MST-Plant and Microbial Sciences

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Phytoplankton and Physicochemical Dynamics of Lake Baringo
(Kenyatta University, 1989) Kiplagat, Wilson; Patterson, Graeme; Muthuri, Francis M.
Some aspects of the decomposition process in the mangrove swamp at Gazi Bay, Kenya
(1993) Boga, Hamadi Iddi
Efficacy of seed-dressing and organic amendments against fusarium root-rot of French beans (phaseolus vulgaris l. Cv. Monel.) in Kenya.
(Kenyatta University, 1998) Wagichungc., A. G. Richard.
Laboratory, greenhouse and field tests were conducted to (i) compare relative pathogenicity of Fusarium solani fsp. phaseoli and F oxysporum f.sp. phaseoli on ,french beans (Phaseolus vulgaris L. cv. Monel), (ii) evaluate the efficacy of seed dressing with various fimgicides singly and in combinations against the two pathogens (iii) compare efficacy of organic matter and / or fungicides on root-rot caused by the two' pathogens and (iv) compare effects of delay ~ planting after application of cowdung and Tithonia diversifolia on the root-rot. Laboratory experiments were arranged in a completely randomised block design while greenhouse and field experiments were arranged in a randomised complete block design. Five replicates were used in all tests. In-vitro fimgitoxicity potential offimgicides and fungicide combinations was assessed by computing % inhibition of mycelial growth, % spore germination and number of spores produced on fungicide treated dishes relative to that of controls. Plant growth assessment was based on plant shoot height, dry weights of seeds, shoots and roots, and number of pods produced .. per plant. Disease severity was determined using mean length of discoloured root tissue (MLDRT) and mean.root rot index (MRRl). The plant growth parameters stated above were significantly (p=O.05) lower on plants infected with the two Fusarium spp. than control. However, slightly higher % loss in nw:nber of pods and seed dry weight were realised on those plants infected with F oxysporum fsp. phaseoli than F solani fsp. phaseoli. No statistical differences were noted between the effects of the two Fusarium spp. on bean, growth. Disease severity on plants inoculated with the two pathogens was similar. MLDRT and MRRI, were the same in both cases. However, MLDRT and MRRI were relatively higher on plants infected by F oxysporum fsp. phaseoli than 1'. so/ani fsp. phaseoli. This suggests that F oxysporum fsp. phaseoli may have been more pathogenic than F solani fsp. phaseoli under the prevailing experimental conditions. Fungitoxicity was based on the eTfect of fungicides on mycelial growth, spore germination and sporulation. Fungicide -treatments significantly (P=O.05) inhibited mycelial growth, spore germination and sporulation of both test fungi. However, fungal sporulation was higher in plates treated with fungicides than in controls an indication that fungicides stimulated sporulation. Although no fungicide treatment was seen to be an inhibitor of all the three measures of fungitoxicity, the ranking of the best three fungicide treatments was be, thiram 50 + captan 50 >triforine > metalaxyl mancozeb. Individual fungicide treatments had significantly (P=O.05) better effects on plant growth and disease development than fungicide combination or control. Significantly (P=O.05) higher plant growth parameters and lower MLD RT and MRRl were recorded from single fungicide treatment on both the greenhouse and field grown plants. Significantly (P=O.05) better plant growth, more pods and heavier seed dry weights were recorded from plants obtained from seeds treated with fungicides triforine or metalaxyl. Integration of cowdung and T diversifolia with the fungicide metalaxyl revealed that cowdung or a combination of cowdung with metalaxyl significantly (P=O.05) reduced disease development and increased plant performance more than T diversifolia or a combination of T diversifolia with metalaxyl. Plants harvested in soils treated with T diversifolia or a combination of T diversifolia with metalaxyl or controls had the poorest seed and pod yields. Germination was low and no pods wereharvested from plants grown in these soils. A delay in planting french bean in soil treated with cowdung significantly (P=O.05) decreased development of root rot when compared to untreated controls. The greatest decrease was obtained with 2 and 4 week delay. Shoot and root dry weights of plants infected by F. oxysporum fsp. phaseoli or F solani fsp. phaseoli significantly (P=O.05) increased with delay in planting and were highest where planting was delayed 2 and 4 weeks, respectively. A delay from 0 to 4 weeks in planting french beans in soil treated with crushed leaves of T diversifolia significantly (p=O.05) increaseddisease severity and reduced plant performance when compared to untreated control.
Molecular characterization and some environmental factors influencing distribution of the endangered and endemic gulella taitensis in Taita hills, Kenya
(2011-05-09) Mwaura, Njeri Ann
Gulella taitensis is a land snail of the family Streptaxidae and genus Gulella. It is endemic to Taita hills and categorized as endangered on the IUCN Red List. The species is threatened by habitat loss and disturbance due to human activities. Two people sampled snails at four sampling plots using standard timed direct search for one hour. Soil samples were collected from four different points within these sampling plots, and its pH, calcium and electrical conductivity obtained using standard soil chemical analysis methods. Litter depth, litter cover, canopy cover, log and tree density were assessed. Distribution of G. taitensis varied significantly between all the forest fragments. Most of the environmental variables investigated recorded a positive association with the G. taitensis densities. Calcium is significant for the snail's survival and has a significant correlation with litter cover, canopy cover and log density and these influences the distribution between the forest fragments. Calcium, canopy cover, log density and tree density form the best combination of environmental variables (model) that influence the distribution and abundance of G. taitensis. Polymerase Chain Reaction of DNA extracted from these snails using microsatellite primers showed there is genetic diversity within this species. We predict that there could be genetic variations within this species since the phylogenetic tree shoed two major clades and samples from the same forest fragment clustered at different clades while others clustered closely together within the same clade. There is a possibility of the presence of a hybrid or sub-species within this species. This requires more specific primers to be used to confirm it as genetic work of this genus is scanty.
Nodulation and nitrogen fixation in promiscuous and non promiscous soybean varieties grown in Kenya
(2011-05-09) Gitonga, M. N.; Gitonga, M. N.
Nitrogen deficiency is a major factor limiting soybean production. Th is problem can be alleviated by the use of nitrogen fertilizers which on the other hand adversely affect the environment, are expensive and unaffordable to most peasant farmers. Alternatively, attention is being paid to improving nitrogen through use of environmental friendly biological nitrogen fixation of soybeans in an attempt to develop sustainable cropping systems. There is however inadequate knowledge on estimates of nitrogen fixation by soybean varieties in Kenya. In light of this, growth experiments were designed to investigate biological nitrogen fixation in promiscuous and non promiscuous soybeans. Non promiscuous and specific Clark was used in greenhouse to confirm presence of Bradyrhizobium japonicum. Results from the field experiment showed that the two promiscuous soybean varieties (TGx 1869 and TGx 1893) nodulated better than the non promiscuous Gazelle. Uninoculated Gazelle did not produced any nodule while uninoculated TGx 1869 and TGx 1893 nodulated (with indigenous soil Rhizobia). Inoculation had effect on nodulation since there was a significant difference (P<0.05) in nodule number and nodule dry weight of inoculated soybeans as compared to uninoculated and N treated soybeans. Nitrogen treated soybeans did not produce any nodule. At early stage of growth, non promiscuous Gazelle was competing effectively with the TGx varieties. Results at podding and final harvest indicated that as growth progressed, the promiscuous soybean varieties fared better than Gazelle in root and shoot dry weight, pod number, pod and stover dry weight. Except in nodule number and nodule dry weight, N treated soybeans had significantly highervalues in all parameters than inoculated and uninoculated soybeans. Inoculated soybeans had higher pod and seed dry weight than uninoculated soybeans. Nitrogen treated soybeans had significantly higher average seed dry weight (22.98g) than inoculated and uninoculated soybeans which had 12.39g and 10.44g respectively. Although there was no significant difference in seed dry weight of inoculated and uninoculated soybeans, inoculated soybeans had higher seed dry weight. The two TGx varieties had higher seed dry weight than Gazelle. Results from greenhouse studies showed that the two TGx varieties nodulated with 85.7% of isolates, Gazelle with 47.6% and Clark 23.8%. Clark was the poorest in nodulation and only nodulated with isolates obtained from soybeans nodulated by Bradyrhizobium japonicum USDA 110. There is need to adopt growing of promiscuous TGx varieties and to exploit biological nitrogen fixation with the view of increasing soybean yields and decreasing overdependence on nitrogen fertilizers for sustainable agriculture.
Association of heavy metal tolerance with multiple antibiotic resistance in bacterial isolated from wetlands of lake Victoria basin Kenya
(2011-05-09) Sikuku, Mutuku Christopher
A study was carried out to determine the heavy metal concentrations in the wetlands of Lake Victoria basin and their effect on bacterial resistance to ten commonly used antibiotics. Water and sediment samples for heavy metal analysis and microbiological assays were randomly collected from sewage outfalls and points bordering heavy metal activity settlements. Heavy metal analysis was done using flame Atomic Absorption Spectrometer (F-AAS). The microbiological assays targeted heterotrophic bacteria, coliforms and enteric pathogens. The isolates were subjected to heavy metals;- mercury, nickel, chromium and copper in the laboratory to assess their levels of tolerance. Antibiotic susceptibility testing was carried out to check for antibiotic resistance. Sediment samples recorded a higher level of heavy metals with a mean range of 31.97109.9, lead; 3.48-183.66, zinc; 3.01-17.03, nickel; 1.93-214.61. copper; 17.01-65.91, cobalt; and 2.08-25.89 mg/g chromium compared to water samples whose mean range was as follows;- lead; 0.77-0.94, manganese; 0.10-3.10, zinc; 0.23-1.16, cadmium 0.020.04, and copper 0.51-0.57 mg/1. There was a significant relationship in the amounts of heavy metals in water samples and the bacterial counts, r = 0.637, p < 0.05. The study showed a significant difference in percentage tolerance to the four, heavy metals tested. (f = 4.25, P = 0.011, P < 0.05). Mercury recorded the highest toxicity followed by chromium and nickel. Copper was the least toxic to the microorganisms. 53.8% of the total isolates showed multidrug resistance. They were significantly resistant (p < 0.05) to cefuroxime (67.7%), cotrimoxazole (65%), tetracycline (62.4%) and ampicillin (53%). They were however highly sensitive to, gentamycin (98.9%), kanamycin (83.9%) and norfloxacin (79.6%). The study showed no significant difference in multidrug resistance in the three bacteria groups (F =1.75 P = 0.191 P > 0.05). however er, multidrug resistance in the enteric pathogenic bacteria (mean % multi drug resistance 65.15%) was higher than in heterotrophs (mean 46.52%) and coliform bacteria (mean 6.36%). There was a significant relationship between chromium tolerance with resistance to cefuroxime (p < 0.000), Nickel tolerance with resistance to cefuroxime (p < 0.05) and mercury tolerance with resistance to ampicillin (p < 0.05). Plasmid DNA finger printing of the metal tolerant and antibiotic resistant isolates showed a positive relationship in number of isolates carrying plasmids and the number having multiple drug resistance (r = 0.372, P = 0.261) suggesting that heavy metal pollution in wetlands induces multidrug resistance. The high degree of resistance to common antibiotics like ampicillin, cotrimoxazole and cefuroxime can therefore be attributed to the inflow of heavy metal rich effluent into the wetlands of the Lake basin resulting to co-selection of both metal tolerant and antibiotic resistant microbial species. This requires intervention measure s to curb the potential health hazard posed by heavy metal pollution in the aquatic ecosystems.
Aspergillus flavus and aflatoxin levels in stored maize in Eastern Kenya and Antifungal activity of some plant extracts
(2011-05-09) Kiswii, Muange Theddeus
Maize is the staple crop in Eastern Province of Kenya especially in Makueni, Kangundo, Kibwezi, Machakos and Kitui South Districts. It has been noted that the abiotic and biotic stresses associated with these Districts promote growth of toxigenic fungi that produce mycotoxins in maize in storage. The aflatoxin producing species of Aspergillus are a common phenomenon in maize contamination that has led to frequent outbreaks of aflatoxicoses in these regions. To address this problem, this study w as carried out between September 2007 and June 2008 to evaluate the incidences of A. flavus, determine the aflatoxin levels in stored maize from 5 Districts and evaluate the efficacy of 15 selected medicinal plants collected from Mwingi District. Four Aspergillus sp. that included A. flavus, A. niger, A. ochraceous and A. tamarii were isolated from maize collected from the 10 Divisions in the 5 Districts. Machakos (mean 58 80%), Kangundo (mean 58.4%), and Kitui South (mean 54.80%) Districts had significantly (P < 0.05) high incidences of total Aspergdlu.s sp. than Makuem (mean 45%) and Kibwezi (mean 24.20%). The incidences of A. flavus from the 5 Districts and the 10 Dix isions indicated a significant difference (P < 0.05). Kangundo District (mean 31.71%) liad the highest incidence of A. flavus while Machakos District (mean 12.92%) had the lowest. Among the 10 Divisions, high incidences of A. flavus were found in Matunguht (mean 33.18%), Kaiti (mean 32.6%) and Kangundo (mean 30.25%) Divisions. The love cst incidence was detected in Kathiani Division (mean 10.82%). Out of the 10 samples analyzed, 2 samples had > 50 ppb, 5 had > 20 ppb and the remaining 3 had < 20ppb of aflatoxin (AF) levels. The highest AF levels were recorded in samples from Kibwezi (60.35 ppb) and Kathiani (50 ppb) Divisions while the lowest was found in Kangundo (Oppb). I 'lie methanol leaf extracts of the 15 plants were evaluated for antifungal activity against A. flavus at different concentrations of 1000mg/ml, 750mg/ml and 400mg/ml rising Agar Well Diffusion Method. Plants found to have inhibition zones of more than 1Omm at 400mg/ml had their bark assayed for antifungal activity. Both the mcthanolic leaf and bark extracts of the fifteen plants assayed displayed concentration depended antifungal activities that were comparable to that of the reference drug Miconazolc at IOmg/ml. The leaf extracts showed better antimicrobial activities than the bark extracts. For the leaf extracts, Boscia coriacea (mean 17.40mm) had the highest zone of inhibition followed by Zanthoxvlem chalybeum (mean 17.20mm). For the bark extracts, Crown megalocarpus (mean 15.Omm) recorded significantly high antifungal activity while 77thonia diversfolia (mean 13.Omm) had the lowest at 400mg/ml. Both the leaf and bark extracts that were found to be effective were assayed for minimum inhibition concentrations (MIC) and minimum fungicidal concentrations (MFC) using broth (SDB) microdilution method. Senna stamea had the lowest MIC and MFC of 6.25mg/ml and 12.51ng/ml respectively. The preliminary phytochemical analysis of the effective plants revealed the presence of bioactive compounds that included tannins, saponins, flavonoids, terpenoids, cardiac glycosides and alkaloids. The results obtained from the study could be used as a viable management strategy against A. flavus.
Evaluation of Antimicrobial activity of some plants used by traditional activity of some plants used by traditional healers for treatment of microbial infections in Kakamega District: Kenya
(2011-07-14) Makhatsa, Wenslaus Luvonga
Resistance to drugs especially antibiotics has become a major challenge facing the medical fraternity today. Most antibiotics that were once effective against pathogenic micro-organisms have now been rendered ineffective owing to resistance developed by these pathogens. This scenario has been complicated by the emergence of HIV/AIDS, which renders the victims immuno-compromised, and open to opportunistic infections. At the same time, most of the African population lives below the poverty line and cannot afford the expensive conventional medicines. These challenges call for renewed strategies on treatment, especially in the development of new antimicrobials. According to the World Health Organization (WHO), medicinal plants can provide the best alternative source to obtain a variety of drugs. Therefore there is need to investigate such plants in order to understand better their properties, safety and efficiency. Renewed interest in plant antimicrobials has also been prompted by the rapid rate of extinction of plant species owing to overharvesting and deforestation. In this study, some 18 plant species used as herbal medicine in Kakamega district were collected and authenticated at the East African Herbarium where voucher specimens are deposited. The plants were soxhlet extracted at the Kenya Medical Research Institute with petroleum ether, dichloromethane and methanol. Total extraction with water was also performed and all extracts screened at 1 g/ml against the organisms such as Escherischia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Candida albicans, Candida parapsilosis, Microsporum gypseum and Trichophyton mentagrophyte by disc diffusion method. MIC tests by agar dilution method were performed on the active extracts at concentration range 0.5 - 0.004 g/ml. MBC and MFC tests were performed on the most active water extracts. Extracts of 6 plants with the highest activity were screened for phytochemicals using methods by Harborne. Of the 144 preliminary tests performed on all the water extracts, activity (inhibition zone >9 mm) was recorded in 44 cases as compared to 35 for methanol, 16 for dichloromethane and 4 for petroleum ether. Entada abyssinica water extract had the widest range of activity, with inhibitions against 7 of the 8 test organisms. However, the aqueous extract of Warbugia ugandensis was most inhibitory with MIC (≤ 12.5 X 103 μg/ml) against 7 (85%) of the 8 tested organisms. Entada abyssinica water extract had the broadest spectrum of bactericidal activity, with MBC (≤50 X 103 μg/ml) against 3 (75%) of the 4 tested bacteria. Only W. ugandensis water extract was fungicidal (MFC ≤50 X 103 μg/ml), with activities against C. parapsilosis and M. gypseum. These activities were not significantly different when compared with ketoconazole (P = 0.217). Phytochemical screening of E. abyssinica, W. ugandensis, N. macrocalyx, Albizia coriaria, Rhamnus prinoides and Albizia amara extracts revealed differences in the presence and abundance of alkaloids, phenolics, terpenoids, anthraquinones, flavonoids and saponins. Phenolics were most abundant and widespread, while anthraquinones were least abundant. Influence of these phytocompounds on the activities of the named plants varied. The ability by some of the plant extracts to inhibit microorganisms that have proved resistant to conventional medicine confirms the therapeutic potential that such plants hold, and the need to incorporate them into our healthcare systems, as well as conserving them.
Incidence of fusarium species and levels of fumonisin B1 in sorghum and finger millet in Western Kenya.
(2011-08-03) Mafurah, J. J.
This study was carried out to determine the incidences of Fusarium species, fumonisin B 1 and aflatoxin B 1 production in sorghum and finger millet grains collected from selected districts of Western Kenya region. The grains were collected randomly from five locations per district in Nyamira, Kakamega, Busia and Teso districts respectively. Ten farmers per location were randomly selected and samples of 0.25 kg of grains were picked per variety. A total of 16 sorghum varieties and 14 finger millet varieties were collected from the four districts. There were 19 morphologically distinct Fusarium species isolated from sorghum and finger millet grains. These included; Fusarium compactum, F. equiseti, F. thapsinum, F. verticillioides, F. longipes, F. andiyazi, F. nygamai, F. pseudonygamai, F. brevicatenulatum, F. chlamydosporum, F. heterosporum, F. napiforme, F. graminearum, F. pseudograminearum, F. oxysporum, F. solani, F. subglutinans, F. semitectum and F. proliferatum. Fusarium compactum and F, equiseti were the most isolated species in all the districts in both grains while F. thapsinum and F. verticillioides were isolated in sorghum varieties from all the districts. Sorghum had significantly higher percentage incidences of Fusarium species than finger millet. Nyamira district had the highest incidences at 65.47% and Kakamega had the lowest at 11.12%. Wagiita variety of sorghum had the highest incidences at 58.95% while Esila had 0% isolation of Fusarium species. All finger millet varieties had low incidences of Fusarium of less than 8%. The levels of fumonisin B1 detected in sorghum grains (48.81 ug/g) were significantly higher than those in finger millet grains (1.13 wg/g). Sorghum from Nyamira district had the highest fumonisin B 1 levels at 76.63 pg/g while low levels of 17.59 gg/g were detected in Busia. Ochuti and Wagiita varieties of sorghum had the highest fumonisin B1 levels, while no fumonisin 131 was detected in KARI Mtama-1 and Esila. Ikhumba variety had levels within the detection limits of 1.75 pg/g. Some finger millet varieties had the lowest levels of fumonisin B 1 which were at the detection limit levels of 1.75 pg/g each while some had no fumonisin B 1 detected. The aflatoxin B 1 in sorghum from Busia and Teso districts had levels below the detection limit of 2 ppb. From the results, finger millet grains are safe for consumption especially when weaning children but sorghum might cause mycotoxicoses. Results also shows that farmers should be sensitized on the dangers of fumonisin B 1 levels in sorghum and be advised to adopt varieties like KARI Mtama-1, Esila (with no fumonisin B 1 detected) and Ikhumba that had very low levels of fumonisin B l . Some of the fumonisin levels found in this study coincide with levels associated with risks to humans and animals reported in other countries.
Physico-chemical and bacteriological quality of water, and antimicrobial susceptibility of pathogenic isolates from selected water sources in Samburu South
(2011-08-09) Mwajuma, Jeophita Mwajuma
Water is the most critical resource in the Samburu District of Northern Kenya. The region has one permanent river, the Uaso Ng'iro. Use pressure by man domestic and wild animals is high in all water sources, which include dams, laggas, and dry river bed wells. The primary objective of this study was to investigate the bacteriological and physicchemical quality of water from these sources, so as to shed some light on the causes of perpetual diarrheal diseases and their likely responses to commonly used antibiotics. Samples were collected quarterly over 1 year. During sample collection, the physicochemical conditions of water such as temperature, pH, conductivity, alkalinity, salinity and water color were recorded. A total of 207 water samples were collected and their microbial quality determined based on the most probable number (MPN) of coliforms, total plate count, feacal coliform counts and presence or absence f E. coli. To isolates water borne bacterial pathogens, samples were inoculated in to appropriate enrichment and selective media and the recovered bacteria characterized using relevant biochemical and serological tests. The pathogenic isolates were subjected to the Kirby Bauer and Epsilometer tests to determine their sensitivity to antibiotics commonly used in first line treatment of enteric infections. The color of water from most source types except boreholes and springs was not aesthetically acceptable for direct drinking. Alkalinity, pH and conductivity values in all water sources except the boreholes were within the recommended limits for potable water, set by World Health Organization (WHO) and Kenya Bureau of Standards (KBS). Dissolved oxygen was within acceptable levels while only springs had salinity levels less than 250 mg L-1, the set limit for potable water. On the basis of total plate count 85% of the water samples were unfit for human consumption. Mean MPN was highest in dams at 643coliforms ml--I and lowest in springs and boreholes at 35coliforms mL-1 As such, the water from clams, rivers, springs, laggas and dry river bed wells was unfit for human consumption. However, on the basis of presence of feacal coliforms, all water sources do not meet the standards for potability. The bacterial pathogens isolated were Shigella flexineri, Shigella boydii, Aeromonas hydrophilla, and Salmonella spp. (non-typhi). Pathogenic isolates from springs and permanent rivers were mainly Klebsiella and Pseudomonas spp., conisidered to be of little clinical significance. Recovered S. flexineri isolates were susceptible to cefotaxime, gentamicin, ciprofloxacin and cefuroxime, but resistant to cotrimoxazole and chloramphenicol. However some of the recovered S. flexineri isolates were resistant to ampicillin, the antibiotic recommended for treatment of dysentery. S.boydii and Aeromonas. spp. displayed complete resistance to ampicillin. S. flexineri isolates were compared using Pulsed-field gel electrophoresis with Notl restriction enzymes. Isolates resistant to ampicillin had similar pulsotypes to those susceptible to it. Although boreholes and springs had better quality water, Samburu District w ater did not meet the WHO and KEBS requirement for potability. Most pathogenic isolates were susceptible to commonly used antibiotics. Pulsed field gel electrophoresis revealed no genetic evidence of difference in strains, where resistance to ampicillin occurred. Water monitoring in Samburu should be carried out over a longer period of time wing more rapid and discriminatory procedures. Appropriate and affordable water disinfection techniques such as boiling and filtration should be designed.
Growth response of seedlings of selected tree species to nutrient enrichment and competiton in Kakamega forest
(2011-08-09) Mwangi, P. Charles; Alexander Njue; Kiplagat, Kotut; Alexander Njue; Kiplagat Kotut
Prunus africana Hook F, Olea capensis Wright and Markhamia lutea 13enth are some of the indigenous tree species growing in Kakamega forest. These species are of great economic importance to the communities living around the forest. However, Kakamega Forest soils on which these species grow are acrisols of low fertility. They are also acidic with a pH value of less than 5.5. Acidic soils are known to have low levels of phosphorus and potassium which are important in improving the availability of microelements. Their deficiency can lead to deficiency of minerals such as iron, manganese, zinc and copper in the soil. Several studies have revealed that soil amendment can improve soil structure, aeration, water retention and nutrient availability. Previous studies have shown that about 80 % of the farms around Kakamega are deficient in phosphorus, nitrogen and potassium. However, limited information is available on the effect of this nutrient deficiency on seedlings of indigenous tree species in Kakamega forest. The aim of this study was to assess the response of the selected tree seedlings to soil amendments with commonly limiting nutrients. Seedlings of the three species were grown individually in tubes containing soil fertilised with nitrogen, phosphorus, potassium and combinations of the three fertilizers. Forest soil, farm soil and a mixture of the two soil types were used for comparison purposes. Nitrogen and phosphorus were found to be co- limiting nutrients to the growth of Prunus africana seedlings. Nitrogen, phosphorus and potassium were all found to be limiting for Markhamia lutea seedlings, while potassium was found to be limiting for Olea capensis seedlings. To determine the effect of intraspecific competition on seedlings growth, three seedlings of each species were grown together in the same tube. To assess the effect of interspecific competition, one seedling of each species were grown in combination in each tube. The seedlings grown individually were used as the control. Prunus africana and Markhamia lutea seedlings grown in absence of competition invested more in mean height and leaves compared to the competing seedling while none competing Olea capensis seedlings invested more on mean height and mean diameter compared to the competing seedlings. Experimental studies conducted in shade houses and in the field have demonstrated that differences in light availability differentially affect seedlings growth and mortality of tree species in wet tropical forests. In Kakamega forest, the seedlings of Prunus africana have recorded unusually high mortality rate of 3.9%. Despite the important role of light in seedlings survival, its effect on seedlings survival in Kakamega forest has not been explored. The findings of this study reveal that seedlings grown in high light regime had larger mean height, mean diameter, mean number of leaves and mean leaf area compared with the seedlings grown in low light regimes.
Evaluation of selected medicinal plant extracts for insecticidal properties against phlebotomine sand flies (Diptera: Psychodidae)
(2011-08-09) Njeru, Ireri Laban; Kongoro, Jedida A.; Tonui, Willy Kiprotich
Phlebotomine sand flies are the vectors of Leishmania species, the causative agents of leishmaniases that are a global health problem and are usually endemic in developing countries affecting predominantly the poor in society. Complementally control measures through use of biodegradable, safe and species selective natural compounds are urgently needed. Plant based compounds are safe in most biosystems. Some synthetic chemical insecticides are toxic, expensive and discriminate use may cause vector resistance. In this study, the aerial parts of Tagetes minuta Linnaeus (Asteraceae), Acalypha fruticosa Forssk (Euphorbiaceae) and Tarchonanthus camphoratus L. (Compositae) currently being used in endemic areas as repellents were extracted using methanol and ethyl acetate prior to being filtered, dried out by rotary evaporation at 30-35°C then dissolved in Dimethylsulphoxide (DMSO) and prepared into 2.5, 5 and l0mg/ml using distilled water. The different concentrations of the extracts were evaluated against Phlebotomus duboscgi while incorporated in filter papers. Other sets of similar concentrations were prepared using 10% sucrose solution and used in feeding technique using cotton wool balls. Pyrethrin incorporated in artificial food was also investigated for insecticidal efficacy using feeding techniques to 10 adult sand flies and 10 larvae and replicated three times. Pyrethrin (0.6 %) was diluted in 10% sucrose solution at 0.08, 0.25 and 0.5 mg/ml and used in the adult feeding bioassays while bioassays on the larvae were conducted using the extracts, powdered plant parts, 0.2% pyrethrin powder and 0.5% EC incorporated in larval food. Mortality was recorded every 24 hours of exposure and the data entered into MS Excel and subjected to computerized probit analysis. The extracts had significant mortality to adult sand flies in both contact and feeding methods but exhibited no larvicidal activity. Extracts of A. fruticosa and T. minuta had significant mortality (P<0.05) to adult sand flies at 48 hours of exposure than those of T. camphoratus. The combination of the extracts yielded weaker insecticidal properties than separate extracts. The feeding techniques had higher LD50 values in all the bioassays at 48 hours of exposure when compared to the contact bioassays using similar concentrations. In the pyrethrin bioassay there was no significant difference between male and female LD50 at 48 hours of exposure. The powdered plant parts were not larvicidal. The two pyrethrin formulations were effective against larvae with a LD50 of 0.1 mg/ml in the third instar larvae groups yielding mortality of about 100% in only 96 hours of exposure. The plant extracts were found to be effective against adult phlebotomine sand flies while pyrethrin products were found to be efficient larvicides when incorporated in artificial sand fly larvae food and are therefore proposed to be harnessed, made into appropriate formulations and used alongside other control strategies.
Entomopathogenicity of hyphomycete fungi to fruit fly bactrocera invadens (Diptera: Tephritidae) and their potential for biological control on mango
(2011-08-10) Ouna, Elizabeth Awuor
Sustainable mango production will rely increasingly on alternatives to conventional chemical insecticides that are environmentally friendly for the management of fruit flies. The use of microbial control agents such as fungi in pest suppression is considered suitable since microorganisms usually exert low environmental impact and are target specific. Application methods of bio-pesticides in the environment, which use minimal amount of inoculum is currently under improvement from inundative to auto-inoculatioe methods. Such devices are usually designed to attract insects into focus of the entomopathogen and use the insect as a vector for transmission of pathogen to other members of its population. Virulence of 24 isolates of metarhizium anisopliae (Metschnik.) Sorok. and Beauveria hassiana (Bals.) Vuill. (Ascomycota: Hypocrales) collected from the microbial culture collection at International Center of Insect Physiology and Ecology and isolates collected pathogen survey of the study was evaluated on immature adult Bactrocera invadens Drews, Tsuruta and White in the laboratory, followed by testing sub-lethal effects of one of the most pathogenic isolates on fecundity and fertility. Out of the 24 isolates of M. anisopliae and B. bassiana screened for pathogenicity against B. invadens, 7 isolates (ICIPE 20, 43, 62, 69, 295, 303 and M. anisopliae var. acridium IMI 330189 or ICIPE 21, available as Green Muscle® induced significantly higher mortalities (ranging from 93.7% to 94.8%) than the rest of the isolates. Lethal time to 50% mortality (LT50) ranged from 2.8 to 3.6 days (M. anisopliae) and 2.7 to 2.8 days (B. bassiana); and slope value estimates of 0.96 and 0.88 respectively at 5 days post treatment. Sub-lethal effect of selected isolate of M. anisopliae (ICIPE 20) on B. invadens exhibited significantly lower egg fecundity and hatchability in treated flies than control at levels of 6.8 and 28.4 eggs per fly per day and a gross of 28.8 eggs against 113.2 eggs/female at 4 days post inoculation; and hatchability of 17.2% and 64.8% respectively. Evaluation of three different bait formulations contaminated with M. anisopliae was carried out based on resident time flies spent on the baits, persistence of conidia on the baits exposed in the field under mango canopy and pathogenicity of fungus contaminated baits to adult B. invadens in the laboratory at 0, 7, 14, 21, and 28 days of exposure in the field under mango canopy.This caused an overall reduction in fecundity between 80.2 - 99.8% and 51- 80% in hatchability. Resident time spent by B. invadens on M. anisopliae-contaminated food baits was not significantly different among the 3 baits (2% NuLure, local bait DuduLure® and a diluted 10% DuduLure®); however germination of conidia from 10% DuduLure and 2% NuLure was significantly higher than that DuduLure® at 14, 21 and 28 days of field exposure and were at the levels of 83%, 96%, 78% (10% DuduLure); 74%, 95%, 96% (2% NuLure); 54% 6%, 6% (DuduLure©). Two-way ANOVA demonstrated there was significant effect of time and environmental factors on conidial germination. Mortality of flies treated with fungus contaminated- 10% DuduLure and 2%NuLure baits was significantly higher than in treated DuduLure®. Mortality of flies exposed treated 10% DuduLure (84%) was significantly higher than 2% NuLure (64%) at 21 days but was significantly lower (48%) than 2% NuLure (90%) by 28 days. In conclusion this study has identified 7 new hyphomycete isolates as highly pathogenic to adult stages of B. invadens. Among the isolates M. anisopliae ICIPE 20 can cause reduction in egg fecundity and hatch rate. Wooden sphere soaked in commercial (NuLure) or locally developed bait (DuduLure) and contaminated with spores of M. anisopliae can provide a viable option for the management of B. invadens on mango. The adaptability of this technology will depend in part on its efficacy to suppress populations of B. invadens in the field.
Ethnobotanical survey, antimicrobial efficacy and preliminary phytochemical screening of selected of selected anti-diarrhoeal medicinal plants used by the Samburu community, Wamba, Samburu districts, Kenya
(2011-08-10) Omwenga, Omori Eric
Despite advances in understanding of the causes, treatment and prevention of diarrhoeal diseases an estimated 4.6 million people die from diarhoreal diseases every year. Diarrhoea can either be acute or chronic, with acute diarhoea being the most common form. The main agents of diarrhoreal diseases include viruses like rotavirus, bacteria such as Enterobacteria e.t.c and parasitic agents like Cryptosporidium. The disease has been found to attack mostly the children who are below the age of 5years old and the elderly. It is also a major opportunistic infection amongst the HIV/AIDS patients. Oral rehydration therapy (ORT), antibiotics and medicinal plants have been used as remedials. But antibiotics have been found to posssess antimicrobial resistence hence need for search of new drugs. Plants have been found to have active compounds (phytochemicals) which have - antidiarrhoreal activity. Therefore this study evaluated the efficacy of the antidiarrhoeal plants used by the Samburu community. By use of simple questionnares a survey was carried out that was targeting the Samburu informants and herbalists on the common antidiarrhoreal plants they use. Thirty three antidiarrhoeal plants were collected, dried, chopped, ground by the laboratory grinding miller. Voucher specimens were prepared and deposited at the Kenyatta University herbarium. Extraction of the selected medicinal plants was done by use of methanol (solvent) and then screened by use of the disk diffusion method against the selected microorganisms that were collected from Kenyatta National Hospital. There was significant difference of the means of the zones of inhibition of the Staplylococcus aureas, Salmonella typhi and Pseudomonas aeruginosa at P< 0.01 except in Fscherichia coli, Bacillus subtilis and Candida albicans that showed no- significancedifference even at P< 0.05. No activity was observed on A. flavus, Aspergillus niger, Penicilhum spp, and Fusarium lateritium. Pseudomonas aeruginosa was affected by most plant extracts in terms of zones of inhibition. The The MICs and the MBCs of the extracts were determined by use of the microtitre plate method with Acacia ethaaica, Acacia nilotica, Acacia tortilis, Albizia anthelmiticq Clerodendrum myriacoides, Plumbago dawei & Thylachium africanum extracts showing good MICS and MBCS results of 9.38- 18.75 (mg/ml) in most test cultures except C. albicans that seemed to be insensitive to any extract. The extracts produced high MICs and MBCs againsts P. aeruginosa and S. aureas. Preliminary phytochemical screening of tannins, terpenoids, cardiac glycosides, alkaloids, and saponins was also carried out. Tannins were the most abundant phytochhemicals present that were found to be present in most plant extracts. Generally the Gram negative isolates were more affected than the Gram positive isolates hence the activity of the extracts can not be ascribed to the cell wall properties but to other factors like the sensitivity of the test organism to the extracts, the diffusion rate of the extract e.t.c. For the fungi isolates the activity of the extracts was very low and thus it can be ascribed to the cell wall properties that may have made the extracts not to diffuse into the test micro-organisms. Also presence of different phyhochemicals may explain the activity of the extracts whereby they can be offering some synergism/additive or antagonism activity against the test isolates. More work needs to be done on the highly active extracts like Acacia nilotica and Plumbago dawei. Thus the results obtained clearly back the use of the medicinal plants selected from the community as they showed good activity against the test cultures.
Efficacy of Medicinal Plants Used by Communities Around Lake Victoria Region and the Samburu Against Mycobacteria, Selecfed Bacteria and Candida Albicans
(2011-08-18) Mariita, Mong'are Richard; Okemo, P. O.; Mbugua, P. K.
In Africa more than 70% of the people refer to ethnomedicine for their health issues. With the emergence of new diseases and drug resistance to infections, traditional medicine should be given more attention in modern research and development. Tuberculosis (TB), a deadly infectious disease that annually kills about 3 million people worldwide is complicated. This is due to significant toxicity, emergence of multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR- TB) and lengthy therapy which creates poor patient compliance. There is also a major therapeutic problem due to emergence of Escherichia coli, Klebsiella pneumoniae and other Blactamase producers. Diarrhoeal diseases are responsible for 4.6 million deaths every year. These highlight the need to develop novel drugs. Natural products provide unlimited opportunities for new drug leads because of the unmatched chemical diversity. This study evaluated the antimicrobial potential of 34 medicinal plants used by communities living around the Lake Victoria region and the Samburu Community of northern Kenya, following an ethnobotanical survey. Plants were collected and identified at the Department of Pharmacy and Complimentary Alternative Medicine, Kenyatta University, Nairobi, Kenya; in whose herbarium voucher specimens were deposited. Methanolic extracts from plants were tested against four strains of Mycobacteria (Mycobacteria tuberculosis, M. kansasii, M. fortuitum, and M. smegmatis) obtained from Kenya Medical Research Institute (KEMRI), Nairobi, Kenya. BACTEC MGIT 960 system was used. Salmonella typhi (clinical isolate), Klebsiella pneumoniae (clinical isolate), Pseudomonas aeruginosa (ATCC 25852), Escherichia coli (ATCC 25922) Staphylococcus aureus (ATCC 20591) and Candida albicans (ATCC EK138), obtained from Kenyatta National Hospital in Nairobi, Kenya, were also screened against using standard procedures. The crude extracts were analyzed for presence of phytochemicals. Croton macrostachyus, Vernonia amygdalina, Toddalia asiatica, Aloe secundiflora, Cordia sinensis, and Euphorbia scarlatina gave strong antimycobacterial activity (zero GUs) against M. kansasii, at all concentrations used. Entada abysinnica, T. asiatica, Salvadora persica, C. sinensis, Scadoxus multiflorus and E. scarlatina extracts were active (zero GUs) against M. tuberculosis. Extracts from Carissa edulis, V. amygdalina, A. secundiora, Pistacia aethiopica, S persica, S. multiflorus, E. scarlatina, and Acacia nilotica were active (zero GUs) against M. fortuitum. Against M. smegmatis, Carissa edulis, V. amygdalina, A. secundiora, S. persica, S. multiflorus, E. scarlatina and A. nilotica were active (zero GUs). Eurphobia scarlatina was active (Zero GUs) against all the strains of mycobacteria. There was significant difference of the means of the zones of inhibition of the S. typhi, K. pneumoniae, P. aeruginosa, E. coli, S aureus and C. albicans at P< 0.05. The MICs and the MBCs of the extracts were determined by use of microtitre plate method with E. abysinnica, T. asiatica, Thylachium africanum, A. secundflora, A. nilotica and Momordica charantia extracts showing good activity with MICS and MBCS of 4.687- 18.75 mg/ml in some test cultures. Klebsiella pneumoniae and C. albicans were mostly insensitive to extracts. Preliminary phytochemistry identified six phytochemicals to which tannins were common in most plant extracts. The data suggests that plant extracts could be a rich source of antimicrobial agents. Results also provide an indication of merit in their ethnomedicinal use
Evaluation of regeneration response in tissue culture of selected groundnut (Arachis Hypogaea L.) varieties adapted to Eastern and southern Africa
(2011-08-18) Maina, Susan Muthoni; Gathara, Moses P.H.; Githuki, Simon T.
Cotyledon explants from mature groundnut seeds (Arachis hypogaea L.) adapted to eastern and southern Africa, were optimized to obtain adventitious shoot buds with high frequencies. This was done using commercial bleach {jik, active ingredient sodium hypochlorite (3.5% v/v NaOCI) at different concentrations and exposure time as compared to 0.1% mercuric chloride. Results showed that 0.525% NaOCI and 1.050% NaOCI concentrations within an exposure time of 10-20 minutes were the optimum active range in all the varieties provided. There was constant contamination of CG-2 even after repeated sterilization indicating endogenous contamination. This showed that surface sterilization was not sufficient to sterilize it. While using mercuric chloride treatment, Chalimbana variety recorded the highest percentage of surviving explants (100%) and CG-2 had the lowest (82%). Regeneration experiments performed using already established optinum NaOCI sterilant concentration in each variety as compared with 0.1% mercuric chloride for 10 minutes (sterilant). Cotyledon explants were cultured in shoot induction medium (with BAP and 2, 4-D), shoot buds started to form along the proximal cut end of the cotyledon since this was the area of high regeneration potential due to presence of high number of meristematic cells. Well-formed shoots transferred to shoot elongation medium (SEM) elongated well in the presence of BAP growth regulator and later developed multiple shoots of about 4-8 shoots per explant. In SEM tubes, growth of all the varieties was rapid and prolific. Results showed that explants regenerated rapidly and with vigour. There was interaction (p>0.05) between variety and treatments in percentage regenerated plants. For example, JL-24 variety regenerated better using NaOCI than mercuric chloride. In terms of rapid growth and number of plants produced using NaOCI treatment, JL-24 variety was the best followed by CG-2, the least was ICGV-12991. Using mercuric chloride JL-24 regenerated the most plants followed by ICGV-90704 while the least was CG-2. All the groundnut varieties introduced into tissue culture in this study responded well, acclimatized, produced normal flowers and seedpods within 4 months. The cotyledon regeneration protocol proved to be a good regeneration system using both NaOCI and mercuric chloride sterilants. Of the selected African adapted varieties evaluated in this study ICGV-90704 performed best using mercuric chloride while CG-2 using NaOCI was the best. These are the varieties recommended for transformation, which could be one of the possible methods to offer resistance to viruses and this should have impact on crop improvement on productivity and food security, especially in the semi-arid tropics
Growth response of seedlings of selected tree species to nutrient enrichment and competition in Kakamega forest
(2011-11-03) Mwangi, Charles P.
Prunus africana Hook F, Olea capensis Wright and Markhamia lutea Benth are some of the indigenous tree species growing in Kakamega forest. These species are of great economic importance to the communities living around the forest. However, Kakamega Forest soils on which these species grow are acrisols of low fertility. They are also acidic with a pH value of less than 5.5. Acidic soils are known to have low levels of phosphorus and potassium which are important in improving the availability of microelements. Their deficiency can lead to deficiency of minerals such as iron, manganese, zinc and copper in the soil. Several studies have revealed that soil amendment can improve soil structure, aeration, water retention and nutrient availability. Previous studies have shown that about 80 % of the farms around Kakamega are deficient in phosphorus, nitrogen and potassium. However, limited information is available on the effect of this nutrient deficiency on seedlings of indigenous tree species in Kakamega forest. The aim of this study was to assess the response of the selected tree seedlings to soil amendments with commonly limiting nutrients. Seedlings of the three species were grown individually in tubes containing soil fertilised with nitrogen, phosphorus, potassium and combinations of the three fertilizers. Forest soil, farm soil and a mixture of the two soil types were used for comparison purposes. Nitrogen and phosphorus were found to be co- limiting nutrients to the growth of Prunus africana seedlings. Nitrogen, phosphorus and potassium were all found to be limiting for Markhamia lutea seedlings, while potassium was found to be limiting for Olea capensis seedlings. To determine the effect of intraspecific competition on seedlings growth, three seedlings of each species were grown together in the same tube. To assess the effect of interspecific competition, one seedling of each species were grown in combination in each tube. The seedlings grown individually were used as the control. Prunus africana and Markhamia lutea seedlings grown in absence of competition invested more in mean height and leaves compared to the competing seedling while none competing Olea capensis seedlings invested more on mean height and mean diameter compared to the competing seedlings. Experimental studies conducted in shade houses and in the field have demonstrated that differences in light availability differentially affect seedlings growth and mortality of tree species in wet tropical forests. In Kakamega forest, the seedlings of Prunus africana have recorded unusually high mortality rate of 3.9%. Despite the important role of light in seedlings survival, its effect on seedlings survival in Kakamega forest has not been explored. The findings of this study reveal that seedlings grown in high light regime had larger mean height, mean diameter, mean number of leaves and mean leaf area compared with the seedlings grown in low light regimes.
Macro-and Micro-propagation of melia volkensii GÜrke (meliaceae) : an indigenous multipurpose drylands tree species
(2011-11-08) Abwao, Stephen Indieka
Melia volkensii GÜrke (Meliaceae) is a popular indigenous multipurpose tree species endemic in the arid and semi arid lands (ASALs) of East Africa. It is a prolific seeder, though its mass-multiplication has been constrained by problems in propagation through seed and conventional stem cuttings. In this study, three vegetative propagation techniques were investigated: (i) macro-propagation using 3-month old rejuvenated leafy stem cuttings, (ii) direct in vitro shoot multiplication using shoot tip explants and (iii) direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants. The effects of fungicide pretreatment, surface sterilization and PGRs (IAA, IBA and NAA) at 0, 2, 4, 6, 8, and 10 g 1-1 on rooting of leafy cuttings in a low cost mist propagator were evaluated. Up to 33 % rooting was achieved on cuttings treated with 8 g 1-1 IBA and 50 % of the rooted cuttings were successfully hardened. In vitro experiments were conducted using Murashige and Skoog (1962) basal medium (MS). The effect of MS supplemented with BAP, kinetin and zeatin (0.25-3.0 mg 1-1) alone and in combination with NAA or IAA (0.1 and 0.2 mg 1"1) on shoot multiplication was investigated. Generally, BAP: IAA combinations induced shoot multiplication and up to 5.4 shoots per explant were achieved in BAP: IAA (0.5: 0.2 mg 1-1) combination. Rooting of in vitro multiplied shoots was achieved only in IBA treatments and up to 40 % success was attained in MS supplemented with 2.0 mg 1-1 IBA. The effect of MS supplemented with BAP, NAA and 2,4-D (0.2-4.0 mg 1"1) alone and in combination on induction of direct somatic embryos was also investigated. Direct somatic embryos were initiated on 30-60 % of the cotyledonary explants from seeds stored for <3 months in BAP (0.5-4.0 mg 1-1) combined with 0.2 mg 1"12,4-D. Further development of somatic embryos into plantlets on MS and 1/2 MS (half macro-elements) without PGRs was unsuccessful but formed clusters of numerous shootlets. Nevertheless plantlets were successfully regenerated from shoot tips obtained from the shoot clusters formed. The results obtained in this study clearly demonstrated that M. volkensii is amenable to propagation by rejuvenated leafy stem cuttings, direct in vitro shoot multiplication and somatic embryogenesis. Optimization of the protocols developed in this study will open up opportunities for mass breeding, conservation and genetic manipulation of this species.
Regeneration Potential and Transient Expression of B-glucuronidase Mediated via Agrobacterium tumefaciens in Immature Embryos of Kenya Maize Genotypes
(2011-11-08) Binott, Jayne Jebichii
Production of maize is constrained by both abiotic and biotic stress factors in the field and by post-harvest pest problems; the most important being the larger grain borer, Prostephanus truncatus (Horn) and maize weevil, Sitophilus zeamais. Developing insect resistance crop varieties through conventional plant breeding is elusive, expensive and time consuming due to the limited genetic variation within the maize genotypes as well as difficulty in maintaining quantitatively controlled traits such as insect resistant. However, by using genetic engineering tools, modified novel genes (e.g. from Bt or plant proteins) can be introduced into maize to produce transgenic maize that confer resistance to these insects pests. The present study investigated regeneration potential of Kenyan maize genotypes as a prerequisite to genetic transformation. Twelve parental inbred lines and their respective single cross hybrids were planted in Kiboko and Kabete and evaluated for callus induction, somatic embryo formation and subsequent plant regeneration. Embryos were excised from surface sterilized kernels harvested at different physiological stages namely 10, 15, 18, 21 and 24 days after pollination (DAP). They were used as explants to initiate callus on N6 induction media with varying levels of 2,4-D (020mg/L) supplemented with 2.87g/L proline, 0.1 g/L casein hydrolysate, 2g/L glycine, 30g/L sucrose and 3g/L gelrite. The pH was adjusted to 5.8 before autoclaving. The induction frequency of primary calli at 2mg/L 2, 4 D was genotype dependent. Callus induction ranged from 80-90% for hybrids and 50-80% inbred lines. Following two biweekly subculture, the embryogenic calli formation was initiated. Three types of calli were initiated: Type I accounted for 17.6%, type II 52.9% while the remaining 29.4% made up type 0. Using this system, somatic embryo competence was demonstrated in 6 inbred lines and 4 hybrids. However, plant regeneration was only achieved in 4 inbred lines and 3 hybrids. The frequency of shoot formation ranged between 4-40%. The development of this efficient and reproducible regeneration system sets a basis for genetic transformation via Agrobacterium tumefaciens. Six Agrobacterium strains carrying two types of plasmids (pBECK2000.4) and (pCAMBIA2301) were used to introduce Gus A (Gus) reporter gene encoding -(3-glucuronidase to maize embryogenic tissues. The expression of gus activity on transformed embryogenic tissues was evaluated by histochemical staining with X-Gluc. Gus staining revealed variation in both intensity and pattern of blue staining in embryos transformed with the same plasmid and bacterial strain. EHA105(pCAMBIA2301) AGL1(pBECK) revealed high infectivity across various genotypes. Fifteen days (15 DAP) was optimal embryo stage for gus expression while incubation for 24 hrs was appropriate for co cultivation stage. There was no gus activity in bacterial strains that were devoid of plasmid.
Isolation, Evaluation and molecular characterisation of bacillus thuringiensis isolates against prostephanus truncatus, a major storage pest in maize
(2011-11-14) Mwathi, J. W.; Mburugu, G.N.; Songa, J. M.
The study was initiated to investigate the potential of locally isolated Bacillus thuringiensis (Bt) isolates for the control of the larger grain borer (LGB) (Prostephanus truncatus), a major storage pest of maize and cassava in Kenya. Samples of soil, grain and dead insects were collected from various maize growing areas in Kenya and screened for the presence of Bt. The isolates were tested for toxic activity against P. truncatus in the laboratory. Bacillus thuringiensis var. tenebrio (Btt), toxic to the Colorado Potato Beetle (CPB) was included in the bioassays as a Standard control reference. Bt precipitate obtained from broth was mixed with maize flour at a ratio of 2:1 to make an artificial diet. Pellets of 2g were cut from the artificial diet and offered to insects placed in plastic containers. A total of 68 isolates of Bt were recovered out of 320 samples screened, and of which 85 % were from grains, 12 % from soils and 3 % from dead insects. Grains proved to be a better source of Bt isolates. The screening for Bt isolates in the laboratory revealed that different Bt isolates from different samples were either bipyramidal, irregular or square shaped. Ninety five (95 %) of the Bt isolates from grain samples were bi-pyramidal shaped and (5 %) from grains were square shaped. Analysis of soil samples showed that 12.5 % were square shaped and 87.5 % were bipyramidal shaped. The Bt isolates from insect samples were all irregular shaped (100 %). Among the Bt isolates, 82 were positive and 18 % were negative for Voges Paskeur test. Methyl Red tests revealed that 24 % of the Bt isolates were negative and 76 % were positive, whilst indole tests showed that 72 % of the Bt isolates were negative and 28 % were positive. The biochemical characteristics of isolated pathogens confirmed that these isolates were Bt. Mortality in the controls was 0 % and 3.3 %, 7 and 30 days after treatment, respectively Isolates of Bt were toxic to adult P. truncatus but mortality varied from 1.7 to 53 % and 3.3 to 85 %, 7 and 30 days after treatment between the isolates, respectively. Seven B. thuringiensis isolates (Bt 41, Bt 61, Bt 51, Bt 60, Bt 46, Bt 45 and Bt 44) were more toxic to adult P. truncatus than other isolates, causing mortalities of 50 and 80 %, 7 and 30 days after treatment. However, there was no significant difference between these isolates (P>0.05). The standard reference isolate, Bt tenebrio was also toxic, causing mortality of 37 % and 60 %, 7 and 30 days after treatment, and was significantly different (P<_0.05) from the most toxic isolates listed above. The lethal time 50 % mortality (LT50) values were calculated for isolates that achieved more than 50 % mortality after 30 days following treatment. The LT50ranged from 7 to 37 days and varied significantly (P<_0.05) among isolates. Bacillus thuringiensis isolates 41, 51, 61, 60, 45, 44, 46 and 23 had the shortest LT50 values of between 8 and 11 days. The standard reference strain, Bt tenebrio, had LT50 of 16 days, which was significantly different (P<0.05) from the most toxic isolates. Bacillus thuringiensis 41 and Bt 51 molecular sizes varied between 67 to 74 KDa while the Standard control isolate, Btt, had a molecular size of 67 KDa. There was a common protein band of 67 KDa in the two isolates (Bt 41 and 51). These results suggest that a further DNA study was necessary to ascertain the presence of cry 111 genes that are known to be toxic to coleopterans. These results demonstrated the potential of discovering isolates of B. thuringiensis that are toxic to adult P. truncatus and could be, therefore, developed as biopesticides. Preservation of these isolates in a germplasm bank for pathogen biodiversity should be considered since these pathogens could be utilized as sources of new cry genes for genetic transformation.

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