Browsing by Author "Adriko, John"

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    Establishment of a transformation protocol for Uganda’s yellow passion fruit using the GUS gene
    (African Journal of Biotechnology, 2019) Tuhaise, Samuel; Nakavuma, Jesca L.; Adriko, John; Ssekatawa, Kenneth; Kiggundu, Andrew
    Horticulture is one of the fastest growing sectors in Uganda, exporting products worth US$100 million annually. Passion fruit (Passiflora edulis) growing and export is one of the critical contributors to this sector employing over a million farmers. However, a number of biotic and abiotic constraints have initiated widespread enterprise abandonment by farmers. Passiflora improvement efforts by conventional breeding has had limited success calling for research into alternative approaches such as genetic engineering. The study aimed at optimizing existing protocols to develop an efficient and reproducible Agrobacterium mediated transformation system to suit Uganda’s Passiflora cultivars. Agrobacterium tumefaciens strain AGL1 (OD600 of 0.5) harbouring pCAMBIA2301 containing the GUS (uidA) reporter gene was used to infect pre-cultured leaf discs. Leaf discs were then vacuum infiltrated for 1.5 min at 750 mmHg followed by a three day co-cultivation period on MS + acetosyringone (100 μml-1). Putatively transgenic yellow passion fruit shoots were induced on Murashige and Skoog (MS) selection media supplemented with benzylaminopurine (BAP) 8.9 μM, kanamycin (100 mgL-1mgl) and cefotaxime (500 mgL-1). Developed shoots were then transferred to elongation media (MS + 0.44 μM BAP) and later rooted on 5.37 μM naphthaleneacetic acid (NAA). Genetic transformation was monitored using GUS staining. A single independently transformed plant was confirmed by polymerase chain reaction (PCR), translating in a transformation efficiency of 0.456%. A viable in vitro transformation protocol for Uganda’s yellow passion fruit directly from leaf discs was developed using GUS reporter gene. Further investigations are required to improve the reported protocols transformation efficiency.
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    Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from groundnuts in selected agroecological zones of Uganda
    (BMC microbiology, 2020) Acur, Amos; Arias, Renée S.; Odongo, Steven; Tuhaise, Samuel; Ssekandi, Joseph; Muhanguzi, Dennis; Adriko, John; Buah, Stephen; Kiggundu, Andrew
    Background Aspergillus is the main fungal genus causing pre- and post-harvest contamination of groundnuts. Aspergillus flavus belongs to section Flavi, a group consisting of both the aflatoxigenic species (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic species (A. oryzae, A. sojae and A. tamarii). Aflatoxins are food-borne toxic secondary metabolites produced by Aspergillus species, causing hepatic carcinoma and stunting in children and are the most toxic carcinogenic mycotoxins ever identified. Despite the well-known public health problems associated with aflatoxicosis in Uganda, information about the genetic diversity of the main aflatoxin causing fungus, Aspergillus flavus in this country is still limited. Results A cross-sectional survey was therefore carried out in three main groundnut-growing agro-ecological zones (AEZs) of Uganda; West Nile farming system, Lake Kyoga basin mixed farming system and Lake Victoria basin farming system. This was to assess the genetic diversity of A. flavus and to establish the contamination rates of groundnuts with Aspergillus species at pre- and post-harvest stages. Out of the 213 A. flavus isolates identified in this study, 96 representative isolates were fingerprinted using 16 insertion/deletion microsatellite markers. Data from fingerprinting were analyzed through Neighbor Joining while polymorphism was determined using Arlequin v 3.5. The pre- and post-harvest contamination rates were; 2.5% and 50.0% (West Nile farming system), 55.0% and 35.0% (Lake Kyoga basin mixed farming system) and 32.5% and 32.5% (Lake Victoria basin farming system) respectively. The Chi-square test showed no significant differences between pre- and post-harvest contamination rates among AEZs (p = 0.199). Only 67 out of 96 isolates produced suitable allele scores for genotypic analysis. Analysis of genetic diversity showed higher variation within populations than among populations. Two major clusters (aflatoxigenic and non-aflatoxigenic isolates) were identified as colonizing groundnuts at pre- and postharvest stages. Conclusions These findings provide a first insight on the existence of non-aflatoxigenic strains of A. flavus in Uganda. These strains are potential candidates for developing local Aspergillus biocontrol agent.
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    Improved PCR for identification of members of the genus Xanthomonas
    (European Journal of Plant Pathology, 2014) Adriko, John; Mbega, Ernest Rashid; Mortensen, Carmen Nieves; Gadelha Wulff, Ednar; Kateera Tushemereirwe, Wilberforce; Kubiriba, Jerome; Lund, Ole Søgaard
    A PCR-based system was developed to reliably and robustly identify group I and II members of the genus Xanthomonas. Primer sets developed from three gene targets namely fyuA, ITS and gumD were evaluated in the study. Primer sets were evaluated using DNA extracted from 45 Xanthomonas strains representing 25 species broadly covering the genus. Fifteen non- Xanthomonas strains of plant-associated bacteria including phylogenetically closely related species Stenotrophomonas maltophilia and Xylella fastidiosa were also tested. The primers targeting fyuA amplified DNA from all xanthomonads except X. theicola, while the ITS primers amplified a DNA fragment of 254 bp in all 45 Xanthomonas strains; whereas no amplification was observed for non-xanthomonads. The gumD primers allowed efficient amplification of DNA in 38 out of 39 isolates from Group II, whereas no or very weak amplification occurred with DNA from Group I members. Internal controls of primers targeting bacterial 16S rDNA or plant 26S mitochondrial rDNA were successfully applied in multiplex PCRs for testing bacterial cultures or plant tissue, respectively. The findings give us a PCR based approach that can reliably and effectively differentiate xanthomonads from nonxanthomonads as well as separating the strains belonging to the two described groups of the genus Xanthomonas. The study thus offers valuable tools for disease surveillance and management. It can effectively be applied in rapid assessment of new disease occurrences, for which no specific detection tools could be in place.
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    Molecular diagnostics of groundnut rosette disease agents in Uganda: Implications on epidemiology and management of groundnut rosette disease
    (Journal of plant breeding and crop science, 2017) Kalule Okello, David; Adrogu Ugen, Michael; Tukamuhabwa, Phinehas; Ochwo- Ssemakula, Mildred; Lapaka Odong, Thomas; Adriko, John; Kiconco, Faith; Male, Allan; Deom, Carl Michael
    The objective of this study was to use molecular diagnostic tools to detect the agents of groundnut rosette disease (GRD) to guide in varietal development and disease management. Samples were collected from both GRD infected and healthy plants and sites geo-referenced. RNA extraction, cDNA synthesis, polymerase chain reaction (PCR) amplification, electrophoresis, staining and visualization were performed according to standard procedures. Molecular diagnosis of the samples showed various combinations of the GRD agents, some in isolation and others a combination of two or three agents. This distribution is attributed to dependence on the aphid feeding behaviour and pathogenicity of GRD agents. Chlorotic and green rosette symptoms were observed throughout the sampling sites signifying the presence of satellite RNA (sat-RNA) variants. Some plants showing GRD symptoms tested negative for GRD, whereas some healthy-looking plants tested positive for the GRD complexes pointing to the ineffectiveness of phenotypic screening and the need for a molecular diagnostic tool that detects all three GRD agents both in absence or presence of disease symptoms. The absence of groundnut rosette assistor virus (GRAV) in some symptomatic samples signifies that they are epidemiologically dead end sources since GRV and sat-RNA must be packaged within the GRAV coat protein to be aphid transmissible. Oyado (Cassia obtusifolia) tested positive for all the GRD agents making it a potential alternative host. There is an urgent need for validation of the phenotypic screening with molecular tools in efficient diagnosis of the multi-pathogenic GRD in guiding both plant breeding and pathology work.