ENDOPHYTIC BACTERIA ASSOCIATED WITH SOLANUM TUBEROSUM (IRISH POTATO) ROOTS

Abstract

The aim of this study was to isolate endophytic bacteria from Solanum tuberosum (Irish Potato) roots. Root tubers of healthy irish potato were bought from the market located in Umudike, Abia state. The samples were surfaced sterilized, isolated and plated. The result shows that water washing from surface-sterilized root samples showed no microbial growth on trypic soy agar after incubation at 25oC. In addition, representative endophytes cultures that were also surface sterilized also failed to grow on trypic Soy agar. This indicates that epiphytic bacteria could not grow after surface sterilization and that any subsequent bacterial isolates were in fact endophytic. The two isolates Pseudomonas sp and Bacillus sp were Gram positive cocci and Gram positive rod-shaped. Morphological and physiological characteristics of the isolates showed that they belonged to the genera Pseudomonas sp and Bacillus sp. The Present study revealed that roots of S tuberosum provided a rich source of endophytic bacteria diversity. Further investigations are required to understand the potential of these endophytes as biocontrol and plant growth promoting agents that may be useful in pharmacological and agricultural fields in future.

TABLE OF CONTENTS

Cover Page                                                                                                                

Title Page                                                                                                                    i

Declaration                                                                                                                 ii

Certification                                                                                                               iii

Dedication                                                                                                                  iv

Acknowledgement                                                                                                      v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              viii

Abstract                                                                                                                      ix

CHAPTER ONE

INTRODUCTION

1.1 Background of the Study                                                                                      1

1.2 Problem Statement                                                                                               5

1.3 Aim Objectives of the Study                                                                                5

1.4 Significance of the Study                                                                                     6

CHAPTER TWO

LITERATURE REVIEW

2.1 General Characteristics of Endophytic bacteria                                                   7

2.2 Distribution of endophytic bacteria in natural habitats                                        8

2.2.1 Endophytic Endophytic bacteria                                                                       8

2.3 Isolation of Endophytics Endophytic bacteria                                                      18

2.3.1 Isolation media                                                                                                  19

2.3.2 Physical or chemical pretreatment method                                                       19

2.3.3 Other method for selective isolation of endophytic endophytic bacteria                       19

2.4 Identification and classification of endophytic bacteria                                      20

2.5 Bioactive metabolites from endophytic bacteria                                                  21

2.5.1 Antibiotics                                                                                                         22

2.5.2 Extracellular enzymes                                                                                       24

2.5.3 Plant growth promoters                                                                                     25

2.6 Endophytic bacteria as Biological Control Agents                                              29

CHAPTER THREE

METHODOLOGY

3.1 Sample Collection                                                                                                            32

3.2 Surface Sterilization and Isolation                                                                                   32

3.2.1 Test for Effectiveness of Surface Sterilization                                                             32

3.3 Selective Isolation of Endophytic Endophytic Bacteria                                                  32

3.4 Biochemical Tests                                                                                                            33

3.4.1 Coagulase Test                                                                                                              33

3.4.2 Catalase Test                                                                                                                  33

3.4.3 Methyle red test                                                                                                             34

3.4.4 Indole test                                                                                                                      34

3.4.5 Oxidase Test                                                                                                                  34

3.4.6 Gram Staining                                                                                                               35

3.4.7 Sugar fermentation test                                                                                                  36

CHAPTER FOUR

RESULTS

4.1 Identification of Bacteria Endophytes                                                                  38

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1 Discussion                                                                                                             41

5.2 Conclusion                                                                                                            42

References                                                                                                                  43

LIST OF TABLES

Table 2.1 Actinomycete antibiotics for medical applications (Kieser et al 2000)               20

Table 4.1 showing morphological characteristics of bacteria isolated from potato

(Solanum tuberosum) root                                                                                                      39

Table 4.2 Biochemical Tests Result for isolates of S tuberosum                                           41

Table 4.3 Frequency of Occurrence of Endophytic Bacteria                                                 42

CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

For decades, microbial natural products have been one of the major resources for discovery of novel drugs. Among the potential sources of natural products, bacteria have been proven to be a prolific source with a surprisingly small group of taxa accounting for the vast majority of compounds discovered. Of the 22,000 known microbial secondary metabolites, 70% are produced by endophytic bacteria, and two thirds of them are contributed by the genus Streptomyces (Subramani and Aalbersberg, 2012).

Endophytic bacteria are Gram positive, filamentous bacteria, with high G+C content (69-78%) in DNA exhibiting highly differentiated developmental, inhabiting a wide range of habitats. Unlike bacteria, endophytic bacteria are unique in their morphology with extensive branching substrate and aerial mycelium bearing chain of arthrospores. The substrate mycelium and spores can be pigmented, which makes them most colourful and attractive among microbes. On agar plates they form lichenoid, leathery or powdery colonies. They possess cell wall characteristic of bacteria and filamentous nature of fungi. They are recognized as prolific producers of secondary metabolites with diverse biological activities. Endophytic bacteria contain about 40 families and over 170 genera and about 2000 species have bee-n validly described and published (Harwani, 2013).

In the early 1950’s, with the isolation of antibiotic actinomycin from the endophytic actinomycete Streptomyces antibioticus, stimulated extensive screening of terrestrial endophytic bacteria for novel drugs. Over the past 70 years, indiscriminate screening of endophytic bacteria has led to the re-isolation of known bioactive compounds from the terrestrial environment. Hence it became crucial that endophytic bacteria from unexplored habitats be pursued as a source of novel secondary metabolites (Hamedi 2013).

Plant root endophytic harbors various kinds of micro-organisms which turned out to be an unlimited source for potential drugs, agrochemicals and biocatalysts (Yarbrough et al., 1993).These micro-organisms have been processed to produce hundreds of industrially valued commercial products in order to provide economic and efficient solution to the problems of increasing cost, pollution and world’s renewable sources (Steele and Stowers 1991).

Endophytic bacteria constitute a significant component of microbial population in different endophytic types and are widely distributed in terrestrial ecosystems. They are Gram-positive bacteria and exist as saprophytes (Takizawa et al 1993).Plant root endophytics are a major habitat for endophytic bacteria, where they aid plant growth by decomposing endophytic organic matter or fixing atmospheric nitrogen (Goodfellow and Williams 1983).They produce antibiotics which are thought to be effective against fungal infections of plants (Weller et al 2002).Most endophyticliving endophytic bacteria belong to the genus Streptomyces (Lazzarini et al 2000).They have the potential to produce a wide range of secondary metabolites and extracellular enzymes that are economical and beneficial to human beings.Roughly 60% of biologically active compounds that have developed for agriculture use originated from Streptomyces sp. with other genera such as Saccharopolyspora, Amycolatopsis, Micromonospora and Actinoplanes producing less (Challis and Hopwood 2003).  

Various groups of bioactive compounds such as macrolides, benzoquinones, amyloglycosides, polyenes and nucleoside antibiotics are examples of agriculturally useful metabolites produced from Streptomyces sp.Agroindustries have a marked interest in endophytic bacteria as a source of agroactive compounds,of PGPR and  of  biocontrol  tools (Behal 2000; Tanaka and Omura 1993).Endophytic bacteria can protect roots against invasion by root pathogenic fungi either by producing enzymes which degrade fungal cell wall or by producing antifungal compounds.Streptomyces griseoviridis strain K61 has been reported to be antagonistic to a variety of plant pathogens,including Alternaria brassicola, Botrytis cinerea and Fusarium oxysporum (Mohammadi and Lahdenpera 1992). Mycostop TM is biofungicide   that   contains   Streptomyces   griseoviridis as the active ingredient which can control some root rots and wilt diseases caused by Pythium sp.,Fusarium sp.,Rhizoctonia and Phytophthora sp. (Mahadeven and Crawford 1997).

Endophytic bacteria can promote plant growth by producing promoters such as indole-3acetic acid (IAA) to help growth of roots or siderophores to improve nutrient uptake (Merckx et al 1987).However, the rate of discovery of new secondary metabolites has been decreasing, so the discovery of endophytic bacteria in several sources increases the chance for the discovery of new secondary metabolites (Hayakawa et al 2004).Endophytic under medicinal plants is also considered an enrichment environment because of addition of root secretions and litters of plants in the rhizospheric endophytics which serve as a nutritive source for the biological activity of microorgnisms. Endophytic bacteria are able to degrade plant, animal and microbial polymers in endophytic and litter. Active endophytic bacteria may be found in medicinal plant root endophytics and may have the ability to produce new inhibitory compounds (Kennedy et al  2004) . 

Actinomycete diversity in plant root endophytics has been widely studied (Prapagdee et al 2008). More recently Thangapandian et al (2007) and Khamna et al., (2009) observed the diversity of endophytic bacteria isolated from different medicinal plants and studied their ability to produce secondary metabolites.However, the biodiversity of endophytic bacteria from plant root endophytics has been the subject of only a few studies in India

The potato is a starchy, tuberous crop from the perennial nightshade Solanum tuberosum. In many contexts, potato refers to the edible tuber, but it can also refer to the plant itself.^[2] Common or slang terms include tater and spud. Potatoes were introduced to Europe in the second half of the 16th century by the Spanish. Today they are a staple food in many parts of the world and an integral part of much of the world's food supply. As of 2014, potatoes were the world's fourth-largest food crop after maize (corn), wheat, and rice.

The potato tuber represents a belowground part of the shoot that clearly differs from the roots anatomically (Fernie & Willmitzer, 2001). While microorganisms colonizing the rhizosphere have access to exudates such as sugars, amino acids and organic acids, which are excreted by the plant (Sørensen, 1997; Bais et al., 2006), nutrient availability on the tuber surface is supposed to be mainly related to cells decaying during tuber growth or lesions (Lottmann et al., 2000). Tubers represent a primary infection source for many pathogens of potato plants that at least temporarily colonize the tuber surface (van der Wolf & De Boer, 2007). Because infected tubers often remain symptomless, pathogens are dispersed over long distances by seed tubers. During the growth of the tuber, its surface might be colonized by a complex microbial community, parts of which have the potential to antagonize phytopathogens (Clulow et al., 1994). However, this has hardly been studied so far. Most previous studies on microorganisms associated with the potato tuber focused on endophytic bacteria (Sturz et al., 1999) or on bacterial and fungal pathogens of potato tuber diseases such as Streptomyces scabies (Loria et al., 1997) or Dickeyaspecies (Dowley & O'Sullivan, 1991; Pérombelon, 2002; Gudmestad et al., 2007). Only Lottmann et al. (1999) studied bacterial isolates from the tuber surface. They characterized 42 bacteria that in vitro antagonized Verticillium dahliae or Erwinia carotovora. Despite the obvious potential to control tuber-disseminated potato diseases through antagonistic communities, serious efforts have not yet been made to study the impact of the potato genotype and other factors on the tuber-associated bacterial communities.

1.2 Problem Statement

The potato genotype was shown to be one of the factors shaping the associated microbial community in the rhizosphere (Becker et al., 2008; Weinert et al., 2009; Andreote et al., 2010). Even minor genotype differences as between genetically modified (GM) and parental potato lines are believed to affect the microbial colonization of the plants (Heuer et al., 2002; Rasche et al., 2006; van Overbeek & van Elsas, 2008; Weinert et al., 2009). Potato plants accumulating the carotenoid zeaxanthin in their tubers were designed to serve as a functional food counteracting age-related macular degeneration (Snodderly, 1995; Römer et al., 2002). Potato plants normally transform zeaxanthin to violaxanthin, which acts as a UV protectant. The fully developed tubers of the GM plants contain high amounts of zeaxanthin (Römer et al., 2002). It is not well understood whether high concentrations of such compounds in the tuber could affect associated bacterial communities. This study therefore is carried out to isolate and characterize endophytic endophytic bacteria (bacteria) from potato roots.

1.3 Aim Objectives of the Study

The aim of this study is the isolation of endophytic bacteria from potato (Solanum tuberosum) roots. Other specific objectives include:

1.     Morphological and biochemical characterization of actinomycete isolates from Potato roots (Solanum tuberosum),

2.     Functional characterization of the actinomycete isolates for exploring its bioactive potential for biotechnological applications.

1.4 Significance of the Study

Endophytic endophytic bacteria has been shown to have warious medicinal importance over the centuries, as like plants, endophytic microorganism has the capacity to produce secondary metabolites thereby it serves as an optimistic source for the novel compound [5]. Although previous studies reported that several bio-pharmacological compounds were isolated from endophytes with activities such as antiviral, anti-inflammatory, antimicrobial and antitumor activities but there is less information about their antioxidant activities. Endophytic are plant-associated bacteria which has valuable effects on the host plants. When compared to rhizospheric bacteria, endophytes are more reliable and specific habitat as it resides within tissues of the plant. Therefore this study tends to explore more plant roots that may be possible houses for large number of endophytic endophytic bacteria.

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