ABSTRACT
Actinomycetes sp. And Streptomyces sp. was isolated from the soil of Michael Okpara University of Agriculture, Umudike, Abia State. Physiological and biochemical analysis strongly suggested that the isolate belonged to the genus of Actinomyces and Streptomyces Temperature is one of the most important environmental factors in soil, exerting not only direct effects on the soil microorganisms. The aim of this study was to select the optimal temperature for the production of antifungal compounds effective against Collectotrichum and Alternaria sp. Activity of the cultivation liquids on Alternaria and Collectotrichum isolates was tested in vitro using Agar well diffusion method. The results indicate that maximum inhibition zone was reached in medium with temperature range with inhibition diameter 1.5mm for Alternaria sp., 16mm for Collectotrichum sp. in isolate 1 (Actinomyces sp) at 300C and 200C and 25mm for Collectotrichum sp., 7mm for Alternaria sp. for isolate 2. The efficiency of the Actinomycetes isolate increased after the optimization of nutritional parameters. Therefore, in order to increase the chances of discovery of new and effective antibiotic against pathogenic bacteria it is necessary to isolate and screen new species from unexplored regions. Improved methodologies and nutritional conditions also plays crucial role in increasing their efficiency.
TABLE
OF CONTENTS
Title
page i
Certification ii
Dedication iii
Acknowledgements
iv
Table
of Contents v
List of
Tables viii
Abstract
ix
CHAPTER ONE
1.0 Introduction 1
1.1 Aim and Objectives 3
CHAPTER TWO
2.0
Literature Review 4
2.1
Actinomycetes 4
2.2 Antifungals 7
2.3 Streptomyces
7
2.3.1 Morphology of Streptomyces 8
2.3.2 Importance of Streptomyces 9
CHAPTER THREE
3.0 Materials And Methods 11
3.1
Sample Collection 11
3.2
Media Used 11
3.3
Sterilization 11
3.4 Bacteriological Procedure 12
3.4.1
Gram Staining 12
3.4.2
Motility Test 12
3.5
Biochemical Cultural Characteristics 13
3.5.1 Starch hydrolysis test 13
3.5.2 Hydrogen sulfide test 13
3.5.3
Gelatin hydrolysis 14
3.5.4 Catalase test 14
3.5.5 Oxidase test 14
3.5.6 Carbohydrate fermentation test 15
3.6 Effect of
Temperature 15
CHAPTER FOUR
4.0 Results
17
CHAPTER FIVE
5.0
Discussion, Conclusion and Recommendation 22
5.1
Discussion 23
5.2
Conclusion 23
5.3
Recommendation
23
References 23 Appendix
LIST
OF TABLES
Table Title Page
4.1 Morphology and Biochemical Characterization
of isolated Organisms 18
4.2
Biochemical Characterization of isolated organisms 19
4.3
The effect of Temperature on antifungal activities of Actinomycetes 20
isolated
from soil (Isolate 1).
4:4
The effect of temperature on antifungal
activities of Actinomycetes 21
isolated
from soil. (Isolate 2).
CHAPTER ONE
1.0 INTRODUCTION
Actinomycetes are the group of gram positive filamentous bacteria
which are ubiquitous various natural and man-made environments. Actinomycetes
are the most economically valuable prokaryotes which are well known to produce
chemically diverse metabolites with wide range of biological activity (Balagurunathan and
Radhakrishnan, 2007).
Recent days the discovery of known metabolites and actinomycetes
are increasing due to the exploitation of natural ecosystems. Exploitation of
less and unexplored ecosystems for actinomycetes is highly necessary for the
discovery of novel bioactive metabolites. Actinomycetes are important sources
of new bioactive compounds such as antibiotics and enzymes (Xu et al., 2005) which have diverse clinical effects
and are active against many organisms (Bacteria, Fungi, Parasites etc.).
In fact more than 50% of the known natural antibiotics are produced from
actinomycetes (Miyadoh, 2003).
The most striking feature of the actinomycetes is their ability to
produce a wide variety of secondary metabolites. These natural products have
been extraordinary sources of lead structures in the development of newer drugs
(Weber et al., 2003).
Actinomycetes
are widely distributed in nature, and are typically useful in the
pharmaceutical industry for their seemingly unlimited capacity to produce
secondary metabolites with diverse chemical structures and biological activities
(Kumar et al.,2014). Agadagba (2014) also reported that the two major groups of
soil actinomycetes that serve as important sources of antibiotics are Streptomyces and Micromonospora. It has
been stated that Streptomyces account
for about 80% of the total antibiotic products; while Micromonospora closely
follows with less than one tenth as much as Streptomyces.
Many
species of actinomycetes, particularly those belonging to the genus Streptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi (Joo,
2005; Errakhi, et al., 2007). Streptomyces species are Gram positive,
aerobic microorganisms with high DNA G + C contents and produce half of all
known antibiotics from microorganisms (Kumar et al., 2014). Keikha et al. (2015) stated that the
antagonistic activity of Streptomyces
to fungal pathogens is usually related to the production of antifungal
compounds and extracellular hydrolytic enzymes. Chitinase and β- 1, 3-glucanase
are considered to be important hydrolytic enzymes in the lysis of fungal cell
walls, as for example, cell walls of Fusarium
oxysporum, Sclerotinia minor, and
S. rolfsii. Approximately, 80% of
known antibiotics have been produced from Streptomyces
(Palla et al., 2018).
Temperature
is one of the most important environmental factors in soil, exerting not only
direct effects on the soil microorganisms, but also indirect effects, for
example on the plant community composition, nutrient conditions and metal
solubility and toxicity (Fernandez-Calvino and Baath, 2010).According to
Guimaraes et al. (2004), the pH of
the culture medium is one of the most important environmental factors, because
it exerts a marked effect on the activity of several enzymes that catalyze
metabolic reactions, as well as exerting significant influence on complex
physiological phenomena such as membrane permeability and cell morphology. The
production of secondary metabolites from the genus Streptomyces can be influenced by optimization of the nutritional
requirements and cultural conditions. These conditions play an important role
in the production of these secondary metabolites (Khattab et al., 2016).
Singh
et al. (2017) suggested that the
ability of Streptomyces to form these
bioactive compounds is not a fixed property but can be greatly increased or
decreased under different conditions of nutrition and cultivation media.
Bundale et al. (2015) stated that
this is because antibiotic biosynthesis is a specific property of
microorganisms which depends greatly on culture conditions. Improvement in the
growth and antibiotic production can be carried out by manipulating the
nutritional and physical parameters of the culturing conditions. Hence media
composition plays a vital role in the efficiency and economics of the ultimate
process. He also reported that several cultural parameters like pH play a major
role in the production of bioactive metabolites and affect the antifungal
activities of Streptomyces. The temperature of the culture medium affects
not only growth but also production of the antibiotic. Optimum Temperature for
production of antimicrobial compound was found to be between 200C
and 300C .
1.1 AIM AND OBJECTIVES
The
aim of this study is to determine the effect of temperature on antifungal activity
of actinomycetes.
The
objectives are:
1. To
determine the antifungal effect of actinomycetes isolated from soil.
2. To
determine the effect of temperature on the antifungal activities of the
isolated actinomycetes.
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