EFFECTS OF CARBON SOURCE ON THE ANTIFUNGAL ACTIVITIES OF SOME ISOLATED ACTINOMYCETES.

ABSTRACT

This study was carried out to determine the effects of carbon source on the antifungal activities of some isolated species of Actinomycetes. Two (2) isolates of Actinomycetes and Streptomyces species were tested against two phytopathogens (Alternaria and Collectotrichum) On screening for its ability to enhance antifungal activity of actinomycetes, basal medium supplemented with glucose as a carbon source was found to be the best for antibiotic production by Actinomyces with the zone of inhibition of (17mm) against Alternaria and (21mm) against Collectrotrichum, this is followed by soluble starch with the zone of inhibition of (15mm) and (18mm) against Alternaria and Collectrotrichum, lactose (5mm and 4mm) and sucrose (2mm and 1mm) whereas fructose had repressed the production of the antimicrobial substance. Basal medium supplemented with soluble starch (12mm and 14mm) as a carbon source was found to be the best for antibiotic production by Streptomyces; this is followed by glucose (10mm and 11mm), lactose (5 and 6) and sucrose (4mm and 6mm) whereas no antimicrobial activity was detected using fructose. The results of this study showed that Actinomyces and Streptomyces species produces antifungal substances which are particularly active against the test organisms; Alternaria and Collectotrichum. Also, soluble starch and glucose were good carbon sources for optimal production of secondary metabolites.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii      

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aims and Objectives                                                                                                  3

CHAPTER TWO

2.0       Literature Review                                                                                                       4

2.1       The Actinomycetes                                                                                                     4

2.2       Streptomyces                                                                                                               6

2.3       Classification of Streptomyces                                                                                   7

2.4       The Life Cycle of Streptomyces                                                                                 7

2.5       Streptomyces Habitats                                                                                                8

2.5.1    Hay and other organic material                                                                                  8

2.5.2    Fresh water and marine habitats                                                                                 9

2.6       Streptomyces Requirements                                                                                       9

2.7       Streptomyces Metabolites                                                                                           10

2.8       Isolation of Streptomyces                                                                                           10

2.9       importances of streptomyces                                                                                      11

2.10     Streptomyces and antibiotics                                                                                     12

2.11     Antibacterial activity of Streptomyces                                                                       14

2.11.1  Sampling                                                                                                                     14

2.12     Nutrient Requirements for Antibiotic Production                                                      14

2.13     Antibacterial Activity Protocols                                                                                 16

2.13.1  Cross streak method                                                                                                   16

2.13.2  Agar overlay method                                                                                                  16

2.13.3  Disc Diffusion assay                                                                                                   16

2.13.4  Agar well diffusion assay                                                                                           17

2.14     Factors Affecting Antibiotic Production                                                                    17

2.14.1  Carbon Source                                                                                                            17

2.14.2  Nitrogen source                                                                                                          18

2.14.3  Inorganic phosphate                                                                                                   18

2.14.4  Trace metals                                                                                                               19

CHAPTER THREE

3.0       Materials and Methods                                                                                               20

3.1       Study Area                                                                                                                  20

3.2       Sample Collection                                                                                                      20

3.3       Preparation of Culture Media                                                                                     20

3.4       Sample Inoculation                                                                                                     21

3.5       Screening for Antimicrobial Activity Of The Isolates                                               21

CHAPTER FOUR

4.0       Result                                                                                                                          23

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                        26

5.1       Discussion                                                                                                                   26

5.2       Conclusion                                                                                                                  27

            References                                                                                                                  28

            Appendix                                                                                                                    36

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Streptomyces, Greek adjective streptos (pliant or bent) myces (fungus) therefore, Streptomyces means pliant or bent fungus (Ambarwati et al., 2012). Streptomyces species are gram-positive filamentous bacteria that belong to the Actinommycetales. They are characterized by the ability to form reproductive mycelium from vegetative mycelium in soil culture. The filamentous growth and the branching of Streptomyces mycelia differentiate these organisms from the true bacteria. Commonly, the genus Streptomyces has slender coenocytic hyphae, the aerial mycelium at maturity forms chains of three to many spores (Bais et al., 2012). Members of the genus are soil inhabitants. They are common in wet than in dry areas having a pH of about 6.5-8.0, and with the exception of few species that cause mycetoma; Streptomyces are saprophytes. They may be found on vegetation, food products, manures, peat, water basins, composts, silage, fresh water and river bottoms, dust and plant residues.

For many years, members of Actinomycetales were classified with fungi, with which they share parallel evolution, but to which they are completely unrelated (Baniasadi, 2009). Various keys for the identification of Streptomyces have been suggested and the most common of them employs four criteria: colour of aerial mycelium, spore chain morphology, structure of spore surface, and melanin formation. Generally, new bioactive products from microbes continue to be discovered at an amazing pace: 500 per year (Baskaran et al., 2011). As a result of the increasing prevalence of antibiotic-resistant pathogens and the pharmacological limitation of antibiotics, there is exigency for new antimicrobial substances. In fact, many of the known antibiotics produced by members of the family Bacillaceae are polypeptides, which have proven generally to be somewhat unstable and difficult to purify. Antibiotics produced by fungi, with a few notable exceptions, are generally found to be too toxic for treatment of eukaryotes including plants (Dehnad et al., 2010).

However, the antibiotics produced by Streptomyces are comparatively recognized as generally safe and stable. For this reason Streptomyces screening for the production of new antibiotics has been intensively pursued for many years by a number of scientists. This resulted in the characterization and purification of about 6000 distinct antibiotic substances from Streptomyces species (Hassan et al., 2011).

Among the different types of drugs prevailing in the market, antifungal antibiotics are very few but significant and have an important role in the control of mycotic plant and animal diseases (Ilić et al., 2010). The search for new, safer, broad- spectrum antifungal agents with greater potency has been progressing. The reason for this is that when compared to antibacterial, fungi, like plant cells, are eukaryotes and therefore agents that inhibit protein, RNA or DNA biosynthesis in fungi have greater potential for toxicity on plant as well (Kariminik and Baniasadi, 2010).

Recent reports have shown that Streptomyces continue to remain an important source of antifungals examples included: 24-Demethylbafilomycin C1, Phenyl - 1- napthyl- phenyl acetamide and DPTB16, and (6S,8aS,9S,11S,12aR)-6-hydroxy-9,10-dimethyldecahydrobenzo [d] azecine- 2,4,12(3H)- trione (Kavitha et al., 2010). About 80% of plant diseases can be traced to fungi. In Sudan; fungi infect some important crops and cause serious diseases that lead to great losses in the production of these crops. Examples include: Alternaria early blight on tomato, Alternaria leaf spot on sesame, Macrophomina charcoal rot on sesame, Drechslera leaf spot on sorghum, Colletotrichum tissue necrosis in beans, D. maydis leaf blight on corn, and Fusarium wilt caused by Fusarium spp. (Kharat et al., 2009). Continuous screening of Streptomyces for secondary metabolites production can possibly reveal a novel antifungal agent which can be used to treat one or more of such plant diseases. Hence the main objective of this study has been screen locally isolated Streptomyces for production of potent antifungal agents that can be used to control some selected important fungal plant pathogens.

1.1       AIMS AND OBJECTIVES

The aim of this study was to determine the effect of different carbon sources on the antifungal activities of some isolated species of Actinomycetes against some test organisms.

The objectives are;

1.     To determine the antifungal activities of Actinomycetes species against some test organisms.

2.     To determine how carbon source could be manipulated to enhance the antifungal substances produced by Actinomycetes species.

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