Browsing by Author "Tushemereirwe, Wilberforce"
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Item Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation(Frontiers in Microbiology, 2017) Onyilo, Francis; Tusiime, Geoffrey; Chen, Li-Hung; Falk, Bryce; Stergiopoulos, Ioannis; Tripathi, Jaindra N.; Tushemereirwe, Wilberforce; Kubiriba, Jerome; Changa, Charles; Tripathi, LeenaBlack Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolutequantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.Item Biofortified East African Highland Bananas to Alleviate Vitamin A Deficiency in Uganda(Gates Open Res, 2019) Buah, Stephen; Tindamanyire, Jimmy M.; Namanya, Priver; Kubiriba, Jerome; Paul, Jean-Yves; Dale, James L.; Tushemereirwe, WilberforceMicronutrient deficiency, also referred to as hidden hunger, is a global public health problem that affects over two billion people (Tulchinsky, 2010). In Uganda, micronutrient deficiency is the third most important public health problem after HIV/AIDS and malaria. Vitamin A deficiency (VAD) and Iron Deficiency Anaemia (IDA) are the major causes of anaemia in Uganda. VAD affects 20-35% of children below five years of age and the same proportion of women of child-bearing age. The aim of the Banana 21 project is to alleviate vitamin A deficiency through biofortification of Uganda’s major staple food, banana.Item Building a bi-directional promoter binary vector from the intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes useful for plant transformation(African Journal of Biotechnology, 2013) Tindamanyire, Jimmy M.; Townsley, Brad; Kiggundu, Andrew; Tushemereirwe, Wilberforce; Sinha, NeelimaThe ability to express genes in a controlled and limited domain is essential to succeed in targeted genetic modification. Having tools by which to rapidly and conveniently generate constructs which can be assayed in a diverse array of plant species expedites research and end-product development. Targeting specifically green plant tissues offers an opportunity to effect changes to diverse processes such as water use efficiency, photosynthesis, predation and nutrition. To facilitate the generation of transgenes to be expressed in this domain, we created a series of plasmids called p2CABA based on the Arabidopsis thaliana chlorophyll a/b gene promoter, a single natural bidirectional promoter that can drive and express two different genes at the same time. Studies we carried out showed reporter gene, GUS expressed in leaves and stems but not in the roots, as expected since this endogenous promoter controls the expression of two photosynthetic genes in A. thaliana. We, therefore, utilized the intergenic region between the A. thaliana cab1 and cab2 divergent genes to design and construct a bidirectional promoter vector containing two multiple cloning sites and a gateway recombination cassette. This in turn will help minimize gene silencing and achieve desirable expression pattern of transgenes, a critical issue in plant genetic engineering and in this report we show their use in Medicago and tomato.Item Corm damage caused by banana weevils Cosmopolites sordidus (Germar) collected from different banana growing regions in Uganda(Agriculture & Food Security, 2018) Twesigye, Charles K.; Ssekatawa, Kenneth; Kiggundu, Andrew; Tushemereirwe, Wilberforce; Matovu, Enock; Karamura, EldadIn this study, both healthy tissue culture plantlets and maiden suckers of the Nakitembe cultivar were used to assess the damage level variation caused by banana weevils collected from different banana growing regions. Seventy-nine (79) tissue culture plantlets and fifty (50) suckers were established in buckets in a randomized complete block design for 5 months. Ten adult weevils (5 females and 5 males) were introduced at the base of each plant, and the buckets were covered with a weevil proof mesh. Weevil damage was estimated as a percentage at 60 days after the weevil introduction by estimating the peripheral damage (PD), total cross section corm damage (XT) and above the collar damage (ACD). Results: The results showed high differences in the PD, XI, XO and XT caused by weevils from the different zones. PD and XT ranged from 4.8–50.4 to 4.2–43.8%, respectively, caused by weevils collected from Kabale and Rakai, Kabale and Wakiso, respectively, while XI and XO varied from 0.0–42.9 to 8.3–40.4%, respectively, caused by banana weevils collected from Kabale and Rakai, Kabale and Rakai, respectively. Banana weevils from Rakai caused the highest ACD of 40.4% and no such damage was caused by banana weevils collected from western Uganda. Average ACD in suckers was 19.6% and significantly higher than that in tissue culture plants (8.5%). Conclusions and recommendations: Corm damage assessment suggests the existence of banana weevil biotypes but it is recommended that follow-up studies be carried out to confirm this phenomenon.Item Golden bananas in the field: elevated fruit pro-vitamin A from the expression of a single banana transgene(Plant Biotechnology Journal, 2017) Jean-Yves, Paul; Harjeet, Khanna; Kleidon, Jennifer; Hoang, Phuong; Geijskes, Jason; Daniells, Jeff; Zaplin, Ella; Rosenberg, Yvonne; James, Anthony; Bulukani, Mlalazi; Pradeep, Deo; Arinaitwe, Geofrey; Namanya, Priver; Becker, Douglas; Tindamanyire, James; Tushemereirwe, Wilberforce; Harding, Robert; Dale, JamesVitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β-carotene equivalent (β-CE) in the fruit. Expression of a Fe'i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.Item Inhibition of cell death as an approach for development of transgenic resistance against Fusarium wilt disease(African Journal of Biotechnology, 2016) Magambo, Betty; Harjeet, Khanna; Arinaitwe, Geofrey; Tendo, Sali; Arinaitwe, Ivan Kabiita; Kubiriba, Jerome; Tushemereirwe, Wilberforce; Dale, JamesFusarium oxysporum f. sp. cubense (Foc) is one of the major threats to dessert banana (Musa spp.) production. In Uganda, ‘Sukali Ndiizi’ is one of the most popular dessert banana cultivars and it is highly susceptible to Fusarium wilt. Development of resistant cultivars through transgenic approaches has shown to offer one of the most effective control options for most diseases. The transgenic approaches for providing plant disease resistance have mainly been through either enzymatic destruction of pathogen structures, neutralization of pathogen and its products or production of metabolites that eventually kill the pathogen. However in recent years, methods that prevent cell death of host plant after infection especially for necrotrophic pathogens like F. oxysporum have registered success in providing resistance in several crops. We investigated whether the transgenic expression of a programmed cell death inhibition gene in Sukali Ndiizi could be used to confer Fusarium resistance to Foc race 1. Embryogenic cell suspensions of cv. ‘Sukali Ndiizi, were stably transformed with a synthetic, plant-codon optimise mCed-9 gene. Twenty-eight independently transformed plant lines were regenerated. The lines were inoculated with Foc race 1 and observed for 13 weeks in small-plant glasshouse. Three transgenic lines showed significantly lower internal and external disease symptoms than the wild-type susceptible ‘Sukali Ndiizi’ banana plants used as controls. This is the first report from Africa on the generation of Fusarium wilt tolerant transgenic ‘Sukali Ndiizi’, a very popular but rapidly diminishing African dessert banana.Item Performance of Narita Banana Hybrids in the Preliminary Yield Trial for Three Cycles in Uganda(NARO, 2015) Tushemereirwe, Wilberforce; Batte, Michael; Nyine, Moses; Tumuhimbise, Robooni; Barekye, Alex; Ssali, Tendo; Talengera, David; Kubiriba, Jerome; Lorenzen, Jim; Swennen, Rony; Brigitte, UwimanaBananas (Musa spp.) are an indispensable part of life in Eastern Africa providing up to one fifth of total calorie consumption per capita. Unlike many staple crops, bananas deliver food throughout the year, making them an ideal crop for household incomes, food and nutrition security. However, banana yields are low due to several factors amongst others pests and diseases: weevils and nematodes, Fusarium wilt, bacterial wilt and black Sigatoka. There are many potential technology-based interventions for increasing banana yields but host plant resistance is the most appropriate and cost effective intervention given the current stage of development of banana systems in the region. Host-plant resistance also offers significant spill over benefits for human health and positive environmental impacts. Therefore, the Ugandan National Agricultural Research Organization (NARO) and the International Institute of Tropical Agriculture (IITA) jointly breed bananas largely for host-plant resistance to improve banana yields. One of the most important current products of their joint banana breeding efforts is secondary triploid hybrids for food and juice herein referred to as NARITA hybrids. This name specifies the contribution of NARO and IITA. An earlier report (NARITA report 1) presented the results of 25 NARITA hybrids for cycles 1 and 2 combined. The current report presents and discusses the results of the same 25 NARITA hybrids (18 for food and seven for juice) evaluated for three crop cycles at Sendusu in central Uganda and analyzed in combined and separate forms. Results of individual NARITA hybrids within cycles showed high degree of variation for the traits assessed, implying a high potential for selection among the NARITA hybrids evaluated. For example, the bunch weight (BWT) of the individual NARITA hybrids ranged from as low as 5 kg for NARITA 19 to as high as 45 kg for NARITA 24 with a mean of 17.8 kg. Averaged across three cycles, BWT ranged from as low as 8.7 kg for NARITA 19 to as a high as 30.4 kg for NARITA 24. Ninety six per cent of the hybrids had a mean BWT greater than the mean of the local check (Mbwazirume) (11.0 kg). Similarly, NARITA hybrids were better than Mbwazirume for most of the other traits assessed. Eighty four per cent of the NARITA hybrids evaluated were better than the best founder parent (NFUUKA) for bunch yield (t ha-1), indicative of the significant breeding progress made by NARO and IITA in this breeding program. This could be confirmed by the positive better founder parent heterosis for BWT recorded by all NARITA hybrids, with NARITA 17, NARITA 18, NARITA 7 (M9), NARITA 21 and NARITA 14 (all food type) exhibiting highest heterosis. Results of combined analysis of variance (ANOVA) showed significant differences among the NARITA hybrids for all the 14 traits assessed including BWT. This indicated the potential for further selection and improvement of the NARITA hybrids for all the 14 traits. Additionally, results of combined ANOVA showed significant differences among three crop cycles for all the traits assessed except days to bunch maturity (DTM) and number of functional leaves at flowering (NFLF), indicating that the selection of banana hybrids could best be done at certain cycle numbers. The performance of NARITA hybrids for most traits was much higher at cycles 2 and 3 than at cycle 1 with the highest performance observed at cycle 3. However, the difference between cycle 2 and cycle 3 was not significantly different for most traits including BWT. The clear implication of this is that selection for banana hybrids should be done at cycle 2 to reduce costs involved in the management of trials since banana trials are always huge considering the size of bananas as well as spacing of 3 x 3 m or 2 x 3 m commonly used. Also, banana performance data analysis should not be based on a combined evaluation of cycle 1 and 2, as was previously done for NARITA report 1, but on an analysis of individual cycles, preferably cycle 2. The limitation of single site and single line plots is acknowledged. Hence, NARITA hybrids will be evaluated in larger and replicated multi-location trials to ascertain their actual performance, adaptability and stability in comparison with the local EAHB cultivars. Nevertheless, based on these preliminary results, potential high yielding banana hybrids combining resistance to black Sigatoka and farmer-preferred quality traits exist within this NARITA population.Item Physical Effectiveness of Soil and Water Conservation Technologies in Drought Prone Areas of Western and Central Uganda(Journal of Agricultural Science and Technology, 2015) Komutunga, Everline; Tushemereirwe, Wilberforce; Kubiriba, Jerome; Namanya, Priver; Oratungye, Kevin John; Akodi, David; Agaba, Choice; Ahumuza, Elizabeth; Kamusingize, DaphineThe effect of soil and water conservation (SWC) practices on controlling surface runoff and soil loss was studied in drought prone banana growing areas of Uganda, during the two major rainy seasons of 2014. The study was conducted at two sites— Ntungamo (Southwest) and Sembabule (Central), with comparable slopes of about 13%-25%. The treatments included mulch, manure, manure + mulch and a control with no conservation. Results indicated that conservation practices of mulch and manure + mulch significantly reduced surface runoff and soil loss by about 72%-85%, when compared to farmers’ up-and-down cultivation practice (control). It was also observed that significantly greater amounts of soil loss occurred from manure and control plots than the ones with mulch. Thus, the combination of manure and mulch is recommended for uptake by crop farmers in the study areas, if they are to overcome drought stress and adapt to changes in climate. More research is needed to quantify nutrient losses resulting from runoff under the different SWC techniques. Modeling such effects is essential in assessing the impacts of SWC practices on soil and crop productivity.Item Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants(Frontiers in Plant Science, 2018) Onyilo, Francis; Tusiime, Geoffrey; Tripathi, Jaindra N.; Chen, Li-Hung; Falk, Bryce; Stergiopoulos, Ioannis; Tushemereirwe, Wilberforce; Kubiriba, Jerome; Tripathi, LeenaPseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.Item Strategies for rehabilitation of banana fields infested with Xanthomonas campestris pv. musacrearum(Journal of Crop Protection, 2014) Kubiriba, Jerome; Muthomi, James; Ndungo, Vigheri; Kwach, Johnson; Erima, Rockefeller; Rwomushana, Ivan; Tushemereirwe, Wilberforce; Opio, FinaXanthomonas campestris pv.musacrearum causes Banana wilt disease (BXW disease) which occurs at different epidemic phases in East and Central Africa (ECA). In the endemic areas, there are many banana fields with over 80% BXW disease incidence. This study aimed at rehabilitating banana fields heavily infected with BXW disease in Uganda, Kenya and DR. Congo. Farmer managed trials were established in BXW disease hotspots in western Kenya and DR. Congo, while in Uganda, similar trials were established at community level i.e. clusters of at least 200 heavily infected banana fields. The control options evaluated included single stem removal, suspension of pruning in affected fields, male bud removal and disinfection of tools with fire or Sodium hypochlorite. Data was collected on the proportion of affected fields (BXW disease prevalence), BXW disease incidence and the number of banana bunches sold at 3-month intervals. BXW disease incidence was reduced by over 80% in 11 months in Kenya and DR. Congo, resulting in yield recovery by up to 70% within one year. In Uganda, the proportion of farmers that effectively controlled BXW disease increased 5% to 60% within a year in some hotspots. Consequently banana sales recovered up to 30% in some hotspots. This study demonstrates that it is possible to effectively control BXW disease within 12 months in previously severely infected fields in various areas of ECA.Item Variation among banana weevil Cosmopolites sordidus (Germar) populations in Uganda as revealed by AFLP markers and corm damage differences(Agriculture & Food Security, 2018) Twesigye, Charles K.; Ssekatawa, Kenneth; Kiggundu, Andrew; Tushemereirwe, Wilberforce; Matovu, EnockThe banana weevil Cosmopolites sordidus (Germar) is a major production constraint of bananas and plantains (Musa spp.) in the world. Differences in damage levels and pesticide response across regions led to the postulation that there might be considerable variation between banana weevil populations (biotypes) with varying levels of virulence. One of the most sustainable options for banana weevil control is the use of host plant resistance. While new resistant varieties are being developed through both conventional crossbreeding and biotechnology, there is a need to assess the genetic variation of banana weevil populations from eastern, central, southern, southwestern and midwest regions of Uganda to determine whether there are biotypes with different virulence levels. This would help guide new control strategies to target all the possible biotypes. The amplified fragment length polymorphism (AFLP) technique was used to analyze population genetic diversity using four primer combinations (EcoRI/MSeI). Results: Analysis of molecular variance results presented no evidence to support significant genetic variability among the banana weevil populations from eastern, central, southern, southwestern and midwest regions. Practically, all the genetic variation was found to reside within populations (97% for sites and 98% for regions), with only approximately 3% and 2% residing among populations of sites and regions, respectively. Conclusions and recommendations: AFLP markers clustered the banana weevils into two distinct populations consequently supporting the hypothesis of possible presence of banana weevil biotypes in Uganda. However, attempts should be made to make follow-up studies on the seemingly unique population of eastern Uganda using more robust molecular techniques to establish whether the eastern Uganda population constitutes a different biotype.