whitefly control and anti-microbiological -...
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WHITEFLY CONTROL AND ANTI-MICROBIOLOGICAL
ACTIVITIES OF ESSENTIAL OILS FROM MEDICINAL
PLANTS FOUND IN FIJI ISLANDS.
By
Ravneel Rajneel Chand
A thesis submitted in fulfillment of the requirements for the degree of
Masters of Science
Copyright © 2016 by Ravneel Chand
School of Biological and Chemical Sciences
Faculty of Science, Technology and Environment
The University of the South Pacific
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.
iv
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.
v
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.
vi
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
vii
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
viii
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
ix
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
x
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
xi
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
xii
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
xiii
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
xiv
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
1
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
2
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 GPP synthase GPP; C10 Monoterpenes FPP synthase (+IPP) 2X FPP; C15 Sesquiterpenes GGPP synthase (+IPP) GGPP; C20 Diterpenes Polyterpenes Figure 1-1: Synthesis of different classes of terpenes in plants.
(Dubey et al., 2003)
Hemiterpenes (isoprene’s)
Prenylated metabolites
� Ubiquinone � Plastoquinone � Abscisic acid � Prenylated proteins
� Gibberellins � Chlorophyll � Prenylated proteins � Carotenoids.
Phytosterols
Triterpenoids
Squalene
Saponins
Essential
oil
2X
� Cytokinins � Anthraquinones
3
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).
4
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).
CH2
CH3
CH3
CH2
CH3CH3
CH3
CH2 CH3
CH3
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
5
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).
O
CH3
CH3
CH3
CH2
H
H
H
CH3
CH2
CH2
CH3
H
CH3
CH3
CH3
CH2
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-
6
insecticidal properties, but they are also recognized sources of antimicrobial,
antiviral, antitumor, antibiotic and amoebicidal activities (Coll & Tandrón, 2007).
CH2
CH3
CH3
CH3CH3
O
OH
CH3
CH3
CH3
CH2
H
H
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).
7
CH3
CH3
CH3
CH3
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).
OHCH3CH2
CH3
CH3
OHCH3 CH3
CH3
O CH3
CH3
Linalool (10) Geraniol (12) α-terpineol (11) Figure 1-6: Selected structures of Alcohol.
8
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)).
OH
CH3 CH3
CH3
OH
CH3
CH3 CH3 CH2
OH
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).
9
CH2 O
O CH3O
CH2
O
CH3
OCH3
CH2
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).
O
CH3
CH3
CH3
O
CH3
CH3 CH3 O
CH3
CH3
CH3
Citral (18) Citronellal (19) Neral (20) Figure 1-9: Selected structures of Aldehydes.
10
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).
CH3
CH2
CH3
O
O
CH3
CH3CH3
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).
11
CH3
O
O
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).
O
CH3
CH3 CH3
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.
12
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
13
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).
14
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
15
(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
16
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
17
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).
18
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.
19
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.
20
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.
21
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.
(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
Figure 2-1: Flowers of C. odorata
22
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
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
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
23
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
Species- koenigii (L) Spreng
(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.
Figure 2-3: Murraya koenigii (L) Spreng
plants.
24
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
Species: tenuiflorum L
(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).
Figure 2-4: Branches of O. tenuiflorum L plants
25
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
(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).
Figure 2-5: Euodia hortensis forma
hortensis plant.
26
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-
27
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.
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
Condenser
3- Neck round bottom flask
Clamp stand with the holder
Collecting tube
Heating Mantle
- H2O in
- H2O out
28
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%).
29
Table 2-1: Physical properties of selected essential oils from medicinal plants found in Fiji.
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:
Medicinal plants found in Fiji
Plant material used
Average Mass (g) taken for extraction *
Average Essential oil content (mL) *
Average Percentage Yield (%)
Essential oil colour
Cananga odorata (Makosoi)
Flowers 215.65 2.60 1.21 light to deep yellow liquid
Cymbopogon citratus (Lemon grass)
Leaves 212.41 2.50 1.17 colourless
Murraya Koenigii (L) (Curry Leaves)
Leaves 300.42 0.50 0.17 yellowish
Ocimum tenuiflorum L (Tulsi)
Leaves 314.35 2.15 0.68 colourless
Euodia hortensis forma hortensis (Uci)
Leaves 219.60 1.40 0.64 pale greenish to colourless
30
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 Time
Percentage Area
Compound* Chemical group
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
acetate ester
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).
31
Lina
lool
Tr
ans,
tran
s-fa
rnes
ol
Ben
zyl b
enzo
ate
Tran
s, tr
ans-
farn
esyl
ace
tate
Figu
re 2
-8: G
C-M
S ch
rom
atog
ram
of e
ssen
tial o
il fr
om th
e flo
wer
s of C
. odo
rata
.
32
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.
*Compounds listed in order of elution from a HP-5MS non polar fused silica capillary column.
Peak # Retention index Percentage Area
Compound* Chemical group
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
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).
33
Sa
bine
ne
ϒ- t
erpi
nene
Terp
inen
e-4-
ol
β- C
aryo
phyl
lene
Figu
re 2
-9: G
C-M
S ch
rom
atog
ram
of e
ssen
tial o
il fr
om th
e le
aves
of M
. koe
nigi
i (L)
Spr
eng.
34
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
acetate ester
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).
35
Lim
onen
e
Men
thof
uran
Evod
one
ϒ- C
urcu
men
e
Figu
re 2
-10:
GC
-MS
chro
mat
ogra
m o
f ess
entia
l oil
from
leav
es o
f E. h
orte
nsis
form
a ho
rten
sis.
36
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 Area
Compound * Chemical 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).
37
CH3
CH3
CH3
CH2
Cis-β-ocimene
O
CH3
OH
CH2
Eugenol
CH3
CH2
CH3
CH3H H
β- Caryophyllene
CH3
CH2
CH3
CH3
Germacrene D
Figure 2-11: GC-MS chromatogram of essential oil from O. tenuiflorum L leaves
38
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.
*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).
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
germacral (10), 5-diene #
sesquiterpenes
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
39
Citronellal
Citronellol
Geraniol
Elemol
Figure 2-12: GC-MS chromatogram of essential oil from C. citratus leaves.
40
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 oils
Percentage Area (%) (Gaydou et al., 1986)
Percentage Area (%) (Murbach Teles Andrade et al., 2013)
Percentage Area (%) (Katague & 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.
41
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.
42
Table 2-8: Comparison of chemical composition of essential oils from M. koenigii.
Components of essential oils
Percentage Area (%) from different locations in India (Walde et al., 2005)
Percentage Area (%) (Chowdhury et al., 2008)
lower Himalayan
Pant Nagar, Uttaranchal
Eastern india Bhubaneshwar, Orissa
Southern India (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) dimethylsilane
2.58
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-none
1.15
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).
43
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 (%) Pino et al. (1998)
Percentage Area (%) 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%).
44
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 essential oils
Percentage Area (%) Matasyoh et al. (2011)
Percentage Area (%) Olivero-Verbel et al. (2010)
Percentage Area (%) (Negrelle & Gomes, 2007) cited in 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-one
2.63%
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).
45
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
46
(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.
47
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 bio-
pesticides 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
Family: Aleyrodidae: (Whiteflies)
Genus: Aleurodicus ©Photograph taken by Ravneel on 9/9/15
Figure 3-1: Whiteflies on cassava
48
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).
49
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).
50
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).
Figure 3-3: Mature pupa (~1.06 mm) of Spiralling whitefly
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.
©Photograph taken by Ravneel on 9/10/15
51
Figure 3-4: Adult (~1.74 mm) of Spiralling whitefly.
Figure 3-5: Life cycle of the Spiralling whitefly
Eggs (~0.3 mm)
Adults (~1.74 mm)
1st instar (~0.32 mm)
4th instar (~1.06 mm)
2nd instar (~0.55 mm)
3rd instars (~0.65 mm)
White waxy flocculants materials
Dark reddish-brown eye, where part the compound eye joined by 3 or 4 facets.
Presence of dark spot on the forewings.
Presence of a pair of antenna
©Photograph taken by Ravneel on 9/10/15
©Photograph taken by Ravneel on 9/10/15
52
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
53
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).
54
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).
55
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).
Limitations of Biological Control 3.1.4.2.1
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 biological control
Explanation Reference
More susceptible to the environment
This means that biological agents are more vulnerable to environmental factors including; sunlight, temperature and rainfall.
(Harper, 2001)
Limits the usage of pesticides subsequently.
If biological agents are used for the controlling of a specific pest on a crop, then it makes it difficult to use insecticides for controlling other insect pests.
(van Emden, 1989)
Biological control has slow action.
It usually takes time to build in numbers 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.
(van Emden, 1989)
Expensive for biological control in field.
It is costly when it comes to selection of biological agents, whereby it requires highly qualified or skilled people.
(Orr & Lahiri, 2014)
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).
56
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
Whitefly Control - Chemicals 3.1.4.3.1
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).
57
Limitations of Chemical Control 3.1.4.3.2
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.
58
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,
59
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%.
60
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.
A
B
61
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).
A
C D
B
62
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).
63
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.
A B
Figure 3-10: T-shaped olfactometer.
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.
64
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.
RI= 0= no effect.
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.
D
A
C
B
65
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).
66
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.
67
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.
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.
b*
d* d*
d*
0
5
10
15
20
25
3hrs 6hrs 9 hrs 12hrs 24 hrs
Mor
talit
y (c
ount
s)
Time
Control
Cananga odorata (a)
Murraya koenigii (b)
Euodia hortensis (c)
Cymbopogon citratus (d)
Ocimum tenuiflorum L (e)
68
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.
d***
d***
d***
d***
e***
e***
e***
e*
e***
0
5
10
15
20
25
30
35
40
45
50
3hrs 6hrs 9 hrs 12hrs 24 hrs
Mor
talit
y (c
ount
s)
Time
Control
Cananga odorata (a)
Murraya koenigii (b)
Euodia hortensis (c)
Cymbopogon citratus (d)
Ocimum tenuiflorum L (e)
69
Figure 3-14: Fumigant effect (Mean ±SE) of 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.
a*
a*
a*
b*
b*
c*
d*
d*** d*** d*** d***
e*** e*** e*** e*** e***
0
10
20
30
40
50
60
3hrs 6hrs 9 hrs 12hrs 24 hrs
Mor
talit
y (c
ount
s)
Time
Control
Cananga odorata (a)
Murraya koenigii (b)
Euodia hortensis (c)
Cymbopogon citratus (d)
Ocimum tenuiflorum L (e)
70
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.
Essential oils Time (hours)
Equation R2 LC50 /EC50 (%)
χ2statistic P-value df
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 forma hortensis
24 y= 3.349+3.887x 0.586 0.114 78.574 <0.0001 1
Cymbopogon citratus
24 y= 8.725+3.764x 0.902 0.004 279.950 <0.0001 1
Ocimum tenuiflorum L
24 y= 12.286+5.020x 0.651 0.003 253.512 <0.0001 1
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
71
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.
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.
***
-40 -20 0 -20 -40 -60
0.25%
0.50%
5%
Repellency index (%)
Conc
entr
atio
n
Murraya koenigiiEuodia hortensisCananga odorataCymbopogon citratusOcimum tenuiflorum L
6040
Attractancy Repellency
72
Table 3-3: Summary of repellent effect (6-8 hours) on adult whiteflies at different concentrations
(Using Probit analysis).
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.
Essential oils
Conc (v/v) %
RI% Equation R2 EC50 (%)
χ2statistic P-value df
Cananga odorata
0.25 -29 y = -0.140+0.290x 0.0795 3.046 5.93 0.015 1 0.5 -17 5 9
Murraya koenigii
0.25 -13 y = 0.260+0.663x 0.3232 0.406
38.214
< 0.0001
1 0.5 8 5 52
Euodia hortensis forma hortensis
0.25 -10 y= 0.003+0.188x
0.028 0.964
3.277
0.070
1 0.5 -3 5 10
Cymbopogon citratus
0.25 -9 y = -0.285+0.953x 0.6111 0.434
27.474
< 0.0001
1 0.5 3 5 52
Ocimum tenuiflorum L
0.25 -18 y = -0.163+0.374x 0.1582 2.728
13.928
0.000
1 0.5 -11 5 12
73
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.
74
Tabl
e 3-
4: S
tudi
es o
f eff
ects
of p
lant
ess
entia
l oils
on
whi
tefli
es.
Plan
t Nam
e E
xtra
cts
Con
cent
ratio
n T
este
d
Eff
ect
Ref
eren
ce
Alliu
m sa
tivum
(Gar
lic).
Esse
ntia
l oil
extra
ctio
n vi
a hy
dro -
dist
illat
ion
usin
g n-
hexa
ne.
50.0
0 μg
/L.
The
esse
ntia
l oi
l sh
owed
stro
nges
t fu
mig
ant
effe
ct a
gain
st B
.taba
ci
adul
ts w
ith a
n LC
50 v
alue
of 0
.11
μg/L
afte
r 24
hour
s.
(Liu
et a
l., 2
008)
Arte
mis
ia si
eber
i Bes
ser
(wor
mw
ood)
. Es
sent
ial o
ils e
xtra
cts
purc
hase
d fr
om B
arij
Esan
s Com
pany
, K
asha
n, Ir
an.
12, 1
25, 1
250
and
2500
ppm
. Th
e re
sults
sh
owed
th
at
the
test
ed
esse
ntia
l oi
l co
ncen
tratio
ns
sign
ifica
ntly
red
uced
the
B. t
abac
i at 2
4 ho
urs.
The
perc
enta
ge m
ean
cont
act t
oxic
ity o
f 12,
125
, 125
0 an
d 25
00 p
pm o
f sel
ecte
d es
sent
ial o
il af
ter
24 h
ours
wer
e 90
, 10
0, 1
00 a
nd 9
8%.
The
rese
arch
er a
lso
high
light
ed th
at th
e co
ncen
tratio
n of
250
0, 1
250,
and
125
ppm
cau
sed
seve
re p
hyto
toxi
city
to th
e pl
ants
that
wer
e us
ed fo
r tre
atin
g w
hite
flies
.
(Yar
ahm
adi e
t al.,
20
13)
Satu
reja
hor
tens
is L
., (S
umm
er sa
vory
) Oci
mum
ba
silic
um L
. (Tu
lsi)
and
Thym
us v
ulga
ris L
. (G
arde
n Th
yme)
.
Esse
ntia
l oil
extra
cts
via
hydr
o-di
still
atio
n us
ing
diet
hyl e
ther
.
1.56
, 3.1
25, 6
.25
and
12.5
µl.
The
fum
igan
t to
xici
ty o
f es
sent
ial
oils
fro
m S
atur
eja
hort
ensi
s L.
, O
cim
um b
asili
cum
L. a
nd T
hym
us v
ulga
ris
L. (
Lam
iaca
e) s
how
ed th
at
mor
talit
y of
B.ta
baci
(whi
tefli
es) w
ere
dire
ctly
link
ed to
the
incr
easi
ng
time
and
conc
entra
tions
. Th
e ov
eral
l m
ultip
le m
ean
for
(N=
10
whi
tefli
es in
eac
h se
t -up)
for t
he fu
mig
ant t
oxic
ity o
f sel
ecte
d es
sent
ial
oils
afte
r 24
hour
s wer
e 44
, 27
and
27 d
ead
coun
ts, r
espe
ctiv
ely.
(Asl
an e
t al.,
20
04)
Can
anga
odo
rata
(M
akos
oi),
C. c
itrat
us
(Lem
on g
rass
), M
. koe
nigi
i (L
) Spr
eng
(Cur
ry L
eave
s),
O. t
enui
floru
m L
(Tul
si) a
nd
E. h
orte
nsis
form
a ho
rten
sis
(Uci
)).
Esse
ntia
l oil
extra
cts
via
hydr
o -di
still
atio
n us
ing
dist
illed
wat
er.
0.12
5%, 0
.25%
and
0.
5% (v
/v).
The
fum
igan
t tox
icity
of s
elec
ted
esse
ntia
l oils
sho
wed
eff
ects
at 3
, 6,
9, 1
2 an
d 24
hou
rs.
The
mos
t ro
bust
eff
ect
was
sh
own
by O
. te
nuifl
orum
L a
s 10
0% m
orta
lity
(50
dead
cou
nts/
50
test
ed)
was
ac
hiev
ed a
t 3
hour
s fo
r 5%
(v/
v) c
once
ntra
tions
, fo
llow
ed b
y C
. ci
trat
us at
6 h
ours
for 5
% c
once
ntra
tions
. Whi
le fo
r oth
ers
at d
iffer
ent
conc
entra
tion s
it
was
rel
ativ
ely
low
as
repo
rted
unde
r re
sult
sect
ion.
Th
e LC
50 f
or O
. te
nuifl
orum
L a
nd C
. ci
trat
us a
fter
24 h
ours
wer
e 0.
003 %
and
0.0
04%
res
pect
ivel
y. N
ote:
no
phyt
otox
icity
was
see
n on
th
e pl
ants
with
the
test
ed so
lutio
n of
ess
entia
l oils
in th
e pr
esen
t stu
dy.
Cur
rent
stud
y
Not
e: N
is re
ferr
ing
to th
e to
tal n
umbe
r of w
hite
flies
test
ed in
eac
h se
t-up
for t
he fu
mig
ant t
est.
75
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
76
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.
77
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.
78
Tabl
e 3-
5: S
tudi
es o
f rep
elle
nt e
ffec
ts o
f pla
nt e
ssen
tial o
ils o
n w
hite
flies
.
Plan
t Nam
e E
xtra
ct ty
pe
Con
cent
ratio
n te
sted
E
ffec
t R
efer
ence
P. c
ablin
(min
t fam
ily),
T. v
ulga
ris (
Gar
den
Thym
e)) a
nd C
. ci
trio
dora
(lem
on-
scen
ted
gum
) oil.
Esse
ntia
l oil
extra
ctio
n.
0.5%
(v/v
). Th
e es
sent
ial o
ils f
rom
P. c
ablin
, T. v
ulga
ris
and
C. c
itrio
dora
res
ulte
d in
74
.5%
, 59
.0%
and
48.
0% f
ewer
egg
s la
id b
y B.
tab
aci
afte
r 5
day
of
obse
rvat
ion .
The
res
earc
her
conc
lude
d th
at P
. cab
lin e
ssen
tial o
ils s
trong
ly
repe
lled
the
B. ta
baci
.
(Yan
g et
al.,
20
10)
Car
ica
papa
ya (p
apay
a),
Bauh
inia
var
iega
ta
(orc
hid
tree)
and
C
hrys
alid
o car
pus
lute
scen
s (ba
mbo
o pa
lm).
Esse
ntia
l oil
extra
ctio
n vi
a hy
dro -
dist
illat
ion
usin
g an
hydr
ous
ethe
r .
20 μ
1of e
ssen
tial
oils
.
The
Y-tu
be o
lfact
omet
er w
as u
sed
for
the
repe
llenc
y te
st.
The
resu
lts
reve
aled
tha
t C
aric
a pa
paya
and
Bau
hini
a va
rieg
ata
repe
lled
Aleu
rodi
cus
disp
ersu
s (S
pira
lling
whi
tefli
es)
as t
he m
ajor
ity o
f th
e w
hite
flies
(N
= 60
w
hite
flies
) wer
e at
con
trolle
d ch
ambe
r (33
whi
tefli
es s
een)
com
pare
d to
the
treat
men
t ch
ambe
r (2
7 w
hite
flies
se
en).
Like
wis
e,
Chr
ysal
idoc
arpu
s lu
tesc
ens
esse
ntia
l oi
ls r
epel
led
the
spira
lling
whi
tefli
es w
here
at
cont
rol
cham
ber
(25
whi
tefli
es s
een)
and
at
the
treat
men
t ch
ambe
r (3
5 w
hite
flies
se
en).
(Zhe
ng e
t al.,
20
14)
Gin
ger (
Fam
ily:
Zing
iber
acea
e).
Gin
ger o
il ex
tract
ed
via
hydr
o -di
still
atio
n .
0.5,
0.7
5, a
nd 1
%
(v/v
) Th
e R
epel
lenc
y ef
fect
on
Bem
isia
arg
entif
olii
(whi
tefli
es)
incr
ease
d w
ith
incr
easi
ng g
inge
r oil
conc
entra
tion.
The
cho
ice
test
(rep
elle
nt te
sts)
sho
wed
th
at t
he n
umbe
r of
whi
tefly
cou
nts
(N=
30 w
hite
flies
) w
ere
high
er (
>16
coun
ts)
in t
he c
ontro
l ch
ambe
r of
the
olfa
ctom
eter
whe
n co
mpa
red
to t
he
treat
men
t cha
mbe
r (<1
3 co
unts
).
(Zha
ng e
t al.,
20
04)
Can
anga
odo
rata
(M
akos
oi),
C. c
itrat
us
(Lem
on g
rass
), M
. ko
enig
ii (L
) Spr
eng
(Cur
ry L
eave
s), O
. te
nuifl
orum
L (T
ulsi
) and
E.
hor
tens
is fo
rma
hort
ensi
s (U
ci))
.
Esse
ntia
l oil
extra
cts v
ia h
ydro
-di
still
atio
n us
ing
dist
i lled
wat
er.
0.25
%, 0
.5%
and
5%
(v/v
) Th
e re
pelle
ncy
activ
ity o
f se
lect
ed e
ssen
tial
oils
in
the
pres
ent
stud
y re
veal
ed t
hat
C.
citr
atus
and
M.
koen
igii
(L)
Spre
ng s
how
ed t
he b
est
repe
llent
eff
ect
as t
he r
epel
lenc
y in
dex
for
both
ess
entia
l oi
ls a
t 5%
co
ncen
t ratio
n w
as 5
2%,
follo
wed
by
O.
tenu
iflor
um L
(R
I= 1
2%),
C.
odor
ata
(RI=
9%
) and
E. h
orte
nsis
form
a ho
rten
sis (
RI=
10%
). B
ased
on
the
obta
ined
res
ults
fro
m t
he T
-sha
ped
olfa
ctor
met
er f
or b
est
repe
llent
eff
ect
(N=
50 w
hite
flies
) th
e C
. citr
atus
had
22
whi
tefli
es in
con
trol a
nd 7
in th
e tre
atm
ent c
ham
ber w
hile
M. k
oeni
gii (
L) S
pren
g ha
d 29
whi
tefli
es in
con
trol
and
9 in
the
treat
men
t cha
mbe
r.
Cur
rent
stud
y
Not
e: N
is re
ferr
ing
to th
e to
tal n
umbe
r of w
hite
flies
test
ed in
eac
h ol
fact
orm
eter
.
79
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
80
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
81
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.
82
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.
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
Octopamine
(C8H11 NO2)
Membrane at post-
synaptic junction Acetylcholine
(C7NH16O2+)
Muscles junction
Insect body fluid
Nerve current.
Modulation at physiological level.
Nerve
impulse
(neurons)
83
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.
84
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
85
natural fumigant and repellent for controlling whitefly needs to be further evaluated as
to their enhanced activity, mode of actions and safety to humans.
86
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
87
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 (Gram (-))
- Causes 21 million cases of typhoid fever and 200,000 deaths each year. These bacteria are known to cause infections in mammalian intestine (intestinal epithelium of the small bowel). - Also resulted in the ineffectiveness of immune system by stopping the oxidative burst of leukocytes.
(Jones et al., 1994; Devi et al., 2010)
Streptococcus (pneumoniae) (Gram (+))
- These bacteria caused many different types of illness, that include; pneumonia (lung infections), ear, sinus, meningitis (covering around spinal cord and the brain) and bacteraemia (blood stream) infections.
(Centers for Disease Control and Prevention, 2004; Buckle, 2015b)
Staphylococcus aureus (Gram (+))
- Caused major infections in skin, soft-tissue and disorders in bone, endovascular and joints.
(Lowy, 1998; Buckle, 2015b)
Pseudomonas aeruginosa (Gram (+))
- Caused infections such as malignant external otitis (outer ear infection), endocarditis (heart valves), meningitis (covering membrane of brain and spinal cord), septicaemia (blood diseases) and pneumonia.
(Bodey et al., 1983; Buckle, 2015b)
Thermus thermophiles (Gram (-))
- The diseases associated with thermophilic are rheumatic heart diseases, immunodeficiency and strokes.
(Rabkin et al., 1985)
88
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
causing spoilage of bread. (Saranraj, 2012)
Penicillum conidia The genus of Penicillum mostly caused
food spoilage; even some produced toxic compounds that had triggered allergic reactions.
(Kung'u, 2016)
Aspergillus conidiophores
Aspergillus species mostly caused invasive pulmonary aspergillosis (type of deficiency in white blood cells). This fungus also affected the food industry through the formation of moulds on fruits, grains, wheats and breads.
(Jahn et al., 2000; Davidson, 2015)
Sodaria wild These species affected the agriculture
industries which are associated with symptoms of brown wood discoloration and leaf spottiness.
(Kavak, 2012) Sodaria gray
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
89
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; Raybaudi Massilia 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).
90
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
91
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.
92
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).
93
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
94
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.
95
Tabl
e 4-
3: M
ean
and
Stan
dard
Err
or (S
E) fo
r eff
ects
of v
aryi
ng c
once
ntra
tion
of th
e es
sent
ial o
ils o
n di
ffer
ent b
acte
ria.
Ess
entia
l oil
Bac
teri
a 0.
25a
0.5b
5c 25
d 50
e 10
0f O
cim
um
tenu
iflor
um L
Ther
mus
ther
mop
hilu
s 0
0 0
8.00
±0.6
3 8.
00±0
.55
17.2
0±0.
20
Pseu
dom
onas
aer
ugin
osa
0 0
0 4.
80±1
.98
8.80
±0.8
6 16
.20±
0.20
St
rept
ococ
cus (
pneu
mon
ia) *
0
0 0
5.60
±1.4
0 7.
80±0
.58
17.8
0±0.
49
Stap
hylo
cocc
us a
ureu
s*
0 0
0 10
.20±
1.11
10
.80±
1.88
25
.60±
0.68
Sa
lmon
ella
0
0 0
3.00
±1.8
4 7.
60±0
.24
14.8
0±0.
20
Cym
bopo
gon
citr
atus
Ther
mus
ther
mop
hilu
s 0
0 5.
40±2
.20
10.6
0±1.
25
13.8
0±0.
37
15.4
0±0.
40
Pseu
dom
onas
aer
ugin
osa
0 0
3.20
±1.9
6 12
.00±
1.10
14
.40±
0.68
15
.00±
0.89
St
rept
ococ
cus (
pneu
mon
ia) *
0
0 0
4.40
±1.8
1 9.
20±0
.66
15.2
0±0.
37
Stap
hylo
cocc
us a
ureu
s*
0 0
0 7.
60±2
.25
9.80
±0.5
8 14
.80±
0.20
Sa
lmon
ella
0
0 0
0 0
0 C
anan
ga
odor
ata
Ther
mus
ther
mop
hilu
s 1.
60±1
.60
2.80
±1.7
1 3.
00±1
.84
8.40
±0.6
0 11
.40±
1.08
12
.20±
1.16
Ps
eudo
mon
as a
erug
inos
a 4.
20±1
.71
4.40
±1.8
1 7.
80±0
.37
8.60
±0.2
4 11
.60±
1.21
12
.60±
0.75
St
rept
ococ
cus (
pneu
mon
ia) *
0
0 0
7.00
±0.0
0 7.
20±0
.20
7.40
±0.4
0 St
aphy
loco
ccus
aur
eus*
0
0 4.
20±1
.71
8.00
±0.5
5 8.
80±0
.20
10.2
0±0.
37
Salm
onel
la
0.0
0.0
0.0
0.0
0.0
0.0
Euod
ia
hort
ensi
s fo
rma
hort
ensi
s
Ther
mus
ther
mop
hilu
s 0
0 0
0 5.
80±1
.46
10.8
0±1.
32
Pseu
dom
onas
aer
ugin
osa
0 0
0 0
5.60
±1.4
0 11
.20±
0.73
St
rept
ococ
cus (
pneu
mon
ia) *
0
0 0
0 0
7.40
±0.2
5 St
aphy
loco
ccus
aur
eus*
0
0 0
0 0
8.40
±0.5
1 Sa
lmon
ella
0
0 0
0 0
0 M
urra
ya
koen
igii
(L)
Spre
ng
Ther
mus
ther
mop
hilu
s 0
0 0
0 1.
40±1
.40
3.00
±1.8
4 Ps
eudo
mon
as a
erug
inos
a 0
0 0
0 1.
40±1
.40
2.80
±1.7
1 St
rept
ococ
cus (
pneu
mon
ia) *
0
0 0
0 0
0 St
aphy
loco
ccus
aur
eus*
0
0 0
0 0
1.60
±1.6
0
Salm
onel
la
0 0
0 0
0 0
Not
e: G
ram
(+) b
acte
ria =
*, 0
= no
dia
met
er o
f inh
ibiti
on z
one,
(Mea
n ±
Stan
dard
Err
or) t
he m
ean
valu
e in
(mm
); a , b , c , d , e a
nd f re
ferr
ed to
con
cent
ratio
ns (%
). Ea
ch te
st re
pres
ents
the
mea
n of
five
repl
icat
es o
f eac
h te
sted
con
cent
ratio
n.
96
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.
Figure 4-1: Anti-bacterial effect of selected essential oils at 5% (v/v) solution.
Figure 4-2: Anti-bacterial effect of selected essential oils at 25% (v/v) solution.
c**
b**
**
0
1
2
3
4
5
6
7
8
9
O. tenuiflorum (a) C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Essential oil solutions
Thermus thermophilusPseudomonas aeruginosaStreptococcus (pneumonia)Staphylococcus aureusSalmonella
b**
a**
***
0
2
4
6
8
10
12
14
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Essential oil solutions
Thermus thermophilusPseudomonas aeruginosaStreptococcus (pneumonia)Staphylococcus aureusSalmonella
97
Figure 4-3: Anti-bacterial effect of selected essential oils at 50% (v/v) solution.
Figure 4-4: Anti-bacterial effect of selected essential oils at 100% (v/v) solution.
e**
e*** d*
e***
e* b*
b*** c*** a** d*
a**
e*** d*
e***
b*
b*** c*** a** d*
c*
b* ***
0
2
4
6
8
10
12
14
16
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Essential oil solutions
Thermus thermophilusPseudomonas aeruginosaStreptococcus (pneumonia)Staphylococcus aureusSalmonella
e***
e***
e***
e**
a*** b*** c*** d**
a*** e***
e*** e***
b*** c*** a*** d***
b** c*** d***
a** c*** d***
a*** b***
a*** b***
e*** b*
C*** d***
e*** a*
e*** a*** e***
d*** a*** c*** b*** d***
***
0
5
10
15
20
25
30
O. tenuiflorum (a) C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Essential oil solutions
Thermus thermophilus
Pseudomonas aeruginosa
Streptococcus (pneumonia)
Staphylococcus aureus
Salmonella
98
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
99
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.
100
Tabl
e 4-
4: M
ean
and
Stan
dard
Err
or (S
E) fo
r effe
cts o
f var
ying
con
cent
ratio
n of
the
esse
ntia
l oils
on
diff
eren
t fun
gi.
Esse
ntia
l oils
Fu
ngi
0.25
a 0.
5b 5c
25d
50e
100f
Oci
mum
tenu
iflor
um L
As
perg
illus
0
0 0
12.8
0±1.
39
13.8
0±2.
52
32.4
0±1.
44
Rhizo
pus
0 0
0 6.
60±1
.75
7.00
±1.7
9 25
.80±
1.80
Pe
nici
llin
0 0
4.80
±3.0
1 7.
40±2
.23
12.4
0±3.
80
34.4
0±5.
01
Sord
aria
wild
0
0 0
11.6
0±5.
05
15.8
0±1.
16
31.6
0±4.
50
Sord
aria
gra
y 0
0 0
12.8
0±0.
66
19.8
0±1.
56
37.4
0±2.
18
Cym
bopo
gon
citr
atus
As
perg
illus
0
0 0
4.20
±1.7
1 9.
20±3
.76
11.0
0±4.
49
Rhizo
pus
0 0
0 4.
60±1
.89
8.20
±0.4
9 14
.40±
0.93
Pe
nici
llin
0 0
0 7.
80±0
.37
9.80
±0.5
8 14
.20±
8.71
So
rdar
ia w
ild
0 0
0 1.
60±1
.60
1.80
±1.8
0 6.
20±3
.83
Sord
aria
gra
y 0
0 0
0 4.
80±2
.96
7.40
±4.6
0 C
anan
ga o
dora
ta
Aspe
rgill
us
0 0
0 0
0 7.
40±0
.24
Rhizo
pus
0 0
0 7.
20±0
.20
7.20
±2.0
1 7.
60±0
.24
Peni
cilli
n 0
0 0
0 0
13.8
0±4.
04
Sord
aria
wild
0
0 0
2.20
±2.0
2 4.
20±1
.71
10.8
0±0.
86
Sord
aria
gra
y 0
0 2.
00±2
.00
4.20
±2.6
2 6.
80±1
.74
9.00
±0.8
4 Eu
odia
hor
tens
is
Aspe
rgill
us
0 0
0 0
8.80
±0.8
6 18
.80±
3.50
Rh
izopu
s 0
0 0
0 4.
80±1
.98
12.6
0±1.
17
Peni
cilli
n 0
0 0
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63
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93
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n ±
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dard
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er fo
r inh
ibiti
on zo
ne; a
, b, c
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and f in
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ns (%
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n of
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101
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.
Figure 4-5: Anti-fungal effect of essential oils at 5% (v/v) solution.
Figure 4-6: Anti-fungal effect of essential oils at 25% (v/v) solution.
0123456789
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis(d)
M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Selected essential oils
Aspergillus
Rhizopus
Pencillin
Sordaria wild
Sordaria grey
b**
a**
c**
a**
0
2
4
6
8
10
12
14
16
18
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Selected essential oils
Aspergillus
Rhizopus
Pencillin
Sordaria wild
Sordaria grey
102
Figure 4-7: Anti-fungal effect of essential oils at 50% (v/v) solution.
Figure 4-8: Anti-fungal effect of essential oils at 100% (v/v) solution
d*
d*
a* b*
b** c* d*
a**
a*
a*
b** d***
a** d*
a***
0
5
10
15
20
25
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Selected essential oils
Aspergillus
Rhizopus
Pencillin
Sordaria wild
Sordaria grey
b* c*
e***
a*
a*
e**
a*** d**
a* c*** d*
e***
e***
e** a***
a* e***
a*** c**
b*** d***
e** b*
a*
a**
e** b**
a** d*
b*
a**
c* b*** e**
a*** d*
a*
b* e*
a** d*
0
5
10
15
20
25
30
35
40
45
O. tenuiflorum(a)
C. citratus (b) C. odorata (c) E. hortensis (d) M. koenigii (e)
Mea
n In
hibi
tion
zone
(mm
)
Selected essential oils
Aspergillus
Rhizopus
Pencillin
Sordaria wild
Sordaria grey
103
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
104
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
105
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
106
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.
107
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).
108
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.
109
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
110
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)
111
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
112
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
113
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
114
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
115
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.
116
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.
117
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
118
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;
� 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.
� 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.
� 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.
119
6. A
PPE
ND
IX
6.
0 C
hem
ical
Ana
lysi
s
Tabl
e 6-
1: C
hem
ical
Ana
lysi
s-gr
oup
of m
ajor
che
mic
al c
ompo
unds
from
sele
cted
ess
entia
l oils
. Ch
emic
al
Maj
or c
hem
ical
con
stitu
ents
O
. ten
uiflo
rum
L
C. c
itrat
us
C. o
dora
ta
E. h
orte
nsis
form
a ho
rten
sis
M. k
oeni
gii
Mon
oter
pene
s Su
btot
al
13.6
4
1.32
60
.18
65.5
1 Ci
s-β-
oci
men
e 10
.79
limon
ene
4.
64
m
enth
ofur
an
55
.17
α-
pin
ene
5.67
sa
bine
ne
43.8
0 α
- ter
pine
ne
2.64
ϒ-
terp
inen
e
4.
82
Sesq
uite
rpen
es
Subt
otal
22
.61
3.44
4.
13
9.17
20
.27
α- c
opae
ne
1.98
β-
car
yoph
ylle
ne
4.31
16.5
2 ge
rmac
rene
D
11.6
8
Al
coho
l and
Ph
enol
Su
btot
al
60.6
1 45
.88
50.8
5 0.
83
9.08
Eu
geno
l 58
.20
citr
onel
lol
19
.11
ge
rani
ol
13
.57
el
emol
6.15
α-ca
rdin
ol
3.
70
lin
aloo
l
16
.65
tran
s, tr
ans-
farn
esol
29
.71
terp
inen
e- 4
-ol
7.20
Es
ter
Subt
otal
1.7
35.7
6 0.
6
met
hyl s
alic
ylat
e
3.
15
benz
yl sa
licyl
ate
2.21
be
nzyl
ben
zoat
e
21.6
9
tr
ans,
tran
s-fa
rnes
yl a
ceta
te
Al
dehy
de
Subt
otal
46.5
1 0.
43
0.2
Ci
tron
ella
l
45.0
9
Keto
ne
Subt
otal
25.9
7
evod
one
25.9
7
Not
e: C
hem
ical
con
stitu
ents
that
are
pre
sent
in la
rge
amou
nt a
re o
nly
show
n (in
per
cent
age
(%))
. The
subt
otal
s are
for a
ll co
mpo
unds
(inc
ludi
ng n
eglig
ible
com
poun
ds).
120
6.1 Whiteflies
Table 6-2: Common Pest Species of Whiteflies with Distinct Nymphs
Spiralling whiteflies (Aleurodicus dispersus)
Host plants: 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 (Siphoninus phillyreae)
Host plants: 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 (Trialeurodes abutilonea)
Host plants: 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 (Aleuroplatus coronata)
Host plants: Oaks and chestnut Characteristics: The nymphs are black and arranged in crown like pattern covered with white wax. The adults are white.
121
Greenhouse whitefly (Trialeurodes vaporariorum)
Host plants: 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 argentifolii and B. tabaci)
Host plants: 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 (Aleyrodes spiraeoides)
Host plants: 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)
122
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.
123
Tabl
e 6-
3: M
ultip
le C
ompa
rison
s (Po
st H
oc T
est)
for C
. odo
rata
.
Plan
t T
ime
Con
cent
ratio
n C
ompa
riso
n P-
valu
e O
vera
ll C
omm
ent
C. o
dora
ta
3 ho
urs
Con
trol
0.25
%
.903
F
(3,1
2) =
3.0
5, p
= .0
7 (n
ot
sign
ifica
nt)
0.5%
.5
47
5%
.058
0.
25%
0.
5%
.903
5%
.1
76
0.5%
5%
.4
51
6
hour
s C
ontro
l 0.
25%
.9
90
F (3
,12)
= 3
.15,
p=
.053
(not
si
gnifi
cant
) 0.
5%
.739
5%
.0
55
0.25
%
0.5%
.8
86
5%
.092
0.
5%
5%
.284
9 ho
urs
Con
trol
0.25
%
.880
F
(3,1
2) =
9.0
2, p
= .0
02
(sig
nific
ant)
0.5%
.9
88
5%
.008
0.
25%
0.
5%
.719
5%
.0
02
0.5%
5%
0.
14
12
hou
rs
Con
trol
0.25
%
.056
F
(3,1
2) =
20.
14, p
= .0
0 (s
igni
fican
t) 0.
5%
.913
5%
.0
03
0.25
%
0.5%
.1
66
5%
.000
0.
5%
5%
.001
24
hou
rs
Con
trol
0.25
%
.666
F
(3,1
2) =
13.
38, p
= .0
0 (s
igni
fican
t) 0.
5%
.998
5%
.0
03
0.25
%
0.5%
.7
64
5%
.000
0.
5%
5%
.002
124
Tabl
e 6-
4: M
ultip
le C
ompa
rison
s (Po
st H
oc T
est)
for M
. koe
nigi
i (L)
Plan
t T
ime
Con
cent
ratio
n C
ompa
riso
n P-
valu
e O
vera
ll C
omm
ent
Mur
raya
ko
enig
ii (L
)
3 ho
urs
Con
trol
0.25
%
1.00
Th
at is
, F (3
,12)
= 4
.24,
p=
.029
(s
igni
fican
t) 0.
5%
.804
5%
.0
41
0.25
%
0.5%
.8
04
5%
.041
0.
5%
5%
.183
6 ho
urs
Con
trol
0.25
%
.908
F
(3,1
2) =
2.0
47, p
= .1
61 (n
ot
sign
ifica
nt)
0.5%
.9
42
5%
.355
0.
25%
0.
5%
.623
5%
.1
32
0.5%
5%
.6
60
9
hour
s C
ontro
l 0.
25%
.7
79
F (3
,12)
= 7
.57,
p=
.004
(s
igni
fican
t) 0.
5%
1.00
0 5%
.0
21
0.25
%
0.5%
.7
79
5%
.004
0.
5%
5%
.021
12 h
ours
C
ontro
l 0.
25%
.0
08
F (3
,12)
= 1
0.05
, p=
.001
(s
igni
fican
t) 0.
5%
1.00
0 5%
.7
31
0.25
%
0.5%
.0
09
5%
.001
0.
5%
5%
.686
24 h
ours
C
ontro
l 0.
25%
.7
12
F (3
,12)
= 1
.64,
p=
.232
( no
t si
gnifi
cant
) 0.
5%
.668
5%
.8
81
0.25
%
0.5%
1.
000
5%
.316
0.
5%
5%
.285
125
Tabl
e 6-
5: M
ultip
le C
ompa
rison
s (Po
st H
oc T
est)
for E
. hor
tens
is fo
rma
hort
ensi
s.
Plan
t T
ime
Con
cent
ratio
n C
ompa
riso
n P-
valu
e O
vera
ll C
omm
ent
Euo
dia
hort
ensi
s for
ma
hort
ensi
s
3 ho
urs
Con
trol
0.25
%
1.00
F
(3,1
2) =
4.2
4, p
= .0
29 (n
ot
sign
ifica
nt)
0.5%
.8
04
5%
.041
0.
25%
0.
5%
.804
5%
.0
41
0.5%
5%
.1
83
6
hour
s C
ontro
l 0.
25%
.4
83
F (3
,12)
= 6
.57,
p=
.007
(s
igni
fican
t) 0.
5%
.989
5%
.0
57
0.25
%
0.5%
.3
29
5%
.004
0.
5%
5%
.097
9 ho
urs
Con
trol
0.25
%
.314
F
(3,1
2) =
5.8
0, p
= .0
11
(sig
nific
ant)
0.5%
.8
63
5%
.158
0.
25%
0.
5%
.095
5%
.0
07
0.5%
5%
.4
66
12
hou
rs
Con
trol
0.25
%
.067
F
(3,1
2) =
5.0
7, p
= .0
17
(sig
nific
ant)
0.5%
.9
99
5%
.809
0.
25%
0.
5%
.082
5%
.0
14
0.5%
5%
.7
45
24
hou
rs
Con
trol
0.25
%
.085
F
(3,1
2) =
4.6
4, p
= .0
22
(sig
nific
ant)
0.5%
.7
08
5%
.815
0.
25%
0.
5%
.427
5%
.0
19
0.5%
5%
.2
56
126
Tabl
e 6-
6: M
ultip
le C
ompa
rison
s (Po
st H
oc T
est)
for C
. citr
atus
.
Plan
t T
ime
Con
cent
ratio
n C
ompa
riso
n P-
valu
e O
vera
ll C
omm
ent
Cym
bopo
gon
citr
atus
3 ho
urs
Con
trol
0.25
%
1.00
F
(3,1
2) =
13.
78, p
= .0
0 (s
igni
fican
t) 0.
5%
.216
5%
.0
01
0.25
%
0.5%
.2
16
5%
.001
0.
5%
5%
.020
6 ho
urs
Con
trol
0.25
%
.001
F
(3,1
2) =
106
7.33
, p=
.00
(sig
nific
ant)
0.5%
.0
00
5%
.000
0.
25%
0.
5%
.000
5%
.0
00
0.5%
5%
.0
00
9
hour
s C
ontro
l 0.
25%
.0
02
F (3
,12)
= 1
603.
48, p
= .0
0 (s
igni
fican
t) 0.
5%
.000
5%
.0
00
0.25
%
0.5%
.0
00
5%
.000
0.
5%
5%
.000
12 h
ours
C
ontro
l 0.
25%
.0
01
F (3
,12)
= 4
28.3
5, p
= .0
0 (s
igni
fican
t) 0.
5%
.000
5%
.0
00
0.25
%
0.5%
.1
10
5%
.000
0.
5%
5%
.000
24 h
ours
C
ontro
l 0.
25%
.1
24
F (3
,12)
= 2
00.9
2, p
= .0
0 (s
igni
fican
t) 0.
5%
.000
5%
.0
00
0.25
%
0.5%
.0
00
5%
.000
0.
5%
5%
.000
127
Tabl
e 6-
7: M
ultip
le C
ompa
rison
s (Po
st H
oc T
est)
for O
. ten
uiflo
rum
L
Plan
t T
ime
Con
cent
ratio
n C
ompa
riso
n P-
valu
e O
vera
ll C
omm
ent
Oci
mum
te
nuifl
orum
L
3 ho
urs
Con
trol
0.25
%
.112
F
(3,1
2) =
293
.94,
p=
.00
(sig
nific
ant)
0.5%
.0
00
5%
.000
0.
25%
0.
5%
.032
5%
.0
00
0.5%
5%
.0
00
6
hour
s C
ontro
l 0.
25%
.0
01
F (3
,12)
= 6
11.4
0, p
= .0
0 (
sign
ifica
nt)
0.5%
.0
00
5%
.000
0.
25%
0.
5%
.003
5%
.0
00
0.5%
5%
.0
00
9
hour
s C
ontro
l 0.
25%
.0
05
F (3
,12)
= 7
72.0
3, p
= .0
0 (s
igni
fican
t) 0.
5%
.000
5%
.0
00
0.25
%
0.5%
.0
00
5%
.000
0.
5%
5%
.000
12 h
ours
C
ontro
l 0.
25%
.3
41
F (3
,12)
= 9
6.34
, p=
.00
(sig
nific
ant)
0.5%
.0
01
5%
.000
0.
25%
0.
5%
.019
5%
.0
00
0.5%
5%
.0
00
24
hou
rs
Con
trol
0.25
%
.382
F
(3,1
2) =
42.
07, p
= .0
0 (s
igni
fican
t) 0.
5%
.000
5%
.0
00
0.25
%
0.5%
.0
02
5%
.000
0.
5%
5%
.018
128
6.1.1.1 Probit Analysis
Ocimum tenuiflorum L Cymbopogon citratus
Cananga odorata E. hortensis forma hortensis
y = 0.4062x + 1.5701 R² = 0.6512
0
0.2
0.4
0.6
0.8
1
-3 -2.5 -2 -1.5 -1 -0.5 0
24 h
rs
Log(Concentration)
Logistic regression of 24 hrs by Log(Concentration)
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
y = 0.4897x + 1.6577 R² = 0.9022
0
0.2
0.4
0.6
0.8
1
-3 -2.5 -2 -1.5 -1 -0.5 0
24 h
rs
Log(Concentration)
Logistic regression of 24 hrs by Log(Concentration)
ActiveModelNatural mortalityLower bound (95%)Upper bound (95%)Linear (Active)
y = 0.2768x + 0.8558 R² = 0.7501
0
0.2
0.4
0.6
0.8
1
-3 -2.5 -2 -1.5 -1 -0.5 0
24 h
rs
Log(Concentration)
Logistic regression of 24 hrs by Log(Concentration)
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
Linear (Active)
y = 0.0685x + 0.3266 R² = 0.5862
0
0.2
0.4
0.6
0.8
1
-3 -2.5 -2 -1.5 -1 -0.5 0
24 h
rs
Log(Concentration)
Logistic regression of 24 hrs by Log(Concentration)
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
129
Murraya koenigii
Figure 6-2: Probit analysis of fumigant test on selected essential oils at different time
interval.
y = 0.0496x + 0.2976 R² = 0.316
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-3 -2.5 -2 -1.5 -1 -0.5 0
24 h
rs
Log(Concentration)
Logistic regression of 24 hrs by Log(Concentration)
Active Model
Natural mortality Lower bound (95%)
Upper bound (95%) Linear (Active)
130
6.1.2 Repellent Test
Table 6-8: Independent Sample t-test for repellent test
Plants Concentration Comparisons
Significant/ Not significant
Murraya koenigii (L)
Control
0.25% t(6)=-1.698, p= .278 (not significant) 0.5% t(6)=.608, p= .919 (not significant) 5% t(6)= 5.286, p= .000 (significant)
Cymbopogon citratus
Control
0.25% t(6)= -.442, p= .102 (not significant) 0.5% t(6)= .164, p= .436 (not significant) 5% t(6)= 2.662, p= .197 (not significant)
C. odorata
Control 0.25% t(6)= -1.101, p= .418 (not significant) 0.5% t(6)= -2.400, p= .248 (not significant) 5% t(6)= .362, p= .541 (not significant)
Ocimum tenuiflorum L
Control
0.25% t(6)= -.880, p= .949 (not significant) 0.5% t(6)= -3.001, p= .710 (not significant) 5% t(6)= 1.709, p= .693 (not significant)
Euodia hortensis forma hortensis
Control
0.25% t(6)= -.618, p= .386 (not significant) 0.5% t(6)= -.179, p= .916 (not significant) 5% t(6)= .607, p= .808 (not significant)
6.1.2.1 Probit Analysis -Graphs
Cymbopogon citratus Cananga odorata
y = 0.2259x + 0.602 R² = 0.3232
0
0.5
1
-0.8 -0.3 0.2 0.7Repe
lled
Log(Concentration(%))
Logistic regression of Repelled by Log(Concentration(%))
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
y = 0.0998x + 0.4364 R² = 0.0795
0
0.5
1
-0.8 -0.3 0.2 0.7Repe
lled
Log(Concentration(%))
Logistic regression of Repelled by Log(Concentration(%))
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
131
Ocimum tenuiflorum L E. hortensis forma hortensis
Murraya koenigii
Figure 6-3: Probit analysis of repellent test on selected essential oils.
y = 0.1484x + 0.4342 R² = 0.1582
0
0.2
0.4
0.6
0.8
1
-0.8 -0.3 0.2 0.7
Repe
lled
Log(Concentration(%))
Logistic regression of Repelled by Log(Concentration(%))
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
y = 0.0541x + 0.5023 R² = 0.028
0
0.2
0.4
0.6
0.8
1
-0.8 -0.3 0.2 0.7
Repe
lled
Log(Concentration(%))
Logistic regression of Repelled by Log(Concentration(%))
Active
Model
Natural mortality
Lower bound (95%)
Upper bound (95%)
Linear (Active)
y = 0.2637x + 0.5984 R² = 0.6111
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-0.8 -0.3 0.2 0.7
Repe
lled
Log(Concentration(%))
Logistic regression of Repelled by Log(Concentration(%))
Active Model
Natural mortality Lower bound (95%)
Upper bound (95%) Linear (Active)
132
6.2 Microbiology
6.2.1 Bacteria
Standard Control - Ampicillin discs
Table 6-9: Effect of control on selected bacteria
Table 6-10: Descriptive statistics for zone of inhibition (mm) across different
concentrations
6.2.1.1 ANOVA analysis using tukey’s test
Bacteria Standard Control (mm) Thermus thermophilus
14.80
Pseudomonas aeruginosa
14.61
Streptococcus (pneumonia)
41.48
Staphylococcus aureus
41.53
Salmonella
24.26
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
133
Bac
teri
a C
once
ntra
tion
(x10
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Pla
nt
com
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son
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val
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134
Bac
teri
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ntra
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Pl
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ci
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Lem
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46
Bac
teri
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once
ntra
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(x10
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Pl
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val
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Bac
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va
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135
Bac
teri
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once
ntra
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(x10
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Pl
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val
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Bac
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Pl
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136
Bac
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Pla
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mus
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Cur
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0.00
25
Mak
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0.
0025
Tu
lsi
1.00
0
0.00
25
Uci
1.
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0025
Le
mon
gr
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Mak
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138
Bac
teri
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(x10
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Pl
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not
Th
erm
us
ther
mop
hile
s 0.
5 C
urry
le
aves
Le
mon
gr
ass
.000
F(
4,20
)=
13.0
11,
p=.0
00
0.
5 M
akas
oi
.000
0.5
Tuls
i .0
01
0.
5 U
ci
.027
0.5
Lem
on
gras
s M
akas
oi
.951
0.5
Tuls
i .3
96
0.
5 U
ci
.035
0.5
Mak
asoi
Tu
lsi
.807
0.5
Uci
.1
42
0.
5 Tu
lsi
Uci
.6
60
Bac
teri
a C
once
ntra
tion
(x10
0%)
Pl
ant c
ompa
riso
n P-
val
ue
Ove
rall
sign
ifica
nt
or n
ot
Ther
mus
th
erm
ophi
les
1 C
urry
le
aves
Le
mon
gr
ass
.000
F(
4,20
)=
14.2
63,
p=.0
00
1
Mak
asoi
.0
00
1
Tuls
i .0
00
1
Uci
.0
01
1
Lem
on
gras
s M
akas
oi
.862
1 Tu
lsi
.987
1 U
ci
.605
1 M
akas
oi
Tuls
i .5
93
1
Uci
.9
89
1
Tuls
i U
ci
.329
139
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
5 125
0.272 1.766 6.59 43.41
25 125
3.32 5.27 1.50 1.86
50 125
5.75 6.51 0.79 -0.37
100 125
14.76 12.13 0.70 -0.15
140
6.2.
2.1
ANO
VA a
naly
sing
usi
ng tu
key’
s tes
t
Fung
i C
once
ntra
tion
Pla
nt c
ompa
riso
n P-
va
lue
Ove
rall
sign
ifica
nt
or n
ot
Aspe
rgill
us
coni
diop
hore
s 0.
25
Cur
ry
leav
es
Lem
on
gras
s .0
10
F(4,
20)=
27
.174
, p=
.000
0.25
M
akas
oi
1.00
0
0.25
Tu
lsi
.000
0.25
U
ci
1.00
0
0.25
Le
mon
gr
ass
Mak
asoi
.0
10
0.
25
Tuls
i .0
01
0.
25
Uci
.0
10
0.
25
Mak
asoi
Tu
lsi
.000
0.25
U
ci
1.00
0
0.25
Tu
lsi
Uci
.0
00
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P-
valu
e O
vera
ll si
gnifi
cant
or
not
As
perg
illus
co
nidi
opho
res
0.5
Cur
ry
leav
es
Lem
on
gras
s .0
14
F(4,
20)=
13
.521
, p=
.000
0.5
Mak
asoi
1.
000
0.
5 Tu
lsi
.000
0.5
Uci
.0
02
0.
5 Le
mon
gr
ass
Mak
asoi
.0
14
0.
5 Tu
lsi
.307
0.5
Uci
.8
87
0.
5 M
akas
oi
Tuls
i .0
00
0.
5 U
ci
.002
0.5
Tuls
i U
ci
.823
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P-
valu
e O
vera
ll si
gnifi
cant
or
not
Pe
nici
llum
co
nidi
a 0.
05
Cur
ry
leav
es
Lem
on
gras
s 1.
000
F(4,
20)=
2.
636,
p=
.065
0.05
M
akas
oi
1.00
0
0.05
Tu
lsi
.115
0.05
U
ci
1.00
0
0.05
Le
mon
gr
ass
Mak
asoi
1.
000
0.
05
Tuls
i .1
15
0.
05
Uci
1.
000
0.
05
Mak
asoi
Tu
lsi
.115
0.05
U
ci
1.00
0
0.05
Tu
lsi
Uci
.1
15
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P-
valu
e O
vera
ll si
gnifi
cant
or
not
As
perg
illus
co
nidi
opho
res
1 C
urry
le
aves
Le
mon
gr
ass
.418
F(
4,20
)=
8.87
7,
p=.0
00
1
Mak
asoi
.3
26
1
Tuls
i .0
00
1
Uci
.0
09
1
Lem
on
gras
s M
akas
oi
1.00
0
1 Tu
lsi
.010
1 U
ci
.291
1 M
akas
oi
Tuls
i .0
15
1
Uci
.3
77
1
Tuls
i U
ci
.447
141
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Pe
nici
llum
co
nidi
a
0.25
C
urry
le
aves
Le
mon
gr
ass
.000
F(
4,20
)=
25.3
22,
p=.0
00
0.
25
Mak
asoi
1.
000
0.
25
Tuls
i .0
00
0.
25
Uci
1.
000
0.
25
Lem
on
gras
s M
akas
oi
.000
0.25
Tu
lsi
.875
0.25
U
ci
.000
0.25
M
akas
oi
Tuls
i .0
00
0.
25
Uci
1.
000
0.
25
Tuls
i U
ci
.000
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Pe
nici
llum
co
nidi
a 0.
5 C
urry
le
aves
Le
mon
gr
ass
.001
F(
4,20
)=
10.9
33,
p=.0
00
0.
5 M
akas
oi
1.00
0
0.5
Tuls
i .0
01
0.
5 U
ci
.529
0.5
Lem
on
gras
s M
akas
oi
.001
0.5
Tuls
i 1.
000
0.
5 U
ci
.042
0.5
Mak
asoi
Tu
lsi
.001
0.5
Uci
.5
29
0.
5 Tu
lsi
Uci
.0
46
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Rh
izop
us
spor
angi
a
0.25
C
urry
le
aves
Le
mon
gr
ass
.056
F(
4,20
)=
9.99
7,
p=.0
00
0.
25
Mak
asoi
.0
01
0.
25
Tuls
i .0
05
0.
25
Uci
1.
000
0.
25
Lem
on
gras
s M
akas
oi
.398
0.25
Tu
lsi
.797
0.25
U
ci
.056
0.25
M
akas
oi
Tuls
i .9
56
0.
25
Uci
.0
01
0.
25
Tuls
i U
ci
.005
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Pe
nici
llum
co
nidi
a
1 C
urry
le
aves
Le
mon
gr
ass
.460
F(
4,20
)=
6.21
6,
p=.0
02
1
Mak
asoi
.0
78
1
Tuls
i .0
01
1
Uci
.0
71
1
Lem
on
gras
s M
akas
oi
.817
1 Tu
lsi
.038
1 U
ci
.790
1 M
akas
oi
Tuls
i .2
79
1
Uci
1.
000
1
Tuls
i U
ci
.302
142
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
So
dari
a gr
ay
0.25
C
urry
le
aves
Le
mon
gr
ass
1.00
0 F(
4,20
)=
18.9
31,
p=.0
00
0.
25
Mak
asoi
.1
18
0.
25
Tuls
i .0
00
0.
25
Uci
1.
000
0.
25
Lem
on
gras
s M
akas
oi
.118
0.25
Tu
lsi
.000
0.25
U
ci
1.00
0
0.25
M
akas
oi
Tuls
i .0
02
0.
25
Uci
.1
18
0.
25
Tuls
i U
ci
.000
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Rh
izop
us
spor
angi
a 1
Cur
ry
leav
es
Lem
on
gras
s .0
00
F(4,
20)=
25
.549
, p=
.000
1 M
akas
oi
.001
1 Tu
lsi
.000
1 U
ci
.000
1 Le
mon
gr
ass
Mak
asoi
.1
96
1
Tuls
i .0
72
1
Uci
.9
80
1
Mak
asoi
Tu
lsi
.000
1 U
ci
.449
1 Tu
lsi
Uci
.0
23
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
R
hizo
pus
spor
angi
a 0.
5 C
urry
le
aves
Le
mon
gr
ass
.005
F(
4,20
)=
5.13
7,
p=.0
05
0.
5 M
akas
oi
.021
0.5
Tuls
i .0
22
0.
5 U
ci
.149
0.5
Lem
on
gras
s M
akas
oi
.959
0.5
Tuls
i .9
52
0.
5 U
ci
.476
0.5
Mak
asoi
Tu
lsi
1.00
0
0.5
Uci
.8
58
0.
5 Tu
lsi
Uci
.8
71
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
So
dari
a gr
ay
0.05
C
urry
le
aves
Le
mon
gr
ass
1.00
0 F(
4,20
)=
1.00
0,
p=.4
31
0.
05
Mak
asoi
.5
25
0.
05
Tuls
i 1.
000
0.
05
Uci
1.
000
0.
05
Lem
on
gras
s M
akas
oi
.525
0.05
Tu
lsi
1.00
0
0.05
U
ci
1.00
0
0.05
M
akas
oi
Tuls
i .5
25
0.
05
Uci
.5
25
0.
05
Tuls
i U
ci
1.00
0
143
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
So
dari
a gr
ay
0.5
Cur
ry
leav
es
Lem
on
gras
s .3
66
F(4,
20)=
11
.123
, p=
.000
0.5
Mak
asoi
.0
37
0.
5 Tu
lsi
.000
0.5
Uci
.9
51
0.
5 Le
mon
gr
ass
Mak
asoi
.7
09
0.
5 Tu
lsi
.004
0.5
Uci
.7
77
0.
5 M
akas
oi
Tuls
i .0
68
0.
5 U
ci
.149
0.5
Tuls
i U
ci
.000
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
So
dari
a gr
ay
1 C
urry
le
aves
Le
mon
gr
ass
.999
F(
4,20
)=
9.89
5,
p=.0
00
1
Mak
asoi
.7
16
1
Tuls
i .0
01
1
Uci
.0
24
1
Lem
on
gras
s M
akas
oi
.578
1 Tu
lsi
.000
1 U
ci
.015
1 M
akas
oi
Tuls
i .0
12
1
Uci
.2
70
1
Tuls
i U
ci
.518
Fu
ngi
Con
cent
ratio
n Pl
ant c
ompa
riso
n P-
val
ue
Ove
rall
sign
ifica
nt
or n
ot
Sord
aria
w
ild
0.5
Cur
ry
leav
es
Lem
on
gras
s .9
20
F(4,
20)=
8.
634,
p=
.000
0.5
Mak
asoi
.2
28
0.
5 Tu
lsi
.000
0.5
Uci
.1
53
0.
5 Le
mon
gr
ass
Mak
asoi
.6
62
0.
5 Tu
lsi
.001
0.5
Uci
.5
21
0.
5 M
akas
oi
Tuls
i .0
29
0.
5 U
ci
.999
0.5
Tuls
i U
ci
.047
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
So
rdar
ia
wild
0.
25
Cur
ry
leav
es
Lem
on
gras
s .9
66
F(4,
20)=
2.
998,
p=
.043
0.25
M
akas
oi
.941
0.25
Tu
lsi
.052
0.25
U
ci
1.00
0
0.25
Le
mon
gr
ass
Mak
asoi
1.
000
0.
25
Tuls
i .1
77
0.
25
Uci
.9
66
0.
25
Mak
asoi
Tu
lsi
.213
0.25
U
ci
.941
0.25
Tu
lsi
Uci
.0
52
144
Fung
i C
once
ntra
tion
Plan
t com
pari
son
P- v
alue
O
vera
ll si
gnifi
cant
or
not
Sord
aria
w
ild
1 C
urry
le
aves
Le
mon
gr
ass
.994
F(
4,20
)=
7.97
7,
p=.0
01
1
Mak
asoi
.4
91
1
Tuls
i .0
02
1
Uci
.0
57
1
Lem
on
gras
s M
akas
oi
.286
1
Tuls
i .0
01
1
Uci
.0
25
1
Mak
asoi
Tu
lsi
.084
1
Uci
.7
00
1
Tuls
i U
ci
.607
145
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