EFFECTS OF DIFFERENT NITROGEN SOURCES ON THE ANTIFUNGAL ACTIVITIES OF ACTINOMYCETE ISOLATED FROM THE SOIL

ABSTRACT

Actinomyces sp. And Streptomyces sp. was isolated from the soil of Okwuta in Abia State. Physiological and biochemical analysis strongly suggested that the isolate belonged to the strains of Actinomycete .Nitrogen source is a critical component of cultivation medium and almost the useful tool for stimulation of the antifungal metabolites production. The aim of this study was to select the best nitrogen sources in medium for the production of antifungal compounds effective against Collectotrichum and Alternaria sp. Activity of the cultivation liquids on Alternairia and Collectotrichum isolates was tested in vitro using Agar well diffusion method. The results  indicate that maximum inhibition zone was reached in medium with soybean meal as nitrogen source with inhibition diameter 25.3mm for Alternaria sp.,15mm for Collectotrichum sp. in isolate 1( Actinomyces sp) and 17.5mm for Collectotrichum sp., 27.7mm for Collectotrichum sp in Isolate 2 (Streptomyces spp). Malt and urea showed the  least minimum  inhibition  zone for both tested fungi and isolates.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgement iv

Table of contents     v

List of tables     viii

Abstract     ix

CHAPTER ONE

1.0 Introduction 1

Aim and objectives 4

Objectives       4

CHAPTER TWO

2.0    Literature Review 4

2.1    Taxanomy             8

2.2    Bioactive Agents In Actinomycetes    10

2.2.1 Antibiotics       10

2.2.2 Antifungals       10

2.3    Plant Growth Bioactive Molecules 11

2.3.1 Indole Acetic Acid    11

2.3.2 Hydrogen Cyanide  12

2.4   Enzyme Production from Actinomycetes  13

2.5   Actinomycetes as Source of Agroactive Compounds 15

2.6   Actinomycetes As Biocontrol Tools 17

2.6.1 Plant colonization and biocontrol 18

2.6.2 Actinomycetes as Biopesticide Agents 19

2.7   Optimization Of Actinomycetes 21

2.8   Colletotrichum 22

2.9   Alternaria 23

CHAPTER THREE

3.0      Materials and Methods 25

3.1 Sample Collection 25

3.2 Sterilization of Materials   25

3.3       Pre-treatment of Soil Samples 25

3.4 Media Used 25

3.4.1 Media Preparation                    25

3.4.2    Test Organisms 26

3.5 Serial Dilution 26

3.5.1 Culturing 26

3.5.2 Identification 27

3.5.3 Sub-culturing 27

3.5.4 Gram Staining 27

3.6 Biochemical Test 28

3.6.1 Starch hydrolysis test 28

3.6.2 Hydrogen sulfide test. 28

3.6.3    Gelatin hydrolysis 29

3.6.4 Catalase test 29

3.6.5 Oxidase test 29

3.6.6 Carbohydrate fermentation test 30

3.7       Optimization of Different Nitrogen Source 30

3.8      Crude Extraction 31

3.9      In-Vitro Antimicrobial Test 31

CHAPTER FOUR

4.0      Results      32

CHAPTER FIVE

5.0   Discussion, Conclusion and Recommendation 37

5.1   Discussion 37

5.2   Conclusion 40

5.3   Recommendation   40

References 41 

Appendix 50

LIST OF TABLES

  Table                                         Title    Page

 1:    Shows the Morphology of isolated Organisms    33

2:    Shows the Biochemical Characterization of isolated organisms   34

3:  Shows the effects of different nitrogen sources on Actinomyces spp 35

(isolate 1) and   their impact on Antimicrobial agent production  

measured by inhibition zones against Alternaria sp. And  Collectotrichum sp.

By agar well diffusion method.

 4   Shows the Effects of Different Nitrogen Sources on Streptomyces spp.   36

(isolate 2) and Their Impacts on Antimicrobial Agent Production Measured By Inhibition Zones        Against  Alternaria sp. and Collectotrichum sp. by Agar well diffusion method.

CHAPTER ONE

1.0 INTRODUCTION

Soil is a complex structure created by the influence of geology, topography, climate time and anthropogenic activities. As with air and water, human life could not be sustained without access to soil since it is the source of food. In addition to producing food, a good quality soil also acts as an environmental filter for cleaning air and water (Lennart and Stenberg, 2008). Soil quality encompasses not only the capacity of a soil for crop productivity but also food safety for humans and other animals. Soil microorganisms including bacteria and fungi are critical in decomposition and recycling and in creating and maintaining good soil structure by proper aeration and formation of humus and particle aggregate (Nolin et al., 2009).

The rhizosphere is the interface between roots and the soil where nutrient absorption for plant growth in agroecosystems is facilitated. An abundant and diverse rhizosphere biome is involved in biogeochemical processes, including bacteria, fungi and soil fauna, driving soil C, N and P dynamics.Plant nitrogen photosynthates allocated to the root and rhizosphere are priming microbial activities important for plant nutrition such as organic matter decomposition, P solubilization, N fixation, mycorrhizal nutrient transport and biocontrol of root pests. While substantial information is available on the role of individual groups of the rhizosphere microbiome in biogeochemical processes, less attention has been given to the interactions between different beneficial rhizosphere microorganisms (Nolin et al., 2009).

Functional traits of the beneficial rhizosphere microbiome in relation to plant nutrition and healthinclude organic matter decomposition, P solubilization and transport, N fixation and biocontrol of root pests (Philippot et al., 2013). Microorganisms are virtually an unlimited source of novel substances with many therapeutic applications and consequently their secondary metabolite screening for pharmaceutically significant novel antibiotic and non - antibiotic compounds and drug lead molecules has assumed greater attention in recent times. Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources (Philippot et al., 2013).

Actinomycetes are the most commonly distributed microbes in nature which largely inhabits the soil environment (Philippot et al., 2013). They form the dominant and significant group among the soil microbial community and comprise about 50% of the uncultivable soil microbes. They play a major role in the recycling of organic matter, production of novel pharmaceuticals, nutritional materials, cosmetics, enzymes, antitumor agents, enzyme inhibitors, immune modulators, and vitamins. Numerous naturally occurring antibiotics have been discovered from actinomycetes ever since the discovery of Selman Waksman,s streptomycin from this group and several studies signify their noteworthy antibiotic production (Drinkwater and Snapp, 2007).

Further, about two thirds of known significant naturally occurring antibiotics are actinomycete derived one and are the prominent candidates receiving number of product and processes related patents. Though ecological studies on soil actinomycetes from various habitats including grasslands, beach sands, under-ground caves, rice-paddies, orchards and sub-glacial ice of Antarctica were reported, only few reports are available on forest soil actinomycete communities. These ubiquitous organisms are deemed to have a preference over the soil constituents such as humus, litter, dung and even rock sur-faces. In fact actinomycetes are the dominant microflora showing viable counts reaching 106 per gram of dry weight soil in relatively dry, humid, and calcareous soils, (Drinkwater and Snapp, 2007).

Screening of microorganisms for the production of novel antibiotics has been intensively pursued for many years by scientists. Antibiotics have been used in many fields including agriculture, veterinary and pharmaceutical industry. Actinomycetes have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic, and enzymes like cellulase and xylanase used in waste treatment. Of these compounds, antibiotics predominate in therapeutic and commercial importance (Jeffery, 2008).

Actinomycetes are the most widely distributed groups of microorganisms in nature. They are attractive, bodacious and charming filamentous gram-positive bacteria. They make up in many cases, especially under dry alkaline conditions, a large part of the microbial population of the soil (Jeffery, 2008).

 Actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products; the Streptomyces are especially prolific (Jeffery, 2008). The ability to produce a large number of chemically different secondary metabolites is associated mostly with the filamentous actinomycetes. They gain special importance, as they are the most potent source for production of antibiotics and other bioactive secondary metabolites. Each actinomycete strain has probably genetic potential for producing 10-20 secondary metabolites (Oskay et al., 2004).

The actinomycetes are noteworthy as antibiotic producers and making three quarters of all known products; the Streptomyces are especially prolific (Oskay et al., 2004). Streptomyces species are widely recognized as industrially important microorganism s because of their ability to produce m any kinds of novel secondary metabolites including antibiotics. The problems of drug resistance, patient's sensitivity and inability to control certain infectious diseases have given an impetus for continuous search of new antibiotics all over the world. To com bat the multidrug resistant organism s, introduction of new antimicrobial com pounds or antibiotics from new source is essential (Anonymous, 2004).

1.1 AIM AND OBJECTIVES

The aim of this study is to determine the effect of nitrogen sources on antimicrobial activity of actinomycetes.

The objectives are:

1. To determine the antimicrobial effect of actinomycetes.

2. To determine the effect of nitrogen source on actinomycetes isolated from soil.

3. To determine the optimization level of nitrogen source on actinomycetes.  

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