IN VITRO ANTIFUNGAL POTENTIAL OF ROSEMARY (ROSMARINUS OFFICINALIS), EUCALYPTUS (EUCALYPTUS GLOBULUS) AND LEMON (CITRUS LIMON) ESSENTIAL OILS ON ANTHRACNOSE OF TOMATO (COLLETOTRICHUM).

ABSTRACT

Antifungal activities of rosemary (Rosmarinus officinalis), Eucalyptus (Eucalyptus globulus) and Lemon (Citrus limon) essential oils evaluated, suppressed the mycelial growth of postharvest pathogenic fungus Colletotrichum. The experiment carried out using the Agar well diffusion method in 75% and 100% concentration of the essential oils on SDA culture at 250C and growth measured after 3days showed that the essential oils did not have much antifungal activities on the test fungus. The Agar incorporation method showed that the three (3) essential oils had effect on the fungus. The antifungal activity was replicated three times with Eucalyptus essential oil having the highest inhibition of 100% on the mycelial growth of Colletotrichum followed by rosemary essential oil which had 95.24% inhibition on the growth of the fungus Colletotrichum, Lemon essential oil had the least antifungal activity of 76.19%. The GC-MS analysis of the essential oils revealed the presence of Eucalyptol, Oleic acid, Bicyclo[2.2.1] heptan-2-one,1, 7,7-trimethylethyl, 1R-alpha-pinene, D-Limonene which could be responsible for the antifungal activities of the different essential oils. These essential oils could be used as possible biofungicides as an alternative to synthetic fungicides against pathogenic fungi on tomato fruits.

TABLE OF CONTENTS

Certification i

Dedication ii

Acknowledgement iii

Table of Contents iv

List of Tables viii

List of Appendix ix

Abstract                                                                                                      x

CHAPTER ONE

INTRODUCTION

 Introduction 1

 Aims and objectives 3

CHAPTER TWO

LITERATURE REVIEW

2.1    Essential oils 4

2.1.1 Extraction of essential oils   4

2.1.2   Therapeutic benefits of essential oils 6

2.1.3   Absorption through the skin 7

2.1.4    Inhalation 7

2.1.5    Ingestion 7

2.1.6    Mechanism of the biological activities of essential oils 8

2.1.7    Antibacterial and antifungal action 8

2.1.8    Antiviral activity 9

2.2       Methods of extraction of essential oils 9

2.2.1    Maceration 10

2.2.2    Cold pressing 10

2.2.3    Solvent extraction 10

2.2.4     Enfleurage 11

2.2.5     Hydrodistillation 11

2.2.6     CO₂ and supercritical CO₂ extraction 11

2.2.7      Steam distillation 12

2.3         Spoilage of tomato 13

2.4         Colletotrichum species 14

2.4.1      General characteristics of Colletotrichum 14

2.5         Description of plant materials and fruit 15

2.5.1      Rosmarinus officinalis          15

2.5.1.1   Description of the oil 16

2.5.1.2    Experimental pharmacology       16

2.5.2       Citrus limon 17

2.5.2.1    Lemon essential oil 17

2.5.3       Description of Eucalyptus globulus 18

CHAPTER THREE

MATERIALS AND METHODS

3.1         The study area 20

3.2         Sample collection and identification 20

3.3         Gas chromatography- Mass Spectometry (GC-MS) 20

3.4         Identification of isolate 20

3.5         Assays for antimicrobial activity 21

3.5.1      Agar well diffusion assay 21

3.5.2      Agar incorporation assay 22

3.6        Statistical analysis 22

CHAPTER FOUR

RESULTS                                  23

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1    Discussion 33

5.2    Conclusion 35

References                                                                                                                36

Appendix

LIST OF TABLES

Table                 Title                  Page

1 Colonial features of isolate        24

2 Microscopic observation of isolate            25

3 Agar well diffusion method        26

4 Agar incorporation method        27

5 Comparison of the mean difference of the test groups and the control group      28

6          Characterization of Rosmarinus officinalis (ROSEMARY) essential oil        29

7          Characterization of Eucalyptus globulus (EUCALYPTUS) essential oil        30

 8         Characterization of Citrus limon (LEMON) essential oil             32

LIST OF APPENDIX

Appendix                Title

1                Chromatogram-Rosmarinus officinalis

2                Chromatogram- Eucalyptus globulus

3                Chromatogram- Citrus limon

4                Zone of inhibition of Citrus limon essential oil

5                Zone of inhibition of Eucalyptus globulus essential oil   

6                Zone of inhibition of Rosmarinus officinalis essential oil      

7                Colonies of Colletotrichum on incorporation of Rosmarinus officinalis essential oil           

8                Colonies of Colletotrichum on incorporation of Citrus limon essential oil    

9                Colonies of Colletotrichum on incorporation of Eucalyptus globulus essential oil

10              Colonies of Colletotrichum on incorporation of Eucalyptus globulus essential oil                                                        

CHAPTER ONE

INTRODUCTION

1.1 INTRODUCTION

Fruits are vital sources of nutrient to human beings. They give the body the necessary vitamins, fats, minerals and oils in the right proportion for human growth and development. Fruits however, have serious challenges to their existence. These include changes in climatic condition, pest, in-adequate rainfall and fungal attack.

Spoilage refers to any change in the condition of food in which the food becomes less palatable, or even toxic; these changes maybe accompanied by alterations in taste, smell, appearance, or texture. Over the years, there has been an increase in the need to identify and isolate the fungi associated with their spoilage (Oyeyipo et al., 2011).

Colletotrichum induced anthracnose is considered an important disease in fruit crops, due to the considerable losses it determines. In tomato fruits, anthracnose directly affects the fruit, producing oily stains that turn black as they grow in size. In commercial crops that receive continuous fungicide applications, losses range from 10 to 25% of the harvested fruit. When management measures are not efficient, losses can go up to 80 or even 100% of the harvest. This is therefore, the most expensive of all the crop’s issues, sometimes determining its abandonment or substitution.

In recent years, there has been an increasing interest in the use of natural substances and some questions concerning the safety of synthetic compounds have encouraged more detailed studies of plant resources. Essential oils, volatile products of plant secondary metabolism, have a wide application. A large number of essential oils and their constituents have been investigated for their antimicrobial properties against some fungi and bacteria in more than 500 reports (Kalemba and Kunicka, 2003). Because of the variability of amount and profiles of the component of essential oils, it is likely that their antimicrobial activities are not due to a single mechanism, but to several sites of action at the cellular level. Then, different modes of action are involved in the antimicrobial activity of essential oils. One of the possibilities for action is the generation of irreversible damage to the membrane of the organism; they include material losses of the organism, leakage of ions, loss of energy substrate and leading to the death of the organism. Another possibility of action is inhibition of amylase and protease and result in coagulation of the cell content (Bakkali et al., 2008; Burt, 2004; Di Pasqua et al., 2007; Hammer et al., 2008).

Essential oils are widely used in the food and pharmaceutical industries because of the antifungal activities. To face the challenge of growing public awareness and concern about food and health safely, studies concerning natural bio-preservatives have become the focus of multidisciplinary research efforts (Jing et al., 2014). This study presents the results of the antimicrobial effect of essential oils from Citrus limon (Lemon), Rosmarinus officinalis (rosemary), and Eucalyptus globulus on the fungal isolate from tomato anthracnose.

1.2 AIM AND OBJECTIVES.

Due to the losses of tomato fruits posed by some microorganisms, there is a great need to reduce this post harvest loss. Therefore, the aim of this work is to determine the effect of eucalyptus (Eucalyptus globulus), rosemary (Rosmarinus officinalis) and lemon (Citrus limon) essential oils on the anthracnose of tomato (Colletotrichum).

The objectives of this research project are:

1. To isolate and identify the fungi that causes anthracnose in tomato fruits.

2. To characterize the essential oils.

3. To evaluate the effect of essential oils of Citrus limon, Rosmarinus officinalis and Eucalyptus globulus against the fungus (Colletotrichum).

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