http://localhost/xmlui/handle/123456789/1280 2026-05-21T13:40:40Z http://localhost/xmlui/handle/123456789/6998 Use of Endophytic Microorganisms in Crop Adaptations to Increased Salinity and Resistance to Pests and Diseases Mutungi, Priscillar Mumo Understanding endophytic microorganisms associated with soda lakes shrubs and building a holistic Kenyan soda lake living library of microorganisms while exploring their potential applications in various industries is crucial in the current climate crisis. Lake Magadi and Lake Bogoria are saline alkaline endorheic lakes, in semi-arid regions with erratic rainfall. Vascular shrubs growing along the shores of these lakes must develop adaptive mechanisms to cope with high alkalinity, salinity and erratic environmental conditions. Previous studies on soda lake microorganisms have focused on isolations from water, soil and sediments, leaving behind the plant associated niche. Plant associated microorganisms from harsh environments have previously been shown to harbor diverse metabolic and genetic profiles that play an important role in plant growth, health and survival under stressful conditions. The aim of this study was to collect shrubs growing along the shores of Lake Bogoria and Lake Magadi, and isolate associated bacteria and fungi. The resultant isolates were identified using molecular techniques and consensus sequences aligned with highly homologous sequences in NCBI database. The isolates were screened for production of exo-enzymes, salinity tolerance and inhibition of bean root rot pathogen, Fusarium solani. The ability of the isolates to endophytically colonize bean and tomato seedlings was also evaluated. The effects of endophytic colonization on salinity stress, Fusarium solani and Tetranycus urticae were assessed in green house experiments. Endophytic bacteria were affiliated to three different phyla, Firmicutes, Proteobacteria and Actinobacteria while fungal endophytes were affiliated to two phyla, Basidiomycetes and Ascomycetes. A larger number of bacterial isolates were more salt tolerant while only few fungal isolates indicated growth at 1M NaCl concentration. All tested fungal and bacterial isolates irrespective of the concentration or the method used, endophytically colonized tomato and bean seedlings respectively.. Two bacterial isolates; Bacillus megaterium and Enterobacter hormaechei subsp. Xiangfangensis showed more than 50% bean root rot pathogen, Fusarium solani mycelial inhibition in dual culture assays, completely inhibited germination of the spores in co-culture assays and resulted in its biocontrol in planta. All the four select fungal endophytes significantly enhanced the germination of tomato seeds by 23% under salinity stress. Endophytic colonization of tomato seedlings significantly decreased the quantity of hydrogen peroxide and enhanced total chlorophyll produced under salinity stress compared to non-inoculated seedlings. Exposure of tomato seedlings colonized with fungal endophytesdid not affect the number of eggs laid or the mortality of spider mites. The study gives a first insight into the bacterial and fungal endophytes associated with saline alkaline adapted shrubs. The isolates also provide the potential use of soda lakes derived microorganisms in the agricultural production, an application that has not been widely explored in Kenya. Doctor of Philosophy in Biotechnology 2026-05-21T00:00:00Z http://localhost/xmlui/handle/123456789/6896 Assessment of Bacterial Diversity and Antibiotic Resistance Genes in Water Used by Eateries and Open Markets in Juja Omune, Alfrick Makori Water is an important bacterial habitat and a major avenue of dissemination of antibiotic resistant bacteria between and among different environments. Contamination of drinking water used in open market eateries with antibiotic resistant bacteria pose a direct threat to human health. This is because antimicrobial resistome monitoring in drinking water is not currently a routine standard inspection protocol of drinking water. This study aimed to investigate the occurrence of antibiotic resistance genes (ARGs) and variation in the composition of bacterial communities in drinking water collected from eighty-two sampling points in Juja. Water samples were collected using purposive sampling. Special media of MacConkey, Thioglycollate and Lauryl Tryptose Broth media were used for isolation of bacteria from the collected water samples. Culture and molecular techniques were used to profile the bacteria and detect antibiotic resistant bacteria (ARBs) and antibiotic resistant genes (ARGs) present in samples. Metagenomics sequencing and analysis was applied to investigate the ARB genetic profiles. Morphological, biochemical characterization, antimicrobial susceptibility and molecular detection of ARGs data (qualitative & quantitative) were analyzed using R software to generate heat maps (Manhattan metric) showing characteristic relationships and antibiotic susceptibility or resistance among bacterial isolates. QIIME (version 2021.4) software and DADA2 were used to analyze microbial profiles in collected water samples. Detection of antibiotic resistance genes data were analyzed using R software to generate heat maps highlighting their presence in water samples. Results showed abundance of bacterial phylum Proteobacteria (above 86%) across all the collected water samples from the open markets and eateries. The dominant phylum documented by this study belonged to Proteobacteria and comprised of; Wakulima Eateries (WE) (Acinetobacter 44.180%), Wakulima Open Markets (WOM) (Duganella 28.201%), Gachororo Eateries (GE) (Acinetobacter 15.189%), Gachororo Open Markets (GOM) (Acinetobacter 30.675%) Mwerevu Eateries (ME) (Acinetobacter 40.823%) and Mwerevu Open Markets (MOM) (Curvibacter 48.785%). This study reported the presence of antibiotic resistant priority pathogens from the WHO list; Priority 1 (Critical) where Pseudomonas spp detected in samples ME (12.163%) and WOM (0.323%), Acinetobacter baumannii detected in samples GE (2.975%) and WE (5.495%), Enterobacteriaceae detected in samples WE (0.125%), MOM (0.326%), WOM (0.875%), GE (0.452%) and ME (0.206%). The genus of Escherichia, Klebsiella, Raoultella and Enterobacter dominated in Enterobacteriaceae. Priority 2 (High) pathogen detected was the genus Staphylococcus in samples MOM (1.826%) and GE (0.962%). The ARGs of qnrD and sul2 were detected in all six samples (WOM, WE, GOM, GE, MOM and ME), int1 and FloR genes were present in five samples except in MOM and GOM samples respectively while those of strB (MOM), catA (MOM) and blaTEM (GOM) were found in one sample each using qualitative PCR assays. Findings of this study form a critical reference point for policy development and rapid mitigation strategies to prevent a possible outbreak of water-borne diseases in the area. Master of Science in Biotechnology 2026-02-16T00:00:00Z http://localhost/xmlui/handle/123456789/6766 Ecological Niche Modeling, Yield Impact Assessment and Improved Detection of SPFMV, SPCSV, and SPLCV in Sweetpotato in Kenya Wanjala, Bramwel Waswa Viral diseases in Kenya and sub-Saharan Africa cause significant crop losses. Over 30 viruses have been reported to infect sweetpotato. Sweet potato cholorotic stunt virus and Sweet potato feathery mottle virus are considered the most wide-spread and devastating. Sweetpotato begomoviruses (sweepoviruses) have increasingly been recognized as common in recent years. They cause no or show mild symptoms but result in 40 to 80% yield losses. This study aimed: i) To develop an ecological niche model for predicting the potential geographic distribution and risk areas of sweepoviruses in Kenya; ii) To assess the yield impact of begomoviruses and their interaction with sweetpotato feathery mottle virus and sweetpotato chlorotic stunt virus; iii) To validate a loop-mediated isothermal amplification assays for on-site detection of the main sweetpotato infecting viruses and iv) To evaluate tube array for the multiplex detection of sweetpotato viruses. The study developed an ecological niche model to predict the geographic distribution and risk areas of sweepoviruses in Kenya. Field surveys were conducted during 2017 and 2018 cropping seasons, targeting the main sweetpotato growing regions. Ecological niche models were constructed using MaxEnt software, incorporating bioclimatic variables and International Soil Reference and Information Centre (ISRIC) factors. MaxEnt consistently surpasses other methods in terms of predicting accuracy, and is highly user-friendly. Results showed high, medium, and low probability for sweepovirus persistence and spatial distribution in regions like Western, Nyanza, and South Rift. Central and Eastern regions showed medium suitability, while the coastal region was predicted to be medium to high risk. The model provided valuable insights for developing effective disease risk management strategies. The impact of Sweet potato leaf curl virus (SPLCV) infection on root yield in Kenyan sweetpotato varieties 'Kakamega' and 'Ejumula' was investigated. Results showed significant differences in yield loss from SPLCV infection, with 'Ejumula' suffering no significant loss, while 'Kakamega', more resistant to Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), suffered an average of 47% loss. The results highlight the variability in sensitivity to SPLCV between sweetpotato cultivars and the lack of correlation between SPLCV-related symptoms and susceptibility to the virus. The study also highlighted the lack of correlation between resistance to RNA viruses SPCSV and SPFMV and DNA virus SPLCV. A simple and rapid loop‐mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to SPLCV (sweepoviruses) was developed. Laboratory validation recorded 100% diagnostic sensitivity for all three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5-30 mins, SPCSV 15-43 mins and begomoviruses 28-45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials. The operational performance of STAR (Sweet potato virus Tube-Array) for simultaneous detection of sweetpotato viruses was evaluated. STAR assay was compared with the conventional method Nitrocellulose Membrane Enzyme-Linked Immunosorbent Assay (NCM-ELISA), with 25 samples tested in parallel in Lima and Nairobi. Cohen's kappa index revealed a disagreement between the two assays, with STAR being more sensitive and detecting more viruses not detected by NCM-ELISA. Concordance analysis accessed by Bland-Altman plot and regression analysis results from the two labs agreed closely. STAR was found to be more sensitive and detected more viruses than NCM-ELISA. STAR's sensitivity, specificity, accuracy, and short turnaround time make it suitable for routine diagnosis, epidemiological studies, quarantine, and certification of the sweetpotato seed system. PhD in Biotechnology 2025-07-16T00:00:00Z http://localhost/xmlui/handle/123456789/6765 Development and comparison of a loop mediated isothermal amplification assay for the rapid diagnosis of lumpy skin disease Macharia, Ednah Serah Wanjiru Lumpy skin disease virus is a poxvirus in the genus Capripoxvirus and is closely related to sheeppox virus and goatpox virus. It’s economically important in cattle and a notifiable disease by World Organization for Animal Health. Lumpy skin disease (LSD) is endemic in most parts of Africa with small-scale farmers experiencing the highest loss during outbreaks due to restricted animal trade and costly control and eradication measures. Serological methods of LSD detection are sensitive, inexpensive but can be laborious and time-consuming while, molecular methods such as Polymerase chain reaction (PCR), and real-time PCR/quantitative PCR (qPCR) are sensitive but require expertise and sophisticated laboratories. Loop-mediated isothermal amplification (LAMP) molecular method is advantageous, as it does not require expertise or sophisticated equipment. Thisstudy aimed to develop a rapid, simple, specific, and sensitive detection method for LSD. Sixtytwo samples that included skin biopsies, whole blood, serum, and cell cultures were used. New LAMP primer (10_LSD) that could detect lumpy skin disease virus, was designed using Genome based LAMP primer designer (GLAPD) software. Samples were analyzed by LAMP assay and a gold standard (real-time PCR). A LAMP field-based extraction method using polyethylene glycol (PEG) was developed and used for the detection of lumpy skin disease virus. The 10_LSD had a kappa value of 0.32 against the qPCR gold standard. In terms of limit of detection, qPCR had a detection limit of 10-3 ng/µl while 10_LSD had a limit of detection of 1 ng/µl and. The 10_LSD assay showed sensitivity of 60% and a specificity of 86 %. The LAMP assay did not cross-react with closely related viruses like camelpox, Orf virus, and Pestes des Petit Ruminants but could amplify sheeppox virus and goatpox virus. The average time to positivity was 14-28 minutes. The study supports the adoption of the LAMP assay for rapid Capripoxvirus diagnosis as a simpler, effective, and rapid method of detection, monitoring, and controlling outbreaks and the spread of disease in a field set up. MSc Research Publication 2025-07-15T00:00:00Z