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Download this complete Project material titled; Antifungal Activities Of Ethyl Acetate Fraction Of Pseudomonas Fluorescens Fermentation Broth Against Phyto-Pathogenic Fungi From Rotten Yam with abstract, chapters 1-5, references, and questionnaire. Preview Abstract or chapter one below

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ABSTRACT

Yam tubers are prone to infestation by microorganism at all stages of growth and in storage. This rot reduces the quality of yam and affects food security. Pseudomonas fluorescens possess varieties of promising antifungal properties through production of secondary metabolites which makes it a biocontrol agent against many phyto-pathogens. In this study, the antifungal activities of metabolite produced by Pseudomonas fluorescens isolated in Zaria was evaluated. Rhizosphere soil was collected from different farms (sugarcane, rice and soybeans) in Zaria, Nigeria and Pseudomonas fluorescens was isolated and identified in accordance with standard microbiological procedure using cultural, microscopical and biochemical characteristics. The four isolate obtained were grown in three (3) fermentation media made from waste such as yam peel, banana peel and sugarcane shaft as carbon sources and groundnut cake as nitrogen source. Crude extract from these fermentations were tested for antifungal activities against Aspergillus niger, Penicillium citrinum, and Rhizopus stolonifer isolated from rotten yam using agar diffusion assay. Only four isolates labelled sugarcane A1, sugarcane A2, Rice B1 and Rice B2 were identified as Pseudomonas fluorescens and only fermentation broth extracts of Sugarcane A2, Rice B1 and Rice B2 had remarkable antifungal activities against the fungi with diameter zones of inhibition ranging from 14.00 – 39.00mm in two (2) of the fermentation media. The crude extracts from P. fluorescens fermentation broth were extracted in this study and were observed to inhibit the mycelial growth of the test fungi with zone of inhibition ranging from 19.50 – 33.50mm. The in vitro & in vivo MICs showed significant antifungal activities against test fungi spores. Rhizopus stolonifer was observed to be the most susceptible test organism in vitro & in vivo (0.15 – 5.00mg/ml). This study showed that the Pseudomonas fluorescens
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isolated in Zaria produced potential antifungal metabolite which can arrest the growth of phyto-pathogenic fungi associated with yam tubers.

 

 

TABLE OF CONTENTS

Cover page ………………………………………………………………………………………………………………… i
Title page ………………………………………………………………………………………………………………….. i
Declaration ……………………………………………………………………………………………………………….. ii
Certification ……………………………………………………………………………………………………………… v
Dedication ……………………………………………………………………………………………………………….. vi
Acknowledgement ……………………………………………………………………………………………………. vii
Abstract ………………………………………………………………………………………………………………….. viii
Table of Content ……………………………………………………………………………………………………….. ix
List of Tables ………………………………………………………………………………………………………….. xiii
List of Figures ………………………………………………………………………………………………………….. xv
List of Plates …………………………………………………………………………………………………………… xvi
List of Appendices …………………………………………………………………………………………………. xvii
Acronyms and Abbreviation …………………………………………………………………………………….. xix
CHAPTER ONE
1.0 INTRODUCTION…………………………………………………………………………1
1.1 Background………………………………………………………………………………..2
1.2 Statement of problem ……………………………………………………………………..3
1.3 Justification of study ………………………………………………………………………3
1.4 Aim and Objectives ……………………………………………………………………….4
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1.4.1 Aim ………………………………………………………………………………………4
1.4.2 Objectives of the study ………………………………………………………………….5
1.5 Hypothesis …………………………………………………………………………………5
1.5.1 Null hypothesis ………………………………………………………………………….5
1.5.2 Alternate hypothesis ……………………………………………………………………5
CHAPTER TWO
2.0 Literature Review …………………………………………………………………………6
2.1 Yam ………………………………………………………………………………………6
2.1.1 Description of yam ………………………………………………………………………6
2.1.2 Species of yam ………………………………………………………………………….7
2.2 Yam Production in Nigeria ……………………………………………………………….9
2.3 Yam Propagation and Harvesting ………………………………………………………..10
2.4 Yam Storage ……………………………………………………………………………..11
2.4.1 Methods of Storage ……………………………………………………………………12
2.4.1.1 Traditional Storage Methods ……………………………………………………….. 12
2.4.1.2 Modern Storage Methods ……………………………………………………………13
2.5 Yam Consumption in Nigeria and around the world ……………………………………13
2.6 Nutritional Value of Yam ……………………………………………………………….14
2.7 Importance of Yam Production …………………………………………………………15
2.8 Problems Associated with Yam Production in Nigeria …………………………………16
2.8.1 Diseases and common pests affecting yam .……………………………………………17
2.8.1.1 Yam Field Diseases ………………………………………………………………….17
2.8.1.2 Yam Storage Diseases ……………………………………………………………….19
2.9 Management and Control of Pests and Diseases of Yams ………………………………20
2.10 Pseudomonas as Biocontrol agents …………………………………………………….21
2.11 Pseudomonas fluorescens ………………………………………………………………26
2.11.1 Bioactive metabolites of Pseudomonas fluorescens …………………………………27
2.11.2 Pyrrolnitrin (PRN) and Pyoluteorin (PLT) ……………………………………………27
2.11.2.1 Biosynthesis of Pyrrolnitrin ………………………………………………………..28
2.11.2.2 Biosynthesis of Pyoluteorin ………………………………………………………..30
2.11.3 Phenazines (PHZ) …………………………………………………………………….31
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2.11.3.1 Phenazine Biosynthesis …………………………………………………………….33
2.11.4 2, 4-Diacetylphloroglucinol …………………………………………………………35
2.11.4.1 Biosynthesis of DAPG ……………………………………………………………..36
2.12 Use of agro-waste as part of media formulation for growth of microorganisms ………37
CHAPTER THREE
3.0 MATERIALS AND METHODS ………………………………………………………..39
3.1 Materials …………………………………………………………………………………39
3.1.1 Equipment ……………………………………………………………………………..39
3.1.2 Glass wares …………………………………………………………………………….39
3.1.3 Reagents and Chemicals ………………………………………………………………39
3.1.4 Culture media …………………………………………………………………………40
3.2 Methods …………………………………………………………………………………40
3.2.1 Isolation of Pseudomonas fluorescens …………………………………………………………………40
3.3 Identification of Pseudomonas fluorescens ………………………………………………..40
3.3.1 Microscopic characteristics ……………………………………………………………40
3.3.2 Cultural characteristics …………………………………………………………………40
3.3.3 Growth at 4°C and 42°C ……………………………………………………………….41
3.3.4 Biochemical tests ……………………………………………………………………….41
3.3.4.1 Oxidase Test …………………………………………………………………………41
3.3.4.2 Catalase Test …………………………………………………………………………41
3.3.4.3 Indole Test ……………………………………………………………………………41
3.3.4.4 Gelatin Hydrolysis Test ………………………………………………………………41
3.3.4.5 Nitrate Reduction Test ………………………………………………………………42
3.3.4.6 Sugar Fermentation Test …………………………………………………………….42
3.3.4.7 Arginine Di-hydrolase Test ………………………………………………………….42
3.4 Isolation and Identification of Phyto-pathogenic fungi ………………………………….43
3.5 Screening for in vitro antifungal activity of crude extract of Pseudomonas fluorescens fermentation broth ……………………………………………………………………………44
3.5.1 Preparation of formulated fermentation media ………………………………………..44
3.5.2 In vitro antifungal activities of crude extract of Pseudomonas fluorescens fermentation broth …………………………………………………………………………………………45
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3.6 Extraction of the crude extract metabolite from Pseudomonas fluorescens in the fermentation broth ……………………………………………………………………………45
3.7 Quantitative Measurement of Metabolite Substance in the Extract ……………………..45
3.8 Determination of Minimum Inhibitory Concentration of the ethyl acetate fraction against test fungal spores …………………………………………………………………………….46
3.8.1 In vitro Minimum inhibitory concentration of ethyl acetate fraction against test fungal spores ………………………………………………………………………………………..46
3.8.2 In vivo minimum inhibitory concentration of ethyl acetate fraction against test fungal spores on yam tuber …………………………………………………………………………46
3.9 Statistical analysis ………………………………………………………………………47
CHAPTER FOUR
4.0 RESULTS ………………………………………………………………………………48
4.1 Isolation and identification of Pseudomonas fluorescens ………………………………48
4.2 Isolation and identification of fungi from rotten yam …………………………………..48
4.3 Antifungal activities of crude extract of Pseudomonas fluorescens fermentation broth against the test fungi spores …………………………………………………………………52
4.4 Antifungal activities of the ethyl acetate fraction of fermentation broth of Pseudomonas fluorescens ……………………………………………………………………………………54
4.5 In vitro Minimum inhibitory concentration (M.I.C.) values for the ethyl acetate fractions of Pseudomonas fluorescens fermentation broth on the test fungi spores …………………..57
4.6 In vivo Minimum inhibitory concentration values for the ethyl acetate fractions of fermentation broth of Pseudomonas fluorescens on the test fungi isolates on yam tuber …..60
CHAPTER FIVE
5.0 DISCUSSION ……………………………………………………………………………63
CHAPTER SIX
6.0 SUMMARY, CONCLUSION AND RECOMMENDATION ………………………….67
6.1 Summary …………………………………………………………………………………67
6.2 Conclusion ……………………………………………………………………………….67
6.3 Recommendations ……………………………………………………………………….67
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CHAPTER ONE

INTRODUCTION
1.1 Background
Yam belongs to the genus Dioscorea (Family Dioscoreaceae) and is the second most important tropical root crop in West Africa after cassava (Adisa et al., 2015). The genus Dioscorea consists of about 600 species (Degras, 1993). The most cultivated species are the Dioscorea rotundata (white yam), Dioscorea alata (water yam), Dioscorea dumetorum (bitter yam), Dioscorea esculentum (loir), Dioscorea cayenensis (yellow or guinea yam) and Dioscorea bulbifera (aerial yam). White or guinea yam (D. rotunduata poir) is the most popular species in Africa (Lawal et al., 2014; Princewill-Ogbonna and Ibeji, 2015).
Yam plays significant roles in the social-cultural and economic wellbeing of thousands of people in Nigeria especially among the people of South Eastern Nigeria (Okigbo et al., 2013). Its cultivation is very profitable despite high costs of production and price fluctuations in the markets (IITA, 2013; Izekor and Olumese, 2010).
The quality of yam tubers is affected by rots, which makes them unappealing to consumers (Ogbo and Agu, 2014b; Agu et al., 2015). Most rots of yam tubers are caused by pathogenic fungi such as Aspergillus flavus, Aspergillus niger (Tiegh), Fusarium oxysporum, Fusarium solani, Botryodiplodia theobromae, Penicilllium chrysogenum, Rhizoctonia spp, Penicillium oxalicum, Trichoderma viride and Rhizopus nodosus (Okigbo and Ikediugwu, 2000; Okigbo et al., 2014; Frank and Kingsley, 2014b; Agu et al., 2014). Several methods have been adopted for controlling losses due to post harvest disease of yam; these include the use of chemicals, biological method of control (Okigbo, 2004) and curing by using natural plant extracts (Amusa et al., (2003).
Pseudomonas is one of the most ubiquitous bacterial genus and Migula designated and described the species associated with the genus in 1985 (Migula, 1985). Pseudomonads are
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well adapted to colonizing roots and the rhizosphere, and they suppress disease by a wide variety of mechanisms (Pieterse et al. 2014; Weller 2007) but the most important are the production of antimicrobial metabolites. These characteristics makes Pseudomonas species good candidates for use as seed inoculant and root dips for biological control of soil borne pathogens.
Pseudomonas fluorescens is a gram negative, rod-shaped, and non-pathogenic bacterium that is known to inhibit primarily the soil, plants and water (Peix et al., 2009). It derives its name from its ability to produce fluorescent pigments under iron-limiting conditions (Baysse et al., 2003). P. fluorescens are well characterized for their ability to produce antimicrobial compounds (Haas and De’ fago, 2005). These metabolites range from simple 2, 4-diacetyl phloroglucinol, phenazine-1-carboxylic acid and pyrrolnitrin [3-chloro-4-(2-nitro-3’-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone 2, 3-de-epoxy-2,3-didehydra-rhizoxin. 2,4-diacetylphloroglucinol (DAPG), is a major factor in control of a range of plant pathogens (Haas and De’ fago, 2005) while Pyrrolnitrin is active against Rhizoctonia spp, Fusarium spp, and other plant pathogen fungi and it has been used as a lead structure in development of a new phenyl pyrrole agricultural fungicide (Ligon et al., 2000).
Wastes are leftovers from production and consumption. It includes plant materials, agricultural, household, industrial and municipal wastes and residues (Okonkwo et al., 2006). The agricultural and agro-industrial activities generate a large amount of lignocellulosic by products such as bagasse, straw, stem, stalk, cobs, fruit peel and husk, among others (Saranraj and Anbu, 2017). These wastes contain simple and complex sugars that are metabolized by microorganism through secretion of extracellular products (Saheed et al., 2013).
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1.2 Statement of Problem
Large quantity of yam losses has been reported and these losses have been due to microbial rot diseases. Yam plants are prone to infection by fungi, bacteria, and viruses at all stages of growth and also during storage of tubers (Okigbo, 2002; Okigbo, 2005; Morse et al., 2000)
Difficulties in yam preservation has compelled majority of the yam farmers are to either consume or sell all their yam products at low prices before the new harvesting season. Consequently, before new harvesting period, they are bound to suffer food security crises.
Chemical agents have been used to control this pre harvest and post-harvest losses in the past decades but these chemical agents are not only expensive for farmers, but they are not eco-friendly and micro-organisms are becoming resistant to them (Okigbo, 2004; Okigbo and Ikediugwu, 2000). This has lead scientist to divert their attention to the use of potential beneficial microbes. Biological agents as biocontrol is gaining ground and has proved to be safer, cheaper, effective and eco-friendly.
As a result of high consumption and industrial processing of the edible parts of fruit, fruit wastes (such as banana, grapes, dragon fruit, orange, strawberry, lemon, watermelon, citrus, Pomegranate, pineapple residues, sugarcane bagasse and other fruit) has become one of the main sources of municipal solid wastes which has become an increasingly tough environmental issue (Saranraj and Anbu, 2017). Insufficient and improper disposal of these solid waste result in scenic blights, increase in rodents and insect vectors of disease, and serious hazards to public health.
1.3 Justification of Study
Nigeria is the largest producer of yam in the world (FAOSTAT, 2017) and yam production is regarded as a source of food security and employer of labour in many areas where it is cultivated. It generates employment for more than 50% of people in places of production either
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through direct involvement in cultivation, marketing, processing, or indirectly through related services in production (i.e. labour both on the field of production and markets or transportation).
Yam tubers are nutritionally rich and a major source of dietary fiber, carbohydrates, vitamin C, and essential minerals (Charles et al., 2005; Polycarp et al., 2012).
The scope of developing microbial metabolites for commercial pesticides as an alternative to chemical control agent has gained importance due to increased concerns on environmental pollution, pathogens resistance and high plant protection cost (Reddy et al., 2007; Uppal et al., 2007)
The agricultural-based industries generate significant quantities of organic wastes including peels from cassava, plantain, banana, oranges and straw from cereals. Rather than allow these wastes to become solid municipal wastes, it is necessary to convert them to useful end products. These wastes could be utilized as cheap raw materials for some industries or as cheap substrates for microbiological processes (Nwabueze and Otowa, 2006). The need to develop alternative media has become imperative as the conventional media are either not readily available or expensive in most developing countries (Saranraj and Anbu, 2017).
1.4 Aim and Objectives
1.4.1 Aim
This study was designed to examine the antifungal activities of ethyl acetate fraction of Pseudomonas fluorescens fermentation broth against some phyto-pathogenic test fungi.
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1.4.2 Objectives of the Study
1. To isolate & identify Pseudomonas fluorescens from rhizosphere of rice, sugar cane and soy beans soil in Zaria, Kaduna state.
2. To Isolate and identify three phyto-pathogenic organisms (Aspergillus niger, Penicillium citrinum and Rhizopus stolonifer) from rotten yam.
3. To formulate and prepare fermentation media using agricultural waste as different carbon and nitrogen sources for the production of active metabolite from Pseudomonas fluorescens.
4. To screen for antifungal activities of the crude extracts using agar well diffusion method.
5. To determine the in vitro and in vivo antifungal activities of the ethyl acetate fraction from Pseudomonas fluorescens fermentation broth on the test fungal spores.
1.5 Hypothesis
1.5.1 Null Hypothesis
The ethyl acetate fraction from Pseudomonas fluorescens fermentation broth does not have antifungal activities against phyto-pathogenic fungi spores of Aspergillus niger, Penicillium citrinum, Rhizopus stonolifer.
1.5.2 Alternate Hypothesis
The ethyl acetate fraction from Pseudomonas fluorescens fermentation broth has antifungal activities against phyto-pathogenic fungi spores of Aspergillus niger, Penicillim citrinum, Rhizopus stonolifer.

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