General Biology

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Characterization of the Causative Agent of Chalkobroood Disease of Honeybee Brood (Apls Melliferal.) in Ethiopia
(Addis Ababa University, 2006-06) Geremew Teshome; Abate Dawit (PhD)
Chalkbrood disease is an invasive mycosis of honeybee larvae (Apis mellifera L) caused by Ascosphaera apis (Maassen ex Claussen) Spiltoir and Olive (1955). The causative agent of this disease occurs in most bee keeping countries of the world. This study aims to characterize the causative agent of the disease and compares it with reference strain (MUCL 34668). The mummies were surface disinfected and inoculated onto Sabouards Dextrose Agar for macroscopic and microscopic identification. The local isolates were also compared with the reference strain, assayed for proteolytic ability, toxicity, and virulence on honeybee larvae in the laboratory and in the hive. Microbiological analysis of 45 samples of dead honeybee larvae collected from the regions during 2004-2006 yielded 28 positive cases for chalkbrood disease incidence (62.22%). Spore cyst, spore ball, and ascospores length to width ratios measured with phase contrast inverted computer fitted microscope were 66.15 –97.66 m, 11.00 –19.27 m and 2.00-2.95 m, respectively. The measurements obtained and examination results enabled us to confirm that the local isolates belongs to the genus Ascosphaera and to the species Ascosphaera apis. Key words: Chalkbrood disease, Ascosphaera apis, spore cyst, spore ball, Ascospores, Apis mellifera.
Characterization of two Nematodedestroying Fungi from Etillopia
(1999-06) Bedelu Theodros; Abate Dawit (PhD)
Under laboratory conditions, ART-l (LDso= 290 conidia/ml) was more efficient in destroying nematodes than ART-2 (LDso= 725 conidia/OIl), Investigation on the growth of MeloidogYlle incognita-infected tomatoes in the greenhouse showed that yield loss was highest in tomatoes treated with only k[, incognita (69.7%). Tomatoes treated with conidia of ART-lor ART·2 had lower losses: 44.5% for ART-l treated & 44% for ART-2 treated tomatoes. ART-2 required 3X the number of conidia of ART-l (180000 conidia/pot) to attain a nematode suppression effect similar to ART-I. Out of 3 solid media compared for their effects on the lateral growths of the isolates at room temperature, oatmeal agar (OA) gave the greatest colony diameters (6.3 cm for ART-l & 7.8 cm for ART-2). Maximal spore yields were also observed on this medium: ART·l, 4960 conidia/ml & ART-2, 31400 conidia/m!. A comparison of lateral growth, on medium A (MA), under different incubation temperatures (room temperature, 28°C, 32°C, & 37°C) showed that greatest colony diameters were attained at 28°C: 5 em & 5.7 cm for ART-l & ART-2 respectively. Broth of MA was found to be suitable for optimal mycelial biomass yield for both isolates (631 mg/IOO ml, ART·l; 755 mg/lOO ml, ART-2). However, ART-I, a slow grower on all solid media tested, gave a higher yit:;ld than ART~::! when grown in c~lej.n ID}ocr:tl :.;:tits·m€rll~~·! (G~S~l) broth (363 mg/lOO ml cr. 273 mg/IOO ml). Optimal mycelial biomass yield in MA broth was found to be in the pH range 4-5 for ART-2 (509·669 mgt 100 ml) & in the range 7·8 for ART-l (487-535 mg/IOO mI). ART·l & ART-2 produced extracellular proteases in CMS-2 broth. The proteases from both fungi had a temperature optimum of 50"C, & were optimally active in the alkaline range (7.5·8, ART-l & 7.5·9, ART- 2). ART-l produced more proteases (19 U/ml/hr) than ART-2 (14 U/ml/hr). ART-l proteases were more efficient in degrading nematodes than those of ART -2 by virtue of being produced in a larger quantity, suggesting that the difference in the nematode-destroying capabilities of ART-l & ART·2 was the result of the difference in the amount of proteases produced.
Evaluation of Ethiopian Isolates of Entomogenous Fungi as Potential Biological Control Agent of the Desert Locust, Schistocerca Gregaria.
(Addis Ababa University, 1998-05) Aysheshim, Seneshaw; Abate Dawit (PhD); Seyoum Emiru (PhD)
Ten native fungal isolates were collected from the different regions of Ethiopia. Eight of the isolates were identified at International Institute of Tropical Agriculture, UTA, Benin. Five of the isolates were identified as Beallveria, two were Paecilomyces and one belonged to Metarhizilllll species. Eight of the tested fungal isolates showed pathogenecity to fifth instar Schistocerca gregaria. The Beallveria spp. isolate FF and MetarhiziulII spp. EE were more virulent compared to the rest. Thus two isolates were further assayed in peanut (Arachis hypogaea) and noug (GlIizotia abyssinica) oil formulations, and compared with a known entomopathogen, M anisopliae ICIPE 30. The results indicated that there were strong doseresponse patterns. Peanut oil formulation enhanced infectivity of both FF and EE. Infectivity of EE was significantly (P<0.05) reduced in noug oil formulation. Comparison of virulence revealed that isolates FF and EE were more pathogenic than ICIPE 30. FF in peanut oil formulation had the lowest LCso value of2.02xlOs conidia/ml followed by EE, 2.93x10s and ICIPE 30 with 4.98x10s conidia/m!. The highest mortality was achieved by FF at Ixl08 conidia/ml, resulting in 50% and 100% mOliality in 4.2 days and seven days, respectively. The median lethal time taken to fOUlih, fifth and adult FF treated insects were 3.47, 4.14 and 4.83 days; respectively that indicated fourth instars are more susceptible. Insects killed as a result of FF infection developed red pigmentation and produced white powdery mass of spores on the external surface. The Metarhizilllll isolates EE and ICIPE 30 produced green sporulation on the integument when the cadavers were kept at high humidity. FF and EE grew at a temperature range of 24°C and 37°C, with peaks at 24°C and 28°C for FF and 28°C for EE. Malt extract agar favoured growth and sporulation of FF, whereas EE grew and sporulated faster on sabouraud dextrose agar. Germination of both isolates was significantly (P<0.05) high in peanut oil formulation than in noug oi!.
Evaluation of Yeast Biomass Production Using Molasses and Supplements
(Addis Ababa University, 2009-08) Milkessa Tamene; Abate Dawit (PhD)
Three yeast strains of saccharomyces cerevisiae, namely commercial baker’s yeast (BA), an isolate from teff dough (TE) and an isolate from tella(TL) were cultivated in the laboratory by submerged method to determine biomass yield. The biomass of these yeast strains was compared with respect to molasses concentrations(3% w/v,5% w/v,8% w/v and 10% w/v), pH(3.5,4.0,4.5,5.0 and 5.5),growth temperatures ( 250C,300C and 370C), duration of incubation( 24,48,72 and 96 hrs) and the effect of addition of supplements as treatments; T1-(NH4)2SO4 (0.5 % w/v),T2-(NH4)2SO4 (0.5 % w/v) and KH2PO4 (0.3 % w/v),T3-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v) and peptone (2% w/v),T4-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v),yeast extract(1%w/v) ,MgSO4.7H2O (0.05 % w/v ) and CaCL2.2H2O (0. 004 % w/v ),T5-(NH4)2SO4 (0.5 % w/v), KH2PO4 (0.3 % w/v), peptone (2%w/v),yeast extract(1%w/v),MgSO4.7H2O (0.05%w/v) and CaCL2.2H2O (0.004% w/v), biotin(0.005%w/v) and calcium panthetonate (0.0001% w/v). The contents of molasses were analyzed before the cultivation process and it was found that the molasses used for this study contains 43.1 % sugar, 0.25% total nitrogen, 1.56 % crude protein, 17.9 % moisture content, 82.1% dry weight and 11.7 % total ash. With respect to molasses concentration, BA isolate showed maximum biomass yield at 5%, 8% and10% concentrations, whereas TE isolates showed the same trend at 5% and 8% concentrations. TL isolate was found to accumulate the maximum yield at 8% molasses concentrations. In all cases, the isolates showed similarity in high biomass accumulation when they were grown at 8% w/v molasses concentration. Concerning the effect of pH on the growth of yeasts, isolate BA was found to be effective at all pH values except pH 5.5; whereas TE isolate was effective at pH 4.0, pH 4.5 and 4.5. Furthermore, isolate BA and isolate TE were also effective at pH 4.5. At pH 3.5 and 5.5, there was a steady decrease in biomass yield by all the isolates. With respect to incubation temperatures, the different isolates displayed biomass yield ranging from 1.27g/l to 3.25g/l. All isolates showed slow growth at 250C, and 370C with subsequent slow increase as the incubation temperatures increased. The highest biomass was observed at 300C by isolates BA (2.98-3.2g/l in 24-72 hrs), TE (2.91-3.1g/l); whereas isolate TLshowed biomass increase of 2.81g/l .Supplementing molasses media with (NH4)2SO4 (0.5% w/v) (Treatment1) increased the biomass of TL (5.6-5.9g/l), TE (5.6-6.2g/l), and BA (6.1-6.4g/l within 24 and 72 hrs. In all cases the maximum biomass was achieved within 48 hrs. When this compared with biomass accumulation on molasses alone, the inclusion of the supplemental nitrogen source showed 1.5-2 fold increase in yeast dry weight by all isolates. Comparing the growth of the isolates on molasses and ammonium sulphate as control the isolates did not show significant difference in biomass with further treatments (T2-T5). The incorporation of all the necessary supplements resulted in maximum biomass production by BA (8.0 g/l), followed by TE (7.5 g/l) and TL (6.5 g/l). In all the biomass propagation processes, the commercial baker’s yeast strain, BA was superior in giving high biomass yield. Further more the leavening action of the two yeast strains, i.e., an isolate from teff dough (TE) and commercial baker’s yeast (BA) was compared at room temperature and 300C. BA was found to be higher than TE both at room temperature and 300C. Key words/phrases/: Baker’s yeast (Saccharomyces cerevisiae), Biomass, Leavening action, Molasses, Supplements
Fusarium and Fusarium Toxins in Maize in Some Regions of Ethiopia.
(Addis Ababa University, 1997-06) Wubet, Tesfaye; Abate Dawit (PhD)
Maize Grain Samples Were Collected from stores and markets in Shashemene and Alemaya regions. Samples were rated as damaged, Hornlal and malted. Nannal and damaged samples were rated based on percentage of kernel discolouration, wrinkledness, floating ofke~le1s in water and attack by insects. Mycofloral study of the three sets of samples showed that the three tox.jgenic fungal genera: Fusarium, Aspergillus and Penicillium were COllunon in maize in Ethiopia. Among these toxigenic fungi Fusarium was the most common genus, comprising 17.5 % of the total fungi isolated and was recovered from 80.5 % of the samples investigated, Species of fusarium were most prevalent in damaged and malted samples than in nonnal samples. TIle ftmgus was represented in 91.7 % and 28 % of the total fungal isolates in damaged and malted samples respectively. In the normal samples, however, Aspergillus and Penicillium spp were more prevalent and they were represented in all the samples examined. Preliminary test result of screening toxigenic fusarium isolates using the brine shrimp (Artemia salina) bioassay showed that 93 % of them were tmdgenic. The frequently occurring toxigenic fusarium were identified to be Fusarium prolijeratum, Fusarium gramil1earum, Fusarium subglutillGns and Fusarium alllhophilum. Isolates of these frequent toxigenic species were grown on sterile shredded maize grain and their extracts were assayed for toxicity on the brine shrimp larvae. The results indicate production of methanol and/or chloroform soluble toxic metabolites. Bioassay directed isolation of toxic principles from chloroform extract of F. graminearum isolates showed production of zearalenonc and trichothecene compounds (TA and TB). Based on their physico· chemical data four compOlUlds that belong to the fumonisins were isolated from methanol extracts of F. proliferatum isolates. TIle results of the study on screening of toxigenic fusarium and bioassay directed isolatfon and purification of toxic principles indicated the importance of brine sluimp bioassay in screeuing toxigenic fusarium and detection of fusarium toxins. Toxicity assay results from extracts of selected grain samples of the three sets of samples indicated that 93 % of the tested sample extracts were toxic. In the natural occurrence study of the toxins, zearatenone and the trichothecenes (TA and TB) were detected in 50, 50, and 75 % of the damaged samples examined respectively. The estimated concentration ranges from 0-0.42 J.tglg for zearalenone, 0-1.5 J.tglg for TA and 0·2.3 J.tglg for TB. The fumonisins, on the other hand, were detected in all the samples examined with the highest concentrations in the malted and damaged samples. The results of this study showed that toxigenic fusarium are associated with maize samples and their toxins are fOlmd on the grain. TIle biological effect and thermostability of some of the toxins indicates the health hazard of consuming mould infected grains, particularly damaged and malted grains. This necessitates the quality control of maize grain destined for human consumption.
Isolation of Bioactive Compounds from a Forest Litter Decomposing Microfungus
(Addis Ababa University, 2009-08) Tsegaw Dawit; Abate Dawit (PhD)
In a screening of fungi isolates associated with litter decomposition (litotrophic) for bioactive metabolite production, LDMFS JP4 was isolated. It was identified to genus level using morphological (cultural) and microscopic characterization to belong to Geotrichum sp. A bioactive metabolite effective against bacteria and fungi, was isolated from this fungus at stationary phase of mycelium culture growing in solid substrate, rice. Through, bioassay guided fractionation of the crude extract using chromatographic methods, the active compound was isolated in pureform. The compound was found to exhibit activity. It was effective against gram positive (S. pnumoneae, S. aureus and B. subtilis) and gram negative bacteria (P. aeruginosa, E. coli and S. boydii) and the yeast, Candidia albicans. The active compound exhibited potent in-vitro antibacterial with a minimal inhibitory concentration (MIC) of 65-75μg/uL against Gram-positive bacteria and 75-80ug/uL against Gram-negative. It was less effective for the yeast, Candidia albicans. The pure compound is promising and further work on chemical characterization is recommended. KEY WORD, LDFs, taxonomic studies, solid substrate, bioactive metabolite, fractionation, minimal inhibitory concentration
Lovastatin: an Inhibitor of Cholesterol Biosynthsis from Pleurotus Mushroom
(Addis Ababa University, 2008-01) Sitotaw, Rediet; Abate Dawit (PhD)
Lovastatin is a secondary metabolite of certain filamentous fungi. It is one of the members of the drug family, statins. Lovastatin, inhibitor of cholesterol biosynthesis, is widely prescribed for the treatment of hypercholesterolemia. Statins have also recently been shown to inhibit HIV-1 infection by targeting cholesterol in the HIV and host membrane. This research was performed to determine the production of lovastatin from fruit body cultivated on sugarcane bagasse and solidstate fermentation on different substrate (Sorghum, Rice and Wheat bran) of two Pleurotus mushrooms species that are edible and commercially cultivated in Ethiopia. The two mushrooms investigated, P. ostreatus and P. sajor-caj, grown in mushroom growing house on sugarcane bagasse (60% moisture content), and yielded an average bioconversion efficiency of 22.58% and 17.67 % respectively. Ethyl acetate was selected as better solvent for extraction of lovastatin in lactone form from acidified samples that were detected on Thin Layer Chromatography (TLC). The crude extracts from fruit bodies of both species show positive result on TLC with same Retention factor (Rf= 0.57and 0.3 for lactone and acid form respectively) which was identical to the standard lovastatin. The TLC result of Solid-State Fermentation (SSF) on rice and wheat bran showed the presence of lovastatin whereas sorghum gives a negative result. For quantification, extraction of lovastatin (β-hydroxyl acid) from fruit body as well as SSF was performed with mixture of methanol: water (1:1 V/V) that was chosen as the most effective solvent for High-performance liquid chromatography (HPLC) analysis at pH 7.7. Highest amount of lovastatin was found from methanol: water (1:1 V/V) extracts of P. ostreatus fruiting body (132μg/g) of dry weight followed by P. sajor-caju (129μg/g). Among SSF, methanol: water extracts of fermented wheat bran and rice gave (11.7μg/g) and (3.69μg/g) of dry weight respectively. The HPLC result of samples prepared with ethyl acetate show low amount of lovastatin. The health benefits of consuming the Pleurotus mushroom particularly for individuals with high plasma cholesterol as well as HIV infection is promising and it grows on a variety of agricultural residues with high bioconversion efficiency.
Mosquito Larvicidal Bacillus Strains From Ethiopia
(Addis Ababa University, 1996-04) Seyoum, Aklilu; Abate Dawit (PhD); Gemetchu Teferi (PhD); Gebremichael, Teshome(PhD)
Four strains of laIVicidal bacilli isolated from soil and dead mosquito lalvae in Ethiopia and two intemational standard strains, Bacillus tituringiensis var. israelensis; IPS-82 and Bacillus sphaericus, SPH-88, were investigated for their lalvicidal potential using laIVae of the mosquito species, Culex: quinquefasciatus and Anopheles arabiensis. Among the four media types selected for cultivation of the strains, medium-2 and medium-3 were found suitable to enhance the toxicity of the Ethiopian strains. Moreover, culture incubation temperature and aeration rate were found to affect the laIVicidal potential of the Ethiopian strains. The laIVicidal efficacy (LCso values) of the Ethiopian strains ranged from 0.0086 mgll for Bacillus NA-82 on second instar laIVae of Cx. quinquefasciallls to 221.6 mgll for Bacillus NA-6 on third instar laIVae of All. arabiensis. IPS-82 showed the highest laIVicidal efficacy with LCso value of 0.0010 mgll against second instar laIVae of All. arabiensis. Two of the Ethiopian strains and IPS-82 showed a higher toxicity on larvae of All. arabiensis than on Cx. quinquefasciallls. All Ethiopian strains have high degree of morphological and biochemical similarities to the reference strain of B. tilllringiensis var. israelensis (IPS-82) and have been taxonomically classified as strains of B. Ihuringiensis. Moreover, serological characterization revealed that two of the Ethiopian strains, Bacillus NA-6 and Bacillus NA-71 are identical to B. tJllIringiensis var. seoulensis. The other two strains, Bacillus NA-IO and Bacillus NA-82 showed low level of titers to two of the type antisera tested and thus are possibly new serotypes of B. IJllIringiensis.
Pathogenicity of Ethiopian Isolates of Entomopathogenic Fungi against Glossina Morsitans Morsitans .
(Addis Ababa University, 1999-05) Hadis Mamuye; Abate Dawit (PhD)
Fungal pathogens of insects have increasingly shown promise as effective control agents. In this work pathogenicity of five local isolates, Mefarrhizilllll anisopliae (EE), Mefarrhizilllll sp. (MM), Beallveria bassiana (FF), B. bassiana (GG), B. bassiana (AK), and two exotic an isolates, M. anisopliae (ICIPE 30) and B. bassiana ( temporarily coded as Bb) were determined against laboratory reared Glossina lIIorsifans lIIorsifans. Fifteen days old cultures of the respective isolates were harvested in sterile distilled water (0.1 % Tween -20). After determining the concentration of spore suspensions using Neubauer improved hemocytometer, concentrations were adjusted to 1 X 108 ( the screening concentration used). Adult flies, less than a week old, were exposed to spore suspensions. Flies were chilled at 1°c for seven minutes prior to exposure. Flies were then exposed to the spores by dipping them in the suspension for 15 seconds, after which they were recovered and put back to their cages and to the insectary (70% RH, 25° C). Mortality was recorded for a maximum duration of 20 days. Among the Mefarrhizilllll species, MefarrhiziulII EE has demonstrated the highest performance (96.67%), followed by MefarrhiziulII ICIPE 30 (90.00%), and Mefarri1izillll1 MM (73.33%). The differences were not significant (P> 0.05). Among the B.bassiana isolates the Kenyan isolate has performed the best (81.67%). Beallveria FF, Beallveria GG, Beallveria AK, have shown per cent moralities 75.00%, 63.33%, 53.33%, respectively. The difference between Beallveria FF and Beallveria Bb was not significant (P<0.05) while the difference between Beallveria Bb and Beallveria GG was significant (P< 0.05). All, but Beallveria GG, were significantly better than Beallveria AK (P< 0.05). Spore production for Mefarri1izilllll MM and }.Iefarrhizilllll EE was determined on solid substrates, whole grains of rice, wheat, barley and sorghum. Both isolates have grown best on rice giving a yield of 1.42 X 109 spores/gram of rice for Metal'l'i1izilllll MM and 1.62 X 109 spore/ gram of rice for Metal'l'i1izilllll EE. The next highest yield was on sorghum, 3.10 X 108 spores/gram for Metal'l'i1izilllll MM and 2.25 X \0' spores/gram of wheat for EE. No correlation was observed between moisture content and the spore yield of grains. Mass production and utilization of selected fungal pathogens against tsetse as a bioinsecticide as a component of Integrated Pest Management is recommended.
Small-Scale Citric Acid Production on Solid-State Fermentation Using Aspergillus Niger
(Addis Ababa University, 2010-06) Feleke Hailemariam; Abate Dawit (PhD)
Citric acid is used in the food, beverage, pharmaceutical, chemical, cosmetic and other industries for applications such as acidulation, anti oxidation, flavour enhancement, preservation, and as a synergistic agent. It is used to flavour the drinks, jams and jellies, candies, water ice and wines. In recent years, a great deal of attention is given to alternative methods for citric acid production using agro-industrial residues. A citric acid producing Aspergillus niger isolate designated as ASGS was isolated from the Faculty of science Addis Ababa University green house soil. The isolate produced citric acid in solid state fermentation (SSF) using different agricultural products and by products (Maize, Rice and wheat bran) as substrates. Out of the three substrates, rice(R) was found to be better followed by the combination of wheat bran and rice (WR). The highest level of citric acid (110.6g/kg DR) was produced in Rice, with Rice-to- moisture ratio of 1:4 (w/v) at 30ºC and 72 hr. The weakest substrate for citrate fermentation was wheat bran with only 7g/kg citrate production. Maximum growth of the isolate was shown at 30◦c, it also showed growth and biomass accumulation at 35◦c. Wrinkled, dense, white mycelium was observed in the flask at 30◦c while at 25◦c was not shown. Citrate accumulation increased exponentially after 48hrs of fermentation and the maximum citric acid (82.6g/kg DR) was obtained at 72hr which declined gradually. Initial rice moisture content had a considerable effect on citric acid production by ASGS. A maximum citric acid production (91g/kg DR) was obtained with 80% moisture content. Citrate accumulation also increased significantly with increase in temperature from 25 to 30◦c and production slightly declined up to 35◦c. The highest value of citric acid concentration (98g/kg DR) was achieved at an initial pH of 4.5. Recovery trial yielded 1.2g citric acid from 10g of dry rice substrate which was white and solid crystalline. Key phrases: Aspergillus niger strain; Solid state fermentation; Citric acid.

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