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PhD / In South Africa, tomato (Solanum /ycopersicum) is an important vegetable crop with considerable nutritional and economic value. Over the last decade, begomovirus (family Geminiviridae) infections associated with an upsurge of the whitefly vector, Bemisia tabaci, on tomato crops has become a serious threat to sustainable tomato production in South Africa. Begomovirus disease control in tomato is challenging and requires an integrated "pest" and "vector" management strategy, primarily based on the use of chemical and cultural practices aimed at reducing the virus vector as well as the use of resistant cultivars. Development of effective disease management practices for South Africa therefore requires detailed information on the complex vector-virus-host cropping system interactions. The aim of the study presented in this thesis was to investigate the South African whitefly vector/begomovirus/tomato-host pathosystem, with emphasis on the virus and vector diversity and distribution, and the identification of possible resistance sources. A survey of tomato-infecting begomoviruses was conducted during a six-year period (2006-2011 ). Techniques used to determine begomoviruses diversity included whole genome amplification using PCR, RCA (rolling circle amplification), conventional as well as next generation sequencing and development of a RCA-RFLP (restriction fragment length polymorphism) for rapid assessment of diversity. Sequence comparisons and phylogenetic analyses revealed the presence of three new monopartite begomovirus species, in addition to ToCSV, all of which belong to the African/South West Indian Ocean (SWIO) begomovirus clade. Recombination analysis indicated that all four tomato-infecting begomovirus species appear to be complex recombinants and suggests that they have evolved within the sub-Saharan Africa region, along with other African begomoviruses and that they are most likely indigenous to the region. Several weed species were also confirmed as symptomless begomovirus reservoirs, supporting their role in the emerging begomovirus epidemics in South Africa.

A thesis presented to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology 2015 / Cassava brown streak disease (CBSD) ranks among the top seven biological threats to global food security and is considered to be a major risk to food security in tropical Africa. In Uganda, overall CBSD incidence has increased by c. 20% since 2004, and persistently reduces cassava yields and storage root quality. Presently the disease negatively impacts the livelihoods of over 80% of the farming families who rely on cassava as a staple food and source of income. Two distinct ipomoviruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) cause CBSD. The viruses systemically infect primary host plants and accumulate, and cause severe disease symptoms as the plant matures, reducing yields through the induction of necrotic lesions in the storage roots and suppressing utility of cassava stems for subsequent vegetative propagation. Effective control strategies require screening of available germplasm for sources of natural resistance in combination with improved understanding of host-virus interaction to facilitate targeted breeding. Due to a lack of known sources of resistance to CBSD in the cassava germplasm, incorporating new virus resistance into existing cassava genotypes through transgenic RNA interference (RNAi) approaches offers an additional, relevant avenue to reduce the increasing impact of CBSD. The research presented in this thesis provides insights into the complex mechanisms of virus-host interactions linking genotype to phenotype in CBSV- and UCBSV-cassava pathosystems and provides proof of principle for CBSD control by RNAi-mediated technology. Both are contributions to progress towards potential control of the CBSD epidemic in East Africa. To correlate CBSD symptoms with virus titer, within-host CBSV and UCBSV accumulation was studied in leaf, stem and storage root samples collected from 10 genotypes of field-grown cassava with varied levels of resistance to CBSD. CBSV was found to be present in 100% of CBSD samples collected from symptomatic plants. Presence of both CBSV and UCBSV was seen in 45.3% of the samples. Quantitative PCR (RT-qPCR) analysis showed that tolerant genotypes were infected with CBSV alone and accumulated lower virus titer compared to susceptible genotypes, which were co-infected with CBSV and UCBSV. To further comprehend the molecular interaction between CBSD viruses and cassava, deep sequencing was performed to compare profiles of virus-derived small RNAs (vsRNAs) in CBSV- and UCBSV-infected cassava genotypes of NASE 3 (CBSD tolerant), TME 204 and 60444 (CBSD susceptible). The results showed an abundance of 21-24 nt sized vsRNAs which when mapped were shown to cover the entire CBSV and UCBSV genomes. The 21- and 22-nt sizes were predominant compared to the 23- and 24-nt size classes. CBSV-infected plants accumulated higher populations of vsRNAs across the genotypes compared to UCBSV-infected plants, which accumulated moderate amounts of UCBSV-derived sRNAs in TME 204 and 60444, and insignificant amounts in UCBSV-challenged NASE 3, respectively. Quantitative RT-PCR analysis was performed to determine transcript levels of cassava homologues of Dicer (DCL) proteins, particularly DCL4 and DCL2, which are involved in the biogenesis of 21- and 22-nt small RNAs, and to correlate to the abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected cassava. Similarly, RT-qPCR was performed to determine the expression of Argonaute (AGO) proteins, specifically AGO2 which preferentially sort and bind sRNAs with 5’ adenine (A) or uracil (U) to effector complexes to target mRNAs repression or cleavage, since in this study a major proportion of the vsRNAs were found to have A or U at the first 5’-end. Expression levels of cassava homologues of AGO2, DCL2 and DCL4, which are core components of the gene-silencing pathway, were found to be affected in virus-infected plants across all three genotypes. The levels of viral RNA and vsRNAs correlated with disease phenotype in infected plants. CBSV-infected plants showed more severe CBSD symptoms compared with UCBSV-infected plants of the same genetic background. These results showed that CBSV is more aggressive compared to UCBSV and supports the hypothesis of occurrence of genotype-specific resistance to CBSD viruses. The abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected plants signifies the viruses activated the RNA-silencing mechanism, referred to as transcriptional or post-transcriptional gene silencing (TGS or PTGS). To test efficacy of RNAi-mediated resistance to control CBSD under field conditions, 14 lines of cassava plants transgenically modified to express, as inverted repeats, two RNAi constructs p718 and p719 targeting near full-length (894 bp) and N-terminal (402 bp) portions of UCBSV coat protein sequence were tested under confined field trial conditions at Namulonge, Uganda. Transgenic plants expressing p718 showed a 3-month delay in CBSD symptom development, while 100% of non-transgenic plants (n = 60) developed CBSD shoot symptoms. Over the 11-month trial duration, 98% of clonal replicates within line 718-001 were found to remain free of CBSD symptoms. RT-PCR analysis detected UCBSV within leaves of 57% of non-transgenic plants compared to only 0.5% across the 14 transgenic lines. Presence of the non-homologous CBSV was detected in all transgenic plants that developed CBSD symptoms. However, 93% of plants of line 718-001 were free of CBSV and UCBSV. At harvest, 90% of storage roots of non-transgenic plants showed severe necrosis, whereas plants of lines 718-001 and 718-005 showed significant suppression of CBSD. Line 718-001 had 95% of roots free from necrosis and was RT-PCR negative for presence of both viral pathogens. To determine durability of RNAi-mediated resistance to CBSD, stem cuttings were obtained from mature plants of lines p718-001, p718-002 and p718-005, replanted and monitored for 11 more months. CBSV but not UCBSV was detected in tissues of plants of lines p718-002 and p718-005, whereas all leaves and roots of p718-001 plants were free of CBSV and UCBSV. Thus, RNAi constructs conferred durable CBSD resistance across the vegetative cropping cycle, providing proof of concept for application of RNAi technology to control CBSD in farmers’ fields. The findings presented in this thesis contribute to understanding the complex interconnected mechanisms involved in CBSV- and UCBSV-host interactions and will contribute to the long-term goals of devising new methods of CBSD control.

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A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy / Greening is a severe disease of citrus and is a major cause of crop loss in many parts of Africa and Asia.Numerous attempts have been made by various workers to isolate the aetiological agent of the disease and although reports claiming the successful culture of the organism have appeared, the isolations were never fully confirmed. ( Abbreviation abstract ) / AC2017

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