Browsing by Author "Alemu, Tesfaye (PhD)"
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Item Antagonistic Activities of Pseudomonas Fluorescens Isolates, As A Bicontrol of Botrytis Fabae (Chocolate Spot Disease), Plant Growth Promoter and Inducer of Physiological Activities of Faba Bean (Vicia Faba)(Addis Ababa University, 2012-06) Alemu, Fekadu; Alemu, Tesfaye (PhD)Plant protection is an important area which needs attention since most of the hazardous inputs added into the agricultural system are in the form of plant protection chemicals. Production of the crop is, however, constrained by several infections including fungal diseases. The present study, Pseudomonas fluorescens isolates possess a variety of promising properties which make it a better biocontrol agent.Twelve Pseudomonas fluorescens isolates from rhizospheric soil of faba bean were evaluated for their antagonistic activity against Botrytis fabae that is known to attack faba bean crops. All Pseudomonas fluorescens isolates are employed in controlling chocolate spot diseases of plant. P. fluorescens10 (88.1%) showed high antagonistic activity against Botrytis fabae. In addition, all isolates were tested for antifungal activity against Botrytis fabae. All isolate of Pseudomonas fluorescens are indicated successfully employed in controlling chocolate spot diseases of plant due to their antifungal metabolites.The antifungal compounds were extracted from all P. fluorescens isolates with equal volume of ethyl acetate, hexane and methanol.The antifungal compounds extracted with ethyl acetate, hexane and methanol from P f3, P f8 and P f3 isolates at 0.1% concentration completely inhibited the pathogen growth respectively. Four fungicides were tested against B. fabae and Curzate ® WP and Sancozeb 80% were prevented mycelia growth at higher concentration (80mg/ml per plate). All isolates Pseudomonas fluorescens were assessed for their plant growth promoting activity based on their ability to produce hydrogen cyanide (HCN), siderophores, indole acetic acid (IAA), ammonia and phosphate solubilization. The results indicated that all most of the isolates tested possess plant growth promoting traits. Bio-primed faba bean seed with P f9 and P f10 for pathogencity test in green house was indicated to show positive result. Two isolates of P f9 and P f10 was reduced both disease severity and incidence, increasing faba bean number of leaves per plant, number of branches per plant, height of plants, root length of plant and lateral roots and number of nodule. The present study was also carried out for estimation of total phenols and flavonoids present in fresh faba bean leaf extract with ethanol. Applying the P f9 and P f10 by bio-primed seed of faba bean treatment enhanced the accumulation of total phenols and flavonoids compared to untreated infected and uninfected untreated faba bean. So it could be concluded that the used P f9 and P f10 could resist the detrimental effects of Botrytis fabae on the plant growth and yield. These isolates can be used as potential biofertilizers and also as biocontrol agents. Key words: Antifungal compounds, Botrytis fabae, Biocontrol, faba bean, Pseudomonas fluorescens, secondary metabolitesItem Biological Control of Anthracnose (Colletotrichum Species) of Sorghum (Sorghum Bicolor L.) Using Trichoderma Isolates Under in Vitro Conditions(Addis Ababa University, 2019-10-10) Hadush, Goitoom; Alemu, Tesfaye (PhD)Biological control with microbial antagonists is considered an alternative approach for controlling plant diseases. Trichoderma species are one of the potential fungal biocontrol agents in suppression of soil-borne and foliar pathogens. The present study aimed to evaluate, test and characterize potential biocontrol agents of Trichoderma isolates against two pathogenic isolates of Colletotrichum species under in vitro condition. Infected sorghum leaf, sheath, stalk and soil samples were collected from different ecological habitat of Welkait district for the isolation of Colletotrichum isolates. The study examined the effect of pH and temperature on the mycelia growth and spore yield of Trichoderma isolates in broth culture. In this study, the in vitro potential of 7 Trichoderma isolates were evaluated against two pathogenic Colletotrichum isolates in dual culture techniques and through production of volatile and non-volatile inhibitors. Pathogenicity test was confirmed Colletotrichum isolates as one of the fungal organisms responsible for sorghum anthracnose disease. It was observed that the optimum pH and temperature for maximum mycelial growth and spore yield produced by Trichoderma isolates in broth cultures was pH 7.5 and 25OC, respectively. In vitro screening results showed that the proportion of isolates with antagonistic activities was the highest for the AUC-1 isolate followed by AUC-2 isolate. In vitro confrontation analysis revealed that all Trichoderma isolates were highly antagonistic against AUC-1 whereas AUT-97, AUT-131, AUT-11 and AUT-12 isolates displayed over 75% inhibition of mycelial growth of AUC-2 isolate (P<0.05). The isolates AUT-11 and AUT-12 showed consistent results in volatile and non-volatile activity under in vitro condition against any of the two pathogen isolates tested. Under in-vitro dual culture test, all isolates of Trichoderma were able to inhibit the growth of both Colletotrichum isolates at rates ranging from 58.92% to 90.29% after 8 days of incubation. The highest mean inhibitory effect on the growth of the test pathogens were achieved by AUT-11 isolate (90.29%) against AUC-1 and AUT-97 isolate (81.1%) against AUC-2 while AUT-32 isolate showed the lowest mean inhibitory effect restricting it almost completely in plates as compared to the control consisting of any of the two test pathogens growing alone. In dual culture, volatile and nonvolatile tests, all the Trichoderma isolates significantly inhibited the radial growth of the two Colletotrichum isolates at (P<0.05) compared with control. Thus, the use of novel isolates of Trichoderma with efficient antagonistic capacity against Colletotrichum isolates is a promising alternative strategy to pesticides for sorghum anthracnose disease management.Item Characterization and Pathogenicity of Blast Pathogen (Pyricularia grisea of Wild Finger Millet (Eleusine Africana and its Control Using Biological Antagonists and Fungicides(Addis Ababa Universty, 2017-02) Ayele, Fantaye; Alemu, Tesfaye (PhD)Blast (Pyricularia grisea) is an important disease of wild finger millet (Eleusine africana). It causes disease on a wide variety of alternative hosts including agriculturally important plants such as finger millet (E. coracana) in Ethiopia. A total of twenty six isolates of P. grisea were isolated from infected wild finger millet plants from 5 different zones of Ethiopia. From the initial twenty six isolates, only nine isolates were selected for the detailed morphological, cultural, physiological and biochemical characterization based on their geographical location and isolate type, whether it is isolated from stem, leaf, neck or seed parts. Among the different media used host seed extract + 2% sucrose agar and oatmeal agar were found to be the best for mycelial growth and sporulation of the isolates, dextrose was the most preferred among the carbon sources and NaNo3 proved to be the best source of nitrogen for all the isolates. The shape, color and compactness of the fungal colonies varied with the media used. At pH 6.5 all the isolates exhibited maximum mean dry mycelial weight, and found optimum pH. The optimum temperature range for the maximum growth of the isolates of P. grisea was found to be 20°C to 30°C. All the six P. grisea studied under green house for their pathogenicity, were able to infect all the tested varieties. Among an In vitro evaluated fungicide against the test pathogen, both Curzate and Sancozeb fungicides showed varied levels of antifungal activity and the highest concentration (800 and 1000ppm) of fungicides were found of inhibiting the growth of the isolates of P. grisea. With regard to the efficacy of the fungicides, Sancozeb was found to be the most effective to inhibit the isolates. Among in vitro tested biocontrol agents viz. Trichoderma viride, Trichoderma harzianum and Pseudomonas fluorescence, the growth of Trichoderma species was fast, the clear inhibition zones were not formed in all incubated Petri plates, therefore it was more effective than the bacterial antagonist. Compatibility of the biocontrol agents with different fungicides (Curzate and Sancozeb) that is., Trichoderma harzianum and Trichoderma viride in an in vitro condition resulted in that, both Curzate and Sancozeb, in lower concentrations (100, 200, and 400ppm) were found well tolerated with both Trichoderma species and hence they were shown effective in managing plant pathogens. Combining antagonists with synthetic chemicals eliminates the chance of resistance development and reduces the fungicide application. Keywords: Finger millet, Pyricularia grisea, biocontrol, fungicide, compatibility, pathogenicityItem Characterization and Testing of Antifungal Extracts from Trichoderma isolates against Fusarium xylarioides, the Causative agent of Coffee Wilt Disease(Addis Ababa University, 2012-06) Mulatu, Afrasa; Alemu, Tesfaye (PhD); Megersa, Negussie (PhD)Coffee Wilt Disease (CWD) is a vascular disease caused by the fungal pathogen; Fusarium xylarioides and one of the most important diseases of coffee that was prevalent in Ethiopia. The use of indigenous antagonistic isolates of Trichoderma would be a nature conserving means to combat this disease. The current research work was designed to isolate, extract, characterize, evaluate and determine the antifungal compounds from Trichoderma isolates against F. xylarioides. An experiment was conducted to extract, characterize, purify, evaluate and determine the antagonistic potential of antifungal compounds from six Trichoderma isolates that were inhibitory towards F. xylarioides. For extraction of antifungal compounds from fungal mycelium or culture media different organic solvents: CHCl3, EtOH, MeOH, EtOAc, n-hexane, and butane were used. The chloroform, ethanol and butane extracts were screened for their antifungal activity. A direct bioautographic procedure, involving spraying suspension of F. xylarioides on TLC plates developed in solvents of varying polarities was used to detect a number of antifungal compounds present in the extracts. Moreover, in-vitro antagonistic bioassays were performed to test, evaluate and determine the potentiality of Trichoderma isolates as biological control agents against F. xylarioides. The study indicated that antifungal compounds were successfully extracted from fungal culture media with all organic solvents used except hexane. For purification and separation of crude extracts on a TLC, the optimum Rf value was obtained by only three extracting solvents: CHCl3, EtOH and Butane using seven pre-screened solvent systems. Bioautography assay revealed 60 zones of inhibition spots and the highest inhibition zone was observed in AUT5 (51 mm) and AUT6 (44 mm) with EtOH extract at Rf value of 0.43. In in-vitro bioassay, the highest mean inhibitory effect on the growth of the pathogen was achieved by AUT2 (77.4%) isolate followed AUT3 (72.9) in dual culture. The minimum inhibitory concentration (MIC) of non-volatile compounds was observed in isolate AUT3 and AUT6 to be 5% of culture filtrate. In general, TLCdirected bioautography assay was useful in isolating active compounds with antifungal activity and all Trichoderma isolates were accounted for reduction in mycelial growth of the test pathogen in-vitro. Keywords: Antagonistic activity, Bioassay methods, Bioautography, Organic solvent polarity, Thin layer chromatographyItem Characterization of antagonistic Trichoderma species against Coffee Wilt Disease (Fusarium xylarioides)(Addis Ababa University, 2010-12) Urbanos, Yonas; Alemu, Tesfaye (PhD)In vitro antagonistic activity of Trichoderma isolates against coffee wilt disease (CWD), Fusarium xylarioides (Gibberella xylarioides) was studied. Out of 32 soil samples and 7 coffee tree parts collected from 6 different coffee growing woredas of Jimma Zone, 7 potential/effective Trichoderma isolates were isolated and characterized out of 74 fungal cultures. Trichoderma isolates were characterized based on spore morphology and vegetative growth characterizations on different growth media, temperature and pH ranges. The most suitable and maximum growth of Trichoderma isolates was obsereved at pH 4.5 and 5.5 in all of the isolates. In vitro evaluation of dual culture test was done for each of the seven Trichoderma isolates against F. xylarioides. It was observed that all of the seven Trichoderma isolates have shown 63.33 to 70.95 percent of mycelial growth inhibition of F. xylarioides after 6 days of incubation at 250C on potato dextrose agar medium. Trichoderma isolate AUT4 was the most effective biocontrol agent which exhibited the maximum inhibition of 70.95%. Whereas, the least percent of mycelial growth inhibition 63.33% was obtained by Trichoderma isolate AUT3. In addition to the above, the seven isolates of Trichoderma were then tested for their production of volatile antifungal compounds against F. xylarioides. They produced volatile compounds which inhibited the mycelial gorwth of the test pathogen from 66 to 80.60%. Trichoderma isolate AUT1 produced the most effective volatile compounds which inhibited growth by 80.60%. Therefore, the application of in vitro evaluations of seven Trichoderma isolates has proved to be highly potential/effective in the control of coffee wilt diseases (Fusarium xylarioides). Key words: Biological Control agents, cofffee wilt disease, Fusarium xylarioides, Trichoderma isolates.Item Characterization of Bacterial Wilt Pathogen (Ralstonia solanacearum L.) of Ginger (Zingiber officinale Roscoe) and Its Controls Using Trichoderma and Pseudomonas Isolates(Addis Ababa Universty, 2017-04) Ibrahim, Abdella; Alemu, Tesfaye (PhD)Ralstonia solanacearum (Rs) race 4 causes bacterial wilt of edible ginger (Zingiber officinale) and limits the crop production in the tropics. The present study was undertaken to isolate and characterize ginger bacterial wilt agent (Ralstonia solanacearum) and to evaluate inhibition of Ralstonia solanacearum by Trichoderma and Pseudomonas isolates in a green house and laboratory experiments in 2014/15 season. Totally, 10 isolates of Trichoderma and 10 isolates of Fluorescent pseudomonas were isolated and characterized. Inhibitory effect of the biocontrol agents was evaluated in vitro following agar well diffusion method for their efficacy against Ralstonia solanacearum. The experimental design was complete randomized design (CRD) with three replications. Data on yield and quality parameters, disease reactions, biocontrol efficacy had been collected and analysed. Based on the result of In vitro inhibition test only four isolates that inhibit the growth of the pathogen were selected and used under the green house experiment. In vitro antagonistic activities of Pseudomonas fluorescens1 and Pseudomonas fluorescens2 showed the maximum inhibition of Ralstonia solanacearum with an inhibition zone of 30 and 39.5 mm respectively. Among antagonists tested P. fluorescens, were found to be more effective in the reduction of disease severity and also increased the biocontrol efficiency when compared to untreated control by 44.4 and 92%, respectively. Also, application of antagonists significantly increased plant yield by 1.6 ton/h. ANOVA showed that a wisely designed combined treatment was found to be more effective than single treatment application. Overall, the result showed that the selected antagonistic isolates managed to suppress the disease caused by the pathogen, R. solanacearum effectively. Thus, it can be concluded that the incidence and severity of bacterial wilt of ginger disease can significantly be reduced by the combined use of antagonists in an equal suspension with foliar spray in order to have a higher yield and with minimum health risk as well as environmental pollution. Cultural characteristics of isolates on Tetrazolium Chloride (TZC) Agar selective medium showed light to red color with the characteristic red center which resembles the pathogen Ralstonia solanascearum. Biochemical and physiological characteristic revealed that similarities among isolates and strains were grouped in biovar III and corresponds to race 4 of the pathogen. Hence, further study is necessary to check the ability of the anatagonists to control the disease under field conditions. Keywords: bacterial wilt, biocontrol, biovar, ginger, race 4, Ralstonia solanacearumItem Effects of Inorganic Fertilizers and Brassica Species Leaf Extract and Green Manure in Controlling Enset Bacterial Wilt in Gurage and Silte Zones, Ethiopia(Addis Ababa University, 2020-06-06) Desta, Bruktawit; Fetene, Masresha (Professor); Alemu, Tesfaye (PhD)Enset (Ensete ventricosum) is one of the most important multipurpose crops grown in Ethiopia that is a staple food for approximately 20 million people in the country. The production and sustainability of enset agriculture is, however, threatened mainly by bacterial wilt of enset, which is caused by Xanthomonas campestris pv. musacearum (Xcm). This study was conducted in Gurage and Silte zones of Ethiopia with the objective of exploring the effects of inorganic fertilizers and Brassica species leaf extract and green manure in controlling enset bacterial wilt. The study began with an assessment of the prevalence and incidence of Xcm in relation to altitude i.e. low, mid and high altitudes. The assessments were done by random sampling of 60 farms. Qualitative analysis of leaf extracts was carried out using standard methods. The role of different levels of NPK fertilizers to control Xcm in tolerant (Yeshrakinkye) and susceptible (Ameratye) enset clones were evaluated under field condition. Again, the effects of selected Brassica species as a green manure and Brassica carinata seed extract residue on growth, physiology of enset clones and their effect on the incidence and severity of Xcm were evaluated under field condition. Field experiments were laid in Randomized Complete Block Design with three replications. Standard procedures were used to collect data for all studies. The data iv were analyzed using descriptive and inferential statistics in SPSS and Microsoft Excel software. The result showed that mid altitude had the highest disease prevalence (DP) in 2014 (55%) and 2015 (43.8%), the highest disese incidence (DI) in 2014 (12.2%) and in 2015 (8.2%) compared to low and high altitude sites. This indicated that there were altitudinal based variations on enset bacterial wilt DP and DI in the study areas. The extracts of Brassica oleracea var capitata and Brassica oleracea var acepala created the widest bacterial growth inhibition zone at (400 and 200 mg/mL) compared to other concentrations. This suggests that Brassica oleracea var capitata and Brassica oleracea var acepala controls the growth and development of Xcm. Qualitative phytochemical screening results revealed that the chemical constituents of extracts vary between Brassica species. The highest total phenolic content was recorded from Brassica carinata seed extracts residue, Brassica oleracea var capitata (Cabbage) and Brassica oleracea var acepala (Tekur Gomen). The result of field experiment showed that inorganic fertilizers treatments with N1/2P1/2K1/2, NPK and N3/2P3/2K3/2 significantly (p<0.05) decreased DI and disease severity (DS) and increased all growth parameters of both enset clones including plant height, pseudostem girth, green leaf number, leaf length, leaf width, leaf area and leaf area index at different measuring periods compared to positive controls. Application of N1/2P1/2K1/2, NPK and N3/2P3/2K3/2 decreased the DI of tolerant enset clones by 6.8%, 7.7% and 13.8%, respectively compared to the positive control. In the same way, the DI of susceptible enset clones was decreased by 22.2%, 27.8%, and 33.1 %, respectively. Similarly, DS of tolerant enset clone was decreased with application of N1/2P1/2K1/2, NPK and N3/2P3/2K3/2 by 12.4%, v 17.3% and 35.2%, respectively as compared to positive control whereas that of susceptible enset clones was decreased by 10.1%, 15.7%, and 17.9 %, respectively. Similarly, the lowest AUDPC value (623) was recorded on tolerant clones treated with N3/2P3/2K3/2 fertilizers while the highest AUDPC value (1455) was recorded in a susceptible clone compared with positive control. Application of Brassica species as green manures such as Brassica oleracea var capitata (G1) and Brassica oleracea var acepala (G2) and Brassica carinata seed extract residue (G3) did not bring significant difference at (P >0.05) all growth parameters compared to positive controls. Similarly, compared to the positive control application of these Brassica plants as green manure and Brassica carinata seed extract residue didn’t bring significant difference (P < 0.05) on all physiological parameters except for assimilation rate and on functional parameters of photosynthetic apparatus (chlorophyll content). However, compared to the positive control, G1, G2 and G3 decreased the DI of tolerant enset clones by 19.4%, 23.3% and 23.1%, respectively. In the same way, application of G1, G2 and G3 treatments decreased the DI of susceptible enset clones by 6.7 %, 12.3 %, and 4.6%, respectively. DS of tolerant enset clone with G1, G2 and G3 decreased by 5.3 %, 7.8% and 11.4%, respectively as compared to positive control. Similarly, application of G1, G2 and G3 treatments decreased the DS of susceptible enset clones by 11.4 %, 8.6 %, and 10 %, respectively. The lowest AUDPC value (976.5) was recorded on tolerant enset clone treated with G3 while the highest AUDPC value (1828.9) was recorded in susceptible clone. In general, the results of the present study showed use of recommended levels of NPK and N3/2P3/2K3/2 amount improved the growth performance of enset and reduce the effect of vi bacterial wilt on enset clones. Moreover, use of Brassica oleracea var capitata and Brassica oleracea var acepala as green manure and Brassica carinata seed extract residue were effective to control enset bacterial wilt. From these results, it can be recommended that combined NPK fertilizer and brassica plants green manuring is crucial to control bacterial wilt of enset.Item Evaluation and Optimization of Agro-industrial Wastes for Conidial Production of Metarhizium and Beauveria Isolates under Solid State Fermentation(Addis Ababa Universty, 2015-01) Tewelde, Abiy; Alemu, Tesfaye (PhD)Two isolates of Beauveria and three isolates of Metarhizium were grown on different agricultural wastes to evaluate their conidia production potential under SSF system. Coffee husk, Tea waste, Wheat bran and Vegetable wastes were used as substrates to determine maximum conidiation of the isolates. Among these vegetable wastes were the best medium to yield 5.77±1.53 (107), 5.80±0.72 (107), 4.44 ± 0.55(107) and 5.58 ± 0.66 (107) conidia/gram of substrate under quantitative assessment for isolates (AUBI2, AUMI1, AUMI2 and AUMI3 respectively) at 60% moisture content. The highest conidia count record of AUBI1 6.34 ± 2.27 (107) conidia/gram was obtained at 60% moisture content on wheat bran. It has been observed that two sample t-Test statistical analyses of conidial yield of vegetable wastes was significantly different from 2mm and 4mm particle size of coffee husk used as substrates. The optimization for temperature indicated that all substrates supported maximum conidia yield with-in the range of temperature from 27 to 300 C. Beauveria isolate AUBI1 was produced 11.63 ± 8.34 (107) conidia/gram of substrate at coffee husk when treated at 3.5 pH value. The pH values used in the present study for optimization best favored only for coffee husk as substrate. The high conidia yielding substrates were best productive at their own natural at 6.29 for Vegetable wastes, 6.63 for Wheat bran and 5.4 for Tea waste pH values. Vegetable wastes supported AUBI1, AUBI2, AUMI1, AUMI2 and AUMI3 to produce high conidia yield of 4.31 ± 1.18 (107), 5.07 ± 0.76 (107), 3.43 ± 0.30 (107), 1.86 ± 0.16 (107) and 5.17 ± 0.61 (107) conidia/gram of substrate at optimum incubation period of 21 days. The optimum incubation period of the fungal isolates on wheat bran is 3 - 4 weeks under sufficient exposure to light. AUBI1, AUBI2 and AUMI1 produce high conidia under exposure to light but AUMI2 and AUMI3 at dark condition when cultivated on vegetable wastes. Therefore, as successful microbial control of insect pests depends on cheap and large scale industrial productivity, entomopathogens cultivation on vegetable wastes and wheat bran can be attained by SSF system. Key words: Beauveria, Metarhizium, Inoculation, Optimization and ConidiaItem Evaluation of Antifungal Activity of Trichoderma Isolates and Their Crude Extracts on Coffee Wilt Pathogen (Gibberella Xylarioides)(Addis Ababa University, 2018-05-05) G/Michael, Gizachew; Alemu, Tesfaye (PhD)Coffee wilt disease (CWD) caused by the fungus Fusarium xylarioides is an important soil-borne disease reducing coffee (Coffea arabica L.) production yields at up to 60%. The present study aimed to evaluate, test and characterize potential biocontrol of rhizosphere Trichoderma isolates and their most active antifungal crude extracts against coffee wilt pathogen (Gibberella xylarioides). In vitro antagonistic bioassays were performed to evaluate and determine the potentiality of Trichoderma isolates as biocontrol agents against F. xylarioides. For extraction of antifungal crude extracts from fungal mycelia organic solvents such as chloroform, ethanol, methanol, ethyl acetate, n-hexane and acetone were used. A direct bioautographic procedure, involving spraying suspension of F. xylarioides on TLC plates developed in solvents of varying polarities to detect a number of antifungal substances present in the extracts. In in-vitro bioassay the experimental results showed that all isolates of Trichoderma were able to inhibit the growth of F. xylarioides under in vitro experiment at rates ranging from 54.8% to 83.2% after 10 days of incubation. The highest mean inhibitory effect on the growth of the test pathogen was achieved by Addis Ababa University Trichoderma isolate 71 (AUT-71) (83.2%) followed by AUT-03 (72.8%) and AUT-86 (67.2%), while AUT-89 isolate showed the lowest (54.8%) mean inhibitory effect restricting it almost completely in plates as compared to the control consisting of F. xylarioides growing alone. AUT-32 and AUT-66 isolates produced zones of inhibition which is an indicative of the production of secondary metabolite(s) inhibiting the mycelial growth of F. xylarioides. Bioautography assay revealed that 94 zones of inhibition spots and the highest inhibition zone was observed by SS1 in isolates AUT-21 (53 mm) at Rf (0.75), and AUT-66 and AUT-89 (35 mm) with chloroform extract at Rf (0.51) and at Rf (0.55), respectively. Thus, the use of novel isolates of Trichoderma with efficient antagonistic capacity against F. xylarioides is a promising alternative strategy to pesticides for coffee wilt disease management.Item Evaluation of Endophytes Associated With Maize and Entomopathogenic Fungi Against the Spotted Stem Borer, Chilo Partellus(Addis Ababa University, 2019-01-02) Kebede, Denberu; Alemu, Tesfaye (PhD); Tefera, Tadele (PhD)Larvicidal effect of maize endophytes isolated from root and stem of maize and pathogenicity of selected entomopathogenic Beauveria spp. and Metarhizium spp. isolates against C. partellus were studied. Among 39 maize endophyte isolates screened for insecticidal activity, 11 isolates (8 bacteria and 3 fungi) showed larvicidal activity against C. partellus. Pathogenicity of crude extracts of the 11 isolates significantly varied in mortality ranging from 4.79% to 50.26%. The Isolate aaueb-11 under the genus Bacillus produced the highest mortality (50.26%) which was at the same significance level to the positive control Bt. Kurtsaki (60.13%). Eight EPF isolates were selected from 27 isolates and there was a significance variation in causing mortality against larvae of C. partellus. EPF isolates APPRC-34GM, S#10H and APPRC-44BC were the most virulent strains with mean percent mortality of 81.39%, 90.00% and 90.00% respectively. Lethal time and lethal concentrations required to kill 50% and 90% of the experimental insects of the most virulent EPF strains were also determined. The isolate S#10H showed relatively short time to kill 50% of the population where the highest time recorded was 6.15 days. There was a general increase in LT50 with decrease in conidial concentration. The highest dose of the isolate S#10H, required to kill 50% and 90% of the larval population were 5.95x104 and 2.2x107 conidia per ml, respectively. The effective isolates could be a promising biocontrol agent against larvae of C. partellus. However, the specific compound contributing on the insecticidal activity from the crude fermentation extracts of endophytes need insecticidal spectra study and further isolation and purification to incorporate in microbial based biopesticide development. Thus, the active ingredients and compound structures should be further studied.Item Evaluation of Trichoderma Species Against Fusarium Wilt and Alternaria Leaf Blight of Sesame (Sesamum Indicum L.) Under In Vitro Condition(Addis Ababa University, 2019-10-10) Goshu, Hadis; Alemu, Tesfaye (PhD)The present study was aimed to evaluate and characterize the potential biocontrol agents of Trichoderma isolates against two pathogenic fungi viz., Alternaria isolates (AUA1) and Fusarium isolates (AUF5) under in vitro condition. Infected leaves, seeds and soil samples of sesame were collected from different habitats of the Wolkait district for the isolation of fungal pathogens. The pathogenicity test of these isolates was confirmed on detached leaves of sesame plants resulting in typical symptoms of leaf blight and wilt disease. In this study, the in vitro potential of 7 Trichoderma isolates were evaluated against two isolates of foliar and soil borne phytopathogenic fungi in dual culture techniques and through production of volatile and non-volatile inhibitors. The effect of pH and temperature on the mycelia growth and spore yield of Trichoderma isolates were determined PDB medium. While, the optimum temperature for mycelial growth and the maximum spore yield was produced at 25OC. Among the seven Trichoderma isolates, AUT-12 (93 %), AUT-158 (91.6 %) and AUT-97 (90.8%), exhibited a significant (P< 0.05) enhancement of germination percentage in sesame seeds under in vitro conditions, while the control treated significantly (p<0.05) decreased these values (55 %). In vitro confrontation analysis revealed that all the Trichoderma isolates were highly antagonistic and AUT-131 isolate displayed over 75% inhibition of mycelial growth against both test pathogens under dual culture. Except isolates AUT-32 and AUT-158, all the other Trichoderma isolates showed consistent results in dual culture test, volatile and non-volatile activity against any of the two pathogens tested. Under in-vitro dual culture test, the experimental results showed that all the isolates of Trichoderma were able to inhibit the growth of both test pathogens at rates ranging from 66.29% to 79.49% after 6 days of incubation. The highest mean inhibitory effect on the growth of the test pathogens were achieved by AUT-131 isolate (79.49%) against AUF5 and AUT-97 isolate (76.42%) against AUA1 while AUT-33 isolate showed the lowest mean inhibitory effect restricting it almost completely in plates compared to the control consisting of any of the two test pathogen growing alone. The highest (90.12%) non –volatile inhibition effect was exhibited by both AUT-11 and AUT-12 against AUF5 isolate, while the lowest (16.38%) inhibition was obtained by AUT-32 isolate. Thus, the use of novel isolates of Trichoderma with efficient antagonistic capacity against Alternaria (AUA-1) and Fusarium (AUF5) isolates is a promising alternative strategy to fungicides for sesame disease management.Item Evaluation of Trichoderma Species as Biological Control Against Fusarium Wilt (Fusarium Spp.) on Cotton Plant (Gossypium Herbaceum) Under in Vitro Condition(Addis Ababa University, 2019-10-10) Asmekash, Tsegay; Alemu, Tesfaye (PhD)In Ethiopia, cotton is one of the most important cash crops that widely grown in the country. The production trend of cotton has been on the decline over the years, mostly as a result of infectious diseases from fungal pathogens. Thus the overall objective of the study was to evaluate local Trichoderma isolates for their pathogenicity potential against Fusarium isolates. The ability of the isolates to antagonize Fusarium isolates was evaluated in invitrodual culture assays and using volatile and non-volatile metabolites produced by the isolates.The effect of temperature and pH on the growth of Trichodermaisolates was also evaluated under a pH value ranging from 4.5-7.5 and level of temperature at 4oC, 25oC and 37oC.The inhibition test effects were analyzed using the analysis of variance (ANOVA) and the significance of the means was evaluated by Tukey‟s HSDtests. Out of27 isolates one isolate wasmorphologicaly identified as Fusarium pathogen isolate then selected and subjected to pathogenicity and antagonize test effects.The pathogenicity result showed the typical symptoms of the pathogen on the inoculated detached leaves and seedlings.Among the fourTrichodermaisolates, T131 showed the highest inhibitory 78.89% effect against the pathogen at based on dual culture assay. Use of volatile and non-volatile metabolites produced by the Trichoderma isolates also confirmed that there was a production of inhibitory substances.TheTrichoderma isolatesT131 (48.14%) and T136 (52.89%) showed the highest inhibitory effect against the pathogen.It is clearly observed fro the results of Trichodermaisolates were successfully grown at 25oC and 37oC.Similarly,Trichodermaisolates sporulated at different pH values. However, the pH change was not significant except in Trichoderma isolate GimT7. In the present study, T 131 were found to be the best isolate against cotton wilt pathogen (Fusarium spp).Item Fungi Associated With Shboot Dieback of Podocarpus Falcatus(Addis Ababa University, 2006-07) Yirgu, Abraham; Gezahgne, Alemu (PhD); Alemu, Tesfaye (PhD)In Ethiopia, information available on tree disease is limited and scanty. In the field survey conducted in Menagesha-Suba and Munesa-shashemene natural forests the shoot dieback was observed on Podocarplls falcalus. Symptoms were initially observed on leaves and two-three small circular brown spots were found at the vety tip of the leading shoot. Gradually the color of the leaves changes into yellow, and to light brown as the shoots die. This symptom of dieback was observed both on seedlings, sapling and mature trees. A study was conducted to understand which fungi was associated with shoot dieback. Isolation was made ii-om the symptomatic plant part. Culture and spore morphology was used to characterize the most common and frequently isolated fungi. Accordingly, it was understood that 3 isolates belong to Alternaria genus, 4 isolates that grouped into genus Phoma, 1 isolate to Pestalotiopsis and I isolate to genus Fusarium was found. Among these isolates of Alternaria, isolates Pf-24 and 32 seem to be close to A.alternala and the other one isolate Pf-53 seems to be close to A. arborescens. Two of the isolates in the Phoma, Pf-42 and Pf-22 seems to be related with P. exigI/a and the other one resemble P.leveillei. The other two isolates Pf-12 and 21 were unidentified. In the inoculation test all the four groups of fungi produced lesion only on wounded leaves. The importance of these fungi in relation with shoot dieback, however, needs further detailed investigation. As far as our knowledge is concerned this is the first report of shoot dieback on P falcatlls in the natural forest of Ethiopia. The impact of these diseases on Pfalcalus should get due attention in the management of P.filicallis forest in Ethiopia. KEY WORDS: Podocarplls filicatus, Alternaria species, Phoma species, Pestaloliopsis species, Fusarium species, Shoot diebackItem Incidence and Phenotypic Characteristics of Coffee Berry Pathogen (Colletotrichum Kahawae) from Sidama and Gedeo Zones and its Control Using Trichoderma Isolates.(Addis Ababa University, 2018-05-04) Dagnew, Wubet; Alemu, Tesfaye (PhD)The experiment was done to assess incidence, severity and variations in Colletotrichum kahawae isolates. Assessment was conducted in Aleta Wendo, Yirgacheffe, Shebedino, Wenago and Dale of Gedeo and Sidama Zones of Southern Region by examining and recording of the incidence and severity of CBD. The survey revealed that CBD was prevalent in all the surveyed districts. The overall mean incidence and severity at district level were 47.9 and 29.0%, respectively in 2015 cropping season and 40.0 and 25.6%, respectively in 2016 cropping season. Seven representatives of Colletotrichum kahawae isolates were isolated from Southern region of Ethiopia and were studied based on their cultural and morphological characters. Isolates were grouped into 4 based on their colony color manifestation on PDA and MEA. The cultures exhibited light gray, gray, dark gray and dark olive green mycelia forms. Mean radial colony growth rate of Colletotrichum kahawae isolates were 4.8, 5.1, 5.0 and 4.4mm/24hr on PDA, MEA, fruit and leaf extracts, respectively. Conidia width and length ranged as 3.5 – 6.7 µ m and 14.0 – 17.8 µ m, respectively. High variation was observed among Colletotrichum kahawae isolates in their sporulation capacity, and varied between 4.83 x 105 and 2.14 x106conidia/ml. An in vitro evaluation of dual culture technique and detached berry test exhibited that the mycelial growth of the pathogenic fungus is suppressed by Trichoderma isolates. Two isolates of Trichoderma showed overgrowth on Colletotrichum kahawae isolates with average inhibition of 74.54 % and 75.75 % by AUTI and AUT2 isolates, respectively; and there was no disease symptom appearance when Colletotrichum kahawae isolates were with Trichoderma isolates inoculated on detached green berries.Item Isolation and Characterization of Endophytic Trichoderma Isolates for Antagonistic Activity Against Coffee Wilt Disease (Gibberella Xylarioides) in Keffa Zone, Southwestern Ethiopia(Addis Ababa University, 2018-09-04) Berihun, Beyene; Alemu, Tesfaye (PhD)The present study aimed to evaluate, test and characterize potential biocontrol of endophytic Trichoderma isolates against coffee wilt pathogen (Gibberella xylarioides). In this study we examined the effect of pH and temperature on the mycelia growth and spore yield of Trichoderma isolates in batch culture. Coffee root samples were collected from different ecological habitat of Kaffa zone for the isolation of endophytic Trichoderma isolates. The ability of Trichoderma isolates to grow at pH 4.5, 6.5 and 7.5 were tested in liquid potato dextrose broth (PDB) medium. The effect of volatile and non-volatile compounds produced by Trichoderma isolates on radial growth of pathogenic fungi, F. xylarioides was also determined. In vitro antagonistic bioassays were performed to evaluate and determine the potentiality of Trichoderma isolates as biocontrol agents against F. xylarioides. The pH and temperature had significant effect on the growth and sporulation of Trichoderma isolates. It was observed that the optimum pH for maximum mycelial growth and spore yield produced by Trichoderma isolates in batch cultures was pH 7.5 while the optimum temperature for mycelial growth and the maximum spore yield was produced at optimum temperature was 25OC. This work has revealed the important role that environmental conditions plays in the mycelia growth and spore yield of Trichoderma isolates, a biocontrol agent. In in-vitro bioassay the experimental results showed that all isolates of Trichoderma were able to inhibit the growth of F. xylarioides under in vitro experiment at rates ranging from 42.2 % to 69 % after 10 days of incubation. The highest mean inhibitory effect on the growth of the test pathogen was achieved by Gimbo Trichoderma isolate 05 (Gim-T05) (69%) followed by Gim-T10 (67.6%) and Gim-05 (66.7%), while Gowata Trichoderma isolate 01 (Gow-01) isolate showed the lowest (42.2%) mean inhibitory effect restricting it almost completely in plates as compared to the control consisting of F. xylarioides growing alone. Gimbo Trichoderma isolate 05 (Gim-T05) produced zones of inhibition which is an indicative of the production of secondary metabolite(s) inhibiting the mycelial growth of F. xylarioides. Thus, the use of novel isolates of Trichoderma with efficient antagonistic capacity against F. xylarioides is a promising alternative strategy to pesticides for coffee wilt disease management.Item Isolation of Α-Amylase Producing Fungi from South Western Part of Ethiopia, Characterization and Evaluation of the Enzyme for Bio-Ethanol Production(Addis Ababa Universty, 2015-04) Endalew, Amare; Alemu, Tesfaye (PhD)Fungi are well known for their ability to excrete enzymes in to the environment. The fungal isolate (Designated as AAUam13) which was isolated from coffee effluent of shesheka (Jimma zone) was the best alpha amylase producer among thirty isolates obtained from south western part of Ethiopia which were screened and evaluated for amylase production. The morphological study of isolate AAUam 13 indicated that the isolate belongs to genus Aspergillus. The optimal initial pH and temperature for amylase production by the same isolate was found to be 5 and 30oC respectively. Yeast extract and soluble starch were found to be the best nitrogen and carbon sources for amylase production respectively. The enzyme alpha amylase was optimally active at 35 oC and pH5. Alpha-amylase production by co-cultivation of the selected isolates showed higher activities than single cultures. The co-culture of isolate AAUam13 and AAUam15 was higher than individual activities of AAUam 13 and AAUam15. Bioethanol production was achieved by separate hydrolysis and fermentation process using the selected fungal isolate and yeast. The bioethanol concentration was measured quantitatively with potassium dichromate and sulphuric acid method. Yeast gave the highest concentration of ethanol as inoculated in the pretreated media than the yeast inoculated in the untreated media after 72 hours. KEY WORDS: Aspergillus, Enzymes, Ethanol, Fermentation, Substrate.Item Isolation, Characterization of Fungal Pathogens of Yam from Seka Chokersa Woreda, Jimma Zone and Its Controls Using Trichoderma Spp and Fungicides(Addis Ababa Universty, 2014-01) Wedajo, Bikila; Alemu, Tesfaye (PhD)In the present study, five different fungal isolates (AUF1, AUA1, AUA2, AUV1 and AUV2) were isolated from infected yam leaves and tubers grown in Seka Chokersa woreda, Jimma Zone, Ethiopia based on cultural and morphological. The ranges of conidia (length by width) of AUF1, AUA1, AUA2, AUV1 and AUV2 were 19.00-26.20×3.20-5.80 μm, 30.50-40.47×10.90-17.37 μm, 27.30-42.50×11.60-16.90 μm, 3.50-11.20×1.80-3.80 μm and 2.50-9.00×1.25-3.20 μm, respectively. The pathogenicity test showed that the most virulent isolates were AUF1, AUA1 and AUV1 with 100% tissues infection. Growth characteristics of the isolates showed that isolate AUF1 and AUV1were best grown on PDA medium with mycelial diameter of 90 mm followed by AUA1, AUV2 and AUA2, respectively. Similarly, the isolates grew best at 25oC, with the exception of AUA1 that displayed maximum growth at 30 oC. From the pH levels studied, maximum dry mycelial weight was attained from AUV2 (697.6±1.6 mg), AUA1 (693.9±3.3 mg) and AUF1 (624.5±4.0 mg), at pH 6.0; whereas, dry mycelial weight of 693.4±4.9 mg and 356.4±1.1 mg at pH 5.0 and 7.0 measured from AUV1 and AUA2, respectively. Dextrose was the most utilized carbon source by AUF1 and AUV1; while maltose was the best carbon source for AUA1, AUA2 and AUV2. Potassium nitrate was the best nitrogen source for all fungal isolates with the exception of AUA2 that utilized ammonium nitrate best. In vitro evaluation of dual culture test with Trichoderma spp against the fungal pathogens revealed that both of them showed effective with 66.75 to 82.59% of mycelial growth inhibition. Similarly, curzate (43.93% WP) and sancozeb (80% WP) were effective at high concentrations of (400-1000 ppm), where sancozeb was more effective than curzate in terms of percent growth inhibition. From the study of combined treatment of the pathogens with fungicides and Trichoderma isolates, the highest percent of inhibition of mycelial growth of 85.6%, 79.7%, 87.5%, 89.3% and 80.2% were detected when AUT2 was combined with sancozeb against AUF1, AUA1, AUA2, AUV1 and AUV2, respectively. So, combination of Trichoderma spp (AUT1 and AUT2) with chemical fungicides (curzate and sancozeb) at lower concentration offers a promising control of fungal pathogens. Key words: Biocontrol agents, Dioscorea spp, Fungal isolates, In vitroItem Isolation, Identification of Fusarium Xylarioides (Gibberella Xylarioides) from Southern Ethiopia and its Response to Fungal Biocontrol Agents(Addis Ababa Universty, 2007-07) Hailu, Negash; Alemu, Tesfaye (PhD)Five isolates of Fusarium xylarioides form five coffee growing Weredas in southwestern Ethiopia were isolated from Arabica coffee trees showing sever symptoms of vascular wilt. These strains were characterized based on vegetative and spore morphology and vegetative growth on different growth media, temperature and pH ranges. These strains produced both microconidia and macroconidia on Potato Dextrose Agar (PDA) and Synthetic low-nutrient Agar (SNA). Microconidia were unicellular, allantoids and had 0-1 septate. Macroconidia were strongly curved with marked foot cells and had 1-4 septate. Pathogenicity test was performed for the subsets of isolates by inoculating 20 ml, 40 ml, and 60 ml of spore suspensions. Symptoms developed on the 30th day of inoculation concentration wisely. All seedlings died on the 90 day of inoculation. The controls were drenched with equivalent amount of distilled water and no symptoms developed until the completion of the study. In vitro evaluation of antagonistic test of dual culture of the test isolates with two Trichoderma species showed that the radial growth inhibition of the pathogens. The cell free culture filtrates of different concentration also showed the radial growth inhibition of the test pathogens. Key words: Fusarium xylarioides, Trichodrma harzianum, Trichoderma viride, Biological control.Item Production and Optimization of cellulase enzyme under Submerged and Solid State Fermentation from Trichoderma Isolates(Addis Ababa University, 2011-10) Gorems, Weldesemayat; Alemu, Tesfaye (PhD)Pure cellulose and agricultural wastes were used as sole carbon sources for the production of cellulase enzymes by Trichoderma isolates in both SmF and SSF, respectively. These isolates (seven) were obtained from Mycology laboratory, Faculty of Life science, Addis Ababa University. Carboxymethyl cellulose (CMC) and Congo red were used to screen four isolates for their ability to produce cellulase enzymes. Cellulase production was assayed by measuring the amount of glucose released in μmol/ml/min by using the DNS assay method at 540nm. To maximize the cellulase production, the critical parameters like carbon source, nitrogen source, cellulose concentration, temperature and pH were optimized under SmF. The highest cellulase enzyme activity was observed after 12 days of incubation on media containing, yeast extract (1%), cellulose concentration (1%) and pH (5.5) from all isolates under SmF whereas under SSF maximum cellulase enzyme was recorded between 5-11 days incubation. WB, RB and WS were found to suitable for high cellulase production whereas CS, CP and BB relatively showed the least cellulase production in SSF. Trichoderma isolates AUT1 produce the highest CMCase on WS (5.68U/g), AUT5 on rice bran (8.15U/g), AUT2 and AUT4 on wheat bran, their enzymatic activities were 4.92U/g and 7.01U/g, respectively. Enzyme synthesis was repressed in the presence of glucose and fructose while it was induced in the presence of maltose and lactose in SmF. The maximum amount of cellulase was observed between 55% to 65% moisture contents. It is evident from the present study, solid state fermentation was better than liquid state fermentation for the production of cellulase by Trichoderma isolates using agricultural wastes. The cellulase enzymes produced by Trichoderma isolates were active at temperature ranges of 40-60oC and pH values 4.5-6.5. Key words:- Agricultural wastes, Carboxymethyl cellulase, Cellulase activity, DNS methods and Congo redItem Protein Enrichment of Orange Wastes by Filamentous Fungi(Addis Ababa University, 2007-07-05) Yalemtesfa, Biniyam; Alemu, Tesfaye (PhD)Orange peel and pulp are rich in carbohydrates but poor in protein and account for approximately 55 to 60% of the weight of the raw/whole fruit. Their high cellulose and low protein content prevents them from being used as non-ruminant feedstuff while their high moisture content leads to difficulty in storage. However, the orange waste is highly susceptible to hydrolysis by mixtures of cellulolytic and pectinolytic enzymes which makes it attractive potential feedstock for biological conversion to value added products. To this effect, two fungal species namely, Aspergillus niger (KA-06) and Chaetomium spp (KC-06) were used to enrich the orange wastes with protein in solid state fermentation. The mycelial biomass of KC-06 was found to have 37.64% protein and KA-06 had 34.201% protein when grown in malt extract broth. The effect of various process parameters were studied in solid media of orange pulp. The optimum duration of fermentation was found to be 5 days and 4 days for KC-06 and KA-06 respectively. The optimum conditions for the enrichment process were found to be moisture content 40%; temperature 250C and substrate load 10gm for both KC-06 and KA-6 . pH 7 gave highest protein yield by KA-06 whereas pH 5.5 was optimum for KC-06. Inoculum concentration 108 spores/ ml was the best dose for KC-06 as 106 spores/ ml was for KA-06. Among the nitrogen supplements studied, (NH4)2SO4 gave the highest protein yield by both fungal species. The growth of KC-06 on orange waste has improved the protein content up to 39.64% and KA-06 was able to enrich the feed with 34% protein.