TRANSIENT STUDIES ON RNA INTERFERENCE AND COAT PROTEIN-MEDIATED RESISTANCE TO CASSAVA BROWN STREAK DISEASE

Cassava (Manihot esculenta Crantz) is an important staple and cash crop in Africa, Latin America and Asia. In east and southern Africa, cassava brown streak disease (CBSD) caused by cassava brown streak virus (CBSV) is associated with significant losses in cassava production. Previously, the disease was prevalent only along coastal eastern and southern Africa, but it recently emerged in Uganda and is spreading rapidly in the country as well as in neighboring countries. Apart from a few cultivars that have shown tolerance to CBSV in Tanzania, no effective resistance to CBSV has been developed and deployed to date. The full genome sequence of CBSV is not yet known, but it is thought to be monopartite, linear, positive sense ssRNA, translated into a polyprotein that is further auto-cleaved into functional proteins with the capsid protein (CP) at the C-terminus. The present study aims to develop transient resistance to CBSV through CP-mediated protection and RNA interference (RNAi) strategies. The entire CBSV CP gene was used to express the CP and thereby trigger CP-mediated protection against CBSV. In addition, the full-length CP gene and its N- and C-terminal regions were used to generate three RNAi constructs, with RNAi-GFP as an internal control for transient studies in sap-inoculated GFP transgenic Nicotiana benthamiana. An efficient protocol for sap transmission of CBSV to N. benthamiana was also developed and used in transient protection studies of the constructs as proof-of-concept for control of CBSV using virus-derived resistance strategies in cassava. The constructs offered high levels of protection against CBSV and are highly recommended for use to transform cassava to generate CBSV resistant cassava plants for the farmers

Cassava brown streak viruses: interactions in cassava and transgenic control

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 2015Cassava 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

A Cross-sectional study to assess the Level of knowledge of Youths aged 18-35 regarding negative Consequences of Cigarette smoking at Napier market, Jinja City.

Background: Cigarette smoking is on the increase among youths, especially in developing countries, and is a leading cause of premature morbidity and mortality worldwide. Youths incorrectly perceive that cigarette smoking is less risky than other behaviors such as alcohol consumption and drug use. The purpose of this study is to assess the determinants of cigarette smoking among youths in Napier market, Jinja city Methodology: A cross-sectional descriptive study design employing both quantitative and qualitative methods of data collection was employed. 30 respondents were selected using a convenient non-probability sampling method and data was collected using semi-structured questionnaires. Results: Regarding knowledge of the dangers of cigarette smoking Majority of the respondents had heard about the dangers of cigarette smoking (80%). Commonly mentioned dangers of cigarette smoking included lung cancer (70%), mouth cancer (36%), heart diseases (30%), hypertension (32%), and stroke (22%). Sources of information were radios (64%) and peers (17%). Of the 57% of the respondents that were willing to quit, 74% did not know the exact time at which they hoped to quit cigarette smoking Conclusion: Respondents’ related factors associated with smoking were lack of employment (66%) non involvement in religious activities (66%) negative life experiences (63%), having a smoking friend (43%), and having smoking parents (36%) Recommendation: Although the majority of respondents had heard about the dangers of cigarette smoking, the majority scored below average on common dangers of cigarette smoking and the majority were not sure of when to quit the smoking habit, therefore there is an urgent need to create awareness about the specific dangers associated with cigarette smoking, the transient nature of its perceived benefits and the fact that the risks associated with smoking are severe

Host and virus effects on reversion in cassava affected by cassava brown streak disease

The phenomenon of virus-infected plants naturally recovering health is known as ‘reversion’, and is a type of resistance mechanism exploited in some crop plants for disease control. Various parameters were investigated that affect reversion from cassava brown streak disease (CBSD) in three cassava varieties (Albert, Kaleso and Kiroba) that differ in levels of resistance to the disease. Cassava plants were inoculated by grafting with two virus species (Ugandan cassava brown streak virus, UCBSV and Cassava brown streak virus, CBSV) that cause CBSD, and the plants grown from them were subsequently assessed for reversion. The rate of reversion depended on the cassava variety, virus species, and the length and position of the stem cuttings used. A significantly high proportion of progenies were virus-free (reverted) for the resistant variety Kaleso (64·1% for UCBSV and 54·9% of CBSV), compared to the tolerant variety Kiroba (56·7 and 45·5%) and the susceptible control Albert (38·9 and 35·1%). The highest number of virus-free plants was generated from short 10 cm long cuttings (e.g. 60·1% for Kaleso for CBSV) compared to 20 cm long stem cuttings (e.g. 21·4% for Albert). Cuttings taken from upper stems of diseased plants produced most virus-free progenies compared to middle and lower parts. More than 50% virus-free plants were obtained in the resistant and tolerant varieties. This is a highly valuable finding and could be exploited for developing strategies to control the current CBSD epidemic in eastern and central Africa

Cassava brown streak disease: historical timeline, current knowledge and future prospects

Cassava is the second most important staple food crop in terms of per capita calories consumed in Africa and holds potential for climate change adaptation. Unfortunately, productivity in East and Central Africa is severely constrained by two viral diseases: cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). CBSD was first reported in 1936 from northeast Tanzania. For approximately seventy years CBSD was restricted to coastal East Africa and so had a relatively low impact on food security compared to CMD. However, at the turn of the 21st century CBSD re-emerged further inland, in areas around Lake Victoria and it has since spread through many East and Central African countries, causing high yield losses and jeopardising the food security of subsistence farmers. This recent re-emergence has attracted intense scientific interest, with studies shedding light on CBSD viral epidemiology, sequence diversity, host interactions and potential sources of resistance within the cassava genome. This review reflects on 80 years of CBSD research history (1936 – 2016) with a timeline of key events. We provide insights into current CBSD knowledge, management efforts and future prospects for improved understanding needed to underpin effective control and mitigation of impacts on food security

Expansion of the cassava brown streak pandemic in Uganda revealed by annual field survey data for 2004 to 2017

Funder: Uganda Government Association for Strengthening Agricultural Research in Eastern and Central AfricaFunder: Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)Abstract: Cassava brown streak disease (CBSD) is currently the most devastating cassava disease in eastern, central and southern Africa affecting a staple crop for over 700 million people on the continent. A major outbreak of CBSD in 2004 near Kampala rapidly spread across Uganda. In the following years, similar CBSD outbreaks were noted in countries across eastern and central Africa, and now the disease poses a threat to West Africa including Nigeria - the biggest cassava producer in the world. A comprehensive dataset with 7,627 locations, annually and consistently sampled between 2004 and 2017 was collated from historic paper and electronic records stored in Uganda. The survey comprises multiple variables including data for incidence and symptom severity of CBSD and abundance of the whitefly vector (Bemisia tabaci). This dataset provides a unique basis to characterize the epidemiology and dynamics of CBSD spread in order to inform disease surveillance and management. We also describe methods used to integrate and verify extensive field records for surveys typical of emerging epidemics in subsistence crops

The process and lessons of exchanging and managing in-vitro elite germplasm to combat CBSD and CMD in Eastern and Southern Africa

Varieties with resistance to both cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) can reverse food and income security threats affecting the rural poor in Eastern and Southern Africa. The International Institute of Tropical Agriculture is leading a partnership of five national (Malawi, Mozambique, Kenya, Tanzania and Uganda) cassava breeding programs to exchange the most elite germplasm resistant to both CMD and CBSD. This poster documents the process and the key learning lessons. Twenty to 25 stem cuttings of 31 clones comprising of 25 elite clones (5 per country), two standard checks (Kibandameno from Kenya and Albert from Tanzania), and four national checks (Kiroba and Mkombozi from Tanzania, Mbundumali from Malawi, and Tomo from Mozambique) were cleaned and indexed for cassava viruses at both the Natural Resources Institute in the United Kingdom and Kenya Plant Health Inspectorate Services, in Kenya. About 75 in-vitro plantlets per clone were sent to Genetic Technologies International Limited, a private tissue culture lab in Kenya, and micro-propagated to ≥1500 plantlets. Formal procedures of material transfer between countries including agreements, import permission and phytosanitary certification were all ensured for germplasm exchange. At least 300 plantlets of each elite and standard check clones were sent to all partner countries, while the national checks were only sent to their respective countries of origin. In each country, the in-vitro plantlets were acclimatized under screen house conditions and transplanted for field multiplication as a basis for multi-site testing. Except for Tomo, a susceptible clone, all the clones were cleaned of the viruses. However, there was varied response to the cleaning process between clones, e.g. FN-19NL, NASE1 and Kibandameno responded slowly. Also, clones responded differently to micro-propagation protocols at GTIL, e.g. Pwani, Tajirika, NASE1, TME204 and Okhumelela responded slowly. Materials are currently being bulked at low disease pressure field sites in preparation for planting at 5-8 evaluation sites per country. The process of cleaning, tissue culture mass propagation, exchange and local hardening off/bulking has been successful for the majority of target varieties. Two key lessons derived from the process are that adequate preparations of infrastructure and trained personnel are required to manage the task, and that a small proportion of varieties are recalcitrant to tissue culture propagation

A CASE STUDY SURVEY ABOUT THE RELATIONSHIP BETWEEN COURSE CONTENT COVERAGE AND ACADEMIC PERFORMANCE OF PUPILS IN PLE IN LIRA CITY.

Background. This study aims to determine the relationship between course content coverage and the academic performance of pupils in PLE in Lira City. Methodology. A case study survey design characterized by both qualitative and quantitative methods was used. The target population of the study comprised 63 participants of which 54 respondents were selected as respondents. Two sampling techniques were employed to select the respondents of the study and these were purposive sampling and simple random sampling. Data was collected from the selected respondents using a structured questionnaire and interview guides.  Results. Teachers provided course outlines to pupils at the start of the course: The mean response for this statement was 2.2, indicating that the majority of pupils disagreed or were not sure about the provision of course outlines. This suggested that there may be a lack of clear direction and guidance for the students at the start of the course.  Findings showed variations in the respondents' perceptions regarding content coverage and academic performance. While some areas, such as the teaching of basic concepts in Mathematics and provision of key concepts received higher agreement scores, areas such as English language instructions and provision of course outlines received lower agreement scores. These findings suggest areas of improvement in terms of providing comprehensive content coverage to enhance academic performance. Conclusion. There was a strong positive correlation between course content and academic performance in PLE, with a Pearson correlation coefficient of 0.871. This suggested that students who had a good understanding of the course content performed better in the PLE. Recommendation. Schools and educators should focus on providing comprehensive course content to enhance academic performance and should prioritize providing clear and detailed course outlines to help students understand the content and structure of their courses, which can improve their academic performance

Risk assessment for the occurrence of Escherichia coli 0157:H7 in indigenous fermented milk (Lee Naga a Agbora) produced in Uganda

Different types of indigenous fermented milks are widely consumed in pastoralist communities of Africa, including Uganda. Local processing of milk generally takes place under uncontrolled hygienic conditions. Lee naga a agbora, a locally fermented milk product in northern Uganda, was evaluated for microbial contamination. The changes in pH, Total Plate Counts (TPC), Total Coliforms (TC), yeasts and moulds, and Escherichia coli 0157:H7, particularly its survival during different stages of  fermentation were studied. The pH was found to decrease with fermentation time (6.54 to 4.54). E. coli was detected in most samples of Lee naga a agbora and its occurrence was observed throughout the fermentation period. The mean value of TPC (6.60± 0.10 log cfu ml-1) at the start of fermentation was significantly different (p0.05) between 48 and 72 hours. The rate of decrease in TC counts in the initial fermentation period (0 to 48 hours) was lower than the later stages of fermentation (48 to 144 hours). Yeast and mould counts increased with fermentation time (6.20 to 7.42 log cfu ml-1). On the whole, there is risk associated with consumption of Lee naga a agbora given the occurrence of E. Coli

Effects of banana peel-ash-extract on cooking time and acceptability of hard-to-cook beans (Phaseolus vulgaris l.)

Beans are among the most important crops that contribute substantially to the agricultural sector and household food security in Uganda. Kawanda (K) 132 (Nambale) is presently a major bean (Phaseolus vulgaris L.) variety of high nutritional value. However, the hard-to-cook (HTC) defect limits its utilization, requiring appropriate processing to preserve the nutrients. Banana peel ash-extract, widely used in indigenous processing of dry beans, was evaluated for chemical composition and potential in reducing cooking time of beans with HTC defect. The peel ash-extract was found to contain substantial amounts of sodium (0.36 g/ml), chloride (0.07 g/ml) and magnesium (0.04g/ml) ions. Calcium, iron, copper, zinc, sodium and phosphorus were also found in detectable amounts. Generally, addition of the peel-ash-extract was found to reduce the cooking time of K132 beans. The cooking time of K132 beans, processed in ash-extract (0.31 g/ml), reduced by 52.3%. Reduction in cooking time was also found to correlate strongly (R2=0.89) with the pH of the ash-solution. Processing of K132 beans in ash-solution of pH G 11 reduced the cooking time by nearly 50%. Sensory attributes of dry beans cooked in banana peel ash-extract were preferred to that cooked in ordinary water. Beans cooked in peel ash-extract at a concentration of 0.23 g/ml were the most preferred (5.81±0.59) to that boiled in ordinary water (4.71±0.26). The taste, flavor and acceptance scores were, in general, higher in beans processed in ash-extract than that of ordinary water. The appearance, consistency and texture of cooked beans were not significantly different (