nymphs associated with the sampled plant species. African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda 2 (EACMV-UG2) were confirmed to be present in the post-epidemic zone in Uganda, as reported previously. As expected, EACMV-UG2 predominated. However, unlike previous observations in which EACMV-UG2 was consistently associated with the severe disease phenotype, in this study EACMV-UG2 occurred almost equally in the severely and mildly diseased plants. Phylogenetic analyses of Ugandan B. tabaci genotypes (mtCOI) revealed that their closest relatives were other Old World genotypes, as might be expected. Two previously reported B. tabaci genotype clusters, Uganda 1 (Ug1) and Uganda 2 (Ug2), at ~8% nt divergence, were confirmed to occur on cassava in the post-epidemic zone. However, Ug1 occurred more frequently (83%) than Ug2 (17%), and no definite association was established of a particular vector genotype with cassava plants exhibiting the severe disease phenotype, in contrast to the B. tabaci genotype distribution and association with the CMGs reported there at the height of the spread of the severe CMD epidemic. Based on the presence of B. tabaci fourth instar nymphs, the Ug1 genotypes colonized five additional non-cassava plant species: Manihot glaziovii, Jatropha gossypifolia, Euphorbia heterophylla, Aspilia africana and Abelmoschus esculentus, suggesting that in Uganda the Ug1 genotypes are not restricted to cassava. However, no Ug2 genotypes were detected on the non-cassava plant species sampled. This study revealed also the presence in Uganda of five distinct previously unrecorded B. tabaci genotype clusters, Uganda 3 (Ug3), Uganda 4 (Ug4), Uganda 5 (Ug5), Uganda 6 (Ug6) and Uganda 7 (Ug7), and a sweetpotato colonizing genotype cluster, designated Uganda 8 (Ug8), among the collective Ugandan B. tabaci populations. Ug3 was the only exemplar representing one cluster, which was unlike any previously described genotype in Uganda or elsewhere, and diverged at 8%, 10% and 17% from Ug1, Ug2 and Ug8, respectively. The Ug3 genotypes colonized a single species, Ocimum gratissimum. Ug4, Ug5, Ug6 and Ug7 formed four closely related sub-clusters (93-97% nt identity), and diverged from one another by 1-7%, and by 15-18% from Ug1, Ug2, Ug3 and Ug8, respectively. The Ug4 genotypes had as their closest relatives (at 97-99% nt identity) previously reported B. tabaci from okra in the Ivory Coast, whereas, the Ug5 and Ug6 genotypes shared 95-99% and 99% nt identity, respectively, with their closest relatives from the Mediterranean-North Africa- Middle East (MED-NAFR-ME) region, which also includes the well studied B and Q biotypes. The Ug7 genotypes were closely related (at 98-99% nt identity) to B. tabaci from Reunion Island in the Indian Ocean. The Ug4, Ug5, Ug6 and Ug7 genotypes were identified on 54%, 8%, 8%, and 31% of the sampled plants species, respectively. Ug4 were most polyphagous, followed by Ug7 and Ug6. However, none of the new five genotypes (Ug3-Ug7) was found associated with, or colonizing, xx cassava or sweetpotato plants in this study. Squash plants colonized by the Ug6 and Ug7 genotypes, both members of the B biotype/B-like cluster, developed the silvering phenotype, while those colonized by the Ug4 genotypes (most closely related to a non-B like genotype from okra in the Ivory Coast) did not. In addition to colonizing sweetpotato, the Ug8 genotypes also colonized Lycopersicon esculentum and L nepetifolia.

Cassava brown streak disease (CBSD) caused by two distinct ssRNA virus species (CBSV and UCBSV of genus Ipomovirus, family Potyviridae) and transmitted by whitefly (Bemisia tabaci), is a major constraint to cassava production in Africa, including Mozambique. In this research, two studies were conducted. First, in order to monitor the incidence, severity and geographical distribution of cassava brown streak disease and associated viruses in Mozambique, field surveys were performed in six cassava major growing provinces. A total of one hundred and fifteen fields and one hundred and forty six fields were surveyed in 2010 and 2012, respectively. The disease was only found in three of six provinces namely Zambezia, Nampula and Cabo Delgado. The CBSD incidence was highest (61.3% and 82.2% in 2010 and 2012, respectively) in Zambezia and lowest (23.6% and 35.1% in 2010 and 2012, respectively) in Cabo Delgado, with cultivars such as Cadri and Robero showing the highest susceptibility to CBSD, while Likonde and Amwalikampiche had relatively low CBSD incidence, illustrating some tolerance to the disease. The results, when compared to previous surveys conducted in 1999 and 2003, demonstrated that the disease is increasing, and replanting new fields with disease-affected cuttings could be responsible for the spread. The second aim of the study was to investigate the genetic diversity of Cassava brown streak viruses, based on analysis of partial sequences of the coat protein gene, in Mozambique. Collections of CBSD-symptomatic leaves were done between June 2010 and June 2012. Diagnostic RT-PCR, using specific primers to screen for the two species, revealed for the first time the presence of Uganda cassava brown streak virus (UCBSV) in Mozambique. UCBSV was found in mixed infections with CBSV, and only confined to a single province of Zambézia, while CBSV species were widely distributed. The phylogenetic analysis revealed two subgroups within CBSV, which were 6.7% divergent in nucleotide sequence. The heterogeneity observed among CBSV isolates in Mozambique suggests that in the future studies more sampling is needed to characterize strains and variants. Addtionally, sequencing of the full CP sequence of CBSaVs isolates is required, which may reveal even more diversity. Infectivity assays of cassava brown streak viruses (CBSV and UCBSV) were established using the host indicator plant Nicotiana benthamiana. Plant sap was extracted from infected cassava leaves and inoculated into N.benthamiana plants. CBSD-like symptoms were observed, and RT-PCR revealed the presence of CBSV in all samples, except for one which was co-infected with UCBSV and CBSV. This study provided further evidence that CBSaVs are efficiently transmitted to N.benthamiana. There is scanty information on alternative hosts, therefore more research is needed to identify other potential hosts of CBSaVs in order to develop an effective strategy to control CBSD.

Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Accurate and efficient detection and identification of plant viruses are fundamental aspects of virus diagnosis leading to sustainable disease management. In the present study I describe two techniques, the first based on a single tube duplex and multiplex polymerase chain reaction (m-PCR), developed for simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) and East African cassava mosaic Malawi virus (EACMMV), and second, a technique based on Restriction Fragment Length Polymorphism (RFLP) analysis of Reverse Transcribed (RT) -PCR amplified Cassava brown streak viruses species, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV). In this work, the single tube duplex and multiplex PCR for simultaneous detection of the four cassava mosaic begomoviruses (CMBs) was developed successfully. Four primer pairs were designed from published DNA-A component sequences targeting specific amplification of the four cassava mosaic begomoviruses (CMBs). Evaluation of the primers sensitivity in serially diluted virus samples revealed that the new primers amplified their target virus to a dilution of 10-4 and 10-3 for uniplex and multiplex PCR respectively. Developed multiplex assay enabled specific amplification of the viruses in producing 950, 503, 435 and 260 base pairs (bp) for ACMV, EACMMV, EACMCV and EACMZV respectively in single and mixed infections of CBSVs. Analysis of 172 field samples from Kenya, Malawi, Mozambique, Rwanda, Tanzania and Zambia detected both single and mixed infections, results which were proved by analysis of the sequenced amplicons. Second, a technique based on 2 Restriction Fragment Length Polymorphism (RFLP) analysis of RT-PCR amplified cassava brown streak viruses, Cassava brown streak virus (CBSV) and cassava brown streak Uganda virus (CBSUV), was performed. A degenerate primer amplifying 785 bp of the coat protein gene (CP) of CBSV and CBSUV was also designed. Two restriction endonucleases, HindIII and EcoR1 (identified by a software package, Vector NTI® Express v1.0 from Life Technologies/Invitrogen), which produce different fragments upon digestion of RT-PCR amplicons from CBSV and CBSUV, were used to distinguish the two viruses RFLP analysis using EcoRI has no site in CBSV producing one fragment (785 bp), two fragments (525 bp and 224 bp) for CBSUV and three fragments (785, 525 and 224 bp) for the mixed infections. On the other hand, HindIII has no site in CBSUV producing one fragment (785 bp), three fragments (437 bp, 267 bp and 81 bp) were produced for CBSV, and four fragments (785, 437, 267 and 81 bp) for CBSV and CBSUV mixed infections. In both multiplex and RFLP analyses, results from the sequenced PCR/RT-PCR amplicons agreed with sequence identities of the respective published virus species. Experience from using developed multiplex and RFLP techniques show that time was saved and amount of reagents used were reduced. RFLPs confirmed the presence of CBSV and CBSUV in RT-PCR amplicons without requirement for sequencing. Additionally, modified protocols from Dellaporta et al. (1983) and Chang et al. (1993), were used to extract DNA and RNA respectively from dry and fresh cassava leaves with comparable results. I also demonstrated a method of collecting and preserving cassava leaf samples to retain their integrity during storage for a period of over one month. The two diagnostic tools can be used routinely in germplasm indexing, disease surveillance, and disease monitoring programs 3 Problem Statement and Rationale In east and southern Africa, cassava (Manihot esculenta Crantz) is one of the leading crops in terms of production and has become an important source of income to households and small-scale farmers. However, the production across the region is greatly affected by Cassava mosaic disease (CMD) and Cassava brown streak disease (CBSD). Reports from different authors (Gibson. 1996; Ogbe et al., 1996; Legg et al., 1999; Fondong et al., 2000; Bisimwa et al., 2012) have reported the occurrence of CMD in different countries in the SSA. In Tanzania, CMD has been reported from many locations. Comprehensive characterization by Ndunguru et al. (2005) showed seven cassava mosaic geminiviruses species occur in Tanzania. Mbanzibwa et al. (2009a) reported prevalence of two potyvirus species causing CBSD in the Lake Victoria basin and along the coastal belt of Indian Ocean. A countrywide survey of all major cassava-growing areas in Kenya by Bull et al. (2006) reported presence of six CMG species with novel begomoviruses and a new recombinant strain of EACMV, demonstrating increasing diversity and geographical distribution of CMGs. Similarly, recent reemergence of CBSD has been reported in many districts in Uganda (Alicai et al., 2007) as well as from Malawi (Winter et al., 2010), Kenya (Mware et al., 2009) and Rwanda (Shirima et al., 2012). No reports of occurrence of CBSD have been reported from Zambia. With the current development of more robust diagnostic tools such as RT-PCR and real-time PCR, the diagnosis of CMD and CBSD has also improved in many cassava- producing countries. Similarly, the challenges to obtain more sensitive broad-spectrum cost-effective diagnostic tools also increase. This is evident following discovery of more 4 virus species causing CMD and CBSD (Mbanzibwa et al., 2009a and Winter et al., 2010) which can easily be overlooked. In the field the co-infections of many CMBs and CBSVs is common. Therefore, it will require several tools to detect the multiple infections using the diagnostic tools currently available. Thus, development of efficient and affordable diagnostic tools for simultaneous detection and identification of CMBs and CBSVs is vital and will have a significant impact on development and implementation of cassava virus disease management. Diagnostics will be used for disease monitoring in cassava multiplication plots production and distribution of disease- free cassava planting materials. Therefore, this research make use of the available sequence information in the database for both CMBs and CBSVs to develop sensitive tools for the simultaneous detection of four species of cassava begomoviruses namely: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava Mosaic Zanzibar Virus (EACMZV) using multiplex PCR. Also identification and differentiation of two species of Cassava brown streak viruses namely Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) by RT-PCR/RFLP approach. This study generated knowledge and new tools that will enhance the diagnosis of both CMD and CBSD. The tools will facilitate deployment of virus-indexed cassava planting materials within the region.

Orientador: Cláudio Cabello / Banca: Waldemar Gastoni Venturini Filho / Banca: Manoel Lima de Menezes / Resumo: Manipueira de extração de fécula de mandioca, em separado da água de lavagem das raízes, através de biodigestores anaeróbios de fluxo ascendente, com separação das fases, sem controle de temperatura ou adição de produtos químicos e, avaliar sua adequabilidade, através das suas características físicas e químicas para lançamento em corpo receptor, sistema público coletor de esgotos ou, aplicação em processo de fertirrigação. Depois dos reatores estabilizados, foram realizados ensaios variando a vazão de alimentação com 8,0; 12,0 e 16,0 Ld-1, correspondentes a um tempo de retenção hidráulica de 8,17; 5,44 e 4,08 dias respectivamente. Os melhores resultados para redução da carga orgânica foram obtidos com os tempos de retenção hidráulica (TRH) de 8,17 e 5,44 dias com eficiências médias de 89,8 e 80,9% respectivamente. As características físicas e químicas dos efluentes tratados foram comparadas com os valores estabelecidos na legislação estadual, federal e, com os parâmetros utilizados pelo órgão ambiental fiscalizador. Os resultados obtidos mostraram que o efluente tratado atende parcialmente aos requisitos legais para o lançamento em corpos receptores, devido ao teor elevado de nitrogênio amoniacal. Por outro lado, foram atendidos integralmente os requisitos legais para o lançamento na rede pública coletora de esgotos. O efluente tratado não atendeu às recomendações requeridas pelo órgão ambiental fiscalizador para a sua disposição através de processo de fertirrigação devido aos teores elevados de ferro (Fe++) e de fluoretos (F-). Considerando os resultados obtidos, concluímos que, devido à simplicidade do sistema utilizado, com a implantação de melhorias como um pós tratamento, poderiam ser atingidos os parâmetros que atenderiam integralmente a legislação. / Abstract: The aim of this paper was to evaluate the efficiency of the treatment of cassava wastewater, separately from the root washing water, by means of ascending flux anaerobic biodigesters, with separation of the phases, without temperature control or addition of chemical products and to evaluate its suitability by means of its physical and chemical characteristics for throwing in receiving body, public sewage system or application in fertilization and irrigation. After reactors had been stabilized, essays were conducted varying feeding flow with 8.0, 12.0 and 16.0 Ld-1, corresponding to a hydraulic retention time of 8.17, 5.44 and 4.08 days, respectively. The best reduction for organic load reduction were obtained with hydraulic retention times (HRT) of 8.17 and 5.44 days with mean efficiencies of 89.8 and 90.9%, respectively. Physical and chemical characteristics of treated effluents were compared with the values established in the current state and federal legislation and with the parameters used by the environmental supervising organ. The results we obtained showed that the treated effluent partially meets the legal requirements for throwing in receiving bodies owing to high contents of ammonia nitrogen. On the other hand, legal requirements were fully met to throw effluents into the public sewage system. The treated effluent did not fulfill the recommendations required by the environmental supervising organ for its disposal by means of fertilization and irrigation due to high concentrations of iron (Fe++) and fluorides (F-). Considering the results obtained, we conclude that, due to the simplicity of the system used, as improvements such as after-treatment are made, parameters fully obeying the legislation could be met. / Mestre

A dissertation submitted to the Faculty of Science of the University of Witwatersrand, Johannesburg, in full fulfilment of the requirements for the degree of Master of Science, 2019 / Cassava, Manihot esculenta Crantz, is categorized as a food security crop, producing large starchy tubers that are gaining interest from both international and local agro-processing industries for products such as bioethanol, textiles, and food additives. However, cassava is currently under threat from a group of begomoviruses that cause cassava mosaic disease (CMD) in all countries in sub-Saharan Africa where cassava is cultivated. CMD can result in up to 100% crop loss. South African cassava mosaic virus (SACMV) is particularly a threat to the growing cassava industry in southern Africa. Despite extensive breeding programs over the past 70 years to develop CMD-resistant farmer-preferred cassava landraces, total resistance has not been achieved. Furthermore, the high mutational rates of begomoviruses, and mixed infections in the field, have exacerbated the problem. TME3 is a West African landrace that displays tolerance to begomoviruses, including SACMV. Infection of TME3 by SACMV leads to recovery, hallmarked by low virus loads and milder symptoms compared to a susceptible southern African landrace T200. The molecular processes that govern tolerance in crops, including cassava, are not well understood. However, systemic immune responses, which are controlled by hormoneresponsive transcription factors (TFs), are required by the plant to successfully combat an invading pathogen. Two different branches of systemic immunity have been described, namely systemic acquired resistance (SAR), facilitated by salicylic acid (SA) signalling, and induced systemic resistance (ISR), which is induced through jasmonic acid (JA) and ethylene (ET) signalling in the presence of beneficial rhizobacteria. In 2014, Allie et al. compared global transcriptomic responses occurring in TME3 and the T200 during early 12 days’ post inoculation (dpi), middle (32 dpi) and late (67 dpi) stages of SACMV infection. In order to give greater context to transcriptomic data, which is inheritably large and complex, network analysis may be implemented. By placing the differentially expressed (DE) gene homologs/orthologs identified from the cassava transcriptome datasets into protein-protein networks, functions of SACMV-responsive genes, interacting partners, and potential hubs, can be derived. Cassava gene functions are based on the model crop Arabidopsis thaliana, as despite the sequencing of the cassava genome, the annotations are incomplete. The aim of this study was to identify potential candidate TFs, and their associated hormones and other network partners, that confer either tolerance (TME3) or susceptibility (T200) to SACMV. / TL (2020)

Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Two of the most widely distributed begomoviruses in East Africa associated with CMD, are East African cassava virus- Uganda2 (EACMV-UG2) and African cassava mosaic virus (ACMV). Despite efforts of generating improved Tropical Manihot Series (TMS) by traditional breeding and using highly resistant geminivirus cassava landraces such as Tropical Manihot Esculenta1 (TME1) and Tropical Manihot Esculenta3 (TME3), more recently two circular single stranded (ss) satellite-like DNA molecules (episomal DNA-II and DNA-III) have been found to be associated with CMD and are able to break resistance to EACMV-UG2 and enhance virus symptoms. The nature of these satellite-like DNA molecules is unknown, and furthermore, the discovery of integration of partial copies of DNA molecules (DNA-II and III fragments), and evidence for transcription from cassava Expressed Sequence Tag (EST) database screening, has led to an even more perplexing disease complex. In the present study, we attempted to further explore the interaction between the satellite-like DNAs and their associated cassava-infecting begomoviruses by investigating the impact of these DNA molecules on disease development in TME3 (tolerant) and cv. 60444 (susceptible) cassava cultivars, and to also gather biological evidence for transcription of integrated genomic and episomal (putative predicted ORFs) sequences in the ACMV and EACMV-UG2-associated DNA-II and DNA-III. Biolistic inoculation of EACMV-UG2, ACMV, and in co-bombardment with DNA-II, DNA-III, DNA-II + DNA-III was successfully performed. CMD symptoms were developed earlier on cassava plants inoculated with ACMV + DNA-II, ACMV + DNA-III, ACMV + DNA-II + DNA-III and EACMV-UG2 + DNA-II, EACMV-UG2 + DNA-III, EACMV-UG2 + DNA-II + DNA-III molecules compared with cassava plants inoculated with begomoviruses alone. Additionally, CMD symptoms were more severe in cv.60444 compared to TME3 when inoculated with begomoviruses alone, or in combination with DNA-II, DNA-III and DNA-II + DNA-III molecules. DNA-II and III were able to break resistance to the highly CMD-tolerant cassava landrace, TME3, and enhance virus symptoms. In order to confirm EST-generated evidence for transcription of DNA-II and III fragments, cDNA was subjected to RT-PCR. RT-PCR of transcripts was successful for only three putative ORFs: ORF C4 of the antisense DNA-II strand, ORF V1 on sense DNA-II strand, and ORF C2 on antisense strand for DNA-III. Primers for transcripts amplified 250 bp and 220 bp for ORF C4 of DNA-II and ORF V1 of DNA-III, respectively. Transcribed ORFs were confirmed by sequencing, and the sequences were similar to the published sequences of Begomovirus associated DNA-II satellite and Begomovirus associated DNA-III satellite, respectively. These results showed that at least two putative ORFs for DNA-II and one (the largest ORF VI) DNA-III can be transcribed. Furthermore, surveys were undertaken in order to ascertain the distribution of episomal and integrated DNA-II and III in cassava germplasm from several countries, namely Tanzania, Uganda, Kenya and Rwanda. Results from this research successfully established genetic diversity and wide geographical distribution of integrated DNA-II and DNA-III molecules. Two primer pairs were designed from a central conserved sequence found in all the integrated DNA-II or III fragments identified from the cDNA libraries (EST database). These primers also amplified integrated sequences of expected size in cassava accessions and wild Manihot species which were similar to satellite-like sequence occurrences in the ESTs. Using designed primers, PCR amplification yielded integrated DNA-II and DNA-III products of ~895 bp and ~306 bp, respectively. Analysis of 363 field leaf samples detected the presence of DNA-II or DNA-III from Kenya (3.3% or 8.3%), Uganda (18% or 2.5%), Rwanda (6.5% or 19.6%) and Tanzania (5.7% or 11.9%) , results which were confirmed by analysis of the sequenced PCR amplicons. Detection of both DNA-II and DNA-III molecules on the samples collected was also found from Kenya (73%), Uganda (69.1%), Rwanda (50%) and Tanzania (69.3%). Interestingly integrated DNA-II and II copies were amplified from healthy, symptomless and infected cassava samples. DNA-II sequences did not vary significantly (93.3% - 99.8%) and were highly similar to the sequences of Begomovirus associated sat DNA-II (AY836366) and 99% with mentha leaf deformity disease associated satellite DNA-II, while DNA-III sequences and Begomovirus associated DNA-II satellite (AY833667). In conclusion, this study has provided useful information that contributes to a further understanding of the biological function of integrated and episomal DNA-II and III molecules in begomoviruses infected cassava plant. However the relationship, if any between episomal and integrated forms needs to be established in future, and investigation into whether the transcribed ORFs can produce functional proteins, needs to be undertaken. How DNA-II and III interact with EACMV-UG2 and ACMV in disease modulation remains to be explored, and the replication of episomal DNA-II and III by these associated begomoviruses needs to be confirmed if these DNA molecules are to truly show a satellite-like relationship. Furthermore, the findings in this study that partial and varied-sized integrated DNA-II and III fragments occur widely in healthy and infected cassava germplasm will enable researchers (plant virologists and breeders) working on cassava in Sub Saharan Africa (SSA) to explore this complex more deeply in order to develop durable management strategies for CMD.

Faculty of Science School of Molecular and Cell Biology 9714718d MMakwarela@arc.agric.za / Cassava (Manihot esculenta Crantz) is a vegetatively propagated root crop used as a staple throughout the tropics and subtropics. It is the fourth most important and cheapest staple food crop after rice, wheat and maize in developing countries, providing food for over 600 million people. However, its production is severely limited by a wide variety of viral and bacterial diseases, especially Cassava Mosaic Disease (CMD) which is caused by several geminivirus species including, South African cassava mosaic virus (SACMV), African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), Indian cassava mosaic virus (ICMV) and the Ugandan recombinant virus (UgV). In South Africa (SA), there has recently been an enormous upsurge of interest in cassava for industrial applications such as the manufacture of starch, animal feeds, and in its potential as a food security crop for marginalised farmers. However, due to serious losses in cassava yields by begomoviruses, such as SACMV, there is an urgent need for the development of appropriate systems that allows for transformation and regeneration of virus-resistant transgenic cassava cultivars suitable for diverse needs and growth requirements in different geographical areas in southern Africa. The potential application of cassava tuber disks as an alternative system to leaf tissue for transformation and regeneration was investigated. Furthermore, the antibiotic, carbenicillin, was tested as a possible shoot inducing factor. Disks from freshly-harvested cassava tubers were cultured on 25 different sets of MS supplemented with zeatin (0.01-5 mgl-1) and indole-3-acetic acid (0.01-5 mgl-1). Carbenicillin at 500 μgl-1 was included in each treatment as a potential viii organogenesis inducing factor. The results observed after 21 days in culture indicated that non-embryogenic friable callus formed readily on MS medium supplemented with MS vitamins, 30 gl-1 sucrose, 0.01 mgl-1 indole-3-acetic acid (IAA), 0.01 mgl-1 zeatin (ZEA), 500 μgml-1 carbenicillin and 0.8% agar, pH 5.8. Shoots or somatic embryos were never formed and only adventitious roots developed at a frequency of 60% on shoot induction medium supplemented with 2μM copper sulphate (CuSO4), 1 mgl-1 6-benzylaminopurine (BAP) and 0.5 mg-1 indole-3-butyric acid (IBA). The current study also investigated infection of cassava and tobacco by the SA begomovirus species SACMV, dimer A and B using the particle inflow gun. Full-length head-to-tail dimers of DNA-A and DNA-B of SACMV were constructed by digestion with SalI or EcoRI, respectively. The DNA-coated particles were used to shoot 3-week-old cassava plantlets (cv. TMS60444) at a pressure of 1500 psi using the Bio-Rad biolistic device. Thirty-day-old N. benthamiana seedlings were also inoculated in the same manner. In both cases young tender uppermost leaves were targeted (five plants inoculated and another 5 as control). Disease symptoms were recorded daily on the first emerging leaves. Cassava plantlets and tobacco seedlings showed infection by visibility of symptoms. On the other hand, control plantlets that were not inoculated were symptomless. Symptoms appeared 7 dpi in tobacco whereas mosaic symptoms became visible 14 dpi in cassava. The pre-requisite for any cassava transformation program that proposes to develop improved plants is the availability of a reliable regeneration system. Presently many laboratories that prioritize cassava research are able to reliably

M.Tech / Mr. E. van Zyl Prof. C. Rey

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