Whitefly Control and Anti-Microbiological Activities of Essential Oils from Medicinal Plants Found in Fiji Islands

WHITEFLY CONTROL AND ANTI-MICROBIOLOGICAL ACTIVITIES OF ESSENTIAL OILS FROM MEDICINAL PLANTS FOUND IN FIJI ISLANDS.

Ravneel Rajneel Chand

A thesis submitted in fulfillment of the requirements for the degree of Masters of Science

August, 2016 DECLARATION OF ORIGINALITY

I, Ravneel Chand, declare that this thesis is my own work and has not been submitted in any other university. The information provided is best of my knowledge, and information derived from the work of others has been acknowledged in the reference list.

Statement by the Principal Supervisor

The research work carried by the principal researcher was solely under my supervision and to my best of knowledge.

Co-Supervisor

Every challenging work needs self-endeavour as well as directions from elders, especially those closest to us. I dedicate this thesis, the fruits of my labour, to my wonderful parents Mr Suresh Chand and Mrs Roshni Chand.

ACKNOWLEDGEMENT Prima facea, I am very thankful to the God for the good wellbeing and health throughout my research journey.

I acknowledge my sincere gratitude to my Principal supervisor, Associate Professor Anjeela Jokhan and my Co-supervisor Dr. Romila Devi Gopalan for their advice, encouragement and continuous support throughout the study. Working with them was the best part of the research. Thank you once again for the constant motivation and assistance in shaping my transitional skills.

A warm thanks to Mr Ashley Dowell and the team from Southern Cross University, Queensland, Australia for assisting me through the identification of compounds in selected essential oils. A special thanks to the Chief Scientist Dr Rajeswara Rao, Dr. Karuna Shanker and the team from Central Institute of Medicinal and Aromatic Plants, India, for sharing their thoughts and ideas throughout the research.

I am grateful to the Chief Technician Mr Dinesh Kumar from the Biology Department (USP) for continuous support and assisting me with the plant materials that I possibly have not identified on my own. A special thanks to Dr. Tamara Osborne and Ms Reema Prakash for continuous support through my research journey especially helping out to structure my thesis. Also, I am thankful to Ms Aradhana Deesh from Koronivia Research Station for the assistance in the identification of the selected plant materials and the whitefly species.

I am thoroughly grateful to my parents for their continuous support, love and understanding through my research journey, it is the faith that they had on me made me complete this thesis.

Finally, I take this opportunity to express my gratitude to one and all, who has directly or indirectly assisted me in completing my Masters study.

ABSTRACT A variety of plant materials contain essential oils that have extensive bioactivity properties. These properties are attributed to the chemical composition of essential oils. In the current research, chemical composition, whitefly control and anti- microbiological activities of essential oils from five medicinal plants found in Fiji; Cananga odorata (Makasoi), Murraya koenigii (L) Spreng (Curry leaves), Euodia hortensis forma hortensis (Uci), Ocimum tenuiflorum L (Tulsi) and Cymbopogon citratus (Lemon grass) were investigated. Firstly, the selected essential oils were analysed using Gas Chromatography Mass Spectrometry (GC-MS). The identified compounds were classified into groups.

For the biological activities, different concentrations of essential oil solutions (0.25%, 0.5% and 5% (v/v)) were subjected to whitefly (Aleurodicus dispersus Russell) control activities in the form of fumigant and repellent test. Essential oils from O. tenuiflorum L were found to be best fumigant agents (100% mortality was achieved at 3 hours after exposure). The significant differences in the mortality for all the tested time (3, 6, 9, 12 and 24 hours) were only shown by O. tenuiflorum L and C. citratus essential oils, as the p<0.05 (5% level of significance). For the repellent test, none of the essential oils obtained 100% repellency based on Repellency index (RI%), however C. citratus and M. koenigii (L) Spreng were found to show the best repelling properties (RI%= 52, 52) compared to the other studied essential oils. In addition, the essential oils exhibited a very interesting antimicrobial profile when tested against five different bacteria and fungi at different concentrations (0.25%, 0.5%, 5%, 25%, 50% and 100% (v/v)). The essential oils from O. tenuiflorum L were considered to have strong antimicrobial properties as it showed the inhibition effect to all test bacteria and fungi.

The trends in the chemical constituents of essential oils revealed that the phenolic and alcoholic compounds were major groups of contributors for the tested activities. Thus, these data suggested that essential oils from selected medicinal plants found in Fiji have potential to be employed in pesticide or anti-microbiological activities.

TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... iv

ABSTRACT ...... v

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... x

LIST OF FIGURES ...... xii

1.CHAPTER 1: ESSENTIAL OILS ...... 1

1.0 Introduction ...... 1

1.1 Formation of Essential Oils ...... 1

1.2 Components of Essential Oils ...... 3

1.2.1 Terpenes hydrocarbons ...... 3

1.2.2 Oxygenated Compounds ...... 7

1.2.3 Ethers...... 8

1.2.4 Aldehydes ...... 9

1.2.5 Ketones ...... 10

1.2.6 Organic acids and esters ...... 10

1.2.7 Oxides ...... 11

1.3 Extraction of Essential Oils ...... 11

1.3.1 Distillation ...... 11

1.3.2 Solvent extraction...... 13

1.3.3 Enfleurage ...... 13

1.4 Methods for Analysis of Chemical Constituents ...... 13

1.4.1 Gas-Liquid Chromatography ...... 14

1.4.2 Gas Chromatography-Mass Spectrometry ...... 14

1.5 Common Uses of Essential Oils ...... 14

1.5.1 Essential Oils Used by Plants ...... 14

1.5.2 Essential Oils Used by Humans ...... 15

1.6 Purpose of this study ...... 18

2.CHAPTER 2: CHEMICAL ANALYSIS OF ESSENTIAL OILS FROM SELECTED MEDICINAL PLANTS FOUND IN FIJI...... 20

2.0 Introduction ...... 20

2.1 Background ...... 21

2.1.1 Description and Common Uses of Selected Medicinal Plants Found in Fiji...... 21

2.2 Methodology ...... 26

2.2.1 Collection of Plant Materials ...... 26

2.2.2 Extraction of Essential Oils ...... 26

2.2.3 Analysis of Chemical Constituents ...... 27

2.3 Results ...... 28

2.3.1 Physical Properties ...... 28

2.3.2 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis ...... 29

2.4 Discussion ...... 40

2.4.1 Cananga odorata (Makosoi) ...... 40

2.4.2 Murraya koenigii (Curry leaves) ...... 41

2.4.3 Euodia hortensis forma hortensis (Uci)...... 42

2.4.4 Ocimum tenuiflorum L (Tulsi) ...... 43

2.4.5 Cymbopogon citratus (Lemon grass) ...... 43

2.5 Factors Responsible for the Essential Oil Composition...... 44

2.6 Conclusion ...... 45

3.CHAPTER 3: FUMIGANT AND REPELLENCY EFFECT OF PLANT ESSENTIAL OILS TO SPIRALLING WHITEFLIES (ALEURODICUS DISPERSUS RUSSELL)...... 47

3.0 Introduction ...... 47

3.1 Background ...... 47 vii

3.1.1 Classification of Spiralling Whitefly ...... 47

3.1.2 The Life Cycle of Spiralling Whiteflies (Aleurodicus dispersus Russell) ...... 49

3.1.3 Spiralling Whitefly- Why Considered a Pest...... 52

3.1.4 Management Strategies of Whiteflies ...... 53

3.2 Methodology ...... 59

3.2.1 Preparation of Essential Oil Solution ...... 59

3.2.2 Whiteflies Breeding -Greenhouse ...... 59

3.2.3 Fumigant Test ...... 62

3.2.4 Repellent Test...... 63

3.2.5 Statistical Analysis ...... 65

3.3 Results ...... 66

3.3.1 Fumigant effect of essential oils on Spiralling whiteflies ...... 66

3.3.2 Repellent Test...... 70

3.4 Discussion ...... 72

3.4.1 Fumigant Test ...... 72

3.4.2 Repellent Test...... 77

3.4.3 Mode of Action of Essential oils in Arthropods (Whiteflies) ...... 80

3.5 Conclusion ...... 84

4.CHAPTER 4: ANTIMICROBIAL ACTIVITIES OF SELECTED ESSENTIAL OILS ...... 86

4.0 Introduction ...... 86

4.1 Background ...... 86

4.1.1 Microorganisms ...... 86

4.1.2 Why Essential Oils as Alternatives for Elimination Pathogenic Micro- organisms? ...... 89

4.2 Methodology ...... 90

viii

4.2.1 Test against Bacteria and Fungi strains ...... 90

4.2.2 Preparation of Essential oil solutions ...... 91

4.2.3 Statistical Analysis ...... 92

4.3 Results ...... 92

4.3.1 Anti-bacterial Activities of Selected Essential oils ...... 92

4.3.2 Anti-fungal Activities of Selected Essential oils ...... 98

4.4 Discussion ...... 103

4.4.1 Anti-bacterial Effect of each Essential oil and its Chemical Perspective ...... 103

4.4.2 Anti-fungal Effects of each Essential oil and its Chemical Perspective ...... 110

4.5 Conclusion ...... 114

5.CHAPTER 5: CONCLUSION AND RECOMMENDATION ...... 117

6.APPENDIX ...... 119

6.0 Chemical Analysis ...... 119

6.1 Whiteflies ...... 120

6.1.1 Results of Fumigant test on whiteflies: ...... 122

6.1.2 Repellent Test...... 130

6.2 Microbiology ...... 132

6.2.1 Bacteria ...... 132

6.2.2 Fungi ...... 139

7.REFERENCE ...... 145

LIST OF TABLES

Table 2-1: Physical properties of selected essential oils from medicinal plants found in Fiji...... 29

Table 2-2: Compounds identified in the essential oil from the flowers of C. odorata...... 30

Table 2-3: Compounds identified in the essential oil from the leaves of M. koenigii (L) Spreng...... 32

Table 2-4: Compounds identified in the essential oil from the leaves of E. hortensis forma hortensis...... 34

Table 2-5: Compounds identified in the essential oil from leaves of O. tenuiflorum L...... 36

Table 2-6: Compounds identified in the essential oil from the leaves of C. citratus ...... 38

Table 2-7: Comparison of major chemical composition of C. odorata essential oils ...... 40

Table 2-8: Comparison of chemical composition of essential oils from M. koenigii...... 42

Table 2-9: Comparison of chemical composition of O. tenuiflorum L essential oils ...... 43

Table 2-10: Comparison of GC-MS analysis of C. citratus essential oils ...... 44

Table 3-1: Limitations of Biological Control...... 55

Table 3-2: Dose-effect analysis of essential oils on the adult Spiralling whiteflies after 24 hours...... 70

Table 3-3: Summary of repellent effect (6-8 hours) on adult whiteflies at different concentrations (Using Probit analysis)...... 72

Table 3-4: Studies of effects of plant essential oils on whiteflies...... 74

Table 3-5: Studies of repellent effects of plant essential oils on whiteflies...... 78

Table 4-1: Harmful effects of selected Gram (+) and (-) bacteria...... 87

Table 4-2: Effects of selected fungi to humans through food and agriculture industries ...... 88

Table 4-3: Mean and Standard Error (SE) for effects of varying concentration of the essential oils on different bacteria...... 95

Table 4-4: Mean and Standard Error (SE) for effects of varying concentration of the essential oils on different fungi...... 100

Table 6-1: Chemical Analysis-group of major chemical compounds from selected essential oils...... 119

Table 6-2: Common Pest Species of Whiteflies with Distinct Nymphs ...... 120

Table 6-3: Multiple Comparisons (Post Hoc Test) for C. odorata...... 123

Table 6-4: Multiple Comparisons (Post Hoc Test) for M. koenigii (L) ...... 124

Table 6-5: Multiple Comparisons (Post Hoc Test) for E. hortensis forma hortensis...... 125

Table 6-6: Multiple Comparisons (Post Hoc Test) for C. citratus...... 126

Table 6-7: Multiple Comparisons (Post Hoc Test) for O. tenuiflorum L ...... 127

Table 6-8: Independent Sample t-test for repellent test ...... 130

Table 6-9: Effect of control on selected bacteria ...... 132

Table 6-10: Descriptive statistics for zone of inhibition (mm) across different concentrations ...... 132

Table 6-11: Effect on control on selected fungi ...... 139

Table 6-12: Descriptive statistics for zone of inhibition (mm) of fungi across different concentration ...... 139

LIST OF FIGURES

Figure 1-1: Synthesis of different classes of terpenes in plants...... 2

Figure 1-2: Selected structures of Monoterpenes...... 4

Figure 1-3: Selected structures of Sesquiterpenes...... 5

Figure 1-4: Selected structures of Diterpenes...... 6

Figure 1-5: Selected structure of Triterpene...... 7

Figure 1-6: Selected structures of Alcohol...... 7

Figure 1-7: Selected structures of Phenols...... 8

Figure 1-8: Selected structures of Ethers...... 9

Figure 1-9: Selected structures of Aldehydes...... 9

Figure 1-10: Selected structures of Ketones...... 10

Figure 1-11: Selected structure of Ester...... 11

Figure 1-12: Selected structure of Oxide...... 11

Figure 1-13: Function of secondary metabolites in plants...... 15

Figure 2-1: Flowers of C. odorata ...... 21

Figure 2-2: Cymbopogon citratus leaves ...... 22

Figure 2-3: Murraya koenigii (L) Spreng plants...... 23

Figure 2-4: Branches of O. tenuiflorum L plants ...... 24

Figure 2-5: Euodia hortensis forma hortensis plant...... 25

Figure 2-6: The sample collection sites in Fiji islands...... 26

Figure 2-7: Set-up for hydro-distillation...... 27

Figure 2-8: GC-MS chromatogram of essential oil from the flowers of C. odorata...... 31

Figure 2-9: GC-MS chromatogram of essential oil from the leaves of M. koenigii (L) Spreng...... 33

xii

Figure 2-10: GC-MS chromatogram of essential oil from leaves of E. hortensis forma hortensis...... 35

Figure 2-11: GC-MS chromatogram of essential oil from O. tenuiflorum L leaves .. 37

Figure 2-12: GC-MS chromatogram of essential oil from C. citratus leaves...... 39

Figure 3-1: Whiteflies on cassava leaves ...... 47

Figure 3-2: Distribution of the Aleurodicus dispersus...... 48

Figure 3-3: Mature pupa (~1.06 mm) of Spiralling whitefly ...... 50

Figure 3-4: Adult (~1.74 mm) of Spiralling whitefly...... 51

Figure 3-5: Life cycle of the Spiralling whitefly ...... 51

Figure 3-6: Electron micrograph of egg pedicel showing insertion of egg stalk into stoma of a plant leaf...... 52

Figure 3-7: Average minimum and maximum temperatures (A) and relative humidity (B) in Suva, Fiji islands for year 2015...... 60

Figure 3-8: Cassava plants for the whitefly experiment...... 61

Figure 3-9: Fumigant test setup (A). Randomised labelled plastic bag (B)...... 63

Figure 3-10: T-shaped olfactometer...... 63

Figure 3-11: Setup for the repellent test in the laboratory...... 64

Figure 3-12: Fumigant effect (Mean ±SE) of 0.25 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals...... 67

Figure 3-13: Fumigant effect (Mean ±SE) of 0.5 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals...... 68

Figure 3-14: Fumigant effect (Mean ±SE) of 5 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals...... 69

Figure 3-15: Repellency Index (%) response of 0.25%, 0.5% and 5% (v/v) essential oil solutions on the adult Spiralling whiteflies...... 71

Figure 3-16: Target sites in insects as possible neurotransmitter mediated toxic action of essential oils...... 82

Figure 4-1: Anti-bacterial effect of selected essential oils at 5% (v/v) solution...... 96 xiii

Figure 4-2: Anti-bacterial effect of selected essential oils at 25% (v/v) solution...... 96

Figure 4-3: Anti-bacterial effect of selected essential oils at 50% (v/v) solution...... 97

Figure 4-4: Anti-bacterial effect of selected essential oils at 100% (v/v) solution. ... 97

Figure 4-5: Anti-fungal effect of essential oils at 5% (v/v) solution...... 101

Figure 4-6: Anti-fungal effect of essential oils at 25% (v/v) solution...... 101

Figure 4-7: Anti-fungal effect of essential oils at 50% (v/v) solution...... 102

Figure 4-8: Anti-fungal effect of essential oils at 100% (v/v) solution ...... 102

Figure 4-9: Mode of action of essential oils on bacterial cell...... 108

Figure 4-10: Envelops of Gram-positive (right side) and Gram-negative (left side) bacteria...... 109

Figure 6-1: General effect of different concentrations (with respect to time factor) on the mean mortality of whiteflies...... 122

Figure 6-2: Probit analysis of fumigant test on selected essential oils at different time interval...... 129

Figure 6-3: Probit analysis of repellent test on selected essential oils...... 131

xiv

1. CHAPTER 1: ESSENTIAL OILS Life on earth began about 4 billion years ago with a single-celled organism that did not have a nucleus. Many of these basic organisms, including algae and bacteria, are still living in our world today. Through gradual evolution a vast range of aromatic plants evolved that presently produces 30,000 known volatile oils (Essential oils). (Elpel (1998) cited in Buckle (2015a)).

1.0 Introduction

Essential oils are diverse groups of natural products which are mainly produced by plants for defence, signalling or part of their secondary metabolism (Charles & Simon, 1990; Bakkali et al., 2008). These oils are volatile liquids which has a lower density than water (Bakkali et al., 2008). Essential oils are also known as ‘essence’ that are strong-smelling liquid components found in aromatic plants, grasses and trees (Ríos, 2016). Essential oils are mostly formed in plants such as flowers, leaves, buds, fruits, seeds, bark and roots (Isman, 2000; Ríos, 2016).The synthesised essential oils are mostly kept in secondary cell cavities, epidermal cells, canals or glandular trichomes (Nazzaro et al., 2013). This chapter focuses on essential oils; their formation, extraction and methods of analysis with its common uses. At the end of this chapter the purpose of the current study is highlighted with a touch stone of subsequent chapters.

1.1 Formation of Essential Oils

Essential oils mostly have a high constituent of terpenes (Farag et al., 1989). Terpenes are usually formed using mevalonate pathways. Mevalonate pathway is also known as isoprenoid pathway which occurs in all higher eukaryotes (Corsini et al., 1993). This biosynthetic pathway is used to produce dimethyl allyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). These two compounds serve as the basis for the biosynthesis of molecules in diverse processes of terpene synthesis, protein prenylation, cell membrane maintenance, hormones, N- glycosylation and protein anchoring (Chaichana, 2009; Cooper & Nicola, 2014).

Terpene biosynthesis involves addition of isopentenyl diphosphate (IPP; C5) to its isomer dimethylallyl diphosphate (DMAPP; C5 - can also form hemiterpenes) synthesizing geranyl diphosphate (GPP; C10) which is a precursor for synthesis of

monoterpenes. GPP and FPP form monoterpenes and sesquiterpenes skeletons

respectively. Further condensation of enzyme-bound geranyl diphosphate (GPP; C10)

with addition of IPP units forms farnasyl diphosphate (FPP; C15). Geranylgeranyl

diphosphate (GGPP; C20), that goes through series of reactions such as cyclization, rearrangement or coupling to form diterpenes and polyterpenes. The Figure 1-1 below shows the parental precursors to synthesise terpenes.

IPP; C5 Isomerase DMAPP; C5

Hemiterpenes (isoprene’s)

GPP synthase Prenylated metabolites Cytokinins Anthraquinones

Phytosterols GPP; C10 Monoterpenes

FPP synthase (+IPP) Essential Triterpenoids2X oil

2X Squalene FPP; C15 Sesquiterpenes Ubiquinone Plastoquinone

Abscisic acid

Prenylated proteins Saponins GGPP synthase (+IPP)

GGPP; C20 Diterpenes Gibberellins Chlorophyll Prenylated proteins Polyterpenes Carotenoids.

Figure 1-1: Synthesis of different classes of terpenes in plants. (Dubey et al., 2003)

Key: DMAPP - Dimethylallyl diphosphate IPP - Isopentenyl diphosphate FPP - Farnesyl diphosphate GPP - Geranyl diphosphate GGPP - Geranylgeranyl diphosphate

1.2 Components of Essential Oils

Essential oils containing between 20-60 components at different concentrations are considered to be very complex natural mixtures (Pandey et al., 2014). Essential oils are characterized by two or more major compounds with few other trace compounds. The percentage composition of essential oils may vary with plants, environmental conditions, soil types and nutrients. The major components of essential oils are mainly composed of terpenes, aromatic and aliphatic constituents (Bakkali et al., 2008; Chamorro et al., 2012; Hossain et al., 2012; Hrckova & Velebny, 2012; Tongnuanchan & Benjakul, 2014). For example, 28 components were identified in the C. citratus (lemongrass) essential oils of which 89.1% were monoterpene hydrocarbons, 7.1% sesquiterpenes hydrocarbons, about 96.4% of the total detected constituents (Tyagi et al., 2014). The chemical constituents of essential oils are mainly grouped by structural formulae as follows:

1.2.1 Terpenes hydrocarbons

Terpenes are the largest constituents of secondary metabolites (Dudareva et al., 2004). These have different structural forms proposed by plants for defence as toxins and feeding deferent for many plant pests and animals (Choi et al., 2006; Bakkali et al., 2008). These defence are mainly due to changes in membrane fluidity of the cell, the enhanced influx of fractional inhibitory concentrations, interference with the membrane bound signalling proteins and the cell cycle arrest (Zore et al., 2011).

The major subclasses of terpenes are mostly monoterpenes, sesquiterpenes, diterpenes and triterpenes (Ríos, 2016).

1.2.1.1 Monoterpenes (C10H16) Monoterpenes contribute to about 90% of the essential oils (Bakkali et al., 2008). These terpenes are considered as secondary metabolites with two isoprene units (Holopainen, 2004). Monoterpenes do not play a role in the basic metabolic processes in plant development and growth (Wise & Croteau, 1999). These molecules have lower boiling points and are insoluble in water, while some monoterpenes such as thymol, thujene and terpinene-4-ol are also toxic to insects (Lee et al., 1997; Choi et al., 2006). There are growing recognitions that these natural products play roles in chemical ecology by producing defence against pathogens, help in the pollination, seed dispersal and allelochemical functions between plants and herbivores (Ibanez et al., 2012). The common examples of monoterpenes are myrcene (1), α-pinene (2) and D-limonene (3) which possess insecticidal properties. The literature showed that Myrcene (5.9%) and α-pinene (27.4%) were the main components identified in the Eleoselinum asclepium essential oils which possessed extensive insecticidal properties against the West Nile virus vector Culex pipiens (Evergetis et al., 2009).

CH3

CH3 CH2 CH3 CH2 H C CH H C CH 3 3 2 3

Myrcene (1) α-pinene (2) D-limonene (3) Figure 1-2: Selected structures of Monoterpenes.

1.2.1.2 Sesquiterpenes (C15H24) Sesquiterpenes consist of three isoprene units and having fifteen carbon atoms (Ghantous et al., 2010). The common examples include; caryophyllene oxide (4), β- selinene (5) and germacrene (6). Sesquiterpenes compounds have contact irritant effects on insects. For example, many species of the Celastraceae family, such as the Chinese bittersweet (Celastrus angulatus) are used traditionally as insecticides in China (Gonzalez-Coloma et al., 2013). Likewise, Caryophyllene oxides from 4

Origanum essential oils showed strong repellency (more than 83% repelled) on Tribolium castaneum (Coleoptera: Tenebrionidae) adults at time 2, 4, and 6 hours (Kim et al., 2010). These sesquiterpenes compounds are also used as analgesic, spasmolytic agents, calming, slight hypotensors and anti-inflammatory (Chaichana, 2009). The examples of plant consisting these compounds are lemongrass, pine, peppermint and mandarin, lavandin, petitgrain, sage and thyme (Bakkali et al., 2008).

H C H3C 2 H CH3 CH3 CH CH3 H 3

CH2 CH2 H H CH3 CH CH CH O 2 3 3

Caryophyllene oxide (4) β-selinene (5) Germacrene (6)

Figure 1-3: Selected structures of Sesquiterpenes.

1.2.1.3 Diterpenes (C20H22) Diterpene compounds are mostly found in leguminous trees and pines in the form of abietic acids (Kemp & Burden, 1986). These are organic compounds mostly composed of four isoprene units. Diterpenes, when compared to monoterpenes and sesquiterpenes, are among the heaviest molecules found in the essential oils (Stewart, 2005b). However, diterpenes are not too heavy to be aromatic and participate in therapeutic activities. The common examples of diterpenes compounds include; para-camphorene (7) and primaric acid (8).

Diterpenes are known to have insecticidal, antimicrobial and anti-inflammatory properties (de Oliveira et al., 2008). For example, demethylsalvicanol (a diterpene) from the roots of Salvia broussonetii has shown strong selective cytotoxicity to insect SF9 cells which is commonly used for recombinant protein production (Fraga et al., 2005). Likewise, of the many diterpene known compounds - clerodane diterpene is the most extensively studied bioactivity for its insect anti-feedant property (Gonzalez-Coloma et al., 2013). Not only clerodane are known for its anti-

5 insecticidal properties, but they are also recognized sources of antimicrobial, antiviral, antitumor, antibiotic and amoebicidal activities (Coll & Tandrón, 2007).

H3C CH3

H2C

CH3

CH2 CH3

H3C H O CH CH 3 3 OH

p-camphorene (7) Pimaric acid (8)

Figure 1-4: Selected structures of Diterpenes.

1.2.1.4 Triterpenes (C20H22) Triterpenes are the most diverse group of plant natural products. These compounds are not regarded as an important component in plants for growth and development (Kemen et al., 2014). However, they exist in plants in unmodified form, more often as conjugate with carbohydrates and other macromolecules (triterpenes glycosides) (Chaichana, 2009; Thimmappa et al., 2014). The common example of this compound includes; Squalene (9). Squalene compounds were also identified as a major component of human sebum (secrete oily matter on skin) that plays a role in promoting oxidative skin damage (Mudiyanselage et al., 2003).

Triterpenes are components of the surface waxes that accumulate in the intra-cuticle layers of stems and leaf surface for protection against dehydrations and herbivores (Thimmappa et al., 2014). The wide ranges of application of these compounds are in food, health, and industrial biotechnology sector (Thimmappa et al., 2014; Hadjimbei et al., 2015).

CH3 CH3 CH3

H3C CH3 CH3 CH3 CH3 Squalene (9)

Figure 1-5: Selected structure of Triterpene.

1.2.2 Oxygenated Compounds

These are compounds that occur less frequently than the terpenes. The principal sources of these compounds are from the families; Apiaceae, Lamiaceae, Myrtaceae and Rutaceae (Janardhanan & Thoppil, 2004). The oxygenated compounds consist of alcohols and phenols.

1.2.2.1 Alcohols Alcohol compounds have hydroxyl group (-OH) attached to the carbon (Chaichana, 2009). The common examples of these compound include; linalool (10), α-terpineol (11), and geraniol (12). The formation of different alcohol compounds are totally dependent on whether the chain to which the (-OH) group attaches in order to give monoterpenes, sesquiterpenes or diterpene alcohols. These compounds have bactericidal, anti-infective and repellent properties. For instance, linalool has shown a good repellent activity against T. castaneum (Red flour beetle), as the insect spent (1.22 min) in test arm as compared to control arms (2.78 min) (Ukeh & Umoetok, 2011).

CH3 CH3

CH3 H3C OH CH2 H C CH H C 3 3 OCH 3 OH 3

Linalool (10) Geraniol (12) α-terpineol (11)

Figure 1-6: Selected structures of Alcohol.

1.2.2.2 Phenols Phenol compounds have -OH group attached to an aromatic ring. These are alcohols that have strong toxic effects, for example thymol (13), carvacrol (14) and chavicol (15). The compounds have antiseptic, bactericidal and insecticidal properties. For instance, thymol has shown to have strong feeding deterrent effect (Effective Concentration (EC)= 10.1 µg/cm2) to Epilachna varivestis (Mexican bean beetle) using disc choice bioassays (Akhtar & Isman, 2004). Similarly, the compounds thymol and carvacrol were found to inhibit the mycelium growth and conidium germination in Corynespora cassiicola (Romero et al. (2013) cited in Pinheiro et al. (2015)).

CH3 CH3 HO OH

H C CH H C CH CH 3 3 3 3 2

Thymol (13) Carvacrol (14) Chavicol (15)

Figure 1-7: Selected structures of Phenols.

1.2.3 Ethers

Ether compounds in the essential oils are known to form phenolic ether derivatives. The common examples include; safrole (16), methyl chavicol (17) and eugenol methyl ether (18). According to He et al. (2009), cineole and citronellol (cyclic and acyclic ethers) had severely affected the speed of germination, seedling growth, chlorophyll content and respiratory activities of a weed; Ageratum conyzoides. These compounds are also known to possess anti-fungal properties. For example, methyl eugenol was used alternatively against fluconazole (drug) for the treatment of Candida infections (Ahmad et al., 2010).

H2C

CH3

H2C O O CH2

H C O O CH 3 3 O

Safrole (16) Methyl chavicol (17) Eugenol methyl ether (18)

Figure 1-8: Selected structures of Ethers.

1.2.4 Aldehydes

Aldehyde compounds have powerful aromas that are mostly used in the making of perfumes. Common examples include; citral (18), citronellal (19) and neral (20). These compounds are also used for antiviral, anti-inflammatory, hypotensive, vasodilators and antipyretic activities (Dorman & Deans, 2000; Djilani & Dicko, 2012). For example, citral along with linalool had strongest inhibiting activity (inhibiting all the isolates at (≤ 0.064% (v/v)) against the fungal species: Candida albicans (Zore et al., 2011). Likewise, citronellal from the Eucalyptus citriodora essential oils has shown complete inhibition against Rhizoctonia solani and Helminthosporium oryzae (rice pathogen) at 10 and 20 ppm (Ramezani et al., 2002).

CH3 CH H3C 3

O CH3 CH3

H3C

O CH H C CH 3 3 3 O

Citral (18) Citronellal (19) Neral (20)

Figure 1-9: Selected structures of Aldehydes.

1.2.5 Ketones

Aromatic ketones rarely occur in essential oils. The rare occurrence is when a carbonyl attaches to a carbon on a chain structure (Chaichana, 2009). Some ketones have sub-divided into monoterpene ketones (such as carvone (21), pulegone, isopulegone, menthone) and sesquiterpene ketone (such as germacrene). These compounds have shown toxic effects to a number of pests. For instance, Fenchone (22) had the strongest toxic effect on the larvae of Colorado potato beetle (Leptinotarsa decemlineata Say) (Kordali et al., 2007). Similarly, carvone compounds have shown strong toxic effects against both species of stored grain insects; S. zeamais (LC50 values were 15.2 µL/mL (LA-13) and 16.7 µL/mL (LA-

57)) and T. castaneum (LC50 values were 28.7 µL/mL (LA-13) and 19.7 µL/mL (LA-57)). The carvone chemotype have the potential in the development of natural insecticides as it was more toxic (LC50 = 8.8 µL/mL) than citral. Some other uses of these compounds include anti-coagulant, anti-inflammatory and digestant (Peixoto et al., 2015).

H3C

H3C O O

CH 2 H3C H C CH 3 3 Carvone (21) Fenchone (22)

Figure 1-10: Selected structures of Ketones.

1.2.6 Organic acids and esters

Esters are formed by addition of organic acid and an alcohol. A very good example of ester is benzyl acetate (23) which is an important component of jasmine and gardenia oils. They hold special properties such as anti-fungal, anti-inflammatory and antispasmodic (Chaichana, 2009). These compounds also have the potential antimicrobial properties, where it is employed in acidic foods for preventing the growth of yeasts and moulds, and bacteria in food with a pH above 4.5 (Stratford & Eklund, 2003). 10

O O H C 3 Benzyl acetate (23)

Figure 1-11: Selected structure of Ester.

1.2.7 Oxides

These compounds are mostly used for aromatherapy, pharmaceuticals and agriculture (Chaichana, 2009). One of the useful oxides found in essential oils is 1, 8-cineole (24) or eucalyptol. For instance, 1, 8-cineole greatly affected the growth of roots and shoots of two weed species (E. crusgalli and C. obtusifolia), which later resulted in the corkscrew shaped morphological distortion (Romagni et al., 2000).

H3C CH3

H C 3 1, 8-cineole (24)

Figure 1-12: Selected structure of Oxide.

1.3 Extraction of Essential Oils

The common methods used for extraction of essential oils are steam distillation, hydro-distillation, hydro-diffusion, extraction with solvents, and enfleurage (Janardhanan & Thoppil, 2004).

1.3.1 Distillation

Distillation is a common method used economically for extracting essential oils. The three common forms of distillation are, steam distillation, water distillation and hydro-diffusion.

1.3.1.1 Steam distillation This method is also known as wet steam distillation as it has both the characteristics of water and steam distillation. In this process the plant materials are placed on a metal grid and water boils at a distance from the grid. There is no direct contact between the plant materials and the boiling water. As the heating progresses, the vapour carries small amounts of the vaporized compounds to the condenser where it cools down and eventually ends up in the collecting tube. It is when the two phases (water and essential oil layers) separates easily (Boutekedjiret et al., 2003; Chaichana, 2009). This method of extraction is appropriate for most of the essential oil extractions except delicate flowers. Steam distillation is mostly used to extract essential oils that are used in the manufacture of perfumes, petroleum refineries and petrochemical plants (Rakesh & Tripathi, 2011). Overall, the steam distillation technique is mostly preferred in the cosmetic industry.

1.3.1.2 Water distillation/Hydro-distillation This technique of extracting essential oils is very ancient and hence gone through centuries of improvement. In this process, samples are boiled in water and the vapour is carried in a vat, through which it enters the condenser. The vapour is cooled in the condenser leading to formation of distillate in the collecting tube (Boutekedjiret et al., 2003; Mohamed, 2005; Chaichana, 2009). The oil can be easily obtained by decantation. Hydro-distillation method when compared to steam distillation is a more rapid and simple in collecting or recovering good yield of essential oils. It is also less time consuming and less labour-intensive process (Charles & Simon, 1990).

1.3.1.3 Hydro-diffusion Hydro-diffusion is a method of extracting essential oils where steam at atmospheric pressure passes through the plant materials from the top of the extraction chamber (Mohamed, 2005). The collection of essential oils retains the original aromas of the plant. This process is favoured over the steam distillation as it requires less time for distillation, low steam consumption, absence of high temperature and high-quality oils. However, co-extraction of other non-volatiles and polar components

12 complicates the whole process of hydro-diffusion (Chaichana, 2009). Thus, it may not be a good technique to obtain a high yield for the desired compounds.

1.3.2 Solvent extraction

One of the extraction methods mostly used for industrial process where the essential oils obtained is very pure. This technique uses organic solvent (e.g. petroleum ether) in order to separate volatile compounds from the plant materials (Mohamed, 2005). The organic compounds are dissolved as the solvent penetrates the plant materials. The collected organic solvent is then transferred to the evaporator, where the solvent is removed at low temperature. The yield is a ‘concrete’ (residual after the solvent has been removed) which is then rapidly washed with alcohol to remove wax and finally resulting in an ‘absolute’ (concentrated form of fragrance). However, this method is not preferred because it is very costly, highly flammable and harmful to the environment (Chaichana, 2009).

1.3.3 Enfleurage

This is one of the oldest techniques used for capturing the true odour of delicate flowers (Rakthaworn et al., 2009). It is where the scented flowers are placed in fixed oil or fat spread out on a glass plate which is left for a few days for absorption. The final product called ‘pomade’ is washed with alcohol before use. This technique preferably holds an advantage to those flowers that form aroma compounds for a few days after they are picked (Mohamed, 2005; Chaichana, 2009).

1.4 Methods for Analysis of Chemical Constituents

The chemical profile of essential oil products differ drastically in terms of quality, quantity and in composition due to climate, soil composition, plant organs, age and vegetative cycle stages (Masotti et al., 2003; Erbil et al., 2015). In order to obtain a detailed chemical analysis, it is important to analyse samples from various locations. The common techniques used in the analysis of essential oils are Gas-Liquid Chromatography and Gas Chromatography-Mass Spectrometry (Bakkali et al., 2008; Falsetto, 2012).

1.4.1 Gas-Liquid Chromatography

One of the useful techniques for analysis of essential oils is Gas Liquid Chromatography (GLC). This technique has two phases: stationary phase and mobile phase. The oils are injected through the injecting port which is carried by the mobile phase through the stationary phase. The speed of the flow is dependent on the affinity of the different components between the stationary phase and the mobile phase. The detection of compounds is usually achieved by the means of flame ionization detector (FID), which then can be identified by comparing the obtained from the known standards (Chaichana, 2009).

1.4.2 Gas Chromatography-Mass Spectrometry

Gas Liquid Chromatography (GLC) is not the only valuable test for the analysis of essential oils; the modern forefront technology is Gas Chromatography-Mass Spectrometry (GC-MS) which is an expensive approach for analysing and identifying individual components of essential oils. In this approach, Gas chromatography bombards (breaking) the molecules with high energy, then using a mass spectrometer detector for possible detection of each component in the complex mixtures (Chaichana, 2009).

1.5 Common Uses of Essential Oils

The use of essential oils is extremely diverse that depends on the source, extraction and quality (Ríos, 2016). The essential oils are primarily used by plants itself, other uses include, agricultural industry, antimicrobial or medicines, food industry and cosmetics.

1.5.1 Essential Oils Used by Plants

Plants respond to herbivore damage by producing secondary metabolites such as essential oils (Bakkali et al., 2008) (see Figure 1-13). A huge diversity of secondary metabolites is produced by plants as a prominent feature of protecting against predators, microbial pathogens and ultra violet protection (Wink, 2006; Oraby & El- Borollosy, 2013). The plants use essential oils against microbial infestations, or inhibiting the growth of other competing plants. For example, Citrus aurantium 14

(orange tree) contain essential oils such as α-pinene and β-pinene, citronellol and limonene ,which inhibits the growth of Amaranthus retrofleuxs - redroot pigweed (Alssadawi & AlRubeaa, 1985).

Figure 1-13: Function of secondary metabolites in plants.

Adapted from: Wink (2006).

Essential oils also aid in pollination, such as attracting insects in the dispersion of pollens or otherwise repel undesirable insects. For instance, James (2003) reported that methyl salicylate a component of essential oils which act as an attractant for the beneficial insects - big-eyed bug and hoverflies.

1.5.2 Essential Oils Used by Humans

1.5.2.1 Agriculture Traditional aromatic plants have a huge impact on agriculture, since plant derived essential oils are considered an integral source of pesticides. It represents a total of US $700.00 million market value with a total production of 45000 tons (Tripathi et al., 2009). According to Bakkali et al. (2008), 300 essential oils out of 3000 known essential oils are commercially used for pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries. The use of natural products dates back to centuries

15 for the protection of crops against insect pests, weeds, fungal, bacterial and viral diseases (Risha et al., 1990; Joel et al., 1991; Lee et al., 1997; Singh et al., 2002; Papachristos & Stamopoulos, 2004; Bakkali et al., 2008). For instance, essential oils from plants consisting of potassium salts are claimed to have herbicidal properties against aphids, whiteflies, squash bugs, caterpillars, earwigs, flea beetle and other relating vegetable and ornamental pests (Copping & Duke, 2007).

The investigations in the area of natural resources have dramatically increased when it comes to public concerns for long term health and environmental effects of synthetic chemicals (Coats, 1994; Regnault-Roger & Hamraoui, 1995; Lee et al., 1997; Akhtar & Isman, 2004; Ukeh & Umoetok, 2011; Khani & Heydarian, 2014; Kumar et al., 2014). Recently, the Government of the United States restricted many synthetic chemicals upon which farmers have depended on for decades. This would create a significant opportunity for other alternatives such as essential oils. Hence, the development of natural insecticides would help to decrease the negative effects of synthetic chemicals. The negative effect is mostly in the form of residues in products, insect resistance and environmental pollution (Kordali et al., 2007).

1.5.2.2 Antimicrobial (Medicine) Essential oil also possesses anti-bacterial and anti-fungal properties in relation to human health (Pattnaik et al., 1997; Burt, 2004; Bakkali et al., 2008; Bassolé & Juliani, 2012). For instance, the medicinal plants; Achyranthus aspera L. commonly known in Tonga as ‘Tamatama’ and Ageratum conyzoides L commonly known as ‘Uchunti’ have shown anti-bacterial activities against certain strains of Staphylococcus aureus bacteria (Sotheeswaran & Sotheeswaran, 1999). Likewise, the essential oils from New Zealand medicinal plants - ‘Kanuka’ and ‘Manuka’ confirmed the antimicrobial activity with minimum inhibitory activities from 0.78% to 3.13% concentrations against M. furfur, T. mucoides, C. albicans and C. tropicalis (Chen et al., 2016).

Essential oils have played a pivotal role in antibiotic drug discoveries. However, the increase in the infectious diseases due to the antimicrobial resistance is in need of a

16 constant supply of new drugs (Chan, 2005). Essential oils are considered an alternative due to a broad spectrum of bioactivities with several chemical constituents making microorganism difficult to develop resistance (Carson et al., 2006; Abad et al., 2013; Hassanshahian et al., 2014).

1.5.2.3 Food industry Essential oils are used for consumer goods, these include, confectionery, food products, distilled alcoholic beverages and soft drinks (Ríos, 2016). Essential oils contain active compounds with antioxidant activities that are used in the preservation of foods such as preventing spoilage of products (Tiwari et al., 2009; Mihai & Popa, 2013). For example, the essential oils from oregano and thyme showed inhibitory activity against Escherichia coli (Burt et al., 2007). These Escherichia coli can lead to a haemolytic uremic syndrome due to destruction of red blood cells especially in children.

In addition, the food industry uses essential oils mainly for flavourings as it has an interesting source of natural antimicrobials for food preservations. For example, eucalyptus oil has showed inhibitory properties towards food spoilage yeasts. The minimum inhibitory concentration varied from 0.56 to 4.50 mg/mL (Kumar Tyagi et al., 2014). The application of essential oils has rapidly increased in recent years due to negative perceptions about the synthetic preservatives. These synthetic preservatives can lead to serious health issues such as allergic reactions (Hyldgaard et al., 2012).

1.5.2.4 Cosmetics Economically, the use of essential oils in cosmetics, perfume, detergent, soap industry is of a great interest. For example, menthol from essential oils is used for flavouring and to give a cooling sensation in refreshing creams and lotions, body rubs, toothpastes, mouthwash (0.25-1% essential oil use), sports creams and massage products (1-10% essential oil use) (Commitee of Experts on Cosmetic Products, 2008). Likewise, the production of perfumes from essential oils had greatly increased the world production of specific aromatic plants. Some of the examples of these novel plants include lavender, thymes and salvia (Ríos, 2016).

The other uses of essential oils include; sanitary industry, home irrigation, post- surgery uses and mouth washes (Seymour, 2003; Adelakun et al., 2016).

1.6 Purpose of this study

Essential oils are studied for many uses such as; in the pest controls, antimicrobial, anti-fungal, anti-viral, food, sanitary and cosmetic industries. Currently, the interest is in agricultural and pharmaceutical industries (George et al., 2014). This attention is due to the diverse use of essential oils and its environmental friendly approach. However, due to variabilities in essential oils mostly due to different locations and climatic conditions had affected the chemical compositions of same or similar species of plant. These variabilities in essential oils have led the researchers to investigate the chemical composition of plants of same or different species all around the world. In Fiji, very little studies have been done on biological activities of essential oils from plants found in Fiji. Hence, this study mainly focuses on three aspects of the essential oils from selected medicinal plants found in Fiji. The emphasis of current research is mainly on the analysis of essential oil composition, pest control and antimicrobial activities.

Firstly, the study aimed to identify the chemical constituents of essential oils from the following selected medicinal plants found in Fiji; C. odorata (Makosoi), C. citratus (Lemon grass), M. koenigii (L) Spreng (Curry Leaves), O. tenuiflorum L (Tulsi) and E. hortensis forma hortensis (Uci). The selection of plant materials for the research was based on the diverse medicinal properties of selected plants that have been used on for ages in the Pacific. The detailed analyses of essential oils from selected plants are presented in Chapter 2.

Secondly, the study aimed to test the fumigant and repellent activities of selected essential oils on the adult whiteflies (Aleurodicus dispersus Russell). A detailed background of Spiralling whiteflies and the effect of essential oils are presented in Chapter 3.

Thirdly, the study investigated the antimicrobial properties of essential oils from the selected medicinal plants found in Fiji. A detailed analysis of the results is discussed in chapter 4.

2. CHAPTER 2: CHEMICAL ANALYSIS OF ESSENTIAL OILS FROM SELECTED MEDICINAL PLANTS FOUND IN FIJI.

2.0 Introduction

Aromatic plants are frequently used by many due to their essential oils and volatile constituents (Crockett, 2010; Mothana et al., 2013). The focus of this chapter was to determine the chemical composition of essential oils from selected medicinal plants found in Fiji that include, C. odorata (Makosoi), C. citratus (Lemon grass), M. koenigii (L) Spreng (Curry Leaves), O. tenuiflorum L (Tulsi) and E. hortensis forma hortensis (Uci) using the technique Gas-Chromatography Mass Spectrometry (GC- MS). The comparisons of selected essential oils analysis were made using available literature.

According to World Health Organization (1998), numerous plant extracts were already known and used by Pacific Islanders for many different purposes. Taking this into account, the selection of medicinal plants for the research was based on the traditional uses by many Pacific islanders. In addition to this, the selection of plant materials was also based on the availability and accessibility.

2.1 Background

2.1.1 Description and Common Uses of Selected Medicinal Plants Found in Fiji.

2.1.1.1 Cananga odorata (Makosoi)

Taxonomical classification Kingdom: Plantae – plants Subkingdom: Tracheobionta – vascular plants Super division: Spermatophyta – seed plants Division: Magnoliophyta – flowering plants Class: Magnoliospida – dicotyledons Subclass: Magnoliidae Order: Magnoliales Family: Annonaceae – custard- apple family

Genus: Cananga (DC.) Hook. f. & Thomson. Species: odorata (Lam.) Hook. f. & Thomson. Figure 2-1: Flowers of C. odorata (Tan et al., 2015)

Cananga odorata belongs to the Annonaceae family, with 125 genera and 2050 species (Saedi & Crawford, 2006; Tan et al., 2015). Cananga odorata is one of the plants that are exploited on a large scale for its essential oils and being a major contributor in the fragrance industry. In the Pacific, C. odorata are known as Makosoi plants. The heights of these plants are mostly 20 m and the leaves are alternate or elliptical in shape.

The flowers are highly fragrant with 6 pointed petals usually yellow to yellowish- brown in colour. The fruits are dark green to black (ripe) in colour and 1.5-2.3 cm in length (Tan et al., 2015). The fruits and the flowers are available throughout the year. This plant is an invasive species that is native to Indonesia. It is widely planted across the South Pacific for its fragrant flowers as well for timber (World Health Organization, 1998).

In addition, these plants are mostly used in the food industry and cosmetics. For instance, people in the South Pacific islands use C. odorata flowers to enhance the

21 scent of coconut oils (Holdsworth, 1991). Traditionally, people used C. odorata for treating malaria, asthma, gout, stomach ailments and rheumatism (Jain & Srivastava, 2005; Tan et al., 2015). According to recent study by Tan et al. (2015), C. odorata had a variety of bioactivities including insect repellent, anti-diabetic, antimicrobial, anti-biofilm, anti-inflammatory, anti-fertility and anti-melanogenesis activities.

2.1.1.2 Cymbopogon citratus (Lemon grass)

Taxonomical Classification

Kingdom: Plantae

Division: Magnoliophyta

Class: Liliopsida

Order: Poales Family: Poaceae Genus: Cymbopogon

Species: citratus

(Shah et al., 2011; Olorunnisola et al., 2014) Figure 2-2: Cymbopogon citratus leaves

Cymbopogon citratus is an herb that is found almost anywhere around the world, 85 species distributed in tropical and sub-tropical countries (Taskinen et al., 1983). The plant is mostly found in the humid and warm climate and grows well with good drainage. It is a perennial herb usually propagate by the roots and the plant grows up to 2 m and 1 m long with leaf height of about 100 cm and 2 cm in width (Naik et al., 2010; Aftab et al., 2011; Skaria et al., 2012; Olorunnisola et al., 2014). The essential oils from squeezed leaves are usually yellow or amber coloured (Adeneye & Agbaje, 2007; Skaria et al., 2012). Cymbopogon citratus are used as fragrance flavouring, cosmetics, soaps, detergents and perfumery (Ganjewala, 2009; Olorunnisola et al., 2014).

Traditionally, people use C. citratus for tea (Onawunmi et al., 1984). Cymbopogon citratus has been used widely for anti-septic, anti-inflammatory, anti-fever and anti- dyspeptic effects (Naik et al., 2010; Skaria et al., 2012). It is also used in major 22 categories of alcoholic and non-alcoholic beverages, food, baked food, pudding, as it promotes digestion of fat.

2.1.1.3 Murraya koenigii (L) Spreng (Curry Leaves)

Taxonomical Classification Kingdom- Plantae Sub-kingdom- Tracheobionta Super-division- Spermatophyta Division- Magnoliophyta Class- Magnoliospida Subclass- Rosidae Order- Sapindales

Family- Rutaceae Genus- Murraya Figure 2-3: Murraya koenigii (L) Spreng Species- koenigii (L) Spreng plants. (Handral et al., 2012; Nishan & Partiban, 2014-2015)

Murraya koenigii (L) Spreng belongs to the family Rutaceae. It is a deciduous semi- evergreen plant found throughout Fiji and native to India and South Asian countries (Handral et al., 2012; Saini & Reddy, 2015). These plants are perennial shrubs that are known as ‘Indian curry tree’, which grows up to 6 m in height and 15-40 cm in diameter (Raina et al., 2002; Muthumani, 2010; Nishan & Partiban, 2014-2015; Rajnikant & Chattree, 2015). In Fiji, Indians refer to this plant as ‘curry leaves’. The leaves are bi-pinnately arranged, 15-30 cm long with 11-25 leaflets alternate on rachis (Handral et al., 2012).

Murraya koenigii (L) Spreng is used as a spice due to its aromatic nature of leaves. Other uses include; febrifuge, stomachic, analgesic and treatment of dysentery (Nishan & Partiban, 2014-2015; Saini & Reddy, 2015). The leaves and the roots are usually bitter, cooling, analgesic, acrid, for curing piles, thirst, inflammation, itching and allays heat of the body (Handral et al., 2012). Carbazole alkaloids are the major constituents of plants which possess cytotoxic, anti-oxidative, anti-mutagenic and anti-inflammatory properties. 23

These plants are also known to have insecticidal properties. For instance, with increased concentrations (0.05 to 1.0 g) of M. koenigii (L) Spreng leaf extract resulted in high mortality, population reduction with delay in development of Tribolium castaneum - pest of stored wheat (Gandhi et al., 2010) Hence, it was suggested that these plants could be employed as an alternative to chemical pesticides.

2.1.1.4 Ocimum tenuiflorum L (Tulsi)

Taxonomical Classification Kingdom: Plantae – Plants Subkingdom: Tracheobionta – Vascular plants Super-division: Spermatophyta – Seed plants Division: Magnoliophyta – Flowering plants Class: Magnoliopsida – Dicotyledons Subclass: Asteridae Order: Lamiales Family: Lamiaceae

Genus: Ocimum Figure 2-4: Branches of O. Species: tenuiflorum L tenuiflorum L plants (Pattanayak et al., 2010; Soni et al., 2012)

Ocimum tenuiflorum L (Tulsi) belongs to the family Lamiaceae, which is an erect plant with ovate (~5 cm long) leaves. The branches are up to 30-60 cm tall with strongly scented and the flowers are purplish in close whorls (Sudesh & Amitabha, 2009). The name ‘Tulsi’ symbolizes the religious bend of Hindu traditions (Pattanayak et al., 2010). Ocimum tenuiflorum L are native to tropical Asia and distributed to South Pacific and other tropical areas. These plants are mostly grown in gardens, villages and enfranchised in waste places (World Health Organization, 1998). The leaves of O. tenuiflorum L were traditionally used for cough and cold, gastric ulcer, sore throat, filariasis and stomach ace (World Health Organization, 1998).

2.1.1.5 Euodia hortensis forma hortensis (Uci)

Taxonomical Classification Kingdom: Plantae Phylum: Magnoliophyta Class: Magnoliopsoda Order: Sapindales Family: Rutaceae

Genus: Euodia Species: hortensis forma hortensis Figure 2-5: Euodia hortensis forma hortensis plant. (Brophy et al., 1985; Global Biodiversity Information Facility, 2014)

Euodia hortnesis forma hortensis is a shrub that belongs to family Rutaceae. The common name for this plant in Fiji is ‘Mata ni raqiqi’ and ‘Uci’. These shrubs grow up to 6 meters in height and the leaves are opposite, aromatic, trifoliate and even compounds. The flowers are very fragrant and the fruits are usually available throughout the year (World Health Organization, 1998).

Traditionally in Fiji, the barks of the E. hortensis forma hortensis were used to treat diseases such as yellow eyes, convulsions in children and yellow urine. In other parts of the Pacific, such as Tonga, Solomon, Niue, people use the leaves as a laxative, for fever reducing, treatment for swelling and curing head-ace. Interestingly, some people believe that the smells of the leaves can cure illnesses that are brought by the spirits (World Health Organization, 1998). Currently, there is no insecticidal activity reported by the researchers on this particular plant.

Overall, the valorisations of these selected medicinal plants were attributed to the extracts (such as essential oils). These oils are highly considered for multi-purpose in agricultural, pharmaceutical, cosmetics and food industries (Smith-Palmer et al., 2001; Edris, 2007; Sparagano et al., 2016).

2.2 Methodology

2.2.1 Collection of Plant Materials

The plant materials from C. odorata (Makosoi flowers), C. citratus (Lemongrass leaves), M. koenigii (L) Spreng (Curry leaves), O. tenuiflorum L (Tulsi leaves) and E. hortensis forma hortensis (Uci leaves) were collected from Fiji islands in April to November, 2015 (Figure 2-6). All the collected samples were verified with the voucher specimens placed at University of the South Pacific Herbarium and Koronivia Research Station, Suva, Fiji Islands.

Figure 2-6: The sample collection sites in Fiji islands.

(Source: https://www.google.com.fj/maps/place/Fiji/@-

17.7836547,178.7340111,549452m/data=!3m1!1e3!4m2!3m1!1s0x6e1990fd703cdc5d:0x9e9c319946ef5b93)

2.2.2 Extraction of Essential Oils

The extraction method of essential oils were depended on the characteristics of the materials from which it was extracted, as these oils were present in different parts of the plant such as leaves, stems, seeds, fruit and roots. The collected fresh plant samples were washed to remove dirt from the surface of selected materials. This was to make sure that no other impurities remained with the samples. The excess moisture from the plant materials were adsorbed using paper towel. The plant materials were then blended in distilled water and the resulting mixtures were hydro-

26 distilled using Clevenger apparatus for 5-7 hours as shown in the Figure 2-7. A meniscus layer (essential oils) was formed in the collecting tube which was then collected in a vial. The samples were dried over anhydrous sodium sulphate

(Na2SO4) and stored at 4 °C.

- H2O in

- H O out 2 Condenser

Clamp stand with the holder

Collecting tube

3- Neck round bottom flask

Heating Mantle

Figure 2-7: Set-up for hydro-distillation.

2.2.3 Analysis of Chemical Constituents

The analysis of essential oils using Gas Chromatography equipped with Mass spectrometry (Agilent Technologies 6890) was performed using an HP-5MS non polar fused silica capillary column (0.25 mm, 30 m, 0.25 μm film thickness; Model Number: 19091S-433) with the following conditions: The oven temperature was programmed from 50 °C to 325 °C over 5 mins, at equilibration time of 0.50 min. The transfer source and quadrupole temperatures were 150 °C, 200 °C, 230 °C and 250 °C respectively, operating at 71 eV ionization energy. For the front inlet the mode used was split with an initial temperature of 250 °C at 42.5 kPa at a split ration of 50:1 and split flow of 43.8 mL/min. Helium was used as a carrier gas at a constant linear velocity of 35 cm/sec, flow rate of 0.9 mL/min; the injected sample volume

27 was 1.0 μL which were diluted in hexane (1000 μL). The analysis was carried at the Southern Cross University, Queensland, Australia.

The constituents of essential oils were identified based on mass spectra comparison of retention indices (RI) with authentic compounds. For the reference purpose, the library search was done using Essoils, Adams and Wiley (6) and the peak locations for unknown were located using Apex. The normalized peak areas of reported compounds were used without any correction factors for establishing abundance for the purpose of semi-quantification.

2.3 Results

In this study, the essential oils from C. odorata (Makosoi flowers), C. citratus (Lemongrass leaves), M. koenigii (L) Spreng (Curry leaves), O. tenuiflorum L (Tulsi leaves) and E. hortensis forma hortensis (Uci leaves) were selected. The plants were selected based on their medicinal properties that many Pacific Islanders have relied on for ages. The essential oils from these plants are listed in the Table 2-1.

The appropriate method used for the extraction of essential oils for this research was a hydro-distillation as shown in Figure 2-7. More importantly, this technique was used as it was less-expensive to obtain high yields of essential oils than other techniques mentioned. Likewise, Gas Chromatography equipped with Mass Spectrometry analysis (GC-MS) was considered the appropriate method for detection and identification of compounds in the selected essential oils. This method was considered appropriate due to its efficiency and simplicity (Havens, 2012). The analyses of essential oils were carried out at Southern Cross University, Australia.

2.3.1 Physical Properties

The essential oils from the selected plants were extracted using hydro-distillation set-up. The average yield obtained for each plant materials were reported in the Table 2-1. The highest yield of essential oils obtained with least amount of plant materials used were C. citratus (1.17%) > C. odorata (1.21%) > O. tenuiflorum (0.68%) > E. hortensis forma hortensis (0.64%) > M. Koenigii (L) (0.17%). 28

Table 2-1: Physical properties of selected essential oils from medicinal plants found in Fiji. Medicinal plants Plant Average Average Average Essential oil found in Fiji material Mass (g) Essential Percentage colour used taken for oil content Yield (%) extraction (mL) * * Cananga odorata Flowers 215.65 2.60 1.21 light to deep (Makosoi) yellow liquid Cymbopogon citratus Leaves 212.41 2.50 1.17 colourless (Lemon grass) Murraya Koenigii (L) Leaves 300.42 0.50 0.17 yellowish (Curry Leaves) Ocimum tenuiflorum L Leaves 314.35 2.15 0.68 colourless (Tulsi) Euodia hortensis Leaves 219.60 1.40 0.64 pale greenish forma hortensis (Uci) to colourless Note: * indicate an estimate on the content of essential oils extracted in one run using hydro- distillation apparatus for 5-7 hours. Murraya koenigii (L) Spreng gave the least content of essential oils as compared to other plant materials.

2.3.2 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Essential oils from selected medicinal plants were analysed for their volatile constituents by Gas Chromatography equipped with Mass Spectrometry technique (GC-MS). The results of their chromatograms and volatile composition with comparison to literature are reported below:

2.3.2.1 Essential oil analysis of C. odorata (Makosoi)

The GC-MS analysis of essential oils from C. odorata (Makosoi) revealed the presence of major compounds such as trans, trans-farnesol (29.71%), benzyl benzoate (21.69%), linalool (16.65%) and trans, trans-farnesyl acetate (6.93%). The analysis also showed the presence of other compounds such as α-thujene (0.31%), sabinene (0.58%), methyl chavicol (0.45%), trans-anethole (0.27%), δ-elemene (0.24%), β-selinene (0.31%), α-germacrene (0.35%) and trans, trans-farnesal (0.43%) as shown in Table 2-2 and Figure 2-8.

Table 2-2: Compounds identified in the essential oil from the flowers of C. odorata. Peak # Retention Percentage Compound* Chemical group Time Area 2 7.35 0.31 α-thujene # monoterpene 3 7.51 0.32 α-pinene monoterpene 4 8.49 0.58 sabinene # monoterpene 5 8.91 0.11 myrcene monoterpene 6 11.02 1.64 methyl benzoate aromatic ester 7 11.14 16.65 linalool monoterpene alcohol 8 12.33 0.14 ethyl benzoate benzyl esters 9 12.46 0.15 terpinen-4-ol monoterpene alcohol 10 12.74 3.15 methyl salicylate phenolic esters 11 12.78 0.45 methyl chavicol # alcohol 12 13.62 0.74 geraniol monoterpene alcohols 13 14.12 0.27 trans-anethole # miscellaneous 14 14.84 0.24 δ-elemene # sesquiterpenes 15 15.14 1.38 eugenol alcohol 19 15.72 1.77 methyl eugenol alcohol 20 16.01 0.49 β-caryophyllene sesquiterpenes 26 16.80 2.74 germacrene D sesquiterpenes 27 16.88 0.31 β-selinene # sesquiterpenes 29 17.11 0.35 α-germacrene # sesquiterpenes 37 17.28 29.71 trans, trans-farnesol sesquiterpenes alcohol 38 19.75 0.43 trans, trans-farnesal aldehyde # 39 20.11 21.69 benzyl benzoate benzyl esters 40 20.75 6.93 trans, trans-farnesyl ester acetate 41 21.18 2.21 benzyl salicyate benzyl ester *Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column. Note: The number sign ‘#’ indicated that the compounds were detected for the first time as compared to the literature (Katague & Kirch, 1963; Gaydou et al., 1986; Murbach Teles Andrade et al., 2013).

Trans, trans-farnesol Benzyl benzoate

Linalool

Trans, trans-farnesyl acetate

Figure 2-8: GC-MS chromatogram of essential oil from the flowers of C. odorata.

2.3.2.2 Essential oil analysis of M. koenigii (L) Spreng (Curry leaves)

The GC-MS analysis of essential oils from M. koenigii (L) Spreng (Curry leaves) revealed the presence of major compounds such as sabinene (43.80%), β- caryophyllene (16.52%), terpinen-4-ol (7.20%) and α-pinene (5.67%). The analysis also reported the presence of other compounds such as isoterpinolene (0.95%), trans-p-menth-2-en-1-ol (0.47%), cis-piperitol (0.12%), trans-piperitol (0.17%), eugenol (0.33%), β-selinene (0.40%), α-selinene (0.78%), α-germacrene (0.18%), trans-nerolidol (0.24%), caryophyllene oxide (0.75%) and intermedeol (0.27%) as shown in Table 2-3 and Figure 2-9. Table 2-3: Compounds identified in the essential oil from the leaves of M. koenigii (L) Spreng. Peak # Retention index Percentage Compound* Chemical group Area 1 7.37 1.79 α-thujene monoterpene 2 7.53 5.67 α-pinene monoterpene 3 8.54 43.80 sabinene monoterpene 4 8.59 1.55 β-pinene monoterpene 5 8.93 1.84 myrcene monoterpene 6 9.46 2.64 α-terpinene monoterpene 7 9.64 0.67 p-cymene monoterpene 8 9.72 0.69 β-phellandrene monoterpene 9 9.91 0.11 cis-β-ocimene monoterpene 10 10.12 0.39 trans-β-ocimene monoterpene 11 10.33 4.82 ϒ-terpinene monoterpene 12 10.53 0.59 trans-sabinene hydrate monoterpene 13 10.88 0.95 isoterpinolene # monoterpene 15 11.53 0.47 trans-p-menth-2-en-1-ol # alcohol 17 12.48 7.20 terpinen-4-ol monoterpene alcohol 18 12.70 0.28 α-terpineol monoterpene alcohol 19 12.77 0.12 cis-piperitol # Monoterpenes alcohol 20 12.96 0.17 trans-piperitol # monoterpene alcohol 22 15.14 0.33 eugenol # monoterpenes alcohol 23 15.61 1.50 β- elemene sesquiterpene 24 16.02 16.52 β-caryophyllene sesquiterpene 26 16.80 0.14 germacrene D sesquiterpene 27 16.88 0.40 β-selinene # sesquiterpene 28 16.98 0.78 α-selinene # sesquiterpene 29 17.12 0.18 α-germacrene # sesquiterpene 30 17.72 0.24 trans-nerolidol # sesquiterpene alcohol 31 18.09 0.75 caryophyllene oxide # sesquiterpene oxide 33 18.92 0.27 intermedeol # alcohol *Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column. Note: The number sign ‘#’ indicated that the compounds were detected for the first time as compared to the literature (Raina et al., 2002; Chowdhury et al., 2008). 32

Terpinene-4-ol

Sabinene ϒ- terpinene

β- Caryophyllene

Figure 2-9: GC-MS chromatogram of essential oil from the leaves of M. koenigii (L) Spreng.

2.3.2.3 Essential oil analysis of E. hortensis forma hortensis (Uci)

The GC-MS analysis of essential oils from E. hortensis forma hortensis (Uci) revealed the presence of major compounds such as menthofuran (55.17%) and evodone (25.91%). The analysis also reported the presence of other compounds such as linalool (0.10%), citronellol (0.13%), α-(2) gurjunene (0.59%), trans-α- bergamotene (0.18%), trans-β-farnesene (0.20%), β-funebrene (0.23%), humulene (0.29%), ϒ-curcumene (3.79%), germacrene D (0.27%), bicyclogermacrene (0.41%), β-curcumene (0.56%) and δ-cardinene (0.46%) as shown in Table 2-4 and Figure 2-10.

Table 2-4: Compounds identified in the essential oil from the leaves of E. hortensis forma hortensis. Peak # Retention Time Percentage Area Compound* Chemical group 1 8.93 0.37 myrcene monoterpene 2 9.72 4.64 limonene monoterpene 3 11.13 0.10 linalool # monoterpene alcohols 4 12.04 0.20 citronellal aldehyde 5 12.25 55.17 menthofuran monoterpene 8 13.24 0.13 citronellol # monoterpene alcohol 9 14.21 0.60 limonene-10-ol monoterpene alcohol 11 14.99 25.97 evodone ketone 14 15.40 0.79 α-copaene sesquiterpene 15 15.58 0.26 β-cubebene sesquiterpene 16 15.82 0.60 limonene-10-yl ester acetate 17 15.93 0.59 α-(2) gurjunene # sesquiterpenes 18 16.01 0.54 β- caryophyllene sesquiterpenes 19 16.15 0.18 trans-α-bergamotene sesquiterpenes # 20 16.36 0.20 trans-β-farnesene # sesquiterpenes 21 16.41 0.23 β-funebrene # sesquiterpenes 22 16.46 0.29 humulene # sesquiterpenes 23 16.71 3.79 ϒ-curcumene # sesquiterpenes 24 16.75 0.60 AR-curcumene sesquiterpenes 25 16.80 0.27 germacrene D # sesquiterpenes 26 17.00 0.41 bicyclogermacrene # sesquiterpenes 27 17.10 0.56 β-curcumene # sesquiterpenes 29 17.28 0.46 δ-cardinene # sesquiterpenes *Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column. Note: The number sign ‘#’ indicated that the compounds were detected for the first time as compared to Brophy et al. (1985).

Menthofuran

Limonene Evodone

ϒ- Curcumene

Figure 2-10: GC-MS chromatogram of essential oil from leaves of E. hortensis forma hortensis.

2.3.2.4 Essential oil analysis of O. tenuiflorum L (Tulsi)

The GC-MS analysis of essential oils from O. tenuiflorum L (Tulsi) revealed the presence of major compounds such as eugenol (58.20%), germacrene D (11.68%) and cis-β-ocimene (10.79%). The analysis also reported the presence of other compounds such as 1-octen-3-ol (0.19%), α-terpinene (0.23%), trans-β-ocimene (0.43%), allo-ocimene (0.17%), α-cubebene (0.18%), α-copaene (1.98%), humulene (0.33%), ϒ-muurolene (0.40%), α-cardinene (0.55%), ϒ-cardinene (0.22%), δ- cadinene (1.44%), epi-1-cubenol (0.13%) and α-cadinol (0.87%) as shown in Table 2-5 and Figure 2-11.

Table 2-5: Compounds identified in the essential oil from leaves of O. tenuiflorum L. Peak # Retention Time Percentage Compound * Chemical Area group 1 7.37 0.61 α- thujene monoterpene 2 8.51 0.43 sabinene monoterpene 3 8.73 0.19 1-octen-3-ol # alcohols 4 8.93 0.38 myrcene monoterpene 5 9.46 0.23 α-terpinene # monoterpene 6 9.64 0.23 p-cymene monoterpene 7 9.92 10.79 cis-β-ocimene monoterpene 8 10.12 0.43 trans-β-ocimene # monoterpene 9 10.33 0.37 ϒ-terpinene monoterpene 11 11.13 0.21 linalool monoterpene alcohol 13 11.61 0.17 allo-ocimene # monoterpene (carotenoid polyenes) 15 12.47 1.01 terpinen-4-ol monoterpene alcohol 18 15.01 0.18 α-cubebene # sesquiterpene 19 15.19 58.20 eugenol monoterpene alcohol 20 15.40 1.98 α-copaene # sesquiterpene 21 15.40 0.93 β-bourbonene sesquiterpene 23 16.02 4.31 β-caryophyllene sesquiterpene 24 16.13 0.35 β-copaene sesquiterpene 25 16.46 0.33 humulene # sesquiterpene 26 16.72 0.40 ϒ-muurolene # sesquiterpene 27 16.81 11.68 germacrene D sesquiterpene 29 17.00 0.55 α-cardinene # sesquiterpene 30 17.20 0.22 ϒ-cardinene # sesquiterpene 32 17.29 1.44 δ-cadinene # sesquiterpene 33 18.09 0.24 caryophyllene oxide sesquiterpene 35 18.58 0.13 epi-1-cubenol # sesquiterpene alcohols 38 18.89 0.87 α-cadinol # sesquiterpene alcohols *Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column. Note: The number sign ‘#’ indicated that the compounds were detected for the first time as compared to the literature (Pino et al., 1998; Naquvi et al., 2012).

CH 3 O CH 2

CH 2 HO CH3 Eugenol H C CH 3 3 Cis-β-ocimene

CH2

H3C CH3 CH 3 Germacrene D CH3

H C 2 H H CH3

CH3 β- Caryophyllene

Figure 2-11: GC-MS chromatogram of essential oil from O. tenuiflorum L leaves

2.3.2.5 Essential oil analysis of C. citratus (Lemon grass)

The GC-MS analysis of essential oils from C. citratus (Lemon grass) revealed the presence of major compounds such as citronellal (45.09%), citronellol (19.11%), geraniol (13.57%) and elemol (6.15%). The analysis also reported the presence of other compounds such as iso iso-pulegol (0.46%), decanal (0.14%), citronellic acid (0.37%), citronellyl acetate (1.05%), β-elemene (0.59%), germacrene D (0.79%), 4- α-hydroxyl germacral (10), 5-diene (1.15%), ϒ-eudesmol (0.72%), δ-cardinol (0.27%), cis, trans-farnesol (0.46%) and benzyl benzoate (0.21%) as shown in Table 2-6 and Figure 2-12.

Table 2-6: Compounds identified in the essential oil from the leaves of C. citratus. Peak # Retention Time Percentage Area Compound * Chemical group 1 11.13 0.27 linalool monoterpene alcohol 2 11.94 1.17 iso-pulegol monoterpene alcohol 3 12.05 45.09 citronellal aldehyde 4 12.12 0.46 iso iso-pulegol # monoterpene alcohol 5 12.86 0.14 decanal # aldehyde 6 13.25 19.11 citronellol alcohol 7 13.43 0.55 neral monoterpene aldehyde 8 13.64 13.57 geraniol alcohol 9 13.87 0.74 geranial monoterpene aldehyde 10 14.52 0.37 citronellic acid # acid 11 14.98 1.05 citronellyl acetate # ester 12 15.40 0.44 geranyl acetate ester 13 15.61 0.59 β-elemene # sesquiterpenes 14 16.80 0.79 germacrene D # sesquiterpenes 16 17.29 0.88 δ-cadinene sesquiterpenes 17 17.63 6.15 elemol alcohol 18 17.98 1.15 4-α-hydroxyl sesquiterpenes germacral (10), 5- diene # 20 18.63 0.72 ϒ-eudesmol # sesquiterpenes alcohol 22 18.78 0.27 δ-cardinol # alcohol 23 18.89 3.70 α-cardinol alcohol 24 19.52 0.46 cis, trans-farnesol # alcohol 25 20.08 0.21 benzyl benzoate # benzyl esters *Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column. Note: The number sign ‘#’ indicated that the compounds were detected for the first time as compared to the literature (Negrelle & Gomes, 2007; Olivero-Verbel et al., 2010; Matasyoh et al., 2011; Tyagi et al., 2014). 38

Citronellal

Citronellol

Geraniol

Elemol

Figure 2-12: GC-MS chromatogram of essential oil from C. citratus leaves.

2.4 Discussion

2.4.1 Cananga odorata (Makosoi)

The essential oils from the flowers of C. odorata were analysed as presented in Table 2-2. The major compounds identified from the current study were trans, trans- farnesol (29.71%), benzyl benzoate (21.69%), linalool (16.65%) and trans, trans- farnesyl acetate (6.93%). Other notable compounds were methyl benzoate (1.64%), methyl salicylate (3.15%), eugenol (1.38%), methyl eugenol (1.77%), germacrene D (2.74%) and benzyl salicyate (2.21%).

Many studies have been conducted on the essential oils of different parts such as leaves, flowers and roots of C. odorata. Cananga odorata oils mostly contained monoterpene hydrocarbons, oxygen-containing monoterpenes, sesquiterpene hydrocarbons, oxygen-containing sesquiterpenes, benzenoids, acetates, benzoates and phenols (Gomes et al., 2006; Tan et al., 2015). It was also stated in the literature that linalool was the main component present in the oxygenated fraction of 28% that gave the floral odour characteristic of C. odorata (Tan et al., 2015). The essential oil of C. odorata was a light to the deep yellow liquid having a harsh floral odour. The GC-MS analysis of C. odorata essential oils by other researchers are shown in Table 2-7 which illustrates the key components.

Table 2-7: Comparison of major chemical composition of C. odorata essential oils Components of essential Percentage Area Percentage Area Percentage oils (%) (%) (Murbach Teles Area (%) (Gaydou et al., Andrade et al., 2013) (Katague & 1986) Kirch, 1963) linalool* 19 11.38 6.5-8.1 P-caryophyllene 10.7 germacrene D* 10.3 11.2 geranyl acetate 7.8 9.87 benzyl acetate 4.6 10.34 19.6-26.5 p-methylanisole 8.4 benzyl benzoate* 7.6 6.2-9.9 methyl benzoate* 3.6 6.9-7.4 trans-β-caryophyllene 12.92 ƥ-cresylmethylether 5.7-6.6 Note: * indicate the presence of same compounds in the present study when compared to the given literature.

Likewise, Benini et al. (2012) reported the variance in the chemical composition of essential oils of C. odorata from different locations in India. The results were very clear as the different composition of compounds (92.47%, 96.38%, 89.42% and 88.81% of total essential oil composition) were obtained from different locations in India (Grande Comore, Mayotte, Nossi Bé and Ambanja). The variation of essential oil composition from different locations could possibly be due to many factors such as growing conditions, genetic differences, soil type and climate as discussed in the Section 2.5 (Factors responsible for variability in the essential oil composition).

2.4.2 Murraya koenigii (Curry leaves)

The essential oils from the leaves of M. koenigii (L) Spreng were analysed using GC-MS (see Table 2-3). The essential oil of M. koenigii (L) Spreng in present study contained 28 identified compounds of which the major compounds were sabinene (43.80%), β-caryophyllene (16.52%), terpinen-4-ol (7.20%), ϒ-terpinene (4.82%) and α-pinene (5.67%). Other notable compounds in the essential oil of M. koenigii (L) Spreng were α-thujene (1.79%), β-pinene (1.55%), myrcene (1.84%), α- terpinene (2.64%) and β-elemene (1.50%). The essential oils from the leaves were mostly yellow in colour and these mostly consisted of monoterpenes hydrocarbons, oxygenated monoterpenes and sesquiterpenes (Walde et al., 2005).

The compounds such as ϒ-eudesmol, (Z,E)-farnesol, piperitone, cada-1,4-diene, Tetradecanoic acid, (Z,Z)-farnesol, hexadecanoic acid and 1,10-di-epi-cubenol were detected for the first time as reported by Rajeswara Rao et al. (2011). Likewise, the Table 2-8 shows the composition difference in the essential oils from different locations as reported in the literature.

Table 2-8: Comparison of chemical composition of essential oils from M. koenigii. Components of Percentage Area (%) from different locations in India Percentage essential oils (Walde et al., 2005) Area (%) lower Pant Nagar, Eastern india Southern (Chowdhury Himalayan Uttaranchal Bhubaneshwar India et al., 2008) , Orissa (Kozhikode , Kerala) β-pinene* 70 65.5 α-caryophyllene 2.81 β-caryophyllene* 6.5 3.3 24 53.9 caryophyllene 9.49 α-pinene* 5.3 15 β -phellandrene* 7.4 30.2 (E)- β -ocimene 5 4 aromadendrene 4.5 10.7 α-selinene 6.3 3-carene 54.22 α-thujene* 1.47 allyl (methoxy) 2.58 dimethylsilane β-myrcene 3.2 α-terpinene* 2.39 γ-terpinene* 2.7 cis-sabinenehydrate 1.46 4- terpineol 2.8 β-elemene* 1.92 γ-elemene 1.96 Caryophyllene oxide* 1.02 3-phenylbutyrophe- 1.15 none Note: * indicate the presence of same compounds in the present study when compared to the given literature.

2.4.3 Euodia hortensis forma hortensis (Uci)

The essential oils from leaves of E. hortensis forma hortensis was analysed using GC-MS as reported in Table 2-4. The major compounds identified were menthofuran (55.17%), evodone (25.97%), limonene (4.64%) and ϒ-curcumene (3.79%). However, Brophy et al. (1985) reported a higher percentage of menthofuran (64%), when compared with other notable compounds such as evodone (17%) and limonene (5%). The difference in the composition of essential oils of E. hortensis forma hortensis from the present study and the literature could be due growing conditions, genetic differences, soil type and climate as discussed in the Section 2.5 (Factors responsible for variability in the essential oil composition).

2.4.4 Ocimum tenuiflorum L (Tulsi)

The GC-MS analysis of essential oils from O. tenuiflorum L was presented in Table 2-5. The major compounds detected were eugenol (58.20%), germacrene D (11.68%), cis-β-ocimene (10.79%) and β-caryophyllene (4.31%). Other notable compounds identified were terpinen-4-ol (1.01%), α-copaene (1.98%) and δ- cadinene (1.44%).

The present study showed sum of 27 compounds in the essential oils of O. tenuiflorum L. However, Pino et al. (1998) showed O. tenuiflorum L contained 40 compounds of which the major compounds present were eugenol (34.3%), β- elemene (18%) and β-caryophyllene (23.1%). Table 2-9 shows the presence of essential oil components analysed by other researchers.

Table 2-9: Comparison of chemical composition of O. tenuiflorum L essential oils. Components present Percentage Area (%) Percentage Area (%) Pino et al. (1998) Naquvi et al. (2012) Myrcene* 0.1 α-pinene 4.2 p-cymene* 0.3 thymol 2.4 eugenol* 34.3 27.4 β-elemene 18.0 β -caryophyllene* 23.1 α -humulene 2.0 (Z)- α -bisabolene 2.2 β -bisabolene 1.1 caryophyllene oxide* 3.8 0.02 Note: * indicate the presence of same compounds in the present study when compared to the given literature.

2.4.5 Cymbopogon citratus (Lemon grass)

The GC-MS analysis of C. citratus essential oil is presented in Table 2-6. The major compounds identified were citronellal (45.09%), citronellol (19.11%), geraniol (13.57%) and elemol (6.15%). Other notable compounds were iso-pulegol (1.17%), citronellyl acetate (1.05%), 4-α-hydroxyl germacral (10), 5-diene (1.15%) and α- cardinol (3.70%).

According to Taskinen et al. (1983), the major chemical constituents detected were citral (neral and geranial) of 60%, hydrocarbons (8%), geraniol (5%) and trace amounts of methyl eugenol. Likewise the comparison of chemical constituents of C. citratus essential oil from literature is presented in Table 2-10.

Table 2-10: Comparison of GC-MS analysis of C. citratus essential oils

Components of Percentage Area Percentage Area (%) Percentage Area (%) essential oils (%) Olivero-Verbel et al. (Negrelle & Gomes, Matasyoh et al. (2010) 2007) cited in (2011) Tyagi et al. (2014) Geranial* 39.53% 34.4% 40.5% Neral* 33.31% 28.4% 30.7% Myrcene 11.41% Geraniol* 3.05% 11.5% Geranyl acetate* 5.1% Caryophyllene 2.5% Trans-geraniol 2% Linalool* 1.68% 6-methyl-5-heptan- 2.63% 2-one Camphene 1.38% Caryophyllene oxide 1.11% Note: * indicate the presence of same compounds in the present study when compared to the given literature.

2.5 Factors Responsible for the Essential Oil Composition.

The results obtained showed variability in chemical composition of essential oils in almost all the selected plants when compared to the literature. This variance could be due to genetic variations, climatic and ecological locations (Pietschmann et al., 1998; Stewart, 2005a; Tchoumbougnang et al., 2005; Koba et al., 2007; Nascimento et al., 2008; Katoch et al., 2013).

The use of different extraction method also contributed towards the variability in essential oils. For instance, the composition of essential oils obtained by Supercritical Carbon dioxide Extraction (SFE) (yield of 1.0-5.8% (w/w)) and hydro- distillation (2.8 (v/w)) differed quantitatively. The detection of compounds in both extraction method differed due to different parameters such as pressure, temperature, and modifier volume and extraction time (Khajeh et al., 2004).

The different seasons also affect the composition of essential oils. The significant amounts of essential oils are accumulated during moderate years in contrast to the hot and dry conditions. The level of trans-anethole in essential oils increased under stress conditions (Acimovic et al., 2014). This could possibly be a way for the plants to produce secondary metabolites in order to prevent oxidation processes in cells. The study done by Janmohammadi et al. (2014) revealed that the use of fertilizers can increase the content and the yield of essential oils, as the researcher used organic fertilizer plant (Dracocephalum moldavica L) in comparison with NPK (Nitrogen- Phosphorus-Potassium) fertilizer to report the effects.

Chemical profile of essential oil product differed drastically in terms of quality, quantity and composition due to different seasons, soil composition, plant organs, age and vegetative cycle stages (Masotti et al., 2003; Angioni et al., 2006; Erbil et al., 2015; Ríos, 2016). The study done by Evan (2009) cited in Ríos (2016) reported the difference in chief constituents in the same plant species of Melaleuca bracteata (black tea-tree) where either methyl eugenol, elemicin and methyl eugenol was their major component due to different climatic conditions.

2.6 Conclusion

The analysis results of essential oils from selected medicinal plants using Gas- Chromatography Mass Spectrometry identified some consistency with the data reported. The results presented in this study are the first given information on the chemical composition of essential oils from Fiji on the selected plant species. So far only E. hortensis forma hortensis (Uci) essential oil component data from Fiji is reported (Brophy et al., 1985).

The results also depicted variation and detection of other compounds in the selected essential oils as compared to the literature. The other compounds detected in the C. odorata (Makasoi) essential oils were α-thujene (0.31%), sabinene (0.58%), methyl chavicol (0.45%), trans-anethole (0.27%), δ-elemene (0.24%), β-selinene (0.31%), α-germacrene (0.35%) and trans, trans-farnesal (0.43%). Likewise, for M. koenigii (curry leaves) the other compounds detected were isoterpinolene (0.95%), trans-p- menth-2-en-1-ol (0.47%), cis-piperitol (0.12%), trans-piperitol (0.17%), eugenol 45

(0.33%), β-selinene (0.40%), α-selinene (0.78%), α-germacrene (0.18%), trans- nerolidol (0.24%), caryophyllene oxide (0.75%) and intermedeol (0.27%). The other compounds reported from E. hortensis forma hortensis (Uci) were linalool (0.10%), citronellol (0.13%), α-(2) gurjunene (0.59%), trans-α-bergamotene (0.18%), trans-β-farnesene (0.20%), β-funebrene (0.23%), humulene (0.29%), ϒ- curcumene (3.79%), germacrene D (0.27%), bicyclogermacrene (0.41%), β- curcumene (0.56%) and δ-cardinene (0.46%). The other compounds reported from O. tenuiflorum L in the present study were 1-octen-3-ol (0.19%), α-terpinene (0.23%), trans-β-ocimene (0.43%), allo-ocimene (0.17%), α-cubebene (0.18%), α- copaene (1.98%), humulene (0.33%), ϒ-muurolene (0.40%), α-cardinene (0.55%), ϒ-cardinene (0.22%), δ-cadinene (1.44%), epi-1-cubenol (0.13%) and α-cadinol (0.87%). The other compounds detected in the C. citratus (Lemon grass) were iso iso-pulegol (0.46%), decanal (0.14%), citronellic acid (0.37%), citronellyl acetate (1.05%), β-elemene (0.59%), germacrene D (0.79%), 4-α-hydroxyl germacral (10), 5-diene (1.15%), ϒ-eudesmol (0.72%), δ-cardinol (0.27%), cis, trans-farnesol (0.46%) and benzyl benzoate (0.21%). The variation in the essential oil composition could be attributed to many reasons such as different seasons, soil composition, plant organs, age, vegetative cycle stages, genetic variations, climatic and ecological locations.

Thus, it is crucial to note that the composition of essential oils are attributed to intrinsic properties for which it is mainly utilised in the pesticidal, pharmaceutical, cosmetics and food industries.

3. CHAPTER 3: FUMIGANT AND REPELLENCY EFFECT OF PLANT ESSENTIAL OILS TO SPIRALLING WHITEFLIES (ALEURODICUS DISPERSUS RUSSELL).

The warm temperatures of the summer bring on a rush of new foliage growth, attracting a wide variety of pests. Whitefly, one of the most difficult pests to control, poses a special challenge to many. Whitefly numbers grow dramatically in the heat, most strains are resistant to pesticides, and the pests infect a huge range of host plants. (Source: http://pioneerthinking.com/gardening/controlling-whitefly-naturally)

3.0 Introduction

In Fiji and the rest of the South Pacific countries, very little work has been done in controlling the Spiralling whiteflies using bio-pesticides. This chapter describes a laboratory study of using essential oils from selected medicinal plants to serve as biopesticides for controlling the whiteflies. The fumigant and repellent effects of selected essential oils were assessed against the adult Spiralling whiteflies (Aleurodicus dispersus Russell) at different times and concentration intervals.

3.1 Background

3.1.1 Classification of Spiralling Whitefly

Kingdom: Animalia (Animals) Phylum: Arthropoda (Arthropods) Subphylum: Hexapoda (Hexapods) Class: Insecta (Insects) Order: Hemiptera Suborder: Sternorrhyncha

Superfamily: Aleyrodoidea Figure 3-1: Whiteflies on cassava Family: Aleyrodidae: (Whiteflies) ©Photograph taken by Ravneel on 9/9/15 Genus: Aleurodicus 47

Species: dispersus Russell. (Belov & Moisset, 2013; vasquez et al., 2015).

THE WHITEFLY, Aleurodicus dispersus Russell is commonly known as Spiralling whitefly, a native to the Caribbean region and Central America. Spiralling whiteflies are thought to be widely spread in countries such as several Pacific islands, America (North and South), Asia and Africa. According to Waterhouse and Norris (1989), the Spiralling whiteflies are thought to be present in the Pacific from Marshall Islands (1986), Cook Islands (1984), Fiji (1986), Nauru (1987), Papua New Guinea (1987), Kiribati (June 1988), Tokelau (late 1988) and Tonga (November 1988) (see Figure 3-2).

Figure 3-2: Distribution of the Aleurodicus dispersus. Adapted from: Waterhouse and Norris (1989).

These Spiralling whiteflies pose extreme threats to the agricultural and horticultural crops in glasshouses and fields worldwide (Oliveira et al., 2001; Mani & Krishnamoorthy, 2002; Stansly & Natwick, 2010). Some specific plants that are usually attacked include; Cassava, pepper, papaya, mango, eggplant, citrus, guava, banana, coconut, breadfruit, tropical almond, sea grape, paper bark and rose (Russell, 1965; Jayma et al., 1993; Neuenschwander, 1994; Reddy, 2015).

3.1.2 The Life Cycle of Spiralling Whiteflies (Aleurodicus dispersus Russell)

In order to understand the pest it is very important to understand the life cycle first. The life cycle of Aleurodicus dispersus Russell is divided into egg, first-fourth instars and adults as illustrated in Figure 3-5.

3.1.2.1 Eggs

The eggs (0.3 mm long) are usually smooth surfaced, yellow and tan elliptical in shape (Reddy, 2015). These are laid at an angle of 90o with spiralling deposits of white flocculence on underside of the leaves.

3.1.2.2 First instars

During the first instar, the tiny crawlers can travel to a short distance to select their feeding sites (Martin, 1987). They are usually 0.32 mm long and settle near the spiral pattern of the eggs from which they were formed. The mid-dorsal waxy tufts are developed as they grow and the secretion of wax is usually from the narrow band of sub-margin.

3.1.2.3 Second and Third instars

The second and third instars are usually 0.5-0.65 mm long that remains feeding at same places. The distinguishing feature about the third instar larvae is the presence of glass- like rods of wax (usually short and evenly-spaced) lined along the body and these cottony secretion is much less abundant than in pupa as stated by Russell (1965) cited in The Centre for Agriculture and Bioscience International (2015). 49

3.1.2.4 Fourth instar /Pupa

In the fourth instar or pupa stage, the embryo is 1.06 mm long and covered with numerous amounts of white materials and long glass-like rods (~8 mm in length); due to fragmentation some are shorter (see Figure 3-3). Overall, from the second to fourth stages, the instars are protected by waxy secretions making them sessile and scale-like (Martin, 1987; Banjo, 2010).

3.1.2.5 Adults

The adults are mobile and most active during the morning. The bodies of males are usually 2.28 mm and females are usually 1.74 mm (3-4 times longer than the body width). The adults develop white translucent powder as covering on their bodies. These whiteflies also have a pair of antenna. The differentiating feature mentioned by Russell (1965) cited in The Centre for Agriculture and Bioscience International (2015) is that, the males have several pores on the abdomen scattered dorsally, laterally and ventrally on the first 2 segments posterior to wax plates while the females are without pores. The eye is reddish-brown in colour. The Spiralling whiteflies also have two characteristic dark spots on their forewings as explained in Figure 3-4. The adult female lay eggs in irregularly spiralling patterns and it is where whiteflies derived their common name, Spiralling whitefly (Reddy, 2015). For details on the comparison of different groups of whiteflies, refer to Appendix (Table 6-2).

Glassy rod emanating from each compound spores. These glassy rods are whitish in colour that is translucent and longer (3-4 times) than the width of the body.

From the dorsum (extending upwards and outwards) of mature pupa, a copious amount of white cottony substance is secreted.

Figure 3-3: Mature pupa (~1.06 mm) of Spiralling whitefly

©Photograph taken by Ravneel on 9/10/15 50

Presence of dark spot on the forewings.

White waxy flocculants materials

Dark reddish-brown eye, where part the compound eye joined by 3 or 4 facets.

Presence of a pair of antenna Figure 3-4: Adult (~1.74 mm) of Spiralling whitefly. ©Photograph taken by Ravneel on 9/10/15

Eggs (~0.3 mm) st 1 instar (~0.32 mm)

nd Adults (~1.74 mm) 2 instar (~0.55 mm)

th rd 4 instar (~1.06 mm) 3 instars (~0.65 mm) Figure 3-5: Life cycle of the Spiralling whitefly ©Photograph taken by Ravneel on 9/10/15 51

3.1.3 Spiralling Whitefly- Why Considered a Pest.

The Spiralling whiteflies (Aleurodicus dispersus) were first discovered in Suva in April 1986 and were regarded as a serious pest. They were thought to be introduced from Guam (Waterhouse & Norris, 1989).

Spiralling whiteflies affect the plants in many ways. The eggs of the whiteflies are usually ovoid with a pedicel for attachment to the leaf surface. The female whiteflies insert the eggs into the plant stomata (Miller et al., 2010). Noting that the purpose of stomata in the leaf is to allow plants to exchange gases that are vital for photosynthesis and respiration. Therefore, if the eggs that are laid by the whiteflies continue to be a barrier in the opening and closing of stomata then obviously it becomes a barrier in plant functions as shown in Figure 3-6.

Figure 3-6: Electron micrograph of egg pedicel showing insertion of egg stalk into stoma of a plant leaf. Adapted from: Paulson and Beardsley (1985).

Whiteflies secrete sticky honey dew which at many times gets deposited, resulting in the formation of dark sooty moulds on leaves. Nymphs secrete white, waxy flocculent materials which spread elsewhere by winds and the honeydews are secreted. The secretion of honey dews causes falling of premature leaves and growth of sooty moulds which interferes with the photosynthesis. The sooty moulds affect the photosynthesis by blocking the entry of carbon dioxide into the leaf cells which greatly reduces the 52 photosynthetic product values (Henneberry et al., 2007; University of Florida, 2015). For example, the nymphs and the adults suck the sap from the leaves, stem and fruits. As a result of heavy infestation the plants wilt or die off or results in yellowish specks on leaves (Al-Shareef, 2011; Reddy, 2015). Thus, these moulds influence the rate of photosynthesis and transpiration as it creates a blocked surface for light penetration, vapour movement and gas exchange (McAuslane et al., 2004).

Spiralling whiteflies have caused detrimental effects in the production of crops and ornamental plants. As a result, there is still a need for development of a new or modification of previous strategies for the management of whiteflies.

3.1.4 Management Strategies of Whiteflies

Whiteflies are very difficult to manage. Some common methods used for the control of whiteflies are: removal and traps, biological control, synthetic and bio-pesticide approaches (Flint, 2015).

3.1.4.1 Removal and Traps

Removal of leaves may be an environmental friendly approach, but it does not completely remove the pest, it rather lessens the level of whitefly population from the plant. A slight infestation can quickly spread to other plants. The removal of leaves is a good approach to get rid of non-mobile nymphal and pupal stages of whiteflies from highly dense leaves.

In addition, yellow sticky traps are used to trap adults since whiteflies are attracted to yellow. It is where a trap consisting strips of paper and sticky substances such as petroleum are placed in the greenhouse. The insects are caught as they fly. The disadvantage of this type of approach is that it only captures specimens that can fly. However it is generally ineffective for the insects that are in their early stages since they are not able to fly (Barbedo, 2014).

This method is used by some farmers since it is a promising tool for Integrated Pest Management (IPM), as it has lower environment impact (Nakamura et al., 2007). This method is not to a complete satisfaction to farmers as this does not eliminate damaging populations, but aims to reduce the whitefly population.

3.1.4.2 Biological Control

There are various methods of biological control of whiteflies. For instance, Technical Centre for Agriculture and Rural Cooperation (1992) reported that a biological control measure using parasitic wasps were able to stop the Spiralling whiteflies in the South Pacific. These were thought to be escaped from Central and South America in the late 1970s and since then has spread across the South Pacific to Asia.

The use of extra fauna, importation of parasitoids of genera Encarsia or Eretmocerus and of various predators that have been successfully used in greenhouse for whitefly control (Gerling et al., 2001). A recent study by Sugiyama et al. (2011) stated that three parasitic species, Eretmocerus mundus (Mercet), Eretmocerus eremicus Rose and Zolnerowich and Encarsia formosa Gahan (Hymenoptera: Aphelinidae) have been used against whiteflies in Japan.

Some common predators of whiteflies are lacewings, big-eyed bugs, minute pirate bugs and several lady beetles (For example; Scymnus or Chilocorus species). A major outbreak of Spiralling whiteflies were reported on Papaya in Samoa in 2005 (Pestnet, 2005). The outbreaks of whiteflies usually happen when their natural enemies are disturbed or destroyed by pesticides, dust build-up and other factors. These flies were common on guavas, palms, ground orchids, and poinsettias (ornamental). A recent study showed that A. swirskii (mite) is increasingly used for the biological control of thrips and whiteflies in many crops (Messelink et al., 2008). The three predators were found to be attacking the Spiralling whiteflies were Megalocaria fijiensis, Serangiella and the neuropteran chrysopa species (Waterhouse & Norris, 1989).

The entomopathogenic fungi of the genus Aschersonia are specific to whiteflies and used especially as a biological control agent against Bemisia argentifolii (silver leaf whitefly) and Trialeurodes vaporariorum (greenhouse whitefly) (Meekes et al., 2002). Isaria species of entomopathogenic fungi are also pathogenic to the nymphal stages of whiteflies (Cabanillas & Jones, 2009).

3.1.4.2.1 Limitations of Biological Control

Biological approach is limited to exotic introduction and might not be appropriate for native natural enemies (Symondson et al., 2002). Some other common limitations of biological control are reported in Table 3-1. These limitations include; greater susceptibility to the environment, limitations to pesticides, slow and expensive approach.

Table 3-1: Limitations of Biological Control The limitations of using Explanation Reference biological control More susceptible to the This means that biological agents are (Harper, 2001) environment more vulnerable to environmental factors including; sunlight, temperature and rainfall.

Limits the usage of pesticides If biological agents are used for the (van Emden, 1989) subsequently. controlling of a specific pest on a crop, then it makes it difficult to use insecticides for controlling other insect pests.

Biological control has slow It usually takes time to build in numbers (van Emden, 1989) action. for the biological agents from the time of release. If the pest infestation is high, it is appropriate to use pesticides. This may also affect the biological system of pest control simultaneously.

Expensive for biological It is costly when it comes to selection of (Orr & Lahiri, 2014) control in field. biological agents, whereby it requires highly qualified or skilled people.

The biological control is considered a population-level process which involves the use of natural enemy to control the targeted pest population (Huffaker & Dahlsten, 1999).

However, this method does not provide complete satisfaction towards pests control even though it has an environmental friendly approach (Zhou et al., 2014).

3.1.4.3 Synthetic Pesticides

3.1.4.3.1 Whitefly Control - Chemicals

There are many chemicals that are used by farmers to control the whiteflies. One of the common chemical used by many farmers is pyriproxyfen. For example, the use of pyriproxyfen (100 ppm) showed 94.5% ovicidal effect (killing eggs) on sweet potato whiteflies as an effective approach to control (Young-Su et al., 2002).

The following are some of the chemical control measures that were reported by Reddy (2015) for the control of Spiralling whiteflies:

- Use of dimethoate 30 EC at 0.05 %. - Insecticidal soap at 2.5 %, which deterred the adults.

The following chemicals; imidacloprid, buprofezin and pyridaben are also used to manage the whiteflies (Bi et al., 2002). Spiromesifen, a novel insecticide inhibited egg hatching in green house by 80% to 100% at the concentrations of 3.1, 3.0, and 10.0 µg mL–1. The insecticide also showed mortality of 100% for the first, second, and third instar nymphs of whiteflies (Toscano & Bi, 2007).

Chemical approach mostly kills only those whiteflies that come in contact with the insecticides (chemicals). The use of the chemical approach showed efficiency towards controlling pests in small and in large scale farms. For instance, farmers in Colombia intensify the use of insecticides, as the whiteflies reduced the crop yield by 79% (Carabalí et al., 2010). Although, plant productions may have increased due to pesticidal applications at the same time these chemicals may have raised detrimental concerns for so many (Aktar et al., 2009).

3.1.4.3.2 Limitations of Chemical Control

There are many concerns raised with the usage of synthetic chemicals for the pest control. With reference to the Food and Agriculture Organization of the United Nations (2015), the results of the high usage of synthetic pesticides in the Pacific island countries had led to threats.

According to Aktar et al. (2009), “if a little is good, a lot more will be better” had greatly influenced and created a havoc to many life forms due to rampant uses of chemicals. Chemical pollutions are a major concern to the environment and humans bodies through food chains, which resulted in severe physiological disorders and diseases (Oliva et al., 2001; Baldi et al., 2003; Briggs, 2003; Saiyed et al., 2003; Lemaire et al., 2004). The extensive uses of synthetic chemicals have led to accumulation of residues in the environment which later becomes pollutants. These pollutants gradually affect the quality of air and water, on which many organisms have relied on.

As a result, alternative search for chemical pesticides has led to global efforts to test the efficacy of various natural products for the pest controls and crop protections. Despite the fact that the chemicals have an efficient rate in pests controls, the use of synthetic chemicals would be contaminating, costly and eventually lead to the development of resistance in the insects (Palumbo et al., 2001; Horowitz et al., 2007; Carabalí et al., 2010; Li et al., 2014b). The insecticides are also ineffective at times; as the adults, eggs and nymphs are located on the underside of the leaves where it is protected from overtop application of insecticides (Palumbo et al., 2001; Mansour et al., 2012).

The spread of whiteflies continue to increase rapidly in many countries since the chemical controls and other measures are generally ineffective (Mani & Krishnamoorthy, 2002). The need for alternative approaches is required in order to tackle the drawbacks highlighted by chemical pesticides and other control measures.

3.1.4.4 Essential Oils- An Alternative

An alternative to pest control is essential oils (Won-II et al., 2003). These essential oils are sources of bioactive compounds which are also used in the food and fragrance industry. It has now been focused on phytochemicals, as potential sources for commercial insect agents due to such diverse use. The essential oils have not only shown effectiveness on the adults of various insects such as mites, flies, beetles but also on larvae and eggs of several insects (Prajapati et al., 2005; Batish et al., 2008).

Using plant materials to control pests may alleviate the burden of heavy reliance on chemical pesticides (Tang & Yang, 1988) for developing countries like Fiji, since agriculture is one of the most important enterprise and the backbone of Fiji’s economic development. Farmers found an effective approach in controlling insect pests, diseases and weeds when first they started applying chemical pesticides. Unfortunately, the effectiveness did not last long as a result of pest resistance and health hazards of pesticides (Food and Fertilizer Technology Center, 1998). Insect resistance to pesticides has led to finding of new molecules as alternative pest-control agents, a well-established approach in control strategies for the pests (Gonzalez-Coloma et al., 2013).

Natural pesticides such as plant essential oils would represent an alternative in crop protection (Coats, 1994; Isman, 2000; Koul et al., 2008). Different plants have been used for the control of pests and the research has worked out well (Gonzalez-Coloma et al., 2013). Medicinal plants can be an alternative to a lot of synthetic chemicals for human health and agriculture. However, people in Fiji and the rest of the South Pacific countries are not very aware of the presences of the great plant diversity surrounding them.

Essential oils from these medicinal plants could be possibly used in agriculture in the form of the pest controls. This could be mainly due to the usage of chemical pesticides, which are becoming increasingly problematic to the environment and the human health (Aktar et al., 2009; Damalas & Eleftherohorinos, 2011; George et al., 2014). Hence,

58 essential oils need to be exploited due to its novel, safe and eco-friendly substitutes for its effective insecticidal properties (Li et al., 2014b; Palanisami et al., 2014).

3.2 Methodology

3.2.1 Preparation of Essential Oil Solution

Essential oils were extracted from C. odorata (Makosoi), C. citratus (Lemon grass), M. koenigii (L) Spreng (Curry Leaves), O. tenuiflorum L (Tulsi) and E. hortensis forma hortensis (Uci) using a hydro-distillation apparatus. The solutions for essential oils were prepared similarly as demonstrated by Yang et al. (2010) and Borrego et al. (2012). For instance, for a prepared solution of 0.25% (v/v); 0.25 mL of essential oils, added to 99.25 mL distilled water and 0.5 mL of Tween 20 (5%) that gave 100 mL of the solution overall. The total solution was about 100 mL, but taking into account of the essential oil content, it was not appropriate to make 100 mL of the solution since the whole experiment did not require that much of the solution. Hence, based on that ratio the final volume of 10 mL was prepared as; 0.025 mL (essential oils) added to 9.925 mL (distilled water) and 0.05 mL (tween 20) that gave overall of 10 mL solution. The solutions for 0.5% and 5% were prepared accordingly and for the control the solution was prepared using distilled water and Tween 20 (5%). The purpose of using Tween 20 was to increase the solubility of hydrophobic compounds from the essential oils (Kim et al., 1995).

3.2.2 Whiteflies Breeding -Greenhouse

3.2.2.1 Climatic Conditions during Breeding.

The climatic conditions in Fiji from last year (2015) were fairly constant in terms of air temperature and relative humidity as shown in Figure 3-7. The temperature for the experimental period (September-December) was mostly in the range of 25-30 °C. Similarly for the relative humidity the range was around 80%.

Figure 3-7: Average minimum and maximum temperatures (A) and relative humidity (B) in Suva, Fiji islands for year 2015.

(Source: weather-and-climate.com)

3.2.2.2 Breeding of Spiralling Whiteflies

The Spiralling whiteflies were bred on cassava plants (Manihot esculenta (Crantz)). The adult Spiralling whiteflies were brought from a nearby farm (Nausori area). According to the farm operator these Spiralling whiteflies were not exposed to any sort of insecticides as they were abandon on a piece of land. The adult Spiralling whiteflies were collected in Petri dish using a small paintbrush. The collected Spiralling whiteflies were brought to the plot land where they were introduced to the cassava plants in order for them to grow and multiply. The plants were maintained in the plot land for appropriately 6-7 months without any pesticide contact before carrying out the actual experiment. 60

B A

C D

Figure 3-8: Cassava plants for the whitefly experiment. (A) - Shows the cassava planting in muslin cage in the greenhouse. (B) - Plants kept in a controlled environment for the experimental test. (C) - Cassava plant (in plot land) on which the Spiralling whiteflies were bred. (D) - The colony of the Spiralling whitefly on cassava leaves.

The Spiralling whiteflies (Aleurodicus Dispersus Russell) bred in the greenhouse were brought into the laboratory when required to carry out the fumigant and repellent test. The conditions that were set in the laboratory were similar to the environment that they were found, that is, under the condition of 28±2 °C, RH - 75±5% and light regime of 14:10 (L:D).

Note: The conditions that were used by the researchers (Yang et al., 2010) were slightly different as the researchers used the true culture, however no laboratory in Fiji has the true culture for whiteflies and it was not possible to even import the cultures as it becomes a Biosecurity issue. Taking into account of this, the local Spiralling whiteflies were considered for the study.

3.2.3 Fumigant Test

The fumigant toxicity of essential oils from M. koenigii (L) Spreng, O. tenuiflorum L, C. odorata, E. hortensis forma hortensis and C. citratus were tested on the adult whiteflies irrespective of their sex. The cassava pot plants used for the assay test (15-29 plants) were grown in the greenhouse. The leaves were enclosed with a clear pocket plastic bag (16 cm in length) with 50 whiteflies in each bag. For each treatment there were 4 replicates. The treatment was introduced into each plastic bag using a filter paper (~2 cm in diameter). The filter paper discs (~2 cm in diameter) were impregnated on the side of the plastic bag.

The three concentrations of essential oils tested were, 0.25%, 0.5% and 5% (v/v). The control was a mixture of Tween 20 (5%) in distilled water. The purpose of Tween 20 in this study was to increase the solubility of hydrophobic compounds in the essential oils (Kim et al., 1995). The treatments and controls for each concentration were carried based on the randomization for placing the treatments. The mortality for each treatment and concentration was recorded at the time of 3, 6, 9, 12 and 24 hours. The experiment was carried out in the laboratory under controlled environment with an external light source (Lamp: E27230-240V -60W- Max) as shown in Figure 3-9 (Abbasipour et al., 2011).

A B

Figure 3-9: Fumigant test setup (A). Randomised labelled plastic bag (B).

3.2.4 Repellent Test

The repellent test assay slightly followed Zhang et al. (2004). A T-shaped olfactometer set was constructed in order to test the repellency on the adult Spiralling whiteflies. The setup consisted of a long glass tube (diameter of 50 cm) as depicted in Figure 3-10. The external light sources were placed between site 1 and site 2. The repellent test was standardized using lamp (E27230-240V -60W- Max) since light is an environmental variable which could have affected the results if not taken into account.

The site where the whiteflies were introduced to. The top surface was covered with muslin (5 cm in diameter) in order to stop whiteflies from escaping as well as to allow air circulations.

Site 1: - The side that had the control. The leaf disc (2 cm in diameter) was dipped in tween 20 (5%) solutions.

Site 2: - The side which had the selected concentrations of essential oils. Figure 3-10: T-shaped olfactometer.

The concentrations tested were 0.25%, 0.5% and 5% (v/v). The test consisted of 50 adult whiteflies with 4 replicates for each concentration. After 6-8 hours the numbers of whiteflies were counted using a hand lens for each site (chamber). The Repellency Index (RI%) were calculated using the formula (Abdellaoui et al., 2009);

RI%= (C-T/ C+T) x 100

C= whitefly counts on the control side of the olfactometer. T= whitefly counts on the treatment side of the olfactometer. If; RI= (-) value= total attractancy. RI= (+) value= total repellency.

A B

C D

Figure 3-11: Setup for the repellent test in the laboratory.

Note: A-Leaf disc (~2 cm in diameter). B-Muslin for ventilation. C & D- Repellent test with aid of external light source. RI= 0= no effect. 64

3.2.5 Statistical Analysis

3.2.5.1 Fumigant Test

In order to test whether each essential oil has any significant difference at 5% level of significance in the mortality between the different concentrations and the controls with respect to time intervals, a Factorial ANOVA (5 x 4 x 5 split plot design) using Tukey’s HSD test was performed (see Appendix; Table 6-3 to Table 6-7). Prior of conducting ANOVA (significant at α= 0.05), the percentage mortalities were transformed by the arcsine of the square root (Prasad, 2013). The total mortalities were converted to % mortality. The main purpose for the transformation of data was mainly due to the raw data being not normally distributed.

Similarly, the effective concentration (EC50) values for mortality after 24 hours were assessed using Probit analysis (see Table 3-2) in XLSTAT software (version 2015.1) (Kabir et al., 2007; Postelnicu, 2011). The morality was corrected using Abbott’s formula for those that exceed 10% by natural mortality (Abbott, 1925). The dose-effect (Probit) analysis was performed in order to find which essential oils can effectively cause 50% death in the Spiralling whiteflies with least concentration (Stephan, 1977; Battaglin & Fairchild, 2002).

3.2.5.2 Repellent test

To evaluate the statistical difference at 5% level of significance between each essential oil with its respective control, an Independent sample t-test was performed. The Probit analysis in XLSTAT software (version 2015.1) was also used to calculate the EC50 for the repelling effect of each essential oil. A similar approach in order to calculate the

EC50 were used by other researchers (Olufayo & Alade, 2012; Padhy & Panigrahi, 2016).

3.3 Results

3.3.1 Fumigant effect of essential oils on Spiralling whiteflies

The fumigant toxicity of selected essential oils on adult whiteflies after 3, 6, 9, 12 and 24 hours are shown in Figure 3-12 to Figure 3-14. The concentration effect of essential oils on adult whiteflies - 0.25%, 0.5% and 5% (v/v) clearly showed a positive linear pattern, with the mortality rates increasing with increasing concentration. Generally, for 0.25% and 0.5% (v/v) solutions of essential oils, the effects on mortality were relatively low as compared to 5% (v/v) solutions. The effects of essential oil at 5% solutions were quite astonishing in all essential oils, as the whitefly mortality rates were achieved rapidly.

The essential oils from O. tenuiflorum L caused 100% mortality after 3 hours at 5% (v/v) concentration. For C. citratus essential oils, 100% whitefly mortality was achieved after 6 hours at 5% (v/v) concentration. Statistically, the results obtained at different concentrations using ANOVA showed that the essential oils of O. tenuiflorum L and C. citratus in relation to mortality after 24 hours had strong significant difference (P<0.00, at 5% level of significance) at 0.5% and 5% (v/v) concentrations. Likewise, for C. odorata, E. hortensis forma hortensis and M. koenigii (L) Spreng essential oils, the effect on mortality was higher at 5% as compared to 0.25% and 0.5% (v/v) concentrations. The mortality rate of C. odorata, E. hortensis forma hortensis and M. koenigii (L) Spreng essential oils did not reach 100%, as it was around 50% for 5% (v/v) solutions. When mortality counts were compared with the control for C. odorata essential oils at 5% (v/v), it showed significant difference of p<0.003 at 5% level of significance. However, the effect of different concentrations of essential oils from M. koenigii and E. hortensis forma hortensis on mortality counts after 24 hours, showed no significant difference as the p>0.05 at 5% level of significance.

In addition, the Table 3-2 reported the EC50 on whiteflies after 24 hours. It was seen that O. tenuiflorum L required the least concentration (0.003% (v/v)) of essential oils in order to kill 50% of the tested population, followed by C. citratus (0.004% (v/v)), C.

66 odorata (0.05% (v/v)), M. koenigii (L) Spreng (0.113% (v/v)) and E. hortensis forma hortensis (0.114% (v/v)). The probit analysis showed that all the tested essential oils after 24 hours had the p<0.0001 at 5% level of significance. This simply means that the significant differences were brought by log (concentration) and whitefly mortality.

The overall fumigation test showed that the most robust effects were shown by O. tenuiflorum L essential oils followed by C. citratus, C. odorata, M. koenigii (L) Spreng and E. hortensis forma hortensis.

Control

15 Cananga odorata (a) d* Murraya koenigii (b)

Euodia hortensis (c) 10 Cymbopogon citratus (d) Mortality (counts) Mortality

Ocimum tenuiflorum L (e)

5 d* d*

b* 0 3hrs 6hrs 9 hrs 12hrs 24 hrs Time Figure 3-12: Fumigant effect (Mean ±SE) of 0.25 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals.

The alphabetical letters representing respective essential oils and the asterisks indicate results statistically different at 5% level of significance from the control at P<0.05 (*), P<0.01 (**), P<0.001 (***) using Tukey’s test.

45 e***

35 Control

30 Cananga odorata (a) e* d*** 25 Murraya koenigii (b)

Euodia hortensis (c) 20 Mortality (counts) Mortality Cymbopogon citratus (d) e*** 15 d*** e*** Ocimum tenuiflorum L (e) 10 d*** e*** d***

0 3hrs 6hrs 9 hrs 12hrs 24 hrs Time Figure 3-13: Fumigant effect (Mean ±SE) of 0.5 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals.

The alphabetical letters representing respective essential oils and the Asterisks indicate results statistically different at 5% level of significance from the control at P<0.05 (*), P<0.01 (**), P<0.001 (***) using Tukey’s test.

e*** e*** e*** e*** e*** 50 d*** d*** d*** d***

40 Control

Cananga odorata (a)

Murraya koenigii (b) 30 a* Euodia hortensis (c)

Mortality (counts) Mortality Cymbopogon citratus (d)

20 Ocimum tenuiflorum L (e)

d* a* 10 b* a*

c* b* 0 3hrs 6hrs 9 hrs 12hrs 24 hrs Time Figure 3-14: Fumigant effect (Mean ±SE) of 5 % (v/v) solutions of selected essential oils on the Spiralling whiteflies over different time intervals.

3.3.1.1 Dose and Effect (Probit) Analysis for the fumigant test.

Table 3-2: Dose-effect analysis of essential oils on the adult Spiralling whiteflies after 24 hours.

2 2 Essential oils Time Equation R LC50 χ statistic P-value df (hours) /EC50 (%) Cananga odorata 24 y= 4.998 + 4.086x 0.750 0.050 118.149 <0.0001 1

Murraya koenigii 24 y= 3.408+ 3.933x 0.316 0.113 76.080 <0.0001 1

Euodia hortensis 24 y= 3.349+3.887x 0.586 0.114 78.574 <0.0001 1 forma hortensis

Cymbopogon 24 y= 8.725+3.764x 0.902 0.004 279.950 <0.0001 1 citratus

Ocimum 24 y= 12.286+5.020x 0.651 0.003 253.512 <0.0001 1 tenuiflorum L

Note: The probability <0.0001, indicated that the significant difference was brought by the log (concentration) variable and mortality. Each test represents the mean of four replicates of 50 whiteflies.

3.3.2 Repellent Test

The repellent effect shown by the selected essential oils was measured based on Repellency index (RI%). Positive values indicated repellent effect and negative values indicated attractant. The higher the positive RI (%) values, the stronger the repellent effect. Repellent compounds are simply when the vapour toxicity is low and most of the insects move towards the control chamber (Maia & Moore, 2011).

The data revealed that none of the essential oils showed a very strong repelling effect on the Spiralling whiteflies as shown in Figure 3-15. However, the ranking based on the Repellency index (RI%) from selected essential oils were; C. citratus (52%) and M. koenigii (L) Spreng (52%), O. tenuiflorum L (12%), E. hortensis forma hortensis (10%) and C. odorata (9%) at 5% (v/v) concentrations. Even at 0.5% (v/v) concentration of 70 essential oils, the effect of positive repellency was only noted for C. citratus (RI= 3%) and M. koenigii (L) Spreng (RI= 8%). Statistically, it was found that only M. koenigii (L) showed strong significant difference at 5% (v/v) concentration; that is, t (6)= 5.286, p= .000 (significant) (refer to Appendix; Table 6-8 for further details).

The EC50 values for the selected essential oils for the repellent effect in percentages were; C. odorata (3.05), O. tenuiflorum L (2.73), E. hortensis forma hortensis (0.96), C. citratus (0.43) and M. koenigii (L) (0.41) (see Table 3-3). The probit analysis also showed that all the tested essential oils had significant difference (p<0.05) expect E. hortensis forma hortensis (P= 0.070) at 5% level of significance. Overall, the data showed that the essential oils from M. koenigii (L) Spreng and C. citratus showed the best repellent effect with increased dose, followed by E. hortensis forma hortensis, O. tenuiflorum L and C. odorata. These effects could be attributed to the chemical constituents of the oils. Attractancy Repellency

*** 5%

0.50% Murraya koenigii Euodia hortensis Cananga odorata Concentration Cymbopogon citratus Ocimum tenuiflorum L 0.25%

-40 -20 0 -20 -4040 -6060 Repellency index (%)

Figure 3-15: Repellency Index (%) response of 0.25%, 0.5% and 5% (v/v) essential oil solutions on the adult Spiralling whiteflies. Note: Asterisks indicate results statistically different at 5% level of significance from its respective control at P<0.05 (*), P<0.01 (**), P<0.001 (***) using Tukey’s test.

Table 3-3: Summary of repellent effect (6-8 hours) on adult whiteflies at different concentrations (Using Probit analysis).

2 2 Essential Conc RI% Equation R EC50 χ statistic P-value df oils (v/v) (%) % Cananga 0.25 -29 y = -0.140+0.290x 0.0795 3.046 5.93 0.015 1 odorata 0.5 -17 5 9

Murraya 0.25 -13 y = 0.260+0.663x 0.3232 0.406 38.214 < 0.0001 1 koenigii 0.5 8 5 52

Euodia 0.25 -10 y= 0.003+0.188x 0.028 0.964 3.277 0.070 1 hortensis 0.5 -3 forma 5 10 hortensis

Cymbopogon 0.25 -9 y = -0.285+0.953x 0.6111 0.434 27.474 < 0.0001 1 citratus 0.5 3 5 52

Ocimum 0.25 -18 y = -0.163+0.374x 0.1582 2.728 13.928 0.000 1 tenuiflorum L 0.5 -11 5 12

Note: The χ² probability < 0.0001, indicated that the significant difference was brought by the log (concentration) variable and the repellency. Each test represents the mean of four replicates of 50 whiteflies.

3.4 Discussion

3.4.1 Fumigant Test

The present study was designed to the test the fumigant toxicity of selected essential oils at concentrations of 0.25%, 0.5% and 5% (v/v) as reported above in the Result (Section: 3.3). Similar studies on the use of plant extracts to control the whiteflies of different species are compared in Table 3-4. The general activities of essential oils on whiteflies based on the available literature have shown that the increasing concentrations of essential oils are directly linked to mortality rates. The fumigant activities of essential oils from selected medicinal plants on local Spiralling whiteflies (Aleurodicus dispersus) are first given information.

All these studies (see Table 3-4) showed that the different forms of fumigant test were done by other researchers in measuring the effectiveness of essential oils at different concentrations. Mostly the researchers have used negligible amounts (μg/L or ppm) of essential oils for the test assay. However, the present study used concentrations of 0.25%, 0.5% and 5% (v/v) which showed no phytotoxicity to the plants. However, the study by Yarahmadi et al. (2013) showed that essential oils at concentration of 125 ppm, 1250 ppm and 2500 ppm (equivalent to 0.0125%, 0.125%, 0.25%) showed phytotoxicity to plants. One of the possibilities of phytotoxicity seen on the plants could be due to use of oil in its pure form (highly concentrated). More importantly, this factor was taken into consideration while deciding the concentrations for the present study.

Overall, the result obtained (fumigant assay test) in the present study is of great interest and could be an excellent contributor to the biopesticides industry. The effectiveness of essential oils especially O. tenuiflorum L and C. citratus is of great interest where the mortality of 100% on Spiralling whiteflies was achieved rapidly at 5% (v/v) concentration with the minimum time range of 3 hours and 6 hours. While the other researchers (see Table 3-4) have shown the effectiveness of essential oils after 24 hours which did not achieved 100% of the mortality in different whitefly species with the given concentrations.

Table 3-4: Studies of effects of plant essential oils on whiteflies.

Plant Name Extracts Concentration Effect Reference Tested

Allium sativum (Garlic). Essential oil extraction 50.00 μg/L. The essential oil showed strongest fumigant effect against B.tabaci (Liu et al., 2008) via hydro-distillation adults with an LC50 value of 0.11 μg/L after 24 hours. using n-hexane.

Artemisia sieberi Besser Essential oils extracts 12, 125, 1250 and The results showed that the tested essential oil concentrations (Yarahmadi et al., (wormwood). purchased from Barij 2500 ppm. significantly reduced the B. tabaci at 24 hours. The percentage mean 2013) Esans Company, contact toxicity of 12, 125, 1250 and 2500 ppm of selected essential oil Kashan, Iran. after 24 hours were 90, 100, 100 and 98%. The researcher also highlighted that the concentration of 2500, 1250, and 125 ppm caused severe phytotoxicity to the plants that were used for treating whiteflies.

Satureja hortensis L., Essential oil extracts 1.56, 3.125, 6.25 and The fumigant toxicity of essential oils from Satureja hortensis L., (Aslan et al., (Summer savory) Ocimum via hydro-distillation 12.5 µl. Ocimum basilicum L. and Thymus vulgaris L. (Lamiacae) showed that 2004) basilicum L. (Tulsi) and using diethyl ether. mortality of B.tabaci (whiteflies) were directly linked to the increasing Thymus vulgaris L. (Garden time and concentrations. The overall multiple mean for (N= 10 Thyme). whiteflies in each set-up) for the fumigant toxicity of selected essential oils after 24 hours were 44, 27 and 27 dead counts, respectively.

Cananga odorata Essential oil extracts 0.125%, 0.25% and The fumigant toxicity of selected essential oils showed effects at 3, 6, Current study (Makosoi), C. citratus via hydro-distillation 0.5% (v/v). 9, 12 and 24 hours. The most robust effect was shown by O. (Lemon grass), M. koenigii using distilled water. tenuiflorum L as 100% mortality (50 dead counts/ 50 tested) was (L) Spreng (Curry Leaves), achieved at 3 hours for 5% (v/v) concentrations, followed by C. O. tenuiflorum L (Tulsi) and citratus at 6 hours for 5 % concentrations. While for others at different E. hortensis forma hortensis concentrations it was relatively low as reported under result section.

(Uci)). The LC50 for O. tenuiflorum L and C. citratus after 24 hours were 0.003% and 0.004% respectively. Note: no phytotoxicity was seen on the plants with the tested solution of essential oils in the present study.

Note: N is referring to the total number of whiteflies tested in each set-up for the fumigant test.

3.4.1.1 Chemical Perspectives for Strong Fumigation Effects

The effects of fumigant activities could be attributed to the chemical constituents in the respective essential oils. The detailed analysis of essential oils as stated in Chapter 2, showed presence of chemical constituents with the respective percentage area (%).

Ocimum tenuiflorum L essential oils showed the most robust fumigant effect which could be attributed to possibilities, such as the presence of high amount of alcohol and phenol groups in the essential oils. O. tenuiflorum L essential oils had 60.61% of alcohol and phenol compounds present as compared to C. citratus (45.88%), C. odorata (50.85%), E. hortensis forma hortensis (0.83%) and M. koenigii (L) Spreng (9.08%) in the present study (See Appendix; Table 6-1). The alcohol and phenol constituents were the major contributors to the fumigant anti-termitic toxicity of sweetgum oil (Park, 2014). Among the tested chemical constituents, benzyl alcohol, acetophenone, 1- phenyl-1-ethanol, hydrocinnamyl alcohol, trans-cinnamyl aldehyde, trans-cinnamyl alcohol, cis-asarone, styrene, and cis-ocimene also showed toxicity against Japanese termites.

More importantly, eugenol compounds in the present study could also have been the cause for such effectiveness in O. tenuiflorum L essential oils (Obeng-Ofori & Reichmuth, 1997; Waliwitiya et al., 2009). The comparative Table 6-1 (See Appendix) showed that eugenol (58.20%) was present in large amounts in the essential oils of O. tenuiflorum L. This could be supported with reference to Ajayi et al. (2014) that eugenol caused > 90% mortality to adult beetles at doses as low as 5 µl/l of air within 24 hours and likewise caused 100% mortality at 20 µl/l and above. Regnault-Roger and Hamraoui (1995) stated that eugenol were among the major compounds that led to inhibition of emergence of Acanthoscelides obrectus (Say) males and females in fumigant test. The toxicity effect may be due to presence of compounds such as eugenol, monoterpenes and sesquiterpenes (Mandal et al. (1993) cited in Pandey et al. (2014)). Similarly, Sosan et al. (2001) carried out larvicidal activity on Aedes aegypti L. (Ae. aegypti) using oils from O. gratissimum (O. tenuiflorum L family) and found that

75 the oil showed 100% mortality at 300 mg/l concentration after 24 hours. A similar test demonstrated that Ocimum suave (O. tenuiflorum L Family) was found to repel and kill all stages of the tick Rhipicephalus appendiculatus. The researcher mentioned that LC50 of the oil was about 0.025% and also stated that 10% solution killed all immatures and 70% of the adults feeding on rabbits (Mwangi et al., 1995). Alcohol and phenolic groups such as linalool and isopulegol compounds showed 100% mortality on stored- product pest insects, Sitophilus oryzae (Lee et al., 2003).

The chemical compounds from the essential oils such as phenols are about 3.5 times more active than the terpenes. Eugenol compounds were found to be 7-9 times more toxic than terpenes and terpinene-4-ol more than twice active than eugenol (Isman, 2000). The statement could clearly support that O. tenuiflorum L essential oils showed a very strong fumigant effect in the present study that could be due to eugenol (58.20%) and terpinene-4-ol (1.01%) compounds.

Likewise, C. citratus essential oils also showed strong fumigant effect. The effect of such activities could be attributed to the major chemical compounds from the present study, especially citronellal (45.09%), citronellol (19.11%) and geraniol (13.57%). These major chemical compounds had showed toxicity and repellent effects on different pest (Fradin & Day, 2002; Ansari et al., 2005; Choochote et al., 2007; Paluch et al., 2009; Sakulku et al., 2009; Maia & Moore, 2011).

The interaction of different chemical compounds could have played a major role in effectiveness or repression of such effects. For instance, the Ocimum family (Basil oil) - when linalool mixed with cuelure (attractant to B. cucurbitae male) its potency for toxicity decreased to the fly species as the culene concentration increased (Ling Chang et al., 2009). The above scenario could clearly explain as to why C. odorata essential oils had the second highest percentage of alcoholic compounds (50.85%) present, but was not able to produce a greater fumigant effect as compared to C. citratus essential oils in the present study. The C. citratus essential oils had 45.88% of alcoholic compounds present. 76

3.4.2 Repellent Test

The Table 3-5 shows the similar studies reported in the literature on the repellent effect of different concentration of essential oils on whiteflies in comparison to the present study. Similar trends of different concentrations of essential oils on the whiteflies were noted both in literature and in the present study. The effects of increasing concentrations of essential oils were directly linked to the repellency effects on the whiteflies.

The activities of essential oils from different plant species reported by other researchers have shown repellency effect on different species of whiteflies. The repellency effects of different concentrations (0.25%, 0.5% and 5% (v/v)) of essential oils on Spiralling whiteflies are first given information. None of the essential oils from selected medicinal plants found in Fiji showed strong repellent property against the Spiralling whiteflies. However the best results were obtained from C. citratus (RI= 52%) and M. koenigii (L) (RI= 52%) at 5% (v/v) concentrations. The repellency effect of different essential oils in the Table 3-5 showed variations in the activities. One of the major causes of variations in the repellency effect of different essential oils could be due to different chemical composition in essential oils as discussed in Chapter 2.

Table 3-5: Studies of repellent effects of plant essential oils on whiteflies.

Plant Name Extract type Concentration Effect Reference tested P. cablin (mint family), Essential oil 0.5% (v/v). The essential oils from P. cablin, T. vulgaris and C. citriodora resulted in (Yang et al., T. vulgaris (Garden extraction. 74.5%, 59.0% and 48.0% fewer eggs laid by B. tabaci after 5 day of 2010) Thyme)) and C. observation. The researcher concluded that P. cablin essential oils strongly citriodora (lemon- repelled the B. tabaci. scented gum) oil. The Y-tube olfactometer was used for the repellency test. The results (Zheng et al., Carica papaya (papaya), Essential oil 20 μ1of essential revealed that Carica papaya and Bauhinia variegata repelled Aleurodicus 2014) Bauhinia variegata extraction via oils. dispersus (Spiralling whiteflies) as the majority of the whiteflies (N= 60 (orchid tree) and hydro-distillation whiteflies) were at controlled chamber (33 whiteflies seen) compared to the Chrysalidocarpus using anhydrous treatment chamber (27 whiteflies seen). Likewise, Chrysalidocarpus lutescens (bamboo ether. lutescens essential oils repelled the spiralling whiteflies where at control palm). chamber (25 whiteflies seen) and at the treatment chamber (35 whiteflies seen).

Ginger (Family: Ginger oil extracted 0.5, 0.75, and 1% The Repellency effect on Bemisia argentifolii (whiteflies) increased with (Zhang et al., Zingiberaceae). via hydro- (v/v) increasing ginger oil concentration. The choice test (repellent tests) showed 2004) distillation . that the number of whitefly counts (N= 30 whiteflies) were higher (>16 counts) in the control chamber of the olfactometer when compared to the treatment chamber (<13 counts).

Cananga odorata Essential oil 0.25%, 0.5% and The repellency activity of selected essential oils in the present study Current study (Makosoi), C. citratus extracts via hydro- 5% (v/v) revealed that C. citratus and M. koenigii (L) Spreng showed the best (Lemon grass), M. distillation using repellent effect as the repellency index for both essential oils at 5% koenigii (L) Spreng distilled water. concentration was 52%, followed by O. tenuiflorum L (RI= 12%), C. (Curry Leaves), O. odorata (RI= 9%) and E. hortensis forma hortensis (RI= 10%). Based on the tenuiflorum L (Tulsi) and obtained results from the T-shaped olfactormeter for best repellent effect E. hortensis forma (N= 50 whiteflies) the C. citratus had 22 whiteflies in control and 7 in the hortensis (Uci)). treatment chamber while M. koenigii (L) Spreng had 29 whiteflies in control and 9 in the treatment chamber. Note: N is referring to the total number of whiteflies tested in each olfactormeter.

3.4.2.1 Chemical Perspectives for Best Repellent effects

The best repellent effects were shown by C. citratus and M. koenigii (L) Spreng followed by E. hortensis forma hortensis, O. tenuiflorum L, and C. odorata essential oils.

The chemical analysis revealed the presence of α-pinene (5.67%), β-pinene (1.55%) and myrcene (1.84%) only in the essential oils of M. koenigii in the present study. These compounds had repelling properties as Debboun et al. (2014) carried out an olfactometer experiment where β-pinene and myrcene caused 60% and 80% repellency to Aedes aegypti among those mosquitoes that responded to either port of olfactometer.

The chemical analysis in the present study revealed that C. citratus had high ester groups (35.76%) and alcohol groups (45.88%) present. Murraya koenigii (L) Spreng had the highest amount of monoterpene (65.81%) compounds in present research, which were identified in literature for effectively repelling the female Aedes aegypti mosquitoes from oviposition (Joel et al., 1991). Cymbopogon citratus is a plant family with promising essential oils used as repellent (Nerio et al., 2010). The researcher also reported that individual compounds such as α-pinene, limonene, citronellol, citronellal, camphor and thymol showed high repellent activity. Citronellol compounds showed higher repellent to ticks (Amblyomma americanum) that resulted in 84%, citronellal (96%) and geraniol (90%) repellency to Aedes aegypti (Debboun et al., 2014). The results from GC-MS analysis for C. citratus in the present study revealed the presence of citronellal (45.09%), citronellol (19.11%) and geraniol (13.57%) which might have contributed towards the best repellent effect against adult whiteflies.

The possibility of M. koenigii (L) Spreng with such effects could also be attributed to the presence of monoterpenes in large amounts, more specifically sabinene (43.80%), α- pinene (5.67%) and ϒ- terpinene (4.82%) from the present study. This could be supported with reference to Regnault-Roger and Hamraoui (1995), that monoterpenes exerted insecticidal effects on adults and on the reproductive development of

Acanthoscelides obrectus (Say) species. The repellent test of M. koenigii (L) Spreng against Callosobruchus chinensis (Coleoptera: Bruchidae) revealed quick knockdown effect causing a maximum of 67% mortality (Haidri et al., 2014). The monoterpene compound such as terpinene-4-ol was essentially important in causing lethal effect to two-spotted spider mites (Lee et al., 1997). Likewise, terpinene-4-ol was found to be more than twice active as eugenol in controlling two spotted spider mite (Isman, 2000). The present study showed both terpinene-4-ol (7.20%) and eugenol (0.33%) in the M. koenigii (L) Spreng essential oils.

One of the possibilities of O. tenuiflorum L essential oils showing a weak repellent activity to Spiralling whiteflies could be attributed to the presence of chemical attractant compounds in the essential oil. The eugenol content in the O. tenuiflorum L essential oils in the present study was 58.20%, which could one of the possibilities of attracting Spiralling whiteflies rather than creating the repellent effect. The above statement was supported with the reference to Isman and Machial (2006), where eugenol and methyl eugenol were used as lure (bait) to trap Japanese beetle Popillia japonica. The other chemical compounds that were found be to attracting the adult corn rootworm beetles (Diabrotica spp.) where Cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and α-terpineol (Hammack, 1996; Petroski & Hammack, 1998).

Likewise, the interaction of different chemical compounds could have a role in the effectiveness or repressiveness for the repellent effect. For instance, the combination of monoterpenoids with thymyl ethyl ether had potentials to reduce monoterpenoids phytotoxicity (Lee et al., 1997).

3.4.3 Mode of Action of Essential oils in Arthropods (Whiteflies)

Many studies of natural insecticides have shown that essential oils are responsible for phyto-protective activities against plant pathogens and pests (Isman, 2000; Li et al., 2014b; Hong et al., 2015). The fumigant and repellent assay tests in the present study

80 were carried out to see the effectiveness of selected essential oils against the Spiralling whiteflies. The fumigant and repellent tests were based on choice and no choice test. For fumigant test (no-choice), the movement of the Spiralling whiteflies were restricted to the plastic bags while for repellent test (choice test) the movement of the Spiralling whiteflies were based on their preference towards control or treatment chamber of the T- shaped olfactometer. The fumigant and repellent test were based on vapour toxicity and repellency of the volatile nature of essential oils (Edris, 2007).

The mode of action of essential oils in the present study could have affected the Spiralling whiteflies through the process of neurotoxicity (damage of nervous tissues) (Isman & Machial, 2006; Koul et al., 2008). The primary target for most of the insecticides - is the insect’s nervous system. The nervous system is the control centre of the body that transduce the activity of nerves into behaviour. The nerve cells also known as neurons that act upon external cues from smell, taste, touch and sound sensors, as well as internal inputs from sources such as, hormones, body temperature and limb position sensors to create control coordination in insects behaviour (Salgado, 2013). The fine-tuned control systems of these insects are disrupted by the volatile nature of the essential oils and other insecticides when applied.

The insecticides lead to poisoning of insects whereby certain cells show alternation of staining properties; while some cells can breakdown (cytolysis) in tissues. Likewise, within the nucleus the chromatin granules results into pycnosis (clump together) and the nissl bodies (granular substances) in the nerve cells dissolves (Tanada & Kaya, 1993; Satar et al., 2008). The symptoms of nerve poisons induce their appearance in four stages; excitation, convulsion, paralysis and death. More importantly, neurotoxic fumigant results only in three stages; excitation, paralysis and death (Tanada & Kaya, 1993). The disturbance of nervous system in the insects often affects the respiratory, muscular and circulatory systems. As a result of disturbance or malfunction in the metabolic system the insect dies.

The compounds such as octopamine and acetylcholine (accumulate in the nerves) in arthropods (whiteflies) have diverse biological roles. The chemical compounds octopamine and acetylcholine function as neurotransmitters, circulating necrohormones and necrohormones (see Figure 3-16). If these compounds get interrupted by any chance, then it could possibly result in the breakdown of nervous system of the insects.

Acetylcholine Membrane at post- Nerve + synaptic junction current. Nerve (C NH O ) 7 16 2 impulse Modulation at Octopamine Muscles junction (neurons) physiological

(C8H11 NO2) level. Insect body fluid

Figure 3-16: Target sites in insects as possible neurotransmitter mediated toxic action of essential oils. Adapted from: Tripathi et al. (2009)

The different components (such as monoterpenes) of essential oils from aromatic plants have shown to inhibit the acetylcholinesterase enzyme activity of different class of arthropods (Houghton et al., 2006; Rajendran & Sriranjini, 2008). For example, azadirachtin (a terpene compound) was found to inhibit the acetylcholinesterase enzyme activity in Nilaparvata lugens S (Brown plant hopper). Chemical compounds such as linalool affected the nervous system of the insects by influencing the release of acetylcholine esterase that functions as neurotransmitters (Re et al., 2000). Likewise, the essential oils from plants have targeted octopamine in insects. The sub-lethal effects on the insects behaviour that is due to the compounds of essential oils is mainly due to the blockage of octopamine receptors (Enan, 2001; Enan, 2005).

Different components of essential oils are mainly considered to have the insecticidal properties (Coats et al., 1991; Regnault-Roger & Hamraoui, 1995). The insecticidal properties of volatile components of essential oils are mainly due to high volatility which makes the fumigant and gaseous action more rapid (Hamza et al., 2016). These

82 essential oils components are not typically volatile but they also have lipophilic (ability of chemical compounds to dissolve in fats, lipids and oils) properties making rapid penetration in insects. This rapid penetration of essential oils in insects creates interference with the physiological functions (Lee et al., 2003). In general, the essential oils with lipophilic nature facilitates its interference with biochemical, basic metabolism, physiological and behavioural functions of the pests (Bakkali et al., 2008). For example, Enan (2001) conducted a research to show the mode of action of insecticidal activities of eugenol, cinnamic alcohol and α-terpineol against P. Americana (cockroaches). The results from this research exhibited that the effects led to hyperactivity in cockroaches followed by hyperextension of the legs and belly, then quick knockdown.

Essential oils not only affect the nervous system of the insects, but also influence the cellular breathing through asphyxiation (deficient supply of oxygen) or respiratory chains. The essential oils form an impermeable film, which covers the insect from the air. The formation of the covering results in suffocation or at many times death in arthropods (Li et al., 2014b). The overall effect of essential oils has led to disruption, dissolution of cell membranes, and blockage of tracheal system (Isman & Machial, 2006; Tehri & Singh, 2015). In addition, Tripathi et al. (2009) reported that essential oil components such as monoterpenes are cytotoxic to tissues of living organisms. Cytotoxic causes reduction in the intact mitochondria and golgi bodies, impairing respiration and reducing cell membrane permeability.

The fumigant and repellent activities of selected essential oils in the present study could slightly explain the mode of action on Spiralling whiteflies based on available literature. The mode of action of essential oils in available literature needs thorough research as how different chemical components affect the whiteflies. Generally, the application of essential oils for both fumigant and repellent test were based on the effect of volatility of the essential oils. Hence, the Spiralling whiteflies in the present study may have been affected by the different concentrations of essential oils via neurotoxicity and respiratory toxicity. 83

3.5 Conclusion

The results of this and earlier studies indicated that essential oils could be used for fumigant or repellent activities against the Spiralling whiteflies (Aleurodicus Dispersus Russell). These whiteflies have affected plants in many ways, such as decreased photosynthesis rates and physical damages to the leaves.

As for the purpose, O. tenuiflorum L essential oils showed the strongest fumigant toxicity, while C. citratus and M. koenigii (L) Spreng showed the best repellent effect. The effect for such activities could be attributed to chemical constituents from the present study. One of the important components that may be responsible for very strong fumigant effect is eugenol. The presence of large amounts of eugenol (58.20%) in Ocimum teniflorum L could have contributed towards the best fumigant effect. Likewise, the presence of monoterpenes in large amounts in M. koenigii (L) Spreng essential oils more specifically sabinene (43.80%), α-pinene (5.67%) and ϒ-terpinene (4.82%) in the present study could have contributed towards the repellency effect. Interestingly, the presence of eugenol in the O. tenuiflorum L essential oils may have contributed towards the best fumigant effect, however the eugenol compound may have attracted the Spiralling whiteflies in the repellent test which was supported with the available literature (Isman & Machial, 2006). This could be one of the reasons why the fumigant and repellent test may have not achieved similar results even though their modes of action were similar. Generally, the biological effects summarized on fumigant and repellent activities reflect the wide spectrum, possibly with individual or interaction of chemical compounds (Joel et al., 1991).

Bio-fumigants had been long touted as an attractive alternative for synthetic fumigants for the arthropod management, as botanicals pose little threat to human health as well as the environment (Pandey et al., 2014). Although economically, synthetic chemicals are more often used as repellents then the essential oils, these essential oils have the potential of providing efficient and safer repellents for the environment as well as for humans (Nerio et al., 2010). Thus, potential need for the development of possible

84 natural fumigant and repellent for controlling whitefly needs to be further evaluated as to their enhanced activity, mode of actions and safety to humans.

85 4. CHAPTER 4: ANTIMICROBIAL ACTIVITIES OF SELECTED ESSENTIAL OILS A large part of the universal action of essential oils lies in their ability to weaken the constant pathogenic aggression to which human beings are subject, while-at the same time–leaving friendly bacteria untouched. Antibiotics, by contrast, are not selective, destroying bacteria indiscriminately. We frequently see fungal infections start to proliferate after treatment with antibiotics. However, such manifestations never appear after treatment with essential oils. Natural Home Health Care Using Essential Oils, Daniel Penoel, MD (Source: http://www.biospiritual-energy-healing.com/essential-oils-affect-the-body.html)

4.0 Introduction

Essential oils are known for their antimicrobial activities since ancient times, which were not scientifically proven till the 20th century (Li et al., 2014a; Dagli et al., 2015). These aromatic compounds are antimicrobial agents that have the ability to fight viruses, bacteria and fungi (Cowan, 1999; Pandey & Kumar, 2013; Hintz et al., 2015). The focus of this chapter is to evaluate the antimicrobial activities of selected medicinal plants used as traditional medicine for the treatment of manifestations resulting from microorganisms. The extracts from five selected plants; C. odorata (Makasoi), C. citratus (Lemon grass), M. koenigii (L) Spreng (Curry Leaves), O. tenuiflorum L (Tulsi) and E. hortensis forma hortensis (Uci) from different families were tested for their inhibitory activities on selected pathogenic bacteria (Salmonella, Streptococcus (pneumoniae), Staphylococcus aureus, Pseudomonas aeruginosa and Thermus thermophiles) and selected fungi (Rhizopus sporangia, Penicillum conidia, Aspergillus conidiophores, Sodaria wild and Sodaria gray).

4.1 Background

4.1.1 Microorganisms

4.1.1.1 Overview of Selected Bacteria

Bacterial infections are widespread and cause much discomfort and sickness. These bacterial pathogens continue to threaten human health and welfare due to new or

86 resistant pathogens (Phillips et al., 2004; Søborg et al., 2013). The present study work to assess the antimicrobial properties of essential oils from five selected medicinal plants found in Fiji under different concentrations. The antimicrobial activities were assessed on; Salmonella, Streptococcus (pneumoniae), Staphylococcus aureus, Pseudomonas aeruginosa and Thermus thermophiles. These selected bacteria were found to be pathogenic to both humans and animals (see Table 4-1) based on the available literature.

Table 4-1: Harmful effects of selected Gram (+) and (-) bacteria.

Bacteria Common harmful effects Reference Salmonella - Causes 21 million cases of typhoid fever and 200,000 (Jones et al., (Gram (-)) deaths each year. These bacteria are known to cause 1994; Devi et infections in mammalian intestine (intestinal epithelium al., 2010) of the small bowel). - Also resulted in the ineffectiveness of immune system by stopping the oxidative burst of leukocytes.

Streptococcus - These bacteria caused many different types of illness, (Centers for (pneumoniae) that include; pneumonia (lung infections), ear, sinus, Disease Control (Gram (+)) meningitis (covering around spinal cord and the brain) and Prevention, and bacteraemia (blood stream) infections. 2004; Buckle, 2015b)

Staphylococcus - Caused major infections in skin, soft-tissue and (Lowy, 1998; aureus disorders in bone, endovascular and joints. Buckle, 2015b) (Gram (+))

Pseudomonas - Caused infections such as malignant external otitis (Bodey et al., aeruginosa (outer ear infection), endocarditis (heart valves), 1983; Buckle, (Gram (+)) meningitis (covering membrane of brain and spinal cord), 2015b) septicaemia (blood diseases) and pneumonia.

Thermus - The diseases associated with thermophilic are rheumatic (Rabkin et al., thermophiles heart diseases, immunodeficiency and strokes. 1985) (Gram (-))

4.1.1.2 Overview of Selected Fungi

Most fungi are destructive agents that affect agricultural commodities around the globe (Palm, 2001). This is mainly due to fungi producing biologically active compounds such as mycotoxins that are particularly toxic to several plants and animals (Souza et al., 2010; Wareing, 2014). These mycotoxins are formed through moulds which cause food spoilage and make mushrooms poisonous (Atanda et al., 2011; Darwish et al., 2014). The selected fungi for the present study were those that were brought to attention through their detrimental effects on the food and agriculture industry as highlighted in Table 4-2.

Table 4-2: Effects of selected fungi to humans through food and agriculture industries Fungi Common harmful effect Reference Rhizopus sporangia Rhizopus species are responsible for (Saranraj, 2012) causing spoilage of bread.

Penicillum conidia The genus of Penicillum mostly caused (Kung'u, 2016) food spoilage; even some produced toxic compounds that had triggered allergic reactions.

Aspergillus Aspergillus species mostly caused (Jahn et al., 2000; conidiophores invasive pulmonary aspergillosis (type of Davidson, 2015) deficiency in white blood cells). This fungus also affected the food industry through the formation of moulds on fruits, grains, wheats and breads.

Sodaria wild These species affected the agriculture (Kavak, 2012) Sodaria gray industries which are associated with symptoms of brown wood discoloration and leaf spottiness.

The selected bacteria and fungi for the present study were found to be pathogenic to humans and animals through health, agriculture or food industry. The synthetic antimicrobial agents and chemical food preservatives have been considered an effective method since ancient times for controlling such pathogens. However, nowadays

88 attention is given more towards natural antimicrobials such as essential oils (Hayek et al., 2013; Bevilacqua, 2014) for eliminating pathogenic microorganisms.

4.1.2 Why Essential Oils as Alternatives for Elimination Pathogenic Micro- organisms?

The main reason for using essential oils over synthetic chemicals is due to consumer concern towards chemical preservatives (Lucera et al., 2011; Fernández et al., 2015). These concerns mostly involve carcinogenic (cancer causing) and teratogenic (disturbance of embryo development) attributes, residual toxicity and microbial resistance (Moreira et al., 2005; RaybaudiǦMassilia et al., 2009).

Essential oils are considered the best and safest alternative for eliminating pathogenic microorganisms (Negi, 2012). This could be possibly due to presence of many different compounds such as phenols, terpenes derivative compounds and other antimicrobial compounds in the essential oils making it very precise in their mode of action against different pathogenic micro-organisms (Faleiro, 2011; Akthar et al., 2014). Hence, the large number of compounds means that there is less chances of pathogens developing resistance.

Some essential oils are thought to cure one or more organ dysfunction or systemic disorders. For example, the essential oils of Eucalyptus citriodora, Eucalyptus tereticornis and Eucalyptus globulus inhibited the neutrophil-dependent (central actions) and inflammatory reaction in rats (Silva et al., 2003). The extracts from Spanish sage (S. lavandulaefolia Vahl.) showed anti-inflammatory, oestrogenic, sedative effects and treatments for Alzheimer’s diseases (Perry et al., 2003). These essential oils affect the cell membrane of pathogenic microorganism by influencing the permeability and leaking of ions and molecules from the cell and disrupting the cell respiration and enzymatic activities (Akthar et al., 2014).

One of the scopes for a new method of eliminating pathogen is the use of essential oils as additives (anti-agents) for anti-bacterial and anti-fungal activities (Kalemba & Kunicka, 2003; Burt, 2004). The reasons for the increase in attention for the essential oils are attributed to the safe status, potential multi-purpose functional use and a greater range of acceptance by the consumers (Srivastava & Sharma, 2003; Dubey et al., 2008; Ahmed, 2013).

4.2 Methodology

The microbiological activities of anti-bacterial and anti-fungal were conducted in a standard microbiology laboratory located at the University of the South Pacific, Suva, Fiji islands. The microbiology laboratory has a laminar flow, which has ISO certification by the New Zealand Accreditation Unit.

4.2.1 Test against Bacteria and Fungi strains

The ratio used to prepare the bacterium culture was 20 g of nutrient agar suspended in 1000 mL of distilled water. The solutions were left in autoclave at 121 °C for 15 mins, after which it was stabilized in S.E.M (CNT number: WB5) water bath (45 °C) for 35 minutes. For the nutrient broth culture (8 g/L), selected bacteria were inoculated for 18 hours at 37 °C. The broth cultures were used to streak the bacteria on the nutrient agar plates using sterile cotton swabs. The following bacteria; Salmonella (Gram (-)), Streptococcus (pneumoniae) (Gram (+)), Staphylococcus aureus (Gram (+)), Pseudomonas aeruginosa (Gram (-)) and Thermus thermophiles (Gram (-)) were used for the anti-bacterial activities.

The Potato Dextrose Agars (PDA) (39.5 g/L solution) was used for the fungi cultures: Rhizopus sporangia, Penicillum conidia and Aspergillus conidiophores. Corn Meal Agar (CMA) (Corn Meal Agar (8.5 g), yeast (0.5 g), glucose (1 g) in 500mL distilled water solution) was used for culturing the fungi; Sodaria wild and Sodaria gray. The pure cultures for the fungi were first cultured in petri dishes. The growth of hypha indicated that it was ready for streak plating using a sterile cotton swab. The stock

90 culture for both bacteria and fungi were maintained at refrigerator temperature from which cultures were used for the actual experiment.

Once the streaking plating was done for bacteria and fungi, the prepared filter paper discs (~6 mm) were dipped in the respective concentrations and placed on the cultured petri dish. For the Standard Control, Ampicillin discs were used for the bacteria test and Nistat discs were used for the fungi (for effects, refer to Appendix: Table 6-9 (Bacteria) and Table 6-11 (Fungi)). The antimicrobial activities of different concentrations of essential oils were assessed using disc diffusion method (Rajendran et al., 2014). After culturing and disc insertion, the petri dishes were left in the incubator (Contherm digital series (Serial number: 05028 and 05025)) at 37 °C (bacteria) for 18-24 hours and 27 °C (fungi) for 1-2 days.

After the specific times, the inhibition zones for bacteria and fungi were calculated by measuring (using a 15 cm ruler) the diameter (mm) of the inhibition zones including the filter paper on which the essential oils were transferred. There were a total of 5 replicates for each bacteria and fungi with its respective concentrations.

4.2.2 Preparation of Essential oil solutions

Solutions for the different concentrations of essential oils were prepared based on the percentage required (Yang et al., 2010). For instance, in order to prepare a solution of 0.25% (v/v); 0.025 mL (essential oils) was added to 9.925 mL (distilled water) and 0.05 mL (Tween 20 (Viscous liquid)) to obtain an overall volume of 10 mL solution. The purpose of Tween 20 in this study was to increase the solubility of hydrophobic compounds and help in their penetration into microorganisms cell wall and membrane (Kim et al., 1995). For all the different concentrations the final volumes of the solutions were 10 mL. The essential oil solutions for 0.5%, 5%, 25% and 50% (v/v) were prepared accordingly.

4.2.3 Statistical Analysis

The software (SPSS) version 21 was used to calculate the Mean and Standard Error (SE) for both bacteria and the fungi as reported in Table 4-3 and Table 4-4. In order to statistically evaluate the difference in the mean diameter (mm) of inhibitory zones between the same species of bacteria and fungi using specific concentrations, an ANOVA using tukey’s test was performed. Prior to using ANOVA, the raw data was transformed using square root (Kim et al., 2000). The transformation step was considered due to the data being not normally distributed.

The results for statistical difference (at 5% level of significance) between selected bacteria and fungi at different concentrations and the essential oils were shown in the bar graphs (under Result section) and the specific p-values were reported under the Appendix section (Bacteria: 6.2.1.1 and Fungi: 6.2.2.1) .

4.3 Results

4.3.1 Anti-bacterial Activities of Selected Essential oils

The effect of lowest concentrations of essential oils (0.25% (v/v)) on different bacteria were associated with the numerically lowest mean diameter (mm) zone of inhibition (µ= 0.23) and the highest concentrations of essential oils (100%) were associated with numerically highest mean diameter (mm) of the zone of inhibition (µ= 9.58) (refer to Appendix: Table 6-10). The anti-bacterial effect of essential oils showed that with increase concentrations of essential oils the diameter (mm) zone of inhibition increased for specific bacteria as shown in Table 4-3. Likewise, the Figure 4-1 to Figure 4-4 showed the effect of essential oils on different bacteria at specific concentrations. The anti-bacterial activities of essential oils at 0.25% and 0.5% were not presented in the graph, as the inhibition zone for all bacteria were zero, except C. odorata essential oils (see Table 4-3).

The anti-bacterial activities showed that O. tenuiflorum L (Tulsi) had the best result whereby the diameter zones of inhibition (mm) were present to all tested Gram (+) and (-) bacteria from 25% (v/v) concentrations. Cananga odorata essential oils showed the diameter (mm) zones of inhibition at the lowest concentrations (0.25% and 0.5% (v/v)) which were not common to other tested essential oils. The inhibitory effect of lowest concentrations of 0.25% and 0.5% were on Thermus thermophiles and Pseudomonas aeruginosa. For Staphylococcus aureus the effect were seen from 25% (v/v). The essential oil at 100% concentration had no effect on Salmonella. Similarly, C. citratus essential oils showed diameter (mm) zones of inhibition to all the tested bacteria except Salmonella. The mean values (mm) for zone of inhibition were obvious at 5%, 25%, 50% and 100% (v/v) concentrations for Pseudomonas aeruginosa. While for Streptococcus (pneumonia) and Staphylococcus aureus, the inhibition zones were seen from 25% (v/v) concentration. Even at 100% concentration, the essential oils had no effect on Salmonella. The essential oils from E. hortensis forma hortensis showed the effects of inhibition activities only at 50 % (v/v) and 100 % (v/v) solutions. The mean values (mm) for diameter zone of inhibition for Thermus thermophiles and Pseudomonas aeruginosa were seen from 50% (v/v). Streptococcus (pneumonia) and Staphylococcus aureus showed inhibitory effect only at 100% (v/v) concentration. Salmonella showed no effect with any of the tested concentrations. The anti-bacterial activities of M. koenigii (L) Spreng essential oils showed the inhibitory effect with increased concentrations, that is for Thermus thermophiles and Pseudomonas aeruginosa; the effect were seen from 50% (v/v) concentrations. Salmonella (Gram- negative bacteria) and Streptococcus (pneumonia) (Gram-positive bacteria) showed no effect with changing concentrations. Staphylococcus aureus showed inhibitory effect at 100% concentration only. There were no diameter zones of inhibition shown at 25% (v/v) concentration and below for any of the tested bacteria.

The Figure 4-1 to Figure 4-4 also showed the comparison of inhibitory activities of each bacterium at specific concentrations. For detailed comparison of anti-bacterial activities (of each bacterium) with different essential oils at specific concentration were reported 93 in the Appendix (Section: 6.2.1.1). The inhibitory activities of all the tested concentrations (0.25%, 0.5%, 5%, 25%, 50%, 100% (v/v)) of essential oils on Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus (pneumonia) resulted in the statistical difference (p<0.05, at 5% level of significance). However, the inhibitory activity of selected essential oils on Salmonella was statistically different (p<0.05, at 5% level of significance) at 50% and 100% (v/v) concentrations. The inhibitory activities of essential oils on bacterium Thermus thermophiles were statistically different (p<0.05, at 5% level of significance) from 5% (v/v) to 100% (v/v) concentrations.

Table 4-3: Mean and Standard Error (SE) for effects of varying concentration of the essential oils on different bacteria. Essential oil Bacteria 0.25a 0.5b 5c 25d 50e 100f Thermus thermophilus 0 0 0 8.00±0.63 8.00±0.55 17.20±0.20 Pseudomonas aeruginosa 0 0 0 4.80±1.98 8.80±0.86 16.20±0.20 Streptococcus (pneumonia) * 0 0 0 5.60±1.40 7.80±0.58 17.80±0.49 Ocimum Staphylococcus aureus* 0 0 0 10.20±1.11 10.80±1.88 25.60±0.68 tenuiflorum L Salmonella 0 0 0 3.00±1.84 7.60±0.24 14.80±0.20 Thermus thermophilus 0 0 5.40±2.20 10.60±1.25 13.80±0.37 15.40±0.40 Pseudomonas aeruginosa 0 0 3.20±1.96 12.00±1.10 14.40±0.68 15.00±0.89 Cymbopogon Streptococcus (pneumonia) * 0 0 0 4.40±1.81 9.20±0.66 15.20±0.37 citratus Staphylococcus aureus* 0 0 0 7.60±2.25 9.80±0.58 14.80±0.20 Salmonella 0 0 0 0 0 0 Thermus thermophilus 1.60±1.60 2.80±1.71 3.00±1.84 8.40±0.60 11.40±1.08 12.20±1.16 Pseudomonas aeruginosa 4.20±1.71 4.40±1.81 7.80±0.37 8.60±0.24 11.60±1.21 12.60±0.75 Cananga Streptococcus (pneumonia) * 0 0 0 7.00±0.00 7.20±0.20 7.40±0.40 odorata Staphylococcus aureus* 0 0 4.20±1.71 8.00±0.55 8.80±0.20 10.20±0.37 Salmonella 0.0 0.0 0.0 0.0 0.0 0.0 Thermus thermophilus 0 0 0 0 5.80±1.46 10.80±1.32 Pseudomonas aeruginosa 0 0 0 0 5.60±1.40 11.20±0.73 Euodia Streptococcus (pneumonia) * 0 0 0 0 0 7.40±0.25 hortensis forma Staphylococcus aureus* 0 0 0 0 0 8.40±0.51 hortensis Salmonella 0 0 0 0 0 0 Thermus thermophilus 0 0 0 0 1.40±1.40 3.00±1.84 Pseudomonas aeruginosa 0 0 0 0 1.40±1.40 2.80±1.71 Murraya Streptococcus (pneumonia) * 0 0 0 0 0 0 koenigii (L) Staphylococcus aureus* 0 0 0 0 0 1.60±1.60 Spreng Salmonella 0 0 0 0 0 0

Note: Gram (+) bacteria = *, 0= no diameter of inhibition zone, (Mean ± Standard Error) the mean value in (mm); a, b, c, d, e and f referred to concentrations (%). Each test represents the mean of five replicates of each tested concentration.

Note: The alphabetical letters and the asterisks on different bars of bacteria indicate statistical difference at 5% level of significance of mean diameter (mm) of inhibition zones of specific bacteria at same concentration of essential oils. For example, at a concentration of 5% (v/v) of essential oils, the inhibitory activities of Pseudomonas aeruginosa in all tested essential oils is statistically compared with each other, that is, P<0.05 (*), P<0.01 (**), P<0.001 (***) using Tukey’s test.

9 b** 8 7 ** 6 Thermus thermophilus c** Pseudomonas aeruginosa 5 Streptococcus (pneumonia) 4 Staphylococcus aureus 3 Salmonella 2 Mean Inhibition Mean Inhibition zone (mm) 1 0 O. tenuiflorum (a) C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) Essential oil solutions

Figure 4-1: Anti-bacterial effect of selected essential oils at 5% (v/v) solution.

14 a**

8 b** Thermus thermophilus 6 Pseudomonas aeruginosa *** Streptococcus (pneumonia) 4 Staphylococcus aureus

Mean Inhibition zone (mm)Mean Salmonella 2

0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) (a) Essential oil solutions

Figure 4-2: Anti-bacterial effect of selected essential oils at 25% (v/v) solution.

16 e*** e*** d* d* 14 e*** e*** 12

10 a** c* e** e* Thermus thermophilus *** 8 b* b* b* Pseudomonas aeruginosa Streptococcus (pneumonia) 6 b*** b*** Staphylococcus aureus c*** c*** Salmonella Mean Inhibition zone (mm)Mean 4 a** a** d* d* 2

0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) (a) Essential oil solutions

Figure 4-3: Anti-bacterial effect of selected essential oils at 50% (v/v) solution.

30 e*** b* C*** d*** 25 Thermus thermophilus

b** Pseudomonas aeruginosa c*** 20 d*** e*** a** Streptococcus (pneumonia) a*** c*** e*** e*** d*** e*** Staphylococcus aureus *** 15 a* e*** e*** Salmonella e*** e** e*** a*** e*** 10 a*** a*** d*** b*** b*** b*** a*** a*** c*** Mean Inhibition zone (mm)Mean b*** c*** a*** c*** b*** d*** 5 d** d***

0 O. tenuiflorum (a) C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) Essential oil solutions

Figure 4-4: Anti-bacterial effect of selected essential oils at 100% (v/v) solution. 97

4.3.2 Anti-fungal Activities of Selected Essential oils

A similar trend was observed for the fungi where the lower mean (µ= 0.27) of diameter (mm) zones of inhibition were associated with lower concentrations (5% (v/v)) and the highest concentrations (100% (v/v)) of essential oils were associated with higher mean (µ= 14.76) of diameter zone (mm) of inhibition (see Appendix: Table 6-12). The Table 4-4 reported the anti-fungal activities of selected essential oils at different concentrations (0.25%, 0.5%, 5%, 25%, 50% and 100% (v/v)). The Figure 4-5 to Figure 4-8 showed the effects of inhibitory activities on fungi with specific concentrations mostly from 25% (v/v) concentrations of different essential oils. The anti-fungal activities below 25% (v/v) concentrations were mostly zero except for Penicillin and Sordaria gray at 5% (v/v) concentration (see Table 4-4).

The essential oils from O. tenuiflorum L showed the best diameter (mm) zones of inhibition to all the selected fungi. Penicillin was the most susceptible out of the tested fungi as the effects were even present at 5% (v/v) concentration while not true for others. Aspergillus, Rhizopus, Sordaria wild and Sordaria gray showed inhibitory effects from 25% (v/v) concentration. Cananga odorata essential oils showed a range of inhibition zones to all the tested fungi. The most susceptible fungus was Sordaria gray, as the inhibition zones (mm) were very clear from 5% to 100% (v/v) concentrations. Rhizopus and Sordaria wild showed inhibitory effects from 25% to 100% (v/v), not much difference was seen with the varying concentrations. For Sordaria wild, the inhibitory effects were from 25% (v/v) and Penicillin was the most resistance one as the effects of inhibition zones were only present at 100% (v/v). Similarly, the essential oils from C. citratus showed inhibitory activities at 25%, 50% and 100% (v/v) concentrations of essential oils. Below 25% (v/v) concentrations, the tested fungi showed resistance. The fungus Sordaria gray showed effect only at 50% and 100% (v/v) concentrations. The anti-fungal activities of E. hortensis forma hortensis essential oils showed the presence of diameter (mm) zone of inhibition only at 50% and 100% (v/v) concentrations. The essential oils from M. koenigii (L) Spreng showed the least

98 inhibitory activities with all the different concentrations of essential oils. All the tested fungi only showed effects at 100% (v/v) concentration.

The Figure 4-5 to Figure 4-8 also showed the statistical difference (at 5% level of significance) of inhibitory activities of selected essential oils with varied concentrations. The inhibitory activities of selected essential oils on Penicillin, Sordaria wild, Sordaria gray and Aspergillus were statistically different (p<0.05, at 5% level of significance) from 25% to 100% (v/v) concentrations. The inhibitory activities of essential oils on Rhizopus showed statistical difference (p<0.05, at 5% level of significance) only at 25% and 100% (v/v) concentrations. The diameter (mm) zones of inhibition for selected fungi below 25% (v/v) concentrations were mostly zero; therefore it was not possible to show statistical difference. The detailed p-values for specific comparison of each essential oil with inhibitory activities were presented in the Appendix (Section: 6.2.2.1).

Overall, the effects of inhibitory activities on selected fungi increased with changing concentrations (that is; 5%, 25%, 50% and 100% (v/v)). The overall trend of selected essential oils for anti-fungal susceptibility were; O. tenuiflorum L > C. odorata > C. citratus > E. hortensis forma hortensis > Murraya koenigii.

Table 4-4: Mean and Standard Error (SE) for effects of varying concentration of the essential oils on different fungi.

Essential oils Fungi 0.25a 0.5b 5c 25d 50e 100f

Ocimum tenuiflorum L Aspergillus 0 0 0 12.80±1.39 13.80±2.52 32.40±1.44 Rhizopus 0 0 0 6.60±1.75 7.00±1.79 25.80±1.80 Penicillin 0 0 4.80±3.01 7.40±2.23 12.40±3.80 34.40±5.01 Sordaria wild 0 0 0 11.60±5.05 15.80±1.16 31.60±4.50 Sordaria gray 0 0 0 12.80±0.66 19.80±1.56 37.40±2.18 Cymbopogon citratus Aspergillus 0 0 0 4.20±1.71 9.20±3.76 11.00±4.49 Rhizopus 0 0 0 4.60±1.89 8.20±0.49 14.40±0.93 Penicillin 0 0 0 7.80±0.37 9.80±0.58 14.20±8.71 Sordaria wild 0 0 0 1.60±1.60 1.80±1.80 6.20±3.83 Sordaria gray 0 0 0 0 4.80±2.96 7.40±4.60 Cananga odorata Aspergillus 0 0 0 0 0 7.40±0.24 Rhizopus 0 0 0 7.20±0.20 7.20±2.01 7.60±0.24 Penicillin 0 0 0 0 0 13.80±4.04 Sordaria wild 0 0 0 2.20±2.02 4.20±1.71 10.80±0.86 Sordaria gray 0 0 2.00±2.00 4.20±2.62 6.80±1.74 9.00±0.84 Euodia hortensis Aspergillus 0 0 0 0 8.80±0.86 18.80±3.50 Rhizopus 0 0 0 0 4.80±1.98 12.60±1.17 Penicillin 0 0 0 0 2.80±1.71 13.60±2.71 Sordaria wild 0 0 0 0 5.20±2.22 19.00±0.63 Sordaria gray 0 0 0 0 1.40±1.40 22.60±0.93 Murraya Koenigii Aspergillus 0 0 0 0 0 3.00±1.84 Rhizopus 0 0 0 0 0 2.20±2.20 Penicillin 0 0 0 0 0 1.40±1.40 Sordaria wild 0 0 0 0 0 6.20±2.85 Sordaria gray 0 0 0 0 0 6.20±2.73 Note :( Mean ± Standard Error); 0= no diameter for inhibition zone; a, b, c, d, e and f indicated different concentrations (%).Each test represents the mean of five replicates of each tested concentration.

100

Note: The alphabetical letters and the asterisks on different bars of bacteria indicate statistical difference at 5% level of significance of mean diameter (mm) of inhibition zones of specific fungus at same concentration of essential oils. For example, at a concentration of 25% (v/v) of essential oils, the inhibitory activities of Aspergillus in all tested essential oils is statistically compared with each other, that is, P<0.05 (*), P<0.01 (**), P<0.001 (***) using Tukey’s test.

9 8 7 Aspergillus 6 Rhizopus 5 Pencillin 4 Sordaria wild 3 Sordaria grey 2

Mean Inhibition zone (mm)Mean 1 0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis M. koenigii (e) (a) (d) Selected essential oils

Figure 4-5: Anti-fungal effect of essential oils at 5% (v/v) solution.

16 b** 14 c**

10 Aspergillus Rhizopus 8 a** a** Pencillin 6 Sordaria wild 4 Mean Inhibition zone (mm)Mean Sordaria grey 2

0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) (a) Selected essential oils

Figure 4-6: Anti-fungal effect of essential oils at 25% (v/v) solution. 101

b** d*** 20 b** Aspergillus c* d* Rhizopus d* Pencillin 15 Sordaria wild Sordaria grey d* 10 a** d* a* a* a* Mean Inhibition zone (mm)Mean b* 5 a** a***

0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) (a) Selected essential oils

Figure 4-7: Anti-fungal effect of essential oils at 50% (v/v) solution.

45 c* e** b*** b* e** 40 b* c* e** e*** b** 35 a* c*** Aspergillus 30 d* e*** Rhizopus b* e* Pencillin 25 a* e** Sordaria wild 20 b* a* Sordaria grey e*** a*** a* 15 d* e*** a** a* a**

Mean Inhibition zone (mm)Mean d* a*** e** a** d* 10 c** a* a*** a*** b*** d*** 5 d** a**

0 O. tenuiflorum C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e) (a) Selected essential oils

Figure 4-8: Anti-fungal effect of essential oils at 100% (v/v) solution

102

4.4 Discussion

4.4.1 Anti-bacterial Effect of each Essential oil and its Chemical Perspective

The different effect of anti-bacterial activities could possibly be due to the presence of different chemical compounds in the respective oils. The essential oils from O. tenuiflorum L (Tulsi) showed the best results whereby the diameter zones of inhibition (mm) were present to all the tested Gram (+) and (-) bacteria from 25% (v/v) concentrations which was in agreement with Pandey et al. (2014). Likewise, another researcher showed that O. tenuiflorum L essential oils had strong anti-bacterial activities to all the tested bacteria; S. aureus (12 mm), P. aeruginosa (10 mm) and E. coli (10 mm) at the concentration of 100 mg/mL (khan et al., 2015). The same researcher concluded that the leaves of Ocimum tenuiflorum had a significant anti-bacterial activity against the selected human bacteria.

The GC-MS analysis in the present study showed that the essential oils of O. tenuiflorum L (Tulsi) consisted alcohol groups (63%) as the major component, followed by 23% sesquiterpenes and 14% monoterpenes. The high percentage of alcohol chemical groups could be one of the possibilities of such obvious effects on both Gram (+) and Gram (-) bacteria (Vaquero et al., 2007). The bacterial species exhibited different sensitivities towards the different concentrations of phenolic compounds (Puupponen-Pimiä et al., 2001; Ng et al., 2014). The rank for the anti-bacterial activities of essential oil components are: phenols > aldehydes > ketones > ethers > hydrocarbons (Kalemba & Kunicka, 2003). In addition, there is a possibility of synergism effect of different compounds as well. For instance, the synergism effect of eugenol and linalool (of sweet basil (Tulsi family)) had the strongest antimicrobial activity (Zengin & Baysal, 2014). The GC-MS analysis results of O. tenuiflorum L essential oils in the present study showed the presence of both compounds; linalool (0.21%) and eugenol (58.20%). Thus, essential oils from O. tenuiflorum L (Tulsi) were found to be the most active in terms of showing diameter (mm) zone of inhibition to all tested bacteria above 25% (v/v) concentrations in the present study. It was also noted in the present study that

103 the diameter of zones of inhibition (mm) was dose dependent which was in agreement with the study by Janssen et al. (1989).

Cananga odorata essential oils showed better anti-bacterial activities against Gram- negative bacteria than Gram-positive bacteria in the present study. However, available literature has shown that C. odorata essential oils are more active in Gram-positive bacteria than Gram-negative bacteria. For instance, C. odorata var. genuine essential oils showed very weak anti-bacterial activity against Escherichia coli (Gram-negative) of mean halo diameter= 8.7±0.3 mm (Thompson et al., 2013). Similarly, the ethanol extraction of oils from the bark of C. odorata showed a broad range of inhibitory effects to almost all the tested concentrations (25, 50, 100, 200 and 400 (µl)) against Propionibacterium acnes (Gram-positive bacteria) ranging from 13-19 mm diameter zone of inhibitions. The difference in the inhibitory activities on different bacteria could possibly be due to the variation in the composition of essential oils in both the present study and the available literature. More importantly, the anti-bacterial effects of essential oils in the present study may have influenced the Gram-negative bacteria more rapidly than the Gram-positive bacteria with strong anti-bacterial compounds such as ester and linalool (Tadtong et al., 2012). The same researcher also expressed that preparation of synergistic antimicrobial effect of accumulative component in the essential oils may have contributed differently on the inhibitory effect to both Gram- positive and Gram-negative bacteria which certainly needs further investigation.

The GC-MS analysis of C. odorata essential oils in the present study revealed the presence of major chemical groups; 54% alcohol, 38% ester and 4% sesquiterpenes. There may be possibilities of chemical compounds such as linalool (16.65%), eugenol (1.38%) and terpinene-4-ol (0.15%) from present study for the effect on the broad spectrum of diameter (mm) zones of inhibition on both Gram-positive and Gram- negative bacteria (Tadtong et al., 2012).

The anti-bacterial activities of C. citratus essential oils showed that Gram-positive bacteria were more susceptible to different concentrations of essential oil when 104 compared to Gram-negative bacteria which was in agreement with the study reported by Onawunmi and Ogunlana (1986). Similarly, C. citratus essential oils from Lucknow (India) were found to resistance to bacterium Pseudomonas aeruginosa (Gram- negative) at all the tested concentrations (5%, 10%, 15%, 20%, 25% and 30% (v/v)) (Naik et al., 2010). While in the present study, Pseudomonas aeruginosa and Thermus thermophilus had shown inhibitory activities from 5% (v/v) both of these bacteria were Gram-negative. The variation in the anti-bacterial activities of C. citratus could be due to the presence of different chemical compounds in the essential oils.

Cymbopogon citratus essential oil analysis in the present study reported that alcohol (47%) and aldehyde (48%) were the major chemical groups. The compound geraniol was found to be effective against Escherichia coli, and Listeria species (Tyagi et al., 2014). The GC-MS analysis in the present study revealed the presence of geraniol (13.57%), linalool (0.27%) and geranial (0.74%). Hence, the variations of anti-bacterial activities are mostly dependent on the presence or absence of strong bacteria inhibitory compounds in essential oils.

Likewise, the GC-MS analysis in the present study for E. hortensis forma hortensis revealed the major presence of monoterpene (62%), ketone (27%) and sesquiterpenes (9%) chemical compounds. The overall effects of essential oils from E. hortensis forma hortensis in the present study were slightly higher than M. koenigii (L) Spreng. The anti- bacterial activities shown by E. hortensis forma hortensis in the present study could be attributed to the presence of linalool compound, a strong anti-bacterial agent (Friedman et al., 2004). The present study showed E. hortensis forma hortensis had (0.10%), while M. koenigii (L) Spreng essential oil had no linalool compound present. Likewise, the essential oils from similar plant species (Evodia lunu-ankenda (Gaertn) Merr) showed anti-bacterial activities to all the tested bacteria especially against Gram-negative bacteria, Salmonella typhi (25 mm) and Klebsiella pneumoniae (10 mm). In contrast, the effect of E. hortensis forma hortensis in the present study revealed no anti-bacterial activities on Salmonella even at the highest concentration (100% (v/v)). This variability

105 in the different anti-bacterial activities in Salmonella and other tested bacteria could have resulted from difference in the essential oil composition of similar plant species.

The anti-bacterial activities of M.koenigii (L) Spreng essential oils in the present study were the least active in the broad range of the zones of inhibition. The anti-bacterial activities of M. koenigii (L) Spreng essential oils on selected bacteria were mostly seen at higher concentration (50% and 100% (v/v)). A similar trend on the anti-bacterial activities of increasing concentration of M. koenigii essential oils was reported by Bisht and Negi (2014). In contrast, the essential oils of M. koenigii (L) in another literature showed that the anti-bacterial effects against B. subtilis, S.aureus, C. pyogenes, P. vulgaris and P. multocida even at a dilution of 1:50033 (Saini & Reddy, 2015). One of the possibilities of M. koenigii (L) showing the least anti-bacterial activities in the present study could be attributed to absence of strong anti-bacterial compounds such as linalool, carvacrol (alcohol) and cinnamaldehyde (Friedman et al., 2004).

The essential oils of M. koenigii (L) Spreng in the present study comprised of three major chemical group; Monoterpenes (69%), Alcohols (10%) and sesquiterpenes (21%). The presence of alcohol compounds in the present study such as; terpinene-4-ol (7.20%) and eugenol (0.33%) were found to have good anti-bacterial properties in the available literature and as a result it may have contributed towards slight anti-bacterial effects that were mostly seen at 50% and 100% (v/v) concentrations in the present study (Friedman et al. (2004); Devi et al., 2010; Vats et al., 2011).

Overall, the anti-bacterial activities of selected essential oils in the present study can be ranked as; O. tenuiflorum L > C. odorata > C. citratus > E. hortensis forma hortensis > M. koenigii. The ranking of essential oils were based on the inhibitory activities on all the tested bacteria.

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4.4.1.1 Mode of Action on Bacteria Cell

The anti-bacterial activities showed a wide range of diameter (mm) zone of inhibition at different concentrations (mm) of essential oils in the present study. The anti-bacterial mode of action of essential oils has a relationship with different constituents of essential oils that may result in different modes of action (Burt, 2004). The mode of action of essential oils on bacteria is not entirely understood as most are based on assumptions (Li et al., 2014a). The effectiveness of anti-bacterial activity is dependent on the different essential oils and different bacterial strains.

Generally, the anti-bacterial action of essential oils mostly occurred in three stages (Carson et al., 2002; Turina et al., 2006; Li et al., 2014a). Firstly, the spread of essential oil on the cell wall of the bacteria intensified the cell membrane permeability that led to loss of cell components subsequently. The second step involved acidification inside the cell which created a blockage in the production of cellular energy (ATP). The blockage in the production of cellular energy was mainly due to loss of ions, reduction and collapse of membrane potential and proton pumps (see Figure 4-9). The last step involved the destruction of genetic materials that led to death of bacteria. Hence, the exposure of essential oils on bacterial cells have caused leakage of cell membrane permeability as this led to depletion of Adenosine Triphosphate pool, loss of ions and proton pumps (Di Pasqua et al., 2006; Turina et al., 2006; Turgis et al., 2009; Saad et al., 2013). The robust effect of O. tenuiflorum L essential oils in the present study could be due to the possibility of eugenol compound which was present in large amounts (58.20%) when compared to the essential oil composition of other selected plant materials. In relation to the available literature, when eugenol (4-allyl-2-methoxyphenol) was exposed to the Salmonella ser. Typhimurium at 1% and 5% (v/v) it resulted into increased cell permeability followed by leakage of cell content (Devi et al., 2010).

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Figure 4-9: Mode of action of essential oils on bacterial cell.

Adapted from: Li et al. (2014a)

Some bacteria appear to be more active with respect to Gram-reaction; the Gram- positive bacteria are more susceptible to antimicrobial activities as compared to Gram- negative bacteria (Trombetta et al., 2005; Lodhia et al., 2009). In the present study, Salmonella (Gram-negative bacteria) was very resistance to all the selected essential oils except in O. tenuiflorum L. Gram-negative bacteria have the presence of lipopolysaccharides (about 90%–95% of peptidoglycan) in their outer membrane. As a result, it has the ability to tolerate components of essential oils that are causing the antimicrobial activities (Nikaido, 2003; Nazzaro et al., 2013). While Gram-positive bacteria have cell walls that easily allow hydrophobic molecules to easily pass through the cells. Despite the fact that, the peptidoglycan layers of Gram-positive bacteria are thicker when compared to Gram-negative bacteria (~2-3 nm thick) as illustrated in Figure 4-10.

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Figure 4-10: Envelops of Gram-positive (right side) and Gram-negative (left side) bacteria. Adapted from: Nazzaro et al. (2013)

The presence of the outer membrane is also a distinguishing feature of the Gram- negative from Gram-positive bacteria (Silhavy et al., 2010). The outer membrane of the Gram-negative bacteria is made up of double layers of phospholipids that are connected to the inner membrane. The outer membrane covering the peptidoglycan layer contains lipids A (polysaccharide) and O-side chain, which makes the bacteria more resistant to the antimicrobial activities of the essential oils and other natural extracts (Weston, 2008).

In contrast, the present study revealed that the anti-bacterial activities were present at the lowest concentration of essential oils. The inhibitory effect of lowest concentrations (0.25% and 0.5% (v/v)) of C. odorata essential oils were on Thermus thermophiles and Pseudomonas aeruginosa, both being Gram-negative bacteria. Although, Gram- negative bacteria being very resistance to the susceptibility of the essential oils effect, the hydrophobic components of essential oils are able to affect the Gram-negative bacteria by gaining the access through the periplasm of the porin protein in the outer membrane which eventually allows essential oils to travel inside the cells of the bacterium (Plésiat & Nikaido, 1992; Helander et al., 1998; O'bryan et al., 2015). According to Deans and Ritchie (1987) and Deans et al. (1995), the inhibitory effect of 109 essential oils is very little dependent on the whether the bacteria is Gram-positive or Gram-negative. This could be supported with the work of Oussalah et al. (2007), where the researcher reported that L. monocytogenes (Gram-positive bacteria) was more resistant than other tested bacteria.

Hence, the present study revealed that the anti-bacterial activities of different concentrations of essential oils were seen on both Gram-positive and Gram-negative bacteria. The mode of action on anti-bacterial activities is not totally dependent on Gram-reactions. It was true for the bacterium Salmonella in the present study, the effect was only noted from O. tenuiflorum L essential oils from 25% (v/v) concentrations while being resistance to other essential oils at different concentrations. However, at the same instance the anti-bacterial activities of Gram-negative bacteria were also seen at the least concentrations (0.25% and 0.5% (v/v)) of C. odorata essential oils. Overall, the anti-bacterial activities of essential oils are not only dependent on the Gram-reaction, other factors that may influence the inhibitory activities include temperature, pH, incubation period, some media and different nitrogen and carbon sources which certainly needs further investigation (Noaman et al., 2004).

4.4.2 Anti-fungal Effects of each Essential oil and its Chemical Perspective

The broad spectrum of anti-fungal activities can also be attributed to different chemical compounds present in essential oils. Based on GC-MS results in the present study, the anti-fungal activities of O. tenuiflorum L essential oils could possibly be due to compounds such as linalool (0.21%) and α-cardinol (0.87%) which possessed strong anti-fungal properties (Chang et al., 2008). The massive presence of eugenol (58.20%) in the present study could have also contributed towards the susceptibility of the selected fungi. Penicillin was the only fungus that was susceptible to the lowest concentration (5%) as this could possibly be due to eugenol compound (Campaniello et al., 2010). A similar study on different species of essential oils from Ocimum L (Lamiaceae) from Uttarakhand, India revealed a broad range of inhibition zone (mm) to different fungi (Sethi et al., 2013). The essential oils from Ocimum basilicum (Sri Tulsi)

110 and Ocimum gratissimum L (clove Basil) exhibited strong inhibitory activities with a minimum inhibitory concentration (MIC) of 62.5 µg/mL. Similarly, the anti-fungal activity of Ocimum basilicum L (Lemon basil) showed inhibitory activities at 31.25 µg/mL concentration. The same researcher also concluded that the anti-fungal activities of essential oils from Ocimum species varied due to the presence or absence of strong anti-fungal agents in the composition of essential oils. More importantly, the Ocimum species in general has a strong potential to act as a good anti-fungal agent which was indeed with the agreement to the present study and the available literature (Pandey & Kumar, 2013; Sethi et al., 2013).

The anti-fungal effect of C. odorata essential oils in the present study was mostly seen at higher concentrations (above 25% (v/v)). According to the literature, C. odorata essential oil has shown a very weak anti-fungal activity which may be due to the presence or absence of different chemical components responsible for inhibitory effects in the essential oils. For example, C. odorata essential oils from Korea (Seoul) showed no inhibitory activities on Malassezia furfur (fungus) even at 2 mg/mL (Lee & Lee, 2010). Similarly, C. odorata essential oils (20 µl) from Tokyo, Japan showed no inhibitory activities against Candida albicans (fungal infection) (Kuspradini et al., 2016). However, the anti-fungal activities in the present study could possibly be attributed to the presence of strong anti-fungal compounds in C. odorata essential oils such as linalool (16.65%), eugenol (1.38%) and α-pinene (0.32%) which may have varied with the available literature (Tan et al., 2015). The presence of such compounds in the current study may have been the cause for inhibitory activities in selected fungi from 5% (v/v) concentrations.

Likewise, C. citratus essential oils hold great potential when it comes to anti-fungal properties. For instance, Cymbopogon khasans and Cymbopogon martini (Lemon grass families) showed potential preservative effects of 93.86% and 88.60% on herbal raw materials in relation to fungal contamination (Mishra et al., 2015). Similarly, C. citratus (DC) Stapf (Gramineae) essential oils showed a broad-spectrum activity against Candida species where the different concentrations (2.0, 4.0 and 8.0 (µl)) showed 111 increasing diameter zone of inhibition (mm) to all the tested species of Candida (Silva et al., 2008). The above study was in agreement to the present study whereby a direct relationship was seen between the different concentrations of C. citratus essential oils and the diameter (mm) zone of inhibition. The essential oils from C. citratus in the present study showed a broad spectrum of diameter (mm) zone of inhibition to all the tested fungi. There may be possibilities of inhibitory compounds in the present study such as linalool (0.21%), citronellal (45.09%) and citronellol (19.11%) that may have contributed towards the strong anti-fungal activities as similarly reported by other researchers (Pauli and Knobloch (1987); Lee et al., 2008; Olorunnisola et al., 2014).

The effect of essential oils from E. hortensis forma hortensis leaves also presented an inhibitory spectrum whereby the zones of inhibition were shown on all the tested fungi mostly at 100% (v/v) concentration in the present study. A similar study showed that Satureja hortensis (Lamiaceae family) essential oils showed inhibitory effect on fungal growth and spore production at highest concentration of 400 ppm, while lower concentrations from 400 ppm reduced the speed of fungal growth (Yazdanpanah & Mohamadi, 2014). Likewise, the effect of hexane extract of Euodia hortensis on Candida albicans (fungal infection) had the increasing diameter zone of inhibition (3%, 6%, and 7%) with the increasing concentrations (125, 250, 500 (µg/mL)) of essential oils (Huish et al., 2014). The gradual increase of strong anti-fungal compounds in the E. hortensis forma hortensis essential oils with respect to the increasing concentrations may have influenced the increasing inhibitory activities that were seen in the literature as well as in the present study. The major chemical groups in the essential oils of E. hortensis forma hortensis in the present study were 54% alcohol and 38% of ester compounds that could have possibly contributed towards the anti-fungal effects in the present study.

The essential oils from M. koenigii (L) Spreng showed relatively small zones of inhibition even at 100% (v/v). In relation to the available literature, a similar study showed that a distilled water extract of essential oils from M. koenigii (L) Spreng showed no inhibitory to Aspergillus niger, Penicillium notatum, Alternaria solani and 112

Helminthosporium solani (Kumar et al., 2010). The same researcher concluded that the essential oils from M. koenigii (L) Spreng had a poor anti-fungal property which was in agreement to the present study. Likewise, the leaf extracts of M. koenigii showed ineffectiveness in inhibiting the growth of S.mutans and C. albicans (Bhuva & Dixit, 2015). There could be many possibilities of this, such as negligible presence or absence of strong anti-fungal compounds that include linalool, eugenol and other phenolic compounds as reported in different plant extracts (Campaniello et al., 2010; Tan et al., 2015).

4.4.2.1 Mode of Action on Fungi Cells

The anti-fungal mode of action of the essential oils could possibly affect the cells in many different ways. Firstly, exposure of the essential oils to the fungus cell could influence the enzymatic activities, which would result in protein denaturation. This statement was clearly supported by Fung et al. (1977), where phenolic compounds have the ability to alter the cell permeability causing leakage of macromolecules and can interact with cell membrane proteins to cause disruptions. The present study revealed that O. tenuiflorum L essential oils had the highest eugenol composition (58.20%) present. This could be one of the possibilities that gave the highest zone of inhibition (mm) to all the selected fungi with changing concentrations of O. tenuiflorum L essential oils.

Likewise, the effect of essential oils on fungal cell can also depolarize the mitochondrial membrane by reducing the membrane potential through affecting the ion channels, proton pump and Adenosine Triphosphate pool (Akthar et al., 2014). For instance, the Cupressus arizonica (coniferous ever trees) leaves affected the Saccharomyces cerevisiae (wild type yeast) in their oxidative stress response and Deoxyribonucleic acid repair pathways (Khouaja et al., 2015). The essential oils from Anethum graveolens L (dill seed) disrupted the permeability in the plasma membrane and the mitochondrial dysfunction (Tian et al., 2012). The mitochondrial dysfunction decreased the Adenosine

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Triphosphate synthesis. This led to cell death through oxidative damage of bio- macromolecule (Wu et al., 2009; Chen et al., 2013).

The present study showed that the selected essential oils had a wide range of inhibitory activities on selected fungi mostly above 25% (v/v) concentrations. The mechanism of essential oils affecting the fungi cells needs detailed investigation, as general assumptions and overview are only available through literature. Generally, the essential oils affect the fungi cells through disruption in the plasma membrane, causing disturbance in the ions and molecules, alteration in the enzymatic and cell organelle activities.

4.5 Conclusion

Plant essential oils have great potentials and hopes, especially when it comes to microbiological studies. As a result, their composition and antimicrobial activities have been studied thoroughly. The screening of selected essential oils had potential sources of antimicrobial properties.

The tested essential oils were active against the Gram (+) and Gram (-) bacteria in the following rank of anti-bacterial activities; O. tenuiflorum L > C. odorata > C. citratus > E. hortensis forma hortensis > M. koenigii. The Gram-negative and Gram-positive bacteria were both found to be susceptible to the increasing concentration of essential oils. Based on some literature, Gram-negative bacteria were found to be less susceptible to the different levels of essential oils due to the presence of strong negative charged lipopolysaccharide (Trombetta et al., 2005). Likewise, the Gram-positive bacteria are more susceptible to drug or exposed chemicals due to the lack of outer membrane even though they have a thicker peptidoglycan layer then Gram-negative bacteria (Silhavy et al., 2010). However, the present study agreed to the above statement to some extent, as it was true for some bacteria (especially Salmonella) which were very resistant to the effect of increasing concentrations of essential oils. Surprisingly, the Gram-negative bacteria also showed susceptibility at lowest concentration (0.25% and 0.5% (v/v)) of

114 essential oils from C. odorata. This activity was clearly supported by other researchers who all highlighted that Gram-reaction is not only the factor that contribute to the susceptibility of bacteria towards the effect of essential oils (Deans & Ritchie, 1987; Deans et al., 1995; Oussalah et al., 2007). The other factors responsible for susceptibility of bacteria could possibly be temperature, pH, incubation period, varied media and different nitrogen and carbon sources which require further investigation (Noaman et al., 2004).

The overall trend of susceptibility for anti-fungal activities were; O. tenuiflorum L > C. odorata > C. citratus > E. hortensis forma hortensis > M. koenigii. One of the possibilities attributed towards the trend was due to the presence of different phenolic compounds in selected essential oils (Alves et al., 2014). The active anti-bacterial and anti-fungal compounds of essential oils are generally terpenes, which are phenolic in nature including; eugenol, α-terpinyl acetate, cymene, thymol, pinene and linalool. These compounds attack the pathogens through the cell wall and cell membrane (Nuzhat & Vidyasagar, 2014).

Generally, a direct relationship was seen in the present study between the increasing concentrations and the diameter (mm) zones of inhibition in bacteria and fungi (Zambonelli et al., 1996; Chen et al., 2001). The anti-bacterial and anti-fungal activities of essential oils from selected medicinal plants in the present study are first given information. The anti-bacterial and anti-fungal activities reported by other researchers on similar plant species showed similar results as the concentrations increased the diameter (mm) zones of inhibition also increased for different microorganisms. However, based on the comparison of diameter (mm) zones of inhibition between the available literature and the present study showed variations that could be due to many factors such as pH, temperature, incubation period and use of different media for culturing (Noaman et al., 2004). The most important factor identified in the present study for the variations in the diameter (mm) zones of inhibition between the present study and the available literature was possibly the variability of chemical composition of selected essential oils. 115

The findings suggested that the encountered beneficial effects of selected essential oils are due to different types of chemical compounds present in the essential oils. The data obtained from the present investigation indicated that the selected essential oils from medicinal plants found in Fiji showed effectiveness in inhibiting the growth of selected bacteria and fungi. Hence, selected essential oils (especially O. tenuiflorum L) represent a good alternative to eliminate microorganisms that can be harmful to human health, food and agricultural industries.

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5. CHAPTER 5: CONCLUSION AND RECOMMENDATION In this research work, essential oils from the five selected medicinal plants found in Fiji were analysed and screened for antimicrobial and whitefly control activities. The studied plants were C. odorata (Makosoi), C. citratus (Lemon grass), M. koenigii (L) Spreng (Curry Leaves), O. tenuiflorum L (Tulsi) and E. hortensis forma hortensis (Uci). The essential oils were extracted using the Hydro-distillation techniques and the volatile compositions were investigated using Gas-chromatography with Mass spectrometry (GC-MS). The chemical profile analysis showed slight variations in the detection of compounds as other compounds were also detected. The importance of GC-MS analysis was to provide a slight justification, as to which chemical groups might have contributed to biological activities tested.

The effect of essential oils was measured on the Spiralling whiteflies (Aleurodicus dispersus Russell) using; fumigant and repellent test. The fumigant test results were recorded at a time interval of 3, 6, 9, 12 and 24 hours. It was found that the most active essential oils at 5% (v/v) solution was O. tenuiflorum L (100% mortality at first recording; 3 hours), followed by C. citratus (100% mortality at second recording; 6 hours), while C. odorata, M. koenigii (L) Spreng and E. hortensis forma hortensis showed increased mortality with the time intervals but 100% mortality were not achieved even at 24 hours. Statistically, the essential oils from C. citratus and O. tenuiflorum L were the only ones that showed a strong significant difference with overall tested time intervals. The p-value was < 0.05 at the 5% level of significance.

The repellent test was carried out using a designed olfactometer. The Repellency Index (RI %) were calculated and it was found that C. citratus (RI= 52%) and M. koenigii (L) Spreng (RI= 52%) showed the best result as compared to O. tenuiflorum L (RI= 12%), C. odorata (RI= 9%) and E. hortensis forma hortensis (RI= 10%) at 5% (v/v) concentrations.

The essential oils showed varied antimicrobial activities at different concentrations. Ocimum tenuiflorum L essential oils showed the best result with all the tested bacteria 117 and fungi. For anti-bacterial activities, the trends were; O. tenuiflorum L > C. odorata > C. citratus > M. koenigii (L) Spreng > E. hortensis forma hortensis. Likewise, the trend for the anti-fungal activities of essential oils were; O. tenuiflorum L > C. odorata > C. citratus > E. hortensis forma hortensis > M. koenigii (L) Spreng. The inhibitory effect on bacteria and fungi increased with the increasing concentrations of essential oils.

The biological activities of selected essential oils have shown a potential source of phytochemicals that can be used to substitute synthetic chemicals in the agriculture, medical and food industries. Hence, the diverse use of essential oils could be both ecologically and economically beneficial.

Future Research Needs: The results available to evaluate the pesticide and antimicrobial activities of selected essential oils from medicinal plants found in Fiji were generally inadequate and there is a bounteous scope (as described below) to generate data in this form;

5 The main chemical compounds of essential oils can be separately tested for antimicrobial or pest controls and then precisely concluding as which compounds might have caused the effect with reference to phenolic and alcoholic compounds, monoterpenes or other present compounds.

5 Additional research into the mode of action of essential oils and other insecticides on whiteflies needs to be studied thoroughly as there seems very little or no data clearly explaining the mode of action on whiteflies and other arthropods. Likewise, the mode of action in bacteria and fungi needs further investigation.

5 The research only involved five medicinal plants found in Fiji. There are many medicinal plants that could substitute many synthetic chemicals in the agriculture, food and health industries. Thus, further studies in the field of biochemistry are strongly recommended for potential plants.

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6. APPENDIX 6.0 Chemical Analysis

Table 6-1: Chemical Analysis-group of major chemical compounds from selected essential oils.

Chemical Major chemical constituents O. tenuiflorum L C. citratus C. odorata E. hortensis forma hortensis M. koenigii Monoterpenes Subtotal 13.64 1.32 60.18 65.51 Cis-β- ocimene 10.79 limonene 4.64 menthofuran 55.17 α- pinene 5.67 sabinene 43.80 α - terpinene 2.64 ϒ- terpinene 4.82 Sesquiterpenes Subtotal 22.61 3.44 4.13 9.17 20.27 α- copaene 1.98 β- caryophyllene 4.31 16.52 germacrene D 11.68 Alcohol and Subtotal 60.61 45.88 50.85 0.83 9.08 Phenol Eugenol 58.20 citronellol 19.11 geraniol 13.57 elemol 6.15 α-cardinol 3.70 linalool 16.65 trans, trans-farnesol 29.71 terpinene- 4-ol 7.20 Ester Subtotal 1.7 35.76 0.6 methyl salicylate 3.15 benzyl salicylate 2.21 benzyl benzoate 21.69 trans, trans-farnesyl acetate Aldehyde Subtotal 46.51 0.43 0.2 Citronellal 45.09 Ketone Subtotal 25.97 evodone 25.97

Note: Chemical constituents that are present in large amount are only shown (in percentage (%)). The subtotals are for all compounds (including negligible compounds).

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6.1 Whiteflies

Table 6-2: Common Pest Species of Whiteflies with Distinct Nymphs Spiralling whiteflies Host plants: (Aleurodicus dispersus) Capsicum, citrus, pawpaw, pepper and cassava.

Characteristics: The distinct feature of having a glass-like waxy rod on nymphs’ lateral surface and the adult having black spots on the forewings.

Ash whitefly Host plants: (Siphoninus phillyreae) Broadleaved trees and shrubs that include citrus, pomegranate and other flowering fruit trees.

Characteristics: Fourth- instar nymphs have fringe of tiny tubes fill with band of wax. The adults are white.

Bandedwinged whitefly Host plants: (Trialeurodes abutilonea) Cottons, cucurbits and other vegetables.

Characteristics: The pupa case has the dark area around the back and the adults have the gray bands across the wings.

Citrus whitefly Host plants: (Aleuroplatus coronata) Oaks and chestnut

Characteristics: The nymphs are black and arranged in crown like pattern covered with white wax. The adults are white.

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Greenhouse whitefly Host plants: (Trialeurodes vaporariorum) Most vegetables and herbaceous ornamentals.

Characteristics: Nymphs have filaments and marginal fringe. The adults have wings with a yellowish surface.

Silverleaf and sweetpotato whiteflies (Bemisia Host plants: argentifolii and B. tabaci) Herbaceous and some woody plants such as cottons, tomatoes, cole crops, hibiscus and pepper.

Characteristics: The nymphs have no waxy filaments or marginal fringe. The adults hold wings slightly tilted to surface.

Iris whitefly Host plants: (Aleyrodes spiraeoides) Vegetables, cotton and other herbaceous plants.

Characteristics: The nymphs have no fridge or waxy filaments and are placed near the distinctive circle of wax. A distinctive feature of adults is that they have dot on each wing and are quite waxy.

(Bellows et al., 2001; Ingram & Recsei, 2014)

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6.1.1 Results of Fumigant test on whiteflies:

Figure 6-1: General effect of different concentrations (with respect to time factor) on the mean mortality of whiteflies.

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Table 6-3: Multiple Comparisons (Post Hoc Test) for C. odorata.

Plant Time Concentration Comparison P-value Overall Comment 3 hours Control 0.25% .903 F (3,12) = 3.05, p= .07 (not 0.5% .547 significant) 5% .058 0.25% 0.5% .903 5% .176 0.5% 5% .451

6 hours Control 0.25% .990 F (3,12) = 3.15, p= .053 (not 0.5% .739 significant) 5% .055 0.25% 0.5% .886 5% .092 0.5% 5% .284

9 hours Control 0.25% .880 F (3,12) = 9.02, p= .002 0.5% .988 (significant) 5% .008 0.25% 0.5% .719 5% .002 C. odorata 0.5% 5% 0.14

12 hours Control 0.25% .056 F (3,12) = 20.14, p= .00 0.5% .913 (significant) 5% .003 0.25% 0.5% .166 5% .000 0.5% 5% .001

24 hours Control 0.25% .666 F (3,12) = 13.38, p= .00 0.5% .998 (significant) 5% .003 0.25% 0.5% .764 5% .000 0.5% 5% .002

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Table 6-4: Multiple Comparisons (Post Hoc Test) for M. koenigii (L)

Plant Time Concentration Comparison P-value Overall Comment 3 hours Control 0.25% 1.00 That is, F (3,12) = 4.24, p= .029 0.5% .804 (significant) 5% .041 0.25% 0.5% .804 5% .041 0.5% 5% .183

6 hours Control 0.25% .908 F (3,12) = 2.047, p= .161 (not 0.5% .942 significant) 5% .355 0.25% 0.5% .623 5% .132 0.5% 5% .660

9 hours Control 0.25% .779 F (3,12) = 7.57, p= .004 0.5% 1.000 (significant) 5% .021 0.25% 0.5% .779 Murraya 5% .004 koenigii (L) 0.5% 5% .021

12 hours Control 0.25% .008 F (3,12) = 10.05, p= .001 0.5% 1.000 (significant) 5% .731 0.25% 0.5% .009 5% .001 0.5% 5% .686

24 hours Control 0.25% .712 F (3,12) = 1.64, p= .232 ( not 0.5% .668 significant) 5% .881 0.25% 0.5% 1.000 5% .316 0.5% 5% .285

124

Table 6-5: Multiple Comparisons (Post Hoc Test) for E. hortensis forma hortensis.

Plant Time Concentration Comparison P-value Overall Comment 3 hours Control 0.25% 1.00 F (3,12) = 4.24, p= .029 (not 0.5% .804 significant) 5% .041 0.25% 0.5% .804 5% .041 0.5% 5% .183

6 hours Control 0.25% .483 F (3,12) = 6.57, p= .007 0.5% .989 (significant) 5% .057 0.25% 0.5% .329 5% .004 0.5% 5% .097

9 hours Control 0.25% .314 F (3,12) = 5.80, p= .011 0.5% .863 (significant) 5% .158 0.25% 0.5% .095 Euodia 5% .007 hortensis forma 0.5% 5% .466 hortensis 12 hours Control 0.25% .067 F (3,12) = 5.07, p= .017 0.5% .999 (significant) 5% .809 0.25% 0.5% .082 5% .014 0.5% 5% .745

24 hours Control 0.25% .085 F (3,12) = 4.64, p= .022 0.5% .708 (significant) 5% .815 0.25% 0.5% .427 5% .019 0.5% 5% .256

125

Table 6-6: Multiple Comparisons (Post Hoc Test) for C. citratus.

Plant Time Concentration Comparison P-value Overall Comment 3 hours Control 0.25% 1.00 F (3,12) = 13.78, p= .00 0.5% .216 (significant) 5% .001 0.25% 0.5% .216 5% .001 0.5% 5% .020

6 hours Control 0.25% .001 F (3,12) = 1067.33, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .000 5% .000 0.5% 5% .000

9 hours Control 0.25% .002 F (3,12) = 1603.48, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .000 5% .000 Cymbopogon 0.5% 5% .000 citratus 12 hours Control 0.25% .001 F (3,12) = 428.35, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .110 5% .000 0.5% 5% .000

24 hours Control 0.25% .124 F (3,12) = 200.92, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .000 5% .000 0.5% 5% .000

126

Table 6-7: Multiple Comparisons (Post Hoc Test) for O. tenuiflorum L

Plant Time Concentration Comparison P-value Overall Comment 3 hours Control 0.25% .112 F (3,12) = 293.94, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .032 5% .000 0.5% 5% .000

6 hours Control 0.25% .001 F (3,12) = 611.40, p= .00 ( 0.5% .000 significant) 5% .000 0.25% 0.5% .003 5% .000 0.5% 5% .000

9 hours Control 0.25% .005 F (3,12) = 772.03, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .000 Ocimum 5% .000 tenuiflorum L 0.5% 5% .000

12 hours Control 0.25% .341 F (3,12) = 96.34, p= .00 0.5% .001 (significant) 5% .000 0.25% 0.5% .019 5% .000 0.5% 5% .000

24 hours Control 0.25% .382 F (3,12) = 42.07, p= .00 0.5% .000 (significant) 5% .000 0.25% 0.5% .002 5% .000 0.5% 5% .018

127

6.1.1.1 Probit Analysis

Ocimum tenuiflorum L Cymbopogon citratus

Logistic regression of 24 hrs by Logistic regression of 24 hrs by Log(Concentration) Log(Concentration)

1 1

y = 0.4062x + 1.5701 0.8 0.8

R² = 0.6512 0.6 0.6 y = 0.4897x + 1.6577 24 hrs 0.4 24 hrs R² = 0.9022 0.4 0.2 0.2

0 0 -3 -2.5 -2 -1.5 -1 -0.5 0 -3 -2.5 -2 -1.5 -1 -0.5 0 Log(Concentration) Log(Concentration)

Active Active Model Model Natural mortality Lower bound (95%) Natural mortality Upper bound (95%) Linear (Active) Lower bound (95%) Upper bound (95%) Linear (Active)

Cananga odorata E. hortensis forma hortensis

Logistic regression of 24 hrs by Logistic regression of 24 hrs by Log(Concentration) Log(Concentration)

1 1 y = 0.2768x + 0.8558 0.8 0.8 R² = 0.7501 0.6 y = 0.0685x + 0.3266 0.6 0.4 24 hrs R² = 0.5862

0.2 24 hrs 0.4 0 0.2 -3 -2.5 -2 -1.5 -1 -0.5 0 0 Log(Concentration) -3 -2.5 -2 -1.5 -1 -0.5 0 Log(Concentration) Active Model Active Natural mortality Model Lower bound (95%) Natural mortality Upper bound (95%) Lower bound (95%) Linear (Active) Upper bound (95%) Linear (Active) Linear (Active)

128

Murraya koenigii

Logistic regression of 24 hrs by Log(Concentration)

1 0.9 0.8 0.7 y = 0.0496x + 0.2976 0.6 R² = 0.316 0.5

24 hrs 0.4 0.3 0.2 0.1 0 -3 -2.5 -2 -1.5 -1 -0.5 0 Log(Concentration)

Active Model Natural mortality Lower bound (95%) Upper bound (95%) Linear (Active)

Figure 6-2: Probit analysis of fumigant test on selected essential oils at different time interval.

129

6.1.2 Repellent Test

Table 6-8: Independent Sample t-test for repellent test

Plants Concentration Significant/ Not significant Comparisons Murraya 0.25% t(6)=-1.698, p= .278 (not significant) koenigii (L) Control 0.5% t(6)=.608, p= .919 (not significant) 5% t(6)= 5.286, p= .000 (significant)

Cymbopogon 0.25% t(6)= -.442, p= .102 (not significant) citratus Control 0.5% t(6)= .164, p= .436 (not significant) 5% t(6)= 2.662, p= .197 (not significant)

C. odorata 0.25% t(6)= -1.101, p= .418 (not significant) Control 0.5% t(6)= -2.400, p= .248 (not significant) 5% t(6)= .362, p= .541 (not significant)

Ocimum 0.25% t(6)= -.880, p= .949 (not significant) tenuiflorum L Control 0.5% t(6)= -3.001, p= .710 (not significant) 5% t(6)= 1.709, p= .693 (not significant)

Euodia 0.25% t(6)= -.618, p= .386 (not significant) hortensis forma Control 0.5% t(6)= -.179, p= .916 (not significant) hortensis 5% t(6)= .607, p= .808 (not significant)

6.1.2.1 Probit Analysis -Graphs

Cymbopogon citratus Cananga odorata

Logistic regression of Repelled by Logistic regression of Repelled by Log(Concentration(%)) Log(Concentration(%)) y = 0.2259x + 0.602 y = 0.0998x + 0.4364 1 1 R² = 0.3232 R² = 0.0795 0.5 0.5 0 0 -0.8 Repelled -0.3 0.2 0.7 -0.8 Repelled -0.3 0.2 0.7 Log(Concentration(%)) Log(Concentration(%))

Active Active Model Model Natural mortality Natural mortality Lower bound (95%) Lower bound (95%) Upper bound (95%) Upper bound (95%) Linear (Active) Linear (Active)

130

Ocimum tenuiflorum L E. hortensis forma hortensis

Logistic regression of Repelled by Logistic regression of Repelled by Log(Concentration(%)) Log(Concentration(%))

1 1 y = 0.0541x + 0.5023 R² = 0.028 0.8 y = 0.1484x + 0.4342 0.8 R² = 0.1582 0.6 0.6

0.4 0.4 Repelled Repelled Repelled 0.2 0.2

0 0 -0.8 -0.3 0.2 0.7 -0.8 -0.3 0.2 0.7 Log(Concentration(%)) Log(Concentration(%))

Active Active Model Model Natural mortality Natural mortality Lower bound (95%) Lower bound (95%) Upper bound (95%) Upper bound (95%) Linear (Active) Linear (Active)

Murraya koenigii

Logistic regression of Repelled by Log(Concentration(%))

1 0.9 0.8 0.7 0.6 0.5 y = 0.2637x + 0.5984

Repelled Repelled 0.4 R² = 0.6111 0.3 0.2 0.1 0 -0.8 -0.3 0.2 0.7 Log(Concentration(%))

Active Model Natural mortality Lower bound (95%) Upper bound (95%) Linear (Active)

Figure 6-3: Probit analysis of repellent test on selected essential oils.

131

6.2 Microbiology

6.2.1 Bacteria

Standard Control - Ampicillin discs Table 6-9: Effect of control on selected bacteria

Bacteria Standard Control (mm) Thermus thermophilus 14.80

Pseudomonas aeruginosa 14.61

Streptococcus (pneumonia) 41.48

Staphylococcus aureus 41.53

Salmonella 24.26

Table 6-10: Descriptive statistics for zone of inhibition (mm) across different concentrations

Concentrations N M SD skew kurtosis (%) 0.25 125 0.23 1.28 5.42 27.92 0.5 125 0.29 1.42 4.78 21.27 5 125 0.94 2.58 2.43 4.05 25 125 3.90 4.61 0.56 1.15 50 125 5.74 5.17 0.19 -1.16 100 125 9.58 7.19 0.04 -0.79

6.2.1.1 ANOVA analysis using tukey’s test

132

Bacteria Concentration Plant P- Overall Bacteria Concentration Plant P- Overall (x100%) comparison value significant or (x100%) comparison value significant not or not Pseudomonas 0.0025 Curry Lemon 1.000 F(4,20)=6.000, Pseudomonas 0.005 Curry Lemon 1.000 F(4,20)= aeruginosa leaves grass p=.002 aeruginosa leaves grass 5.985 0.0025 Makasoi .007 0.005 Makasoi .008 p=.002 0.0025 Tulsi 1.000 0.005 Tulsi 1.000 0.0025 Uci 1.000 0.005 Uci 1.000 0.0025 Lemon Makasoi .007 0.005 Lemon Makasoi .008 0.0025 grass Tulsi 1.000 0.005 grass Tulsi 1.000 0.0025 Uci 1.000 0.005 Uci 1.000 0.0025 Makasoi Tulsi .007 0.005 Makasoi Tulsi .008 0.0025 Uci .007 0.005 Uci .008 0.0025 Tulsi Uci 1.000 0.005 Tulsi Uci 1.000

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- value Overall (x100%) significant (x100%) significant or not or not Pseudomonas 0.05 Curry Lemon .115 F(4,20)= Pseudomonas 0.25 Curry Lemon .000 F(4,20)= aeruginosa leaves grass 15.449, aeruginosa leaves grass 25.209,

0.05 Makasoi .000 p=.000 0.25 Makasoi .000 p=.000 0.05 Tulsi 1.000 0.25 Tulsi .010

0.05 Uci 1.000 0.25 Uci 1.000 0.25 Lemon Makasoi .783 0.05 Lemon Makasoi .009 0.05 grass Tulsi .115 0.25 grass Tulsi .007 0.05 Uci .115 0.25 Uci .000 0.05 Makasoi Tulsi .000 0.25 Makasoi Tulsi .084 0.05 Uci .000 0.25 Uci .000 0.05 Tulsi Uci 1.000 0.25 Tulsi Uci .010

133

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- Overall (x100%) significant (x100%) value significant or not or not Pseudomonas 0.5 Curry Lemon .000 F(4,20)= Pseudomonas 1 Curry Lemon .000 F(4,20)= aeruginosa leaves grass 13.501, aeruginosa leaves grass 15.946, 0.5 Makasoi .000 p=.000 1 Makasoi .000 p=.000 0.5 Tulsi .001 1 Tulsi .000 0.5 Uci .033 1 Uci .000 0.5 Lemon Makasoi .924 1 Lemon Makasoi .941 0.5 grass Tulsi .465 1 grass Tulsi .996 0.5 Uci .023 1 Uci .734 0.5 Makasoi Tulsi .903 1 Makasoi Tulsi .792 0.5 Uci .117 1 Uci .989 0.5 Tulsi Uci .446 1 Tulsi Uci .513

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- value Overall (x100%) significant (x100%) significant or not or not Salmonella 0.25 Curry Lemon 1.000 F(4,20)= Salmonella 0.5 Curry Lemon 1.000 F(4,20)= leaves grass 2.662, leaves grass 3791.773, 0.25 Makasoi 1.000 p=.063 0.5 Makasoi 1.000 p=.000 0.25 Tulsi .113 0.5 Tulsi .000 0.25 Uci 1.000 0.5 Uci 1.000 0.25 Lemon Makasoi 1.000 0.5 Lemon Makasoi 1.000 0.25 grass Tulsi .113 0.5 grass Tulsi .000 0.25 Uci 1.000 0.5 Uci 1.000 0.25 Makasoi Tulsi .113 0.5 Makasoi Tulsi .000 0.25 Uci 1.000 0.5 Uci 1.000 0.25 Tulsi Uci .113 0.5 Tulsi Uci .000

134

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- value Overall (x100%) significant (x100%) significant or not or not Salmonella 1 Curry Lemon 1.000 F(4,20)= Staphylococcus 0.05 Curry Lemon 1.000 F(4,20)= leaves grass 21449.619, aureus leaves grass 6.00, 1 Makasoi 1.000 p=.000 0.05 Makasoi .007 p=.002 1 Tulsi .000 0.05 Tulsi 1.000 1 Uci 1.000 0.05 Uci 1.000 1 Lemon Makasoi 1.000 0.05 Lemon Makasoi .007 grass 1 grass Tulsi .000 0.05 Tulsi 1.000 1 Uci 1.000 0.05 Uci 1.000 1 Makasoi Tulsi .000 0.05 Makasoi Tulsi .007 1 Uci 1.000 0.05 Uci .007 1 Tulsi Uci .000 0.05 Tulsi Uci 1.000

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- value Overall

(x100%) significant (x100%) significant or not or not Staphylococcus 0.25 Curry Lemon .000 F(4,20)= Staphylococcus 0.5 Curry Lemon .000 F(4,20)= aureus leaves grass 27.327, aureus leaves grass 181.032, 0.25 Makasoi .000 p=.000 0.5 Makasoi .000 p=.000 0.25 Tulsi .000 0.5 Tulsi .000 0.25 Uci 1.000 0.5 Uci 1.000 0.25 Lemon Makasoi .895 0.5 Lemon Makasoi .898 0.25 grass Tulsi .439 0.5 grass Tulsi .963 0.25 Uci .000 0.5 Uci .000 0.25 Makasoi Tulsi .917 0.5 Makasoi Tulsi .546 0.25 Uci .000 0.5 Uci .000 0.25 Tulsi Uci .000 0.5 Tulsi Uci .000

135

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- Overall (x100%) significant (x100%) value significant or not or not Staphylococcus 1 Curry Lemon .000 F(4,20)= Streptococcus 0.25 Curry Lemon .047 F(4,20)= aureus leaves grass 40.373, (pneumoniae) leaves grass 10.338, 1 Makasoi .000 p=.000 0.25 Makasoi .001 p=.000 1 Tulsi .000 0.25 Tulsi .006 1 Uci .000 0.25 Uci 1.000 1 Lemon Makasoi .408 0.25 Lemon Makasoi .348 1 grass Tulsi .026 0.25 grass Tulsi .887 1 Uci .108 0.25 Uci .047 1 Makasoi Tulsi .000 0.25 Makasoi Tulsi .859 1 Uci .923 0.25 Uci .001 1 Tulsi Uci .000 0.25 Tulsi Uci .006

Bacteria Concentration Plant P- value Overall Bacteria Concentration Plant comparison P- value Overall (x100%) comparison significant (x100%) significant or not or not Streptococcus 0.5 Curry Lemon .000 F(4,20)= Streptococcus 1 Curry Lemon .000 F(4,20)= (pneumoniae) leaves grass 506.152, (pneumoniae) leaves grass 1086.774, 0.5 Makasoi .000 p=.000 1 Makasoi .000 p=.000 0.5 Tulsi .000 1 Tulsi .000 0.5 Uci 1.000 1 Uci .000 0.5 Lemon Makasoi .017 1 Lemon Makasoi .000 0.5 grass Tulsi .143 1 grass Tulsi .002 0.5 Uci .000 1 Uci .000 0.5 Makasoi Tulsi .826 1 Makasoi Tulsi .000 0.5 Uci .000 1 Uci 1.000 0.5 Tulsi Uci .000 1 Tulsi Uci .000

136

Bacteria Concentration Plant comparison P- Overall Bacteria Concentration Plant comparison P- value Overall (x100%) value significant (x100%) significant or not or not Thermus 0.0025 Curry Lemon 1.000 F(4,20)= Thermus 0.005 Curry Lemon 1.000 F(4,20)= thermophiles leaves grass 1.000, thermophiles leaves grass 2.667,

0.0025 Makasoi .525 p=.431 0.005 Makasoi .112 p=.062 0.0025 Tulsi 1.000 0.005 Tulsi 1.000 0.0025 Uci 1.000 0.005 Uci 1.000 0.0025 Lemon Makasoi .525 0.005 Lemon Makasoi .112 0.0025 grass Tulsi 1.000 0.005 grass Tulsi 1.000 0.0025 Uci 1.000 0.005 Uci 1.000 0.0025 Makasoi Tulsi .525 0.005 Makasoi Tulsi .112

0.0025 Uci .525 0.005 Uci .112 0.0025 Tulsi Uci 1.000 0.005 Tulsi Uci 1.000

Bacteria Concentration Plant comparison P- value Overall Bacteria Concentration Plant comparison P- value Overall (x100%) significant (x100%) significant or not or not Thermus 0.05 Curry Lemon .065 F(4,20)= Thermus 0.25 Curry Lemon .000 F(4,20)= thermophiles leaves grass 3.487, thermophiles leaves grass 226.473, 0.05 Makasoi .434 p=.026 0.25 Makasoi .000 p=.000 0.05 Tulsi 1.000 0.25 Tulsi .000 0.05 Uci 1.000 0.25 Uci 1.000 0.05 Lemon Makasoi .794 0.25 Lemon Makasoi .212 0.05 grass Tulsi .065 0.25 grass Tulsi .093 0.05 Uci .065 0.25 Uci .000 0.05 Makasoi Tulsi .434 0.25 Makasoi Tulsi .990 0.05 Uci .434 0.25 Uci .000 0.05 Tulsi Uci 1.000 0.25 Tulsi Uci .000

137

Bacteria Concentration Plant P- Overall Bacteria Concentration Plant comparison P- value Overall (x100%) comparison value significant (x100%) significant or not or not Thermus 0.5 Curry Lemon .000 F(4,20)= Thermus 1 Curry Lemon .000 F(4,20)= thermophiles leaves grass 13.011, thermophiles leaves grass 14.263, 0.5 Makasoi .000 p=.000 1 Makasoi .000 p=.000 0.5 Tulsi .001 1 Tulsi .000 0.5 Uci .027 1 Uci .001 0.5 Lemon Makasoi .951 1 Lemon Makasoi .862 0.5 grass Tulsi .396 1 grass Tulsi .987 0.5 Uci .035 1 Uci .605 0.5 Makasoi Tulsi .807 1 Makasoi Tulsi .593 0.5 Uci .142 1 Uci .989 0.5 Tulsi Uci .660 1 Tulsi Uci .329

138

6.2.2 Fungi

Standard Control - Nistat discs Table 6-11: Effect on control on selected fungi Fungi Standard Control (mm) Aspergillus 10.17

Rhizopus 0

Pencillin 12.10

Sordaria wild 11.73

Sordaria gray 14.83

Table 6-12: Descriptive statistics for zone of inhibition (mm) of fungi across different concentration

Concentrations (%) N M SD Skew kurtosis

5 12 0.272 1.766 6.59 43.41 5 25 12 3.32 5.27 1.50 1.86 5 50 12 5.75 6.51 0.79 -0.37 5 100 12 14.76 12.13 0.70 -0.15 5

139

6.2.2.1 ANOVA analysing using tukey’s test

Fungi Concentration Plant comparison P- Overall Fungi Concentration Plant comparison P- Overall value significant value significant or not or not Aspergillus 0.25 Curry Lemon .010 F(4,20)= Aspergillus 0.5 Curry Lemon .014 F(4,20)= conidiophores leaves grass 27.174, conidiophores leaves grass 13.521, 0.25 Makasoi 1.000 p=.000 0.5 Makasoi 1.000 p=.000 0.25 Tulsi .000 0.5 Tulsi .000 0.25 Uci 1.000 0.5 Uci .002 0.25 Lemon Makasoi .010 0.5 Lemon Makasoi .014 0.25 grass Tulsi .001 0.5 grass Tulsi .307 0.25 Uci .010 0.5 Uci .887 0.25 Makasoi Tulsi .000 0.5 Makasoi Tulsi .000 0.25 Uci 1.000 0.5 Uci .002 0.25 Tulsi Uci .000 0.5 Tulsi Uci .823

Fungi Concentration Plant comparison P- Overall Fungi Concentration Plant comparison P- Overall value significant value significant or not or not Aspergillus 1 Curry Lemon .418 F(4,20)= Penicillum 0.05 Curry Lemon 1.000 F(4,20)= conidiophores leaves grass 8.877, conidia leaves grass 2.636, 1 Makasoi .326 p=.000 0.05 Makasoi 1.000 p=.065 1 Tulsi .000 0.05 Tulsi .115 1 Uci .009 0.05 Uci 1.000 1 Lemon Makasoi 1.000 0.05 Lemon Makasoi 1.000 1 grass Tulsi .010 0.05 grass Tulsi .115 1 Uci .291 0.05 Uci 1.000 1 Makasoi Tulsi .015 0.05 Makasoi Tulsi .115 1 Uci .377 0.05 Uci 1.000 1 Tulsi Uci .447 0.05 Tulsi Uci .115

140

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall significant significant or not or not Penicillum 0.25 Curry Lemon .000 F(4,20)= Penicillum 0.5 Curry Lemon .001 F(4,20)= conidia leaves grass 25.322, conidia leaves grass 10.933, 0.25 Makasoi 1.000 p=.000 0.5 Makasoi 1.000 p=.000 0.25 Tulsi .000 0.5 Tulsi .001 0.25 Uci 1.000 0.5 Uci .529 0.25 Lemon Makasoi .000 0.5 Lemon Makasoi .001 0.25 grass Tulsi .875 0.5 grass Tulsi 1.000 0.25 Uci .000 0.5 Uci .042 0.25 Makasoi Tulsi .000 0.5 Makasoi Tulsi .001 0.25 Uci 1.000 0.5 Uci .529 0.25 Tulsi Uci .000 0.5 Tulsi Uci .046

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall

significant significant or not or not Penicillum 1 Curry Lemon .460 F(4,20)= Rhizopus 0.25 Curry Lemon .056 F(4,20)= conidia leaves grass 6.216, sporangia leaves grass 9.997, 1 Makasoi .078 p=.002 0.25 Makasoi .001 p=.000 1 Tulsi .001 0.25 Tulsi .005 1 Uci .071 0.25 Uci 1.000 1 Lemon Makasoi .817 0.25 Lemon Makasoi .398 1 grass Tulsi .038 0.25 grass Tulsi .797 1 Uci .790 0.25 Uci .056 1 Makasoi Tulsi .279 0.25 Makasoi Tulsi .956 1 Uci 1.000 0.25 Uci .001 1 Tulsi Uci .302 0.25 Tulsi Uci .005

141

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall significant significant or not or not Rhizopus 0.5 Curry Lemon .005 F(4,20)= Rhizopus 1 Curry Lemon .000 F(4,20)= sporangia leaves grass 5.137, sporangia leaves grass 25.549, 0.5 Makasoi .021 p=.005 1 Makasoi .001 p=.000 0.5 Tulsi .022 1 Tulsi .000 0.5 Uci .149 1 Uci .000 0.5 Lemon Makasoi .959 1 Lemon Makasoi .196 0.5 grass Tulsi .952 1 grass Tulsi .072 0.5 Uci .476 1 Uci .980 0.5 Makasoi Tulsi 1.000 1 Makasoi Tulsi .000 0.5 Uci .858 1 Uci .449 0.5 Tulsi Uci .871 1 Tulsi Uci .023

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall significant significant or not or not Sodaria 0.05 Curry Lemon 1.000 F(4,20)= Sodaria 0.25 Curry Lemon 1.000 F(4,20)= gray leaves grass 1.000, gray leaves grass 18.931, p=.431 0.05 Makasoi .525 0.25 Makasoi .118 p=.000 0.05 Tulsi 1.000 0.25 Tulsi .000 0.05 Uci 1.000 0.25 Uci 1.000 0.05 Lemon Makasoi .525 0.25 Lemon Makasoi .118 grass 0.05 Tulsi 1.000 0.25 grass Tulsi .000 0.05 Uci 1.000 0.25 Uci 1.000 0.05 Makasoi Tulsi .525 0.25 Makasoi Tulsi .002 0.05 Uci .525 0.25 Uci .118 0.05 Tulsi Uci 1.000 0.25 Tulsi Uci .000 142

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall significant significant or not or not Sodaria 0.5 Curry Lemon .366 F(4,20)= Sodaria 1 Curry Lemon .999 F(4,20)= gray leaves grass 11.123, gray leaves grass 9.895, 0.5 Makasoi .037 p=.000 1 Makasoi .716 p=.000 0.5 Tulsi .000 1 Tulsi .001 0.5 Uci .951 1 Uci .024 0.5 Lemon Makasoi .709 1 Lemon Makasoi .578 0.5 grass Tulsi .004 1 grass Tulsi .000 0.5 Uci .777 1 Uci .015 0.5 Makasoi Tulsi .068 1 Makasoi Tulsi .012 0.5 Uci .149 1 Uci .270 0.5 Tulsi Uci .000 1 Tulsi Uci .518

Fungi Concentration Plant comparison P- value Overall Fungi Concentration Plant comparison P- value Overall significant significant or not or not Sordaria 0.25 Curry Lemon .966 F(4,20)= Sordaria 0.5 Curry Lemon .920 F(4,20)= wild leaves grass 2.998, wild leaves grass 8.634, 0.25 Makasoi .941 p=.043 0.5 Makasoi .228 p=.000 0.25 Tulsi .052 0.5 Tulsi .000 0.25 Uci 1.000 0.5 Uci .153 0.25 Lemon Makasoi 1.000 0.5 Lemon Makasoi .662 0.25 grass Tulsi .177 0.5 grass Tulsi .001 0.25 Uci .966 0.5 Uci .521 0.25 Makasoi Tulsi .213 0.5 Makasoi Tulsi .029 0.25 Uci .941 0.5 Uci .999 0.25 Tulsi Uci .052 0.5 Tulsi Uci .047

143

Fungi Concentration Plant comparison P- value Overall significant or not Sordaria 1 Curry Lemon .994 F(4,20)= wild leaves grass 7.977, 1 Makasoi .491 p=.001 1 Tulsi .002 1 Uci .057 1 Lemon Makasoi .286 grass 1 Tulsi .001 1 Uci .025 1 Makasoi Tulsi .084 1 Uci .700 1 Tulsi Uci .607

144

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Abbasipour, H., Mahmoudvand, M., Rastegar, F., & Hosseinpour, M. H. (2011). Fumigant toxicity and oviposition deterrency of the essential oil from cardamom, Elettaria cardamomum, against three stored–product insects. Journal of Insect Science, 11(165), 1-10.

Abbott, W. S. (1925). A Method of Computing the Effectiveness of an Insecticide. Journal of Economic Entomology, 18(2), 265-267.

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