EVALUATION OF DIFFERENT METHODS OF ISOLATION OF ACTINOMYCETES

ABSTRACT

This study evaluated various methods of isolation of actinomycetes species from soil of which one soil sample was collected from designated area in Umudike for the purpose of the work. A total of five (5) actinomycetes species were isolated from the soil using the various methods of isolation which included Direct inoculation, Membrane filter and Sprinkle plate methods. The cultural characteristics from this study, using direct inoculation method showed no growth on the media while there was Cottony white appearance on media using both Membrane filter and Sprinkle plate methods. The results from this study showed that a total number of five (5) actinomycetes species were isolated using various isolation methods. Sprinkle plate method had the highest percentage and number of actinomycetes species 4(80%), while the least number of isolates employed in the Membrane filter method 1(20%). No actinomycetes species was isolated using the direct inoculation method. Conclusively, these methods can provide significant impetus towards the isolation and screening of novel actinomycetes which will be ultimately significant for discoveries and other industrially important bioactive compounds like antibiotics. Isolation of rare actinomycetes is difficult using conventional isolation techniques and hence advanced techniques and high screening techniques have been adopted for their isolations.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    3

CHAPTER TWO

2.1       Literature Review                                                                                                       4

2.2       Brief Description of Actinomycetes                                                                           4

2.3       Structure of Actinomycetes                                                                                        5

2.4       Plant Growth Promoting Activity of Actinomycetes                                                 6

2.5       Production of Plant Growth Hormone by Actinomycetes                                         8

2.6       Filamentous Actinomycetes                                                                                       9

2.7       Various Methods of Isolating Rhizospheric Actinomycetes                                      10

2.7.1    Bacterial Isolation Methods                                                                                        10

2.7.2    Selective Isolation Methods                                                                                       10

2.7.3    Nutritional Selection                                                                                                   11

2.7.4    Selective Inhibition                                                                                                     12

2.7.5    Pretreatment of Sample                                                                                              12

2.7.6    Physical Treatments                                                                                                   13

2.7.7    Chemical Treatments                                                                                                  14

2.7.8    Membrane Filter Method                                                                                            14

CHAPTER THREE

3.0       Materials and Methods                                                                                               16

3.1       Sample Collection                                                                                                      16

3.2       Processing of Samples                                                                                                16

3.3       Microbial Analysis                                                                                                     16

3.3.1    Sterilization Method                                                                                                   16

3.4       Methods of Isolating Actinomycetes from the Soil                                                    16

3.4.1    Serial Dilution Technique                                                                                          16

3.4.2    Membrane Filter Technique                                                                                       17

3.4.3    Direct Inoculation Technique                                                                                     17

3.5       Microbial Characterization and Identification                                                           18

3.5.1    Identification of Rhizospheric Actinomycetes                                                           18

3.5.1.1 Gram Staining                                                                                                             18

3.6.2    Biochemical Tests                                                                                                      18

3.6.2.1 Indole Test                                                                                                                  18

3.6.2.2 Carbohydrate Utilization Analysis                                                                             18

3.6.2.3 Catalase Test                                                                                                               19

3.6.2.4 Oxidase Test                                                                                                               19

3.6.2.5 Coagulase Test                                                                                                           19

3.6.2.6 Citrate Utilization Test                                                                                               19

3.6.2.7 Motility Test                                                                                                               20

CHAPTER FOUR

4.0       Results                                                                                                                        21

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                        26

5.1       Discussion                                                                                                                   26

5.2       Conclusion                                                                                                                  27

            References

LIST OF TABLES

CHAPTER ONE

1.0   INTRODUCTION

Actinomycetes have been and remain the most fruitful source of microorganisms for all types of bioactive metabolites, including agroactive type. Over one thousand secondary metabolites from actinomycetes were discovered during 1988-1992. Most of these compounds are produced by various species of the genus Streptomyces. In fact, about 60% of the new insecticides and herbicides reported in the past 5 years originate from Streptomyces (Tanaka and Omura, 2003). It is also estimated that as many as three-quarters of all streptomycete species are capable of antibiotic production (Alexander, 2007). Actinomycetes produce a variety of antibiotics with diverse chemical structures such as polyketides, b-lactams and peptides in addition to a variety of other secondary metabolites that have antifungal, anti-tumor and immunosuppressive activities. Actinomycetes can promote plant growth by producing promoters such as indole-3-acetic acid (IAA) to help growth of roots or produce siderophores to improve nutrient uptake (Merckx et al., 2007).

However, the rate of discovery of new secondary metabolites has been decreasing, so the discovery of actinomycetes from several sources increases the chance for the discovery of new secondary metabolites (Hayakawa et al., 2004). Active actinomycetes may be found in medicinal plant root rhizosphere soils and may have the ability to produce new inhibitory compounds. In attempts to develop commercial biocontrol and plant growth promoting products using rhizobacteria, it is important to recognize the specific challenges they present. To begin with, the interaction between plant growth promoting actinomycetes species and their plant symbionts appears to be specific, even within a crop or cultivar (Glick, 2005). While a rhizobacterium screened for growth promotion may reveal positive effects on one crop, it may have no effect, or even retard growth of another crop (O'Neill et al., 2002).

Plant growth promoting rhizobacteria (PGPR) is a group of naturally occurring, free living rhizosphere colonizing bacteria that improve plant growth, increase yield, enhance soil fertility, and reduce pathogens as well as biotic or abiotic stresses (Vessey, 2003). Plant growth promoting rhizobacteria help the plants by producing plant growth phytohormones such as indole acetic acid (IAA), cytokinins, and gibberellins (Marques et al., 2010), solubilization of inorganic phosphate (Jeon et al., 2003), asymbiotic nitrogen fixation (Khan, 2005), antagonistic effect against phytopathogenic microorganisms by producing siderophore, antibiotics, and fungicidal compounds (Majeed et al., 2015). Actinomycetes are present extensively in the plant rhizosphere and produce various agroactive compounds.

In the last few years, this group of bacteria, due to its strong antimicrobial potential, and soil dominant saprophytic nature, gained much attention as plant growth promoters (Franco-Correa et al., 2010).

Actinomycetes can actively colonize plant root systems, can degrade a wide range of biopolymers by secreting several hydrolytic enzymes and tolerate hostile conditions by forming spores. Actinomycetes, especially Streptomyces, also exhibit immense biocontrol action against a range of phytopathogens (Wang et al., 2013). Actinomycetes can produce phytohormones indole acetic acid (IAA) and siderophore as well as solubilize phosphate and promote plant growth (Jeon et al., 2003). Actinomycetes have been mainly exploited in pharmaceutical industry since 1940s, Whereas, only a few have been developed as commercial products for plant application in agriculture (Minuto et al., 2006). Streptomycetes have been long considered simply as free-living soil inhabitants, but recently the importance of their complex interactions with plants, and other organisms is being uncovered (Seipke et al., 2011).

1.1  AIM AND OBJECTIVES

To evaluate various methods for of isolation of actinomycetes, while the specific objectives included;

      i.         The determination of the effectiveness of the various methods employed in the isolation of rhizospeheric actinomycetes

     ii.         The determination of the identities of the actinomycetes isolates.

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