use of plant essential oils for the control of...

USE OF PLANT ESSENTIAL OILS FOR THE

CONTROL OF FUNGAL DETERIORATION

IN CITRUS DURING STORAGE

NOSHEEN AKHTAR

INSTITUTE OF AGRICULTURAL SCIENCES,

UNIVERSITY OF THE PUNJAB,

LAHORE, PAKISTAN

USE OF PLANT ESSENTIAL OILS FOR THE

CONTROL OF FUNGAL DETERIORATION

IN CITRUS DURING STORAGE

By

Nosheen Akhtar

A THESIS SUBMITTED FOR THE FULFILLMENT OF DEGREE OF

Doctor of Philosophy in

Institute of Agricultural Sciences

Supervisor Asst. Prof. Dr. Tehmina Anjum

Institute of Agricultural Sciences,

University of the Punjab, Lahore, Pakistan

September 2014

i

Certificate

This is to certify that the research work entitled “Use of Plant Essential Oils for the

Control of Fungal Deterioration in Citrus during Storage” described in this thesis by

Miss. Nosheen Akhtar, is an original work of the author and has been carried out under

my direct supervision. We have personally gone through all the data, results, materials

reported in the manuscript and certify their correctness and authenticity. We further

certify that the material included in this thesis has not been used in part or full in a

manuscript already submitted or in the process of submission in partial or complete

fulfillment of the award of any other degree from any institution. I also certify that the

thesis has been prepared under my supervision according to the prescribed format and we

endorse its evaluation for the award of Ph.D. degree through the official procedure of the

University of the Punjab, Lahore.

Supervisor

(Dr. Tehmina Anjum)

Assistant Professor

Institute of Agricultural

Sciences

University of the Punjab,

Lahore, Pakistan.

Date: ______________________

ii

Declaration Certificate

This thesis entitled “Use of Plant Essential Oils for the Control of Fungal

Deterioration in Citrus during Storage” is being submitted for the degree of Ph.D. in

the University of the Punjab does not contain any material which has been submitted for

the award of Ph.D. degree in any other university and, to the best of my knowledge and

belief, neither does this thesis contain any material published or written previously by

another person, except when due reference is made to the source in the text of the thesis.

(Nosheen Akhtar)

Ph.D. Scholar

iii

Dedication

This work is dedicated to:

My Parents

For their unconditional love

My Husband & my Son For their encouragement and love

iv

ACKNOWLEDGEMENT

All praises and thanks are for almighty ALLAH who blessed us with knowledge,

courage, wisdom and all other bounties of life and enormous blessings on Prophet Hazrat

Muhammad (Peace Be Upon Him), the most perfect and exhaled among ever born on

earth.

I am under immense gratitude to my supervisor, Assistant Professor, Dr. Tehmina

Anjum for her skillful guidance, golden suggestions and untiring help whenever I needed

that. Her generous response to my difficulties will never be forgotten. It was only because

of her kind cooperation that writing this manuscript was very easy for me.

Special thanks are for Professor Dr. Saleem Haider, Director, Institute of

Agricultural Sciences, University of the Punjab, for providing me all the possible

facilities to complete this research work.

I would like to pay a humble thanks and appreciation to Dr. Mateen Abbas,

Assistant Professor University of Vetnary Lahore, for giving me a chance to work in his

well-equipped laboratory. There is a pleasure in recording my humble thanks to Mr.

Abdul Muqeet, (Research officer) for their illuminating and inspiring guidance and ever

encouraging attitude in carrying out the research work on analytical analysis of essential

oil. Without their help, these investigations would never have been possible.

I am also grateful to Higher Education Commission (HEC), Pakistan for awarding

me the Indigenous PhD Fellowship to carry out my research work smoothly.

I have the honor to express my deep sense of gratitude to Miss Rasheda jabeen,

under who’s extremely sincere, unfathomable, cooperative nature, incalculable guidance,

subtle direction, this project was successfully executed.

I am quite thankful to all the non-academic staff of IAGS, Mr. Muhammad Aslam

(PS to director), Mr. Irfan, Mr. Taufeeq, Mr. Ahsan Zaidi, Mr. Nasir, Miss Aliya Ahmed

and Miss Faiza, who’s “always ready to help” attitude ensured my research

accomplishment.

I am delighted to express my heartiest thanks to friends Sana Hanif, Wajiha Iram,

Amna Ali and Mehreen Hassan, who never failed to help me whenever their help was

required I would also like to thank Mr. Waheed Akram for his advices during statistical

analyses of data presented in this thesis.

v

I express my greatest gratitude to my beloved husband Mr. Wasim Iqbal and son

Mr. Azlan Wasim for their support, huge sacrifices, cooperation, and encouragement

throughout to keep me going when frustrations and other problems drained my

motivation. A profound thanks to all other members of my family especially father in

law, Saima, Saleem Raza and Qaiser Nawaz for their support and patients.

Last but not least, I really acknowledge and offer my heartiest gratitude to my

loving parents for their huge sacrifice, moral support, encouragement and mellifluous

affection. My brothers, Amir Zahoor and Imran Zahoor and my sister Noreen Shehzal supported

me in all respects to achieve this milestone.

Nosheen Akhtar

vi

SUMMARY

Citrus stands first in area and production among the world’s tree fruits. Pakistan is

an important citrus producer across the globe. In Pakistan, only 10% of the fruit is

exported, 2% is processed, 40% is lost in post-harvest process and remaining 48% is

supplied to domestic markets. A significant portion of losses during post-harvest period is

attributed to diseases caused by fungi. This not only destroys the fruit but also affect the

marketing of fruit leading to overall reduced GDP of the country. Globally many

postharvest strategies have been developed which includes use of different chemicals

such as fungicides. These chemicals are not biologically safe and this necessitates a more

intensive search for alternative management strategies that are economic, sustainable and

environment friendly with minimal risks to health and life.

In present study essential oils derived from natural sources were used to enhance the shelf

life of mandarin fruit and to improve their quality during cold storage conditions. Brief

stepwise investigations, consequent findings and conclusions are as follows:

A survey of citrus producing areas of Punjab, Pakistan was conducted and

mandarin samples were collected to isolate citrus deteriorating fungi during cold

storage conditions. Twenty two storage houses were sampled in eight major citrus

growing cities. A total of sixteen different fungal species belonging to seven

different genera were identified on the basis of their morphological

characteristics. Penicillium italicum was found with maximum percentage of

occurrence (29.75%).

Genetic analysis among eight isolates of P. italicum was done by using Random

Amplified Polymorphic DNA (RAPD) finger printing technique, in which 20

decamers were used for testing the genetic variability. These isolates found to fall

in three major groups belonging to different geographical regions of citrus

producing areas. G1 comprised of five isolates out of eight i.e. isolates from

Lahore, Multan, Jhang, Bhakar and Sargodha.

Genetic differentiation also accounts for the difference in pathogenicity levels of

isolates. After screening of isolates, Lahore (Lhr) isolate was found to be the most

vii

pathogenic and in turn different antifungal assays were performed using essential

oils of ten plants against P. italicum Lhr isolate.

Essential oils were extracted from seeds of Trachyspermum captivum L.,

Foeniculum vulgare M., Cuminum cyminum L., buds of Eugenia caryophyllata

T., bark of Cinnnamomum zylanicum J. and leaves of Azadirachta indica L.,

Ocimum basilicum L., Ocimum sanctum L. and Eucalyptus globulus L. by using

hydro-distillation process. The In Vitro antifungal efficacy was first checked by

both agar dilution and volatile assays and then through In Vivo assays at 25oC.

Comparative evaluation of ten essential oils revealed T. captivum, F. vulgare and

E. caryophyllata as three most effective antifungal oils. At lowest tested

concentration of 3µL mL-1

maximum In Vitro inhibition of 100% was caused by

T. captivum essential oil which was followed by 98 and 84% inhibition by F.

vulgare and E. caryophallata essential oil. In In Vivo assays the three essential

oils resulted in 96, 93.2 and 62.7% inhibition respectively.

All essential oils were then subjected to chemical profiling by using Gas

Chromatography-Mass Spectrometery (GC-MS) and Gas Chromatography- Flame

Ionization Detector (GC-FID). The chromatograms analyzed confirmed the

presence of thymol, anethole, eugenol, citral, linalool, limonene, 1,8-cineole,

eucalyptol, ɣ-terpinene and β-pinene as major constituents of selected essential

oils.

Among these compounds thymol was found to be the most effective chemical

component that highly significantly inhibited the fungal growth. This was

followed by anethole and eugenol. Maximum quantity of thymol was recorded in

T. captivum, whereas, anethole and eugenol was found highest in F. vulgare and

E. caryophyllata respectively.

Mandarin fruits were treated with top three essential oils (T. captivum, F.

vulgare and E. caryophyllata) with different concentrations i.e. 100 and 200µL L-

1 for 10 and 20 minutes to study their effect on physicochemical properties of the

fruit when stored at 4oC for eight weeks. At the end of the storage period weight

loss, juice content, total soluble solids, titratable acidity, pH, and ascorbic acid

content were determined. Results obtained in the present study indicated that T.

viii

captivum essential oil effectively enhanced the quality and shelf life of the fruit

during cold storage.

To assess the significance of the results obtained during various experiments data

was subjected to a number of statistical analyses. These analyses include Duncan’s

Multiple Range Test (DMRT) and Fisher’s Least Significant Differences (LSD) test.

The findings have been further argued and discussed with reference to isolation

and identification of storage fungi, In Vitro and In Vivo antifungal potential of essential

oils against blue mold fungus and interaction of these essential oils in rendering various

physicochemical changes in mandarin fruits during cold storage.

The study justifies its credibility of using essential oils as an environment safe

strategy against citrus spoiling fungi during cold storage which can be a substitute for

synthetic chemical fungicides.

ix

CONTENTS

TITLE PAGE #

Certificate I

Declaration Certificate II

Dedication III

Acknowledgement IV

Summary

VI

Table of Contents

IX

List of Illustration XIII

List of Tables XVI

List of Plates XVII

List of Abbreviations XVIII

CHAPTER 1: INTRODUCTION

1.1. Citrus production 2

1.1.1. Citrus production worldwide 2

1.1.2. Citrus production in Pakistan 3

1.2. Physicochemical changes in fruit during cold storage 6

1.3. Citrus post-harvest losses 7

1.3.1. Blue mold disease in citrus 8

1.4. Control measures 9

1.5. Essential oils 11

1.5.1. Extraction of essential oils 12

1.5.2. Biological activities of essential oils 14

1.6. Antifungal mechanism of essential oils 18

1.7. Characterization of essential oils 19

1.8. Aims and objectives 20

CHAPTER 2: MATERIAL AND METHODS

2.1. Sample collection 21

2.2. Isolation of storage fungi 23

2.2.1. Media composition 23

2.3. Morphological characterization 24

2.4. Analysis of genetic diversity among fungal isolates 24

2.4.1. DNA Extraction 24

2.4.2. Estimation of extracted DNA 25

2.4.3. DNA Quantification and DNA quality analysis 25

2.4.4. Agarose gel electrophoresis 26

2.4.5. Random amplification of polymorphic DNA analysis 26

2.4.5.1. Random primer screening 27

2.4.5.2. RAPD reaction mixture 28

2.4.5.3. RAPD temperature cycling conditions 28

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2.4.5.4. Analysis of amplified DNA fragments 29

2.4.5.5. Data analysis 29

2.5. Screening of most pathogenic isolate of P. italicum 30

2.6. Collection of plant material 30

2.7. Extraction of essential oils 31

2.8. Analysis of essential oils 31

2.8.1. Chemical analysis 31

2.8.1.1. GC-MS analysis 32

2.8.1.2. GC-FID analysis 32

2.9. Antifungal activity 33

2.9.1. Concentration of essential oils 33

2.9.2. Preparation of spore suspension 33

2.9.3. In Vitro antifungal assay 33

2.9.3.1. Agar dilution assay 33

2.9.3.2. Volatile assay 33

2.9.4. In Vivo antifungal assay 34

2.9.4.1. Surface sterilization of mandarin fruit 34

2.9.4.2. Fruit inoculation with fungal spores and plant

EOs 34

2.10. In Vitro antifungal activity of major components of essential oils

against P. italicum 35

2.11. Effect of selected essential oils on post-harvest physicochemical

changes in citrus fruit during cold storage 36

2.11.1. Weight loss 36

2.11.2. Juice content 37

2.11.3. pH 37

2.11.4. Total soluble solids 37

2.11.5. Ascorbic acid 37

2.11.6. Titratable acidity (TA) 38

2.12. Statistical analysis 38

CHAPTER 3: RESULTS

3.1. Isolation and identification of storage fungi 39

3.1.1. Morphological identification 39

3.1.2 Analysis of genetic diversity among fungal isolates 39

3.1.3. Genetic analysis of P. italicum isolates 44

3.1.4. Screening of most pathogenic isolate of P. italicum 49

3.2. Antifungal activity of essential oils 51

3.2.1. AZADIRACHTA INDICA 51

3.2.1.1. In Vitro antifungal efficacy 51

3.2.1.2. In Vivo antifungal efficacy 51

3.2.1.3. Chemical composition 52

3.2.1.4. In Vitro antifungal efficacy of major components

of A. indica essential oil 56

3.2.2. CINNAMOMUM ZYLANICUM 57




xi

3.2.2.4. In Vitro antifungal efficacy of major components of

C. zylanicum essential oil 62

3.2.3. CUMINUM CYMINUM 62





C. cyminium essential oil 67

3.2.4. CYMBOPOGON CITRATUS 68





C. citratus essential oil 72

3.2.5. EUCALYPTUS GLOBULUS 73





E. globulus essential oil 78

3.2.6. EUGENIA CARYOPHYLLATA 79





E. caryophyllata essential oil 84

3.2.7. FOENICULUM VULGARE 85





F. vulgare essential oil 89

3.2.8. OCIMUM BASILICUM 90





O. basilicum essential oil 95

3.2.9. OCIMUM SANCTUM 96





O. sanctum essential oil 100

3.2.10. TRACHYSPERMUM CAPTIVUM 101





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T. captivum essential oil 106

3.2.11. Selection of most effective essential oil 107

3.2.11.1. In Vitro antifungal assay 107

3.2.11.2. In Vivo antifungal assay 108

3.2.12. Effect of essential oils on post-harvest physicochemical changes

in citrus fruit during cold storage 114

3.2.12.1. Weight loss of fruit 114

3.2.12.2. Juice content 117

3.2.12.3. pH 120

3.2.12.4. Total soluble solids 123

3.2.12.5. Ascorbic acid content 126

3.2.12.6.Titratable acidity 129

CHAPTER 4: Discussion 136

Conclusion 149

Future prospects 149

References 150

xiii

LIST OF ILLUSTRATIONS

TITLE PAGE #


1.1. Export of kinnow from Pakistan ................................................................................ 4

1.2: Penicillium Italicum ................................................................................................... 9


2.1: Map of Punjab Pakistan arrows showing major sampling cities. ........................................... 21

2.2: Three PCR heat steps were involved in temperature optimization ...................................... 29

CHAPTER 3: RESULTS

3.1: RAPD profile of eight isolates of P. italicum produced by random primers .......................... 47

3.2: UPGMA cluster analysis based dendogram depicting the genetic relationship among selected

P. italicum isolates. ................................................................................. 48

3.3: Screening of most pathogenic isolate Of Penicillium italicum. .............................................. 49

3.4: In Vitro antifungal activity of A. indica essential oil, used at various concentrations on

mycelial growth of P. italicum ............................................................................... 53

3.5: In Vivo antifungal activity of a. indica essential oil, used in various concentrations, on

mycelial growth of P. italicum. ................................................................................. 53

3.6: GC-MS chromatogram of essential oil extracted from leaves of A. indica. ........................... 54

3.7: Mycelial growth of P. italicum measured after 7 days of incubation on MEA medium, treated

with different concentrations of major components of A. indica essential oil.. ...................56 3.8: In Vitro antifungal activity of C. zylanicum essential oil, used at various concentrations on

mycelial growth of P. italicum.. ................................................................................. 58

3.9: In Vivo antifungal activity of C. zylanicum essential oil, used in various concentrations, on

mycelial growth of P. iItalicum ................................................................................. 58

3.10: GC-MS chromatogram of essential oil extracted from bark of C. zylanicum. ...................... 59

3.11: Mycelial growth of P. italicum measured after 7 days of incubation on MEA medium,

treated with different concentrations of major components of C. zylanicum essential oil.. .. 61

3.12: In Vitro antifungal activity of C. cyminum essential oil, used at various concentrations on

mycelial growth of P. italicum ................................................................................. 63

3.13: In Vivo antifungal activity of C. cyminum essential oil, used in various concentrations, on


3.14: GC-MS chromatogram of essential oil extracted from seeds of C. cyminium. ..................... 64

3.145: Mycelial growth of P. italicum measured after 7 days of incubation on mea medium,

treated with different concentrations of major components of C. cyminium essential oil.. ... 67

3.16: In Vitro antifungal activity of C. citratus essential oil, used at various concentrations on


3.17: In Vivo antifungal activity of C. citratus essential oil, used in various concentrations, on

mycelial growth of P. italicum.. ................................................................................. 69

3.18: GC-MS chromatogram of essential oil extracted from leaves of C. citratus. .....................70 3.19: Mycelial growth of P.italicum measured after 7 days of incubation on MEA medium,

treated with different concentrations of major components of C. citratus Essential Oil. ...... 72

3.20: In Vitro antifungal activity of E. globulus essential oil, used at various concentrations on


3.21: In Vivo antifungal activity of E. globulus essential oil, used in various concentrations, on

mycelial growth of P. Italicum. ................................................................................ 74

xiv

3.22: GC-MS chromatogram of essential oil extracted from leaves of E. globulus. ..................... 75

3.23: Mycelial growth of P. Italicum measured after 7 days of incubation on MEAmedium,

treated with different concentrations of major components of E. globulus essential oil.. ..... 78

3.24: In Vitro antifungal activity of E. caryophyllata essential oil, used at various concentrations

on mycelial growth of P. Italicum.. ................................................................................. 81

3.25: In Vivo antifungal activity of E. caryophyllata essential oil, used in various concentrations,

on mycelial growth of P. ialicum. ................................................................................. 81

3.26: GC-MS chromatogram of essential oil extracted from buds of E. caryophyllata. ............... 82


treated with different concentrations of major components of E. caryophyllata.. ................. 84

3.28: In Vitro antifungal activity of F. vulgare essential oil, used at various concentrations on


3.29: In Vivo antifungal activity of F. vulgare essential oil, used in various concentrations, on


3.30: GC-MS chromatogram of essential oil extracted from leaves of F. vulgare. ....................... 87


treated with different concentrations of major components of F. vulgare. ............................ 89

3.32: In Vitro antifungal activity of O. basilicum essential oil, used at various concentrations on


3.33: In Vivo antifungal activity of O. basilicum essential oil, used in various concentrations, on


3.34: GC-MS chromatogram of essential oil extracted from leaves of O. basilicum. ................... 92

3.35: Mycelial growth of P. italicum was measured after 7 days of incubation on MEA medium,

treated with different concentrations of major components of O. basilicum ......................... 95

3.36: in Vitro antifungal activity of O. sanctum essential oil, used at various concentrations on


3.37: In Vivo antifungal activity of O. sanctum essential oil, used in various concentrations, on

mycelial growth of P. Italicum. ................................................................................. 97

3.38: GC-MS chromatogram of essential oil extracted from leaves of O. Sanctum. ..................... 98

3.39: Mycelial growth of P. italicum measured after 7 days of incubation on mea medium, treated

with different concentrations of major components of O. sanctum essential oil. ................ 100

3.40: In Vitro antifungal activity of T. captivum essential oil, used at various concentrations on

mycelial growth of P. Italicum ............................................................................... 102

3.41: In Vivo antifungal activity of T. captivum essential oil, used in various concentrations, on

mycelial growth of P. italicum. ............................................................................... 102

3.42: GC-MS chromatogram of essential oil extracted from leaves of T. captivum .................... 103

3.43: Mycelial growth of measured after 7 days of incubation on MEA medium, treated with

different concentrations of major components of T. captivum essential oil.. ...................... 106

3.44: In Vitro antifungal efficacy of various essential oils against P. italicum at various

concentrations. ............................................................................... 109

3.45: In Vivo antifungal efficacy of various essential oils against P. italicum at various

concentrations. ............................................................................... 110

3.46: Linear regression fitted for the effect of different concentrations of essential oils on weight

loss (%) of mandarin fruit during cold storage at 4ºc for 8 weeks. .................................... 115

3.47: Linear regression fitted for the effect of different concentrations of essential oils on weight

loss (%) of mandarin fruit during cold storage at 4ºc for 8 weeks. .................................... 116

3.48: Linear regression fitted for the effect of different concentrations of essential oils on juice

content of mandarin fruit during cold storage at 4ºC For 8 weeks ..................................... 118

3.49: Linear regression fitted for the effect of concentrations of essential oils on juice content ff

mandarin fruit durring Different cold storage at 4ºc for 8 weeks. ...................................... 119

3.50: Linear regression fitted for the effect of different concentrations of essential oils on pH of

mandarin fruit during cold storage at 4ºc for 8 weeks. ...................................................... 121

xv

3.51: Linear regression fitted for the effect of different concentrations of essential oils on pH of

mandarin fruit during cold storage at 4ºc for 8 weeks. ...................................................... 122

3.52: Linear regression fitted for the effect of different concentrations of essential oils on Total

Soluble Solids (Tss) of mandarin fruit during cold storage at 4ºC for 8 Weeks. ............... 124

3.53: Linear regression fitted for the effect of different concentrations of essential oils on Total

Soluble Solids (Tss) of mandarin fruit during cold storage at 4ºC for 8 weeks. ................ 125

3.54: Linear regression fitted for the effect of different concentrations of essential oils on ascorbic

acid of mandarin fruit during cold storage at 4ºc for 8 weeks. .......................................... 127

3.55: Linear regression fitted for the effect of different concentrations of essential oils on ascorbic

acid of mandarin fruit during cold storage at 4ºC for 8 weeks. .......................................... 128

3.56: Linear regression fitted for the effect of different concentrations of essential oils on

titratable acidity of mandarin Fruit during cold storage at 4ºC for 8 weeks. ..................... 130

3.57: Linear regression fitted for the effect of different concentration of essential oils on titratable

acidity of mandarin fruit during cold storage at 4ºC for 8 weeks. ..................................... 131

xvi

LIST OF TABLES

TITLE PAGE #


1.1 Citrus area under cultivation in Pakistan from 2001-2011. .......................................... 5

1.2 Province wise citrus production in Pakistan from 2001-2011. ..................................... 5


2.1 List of cold storage houses and sampling date ............................................................ 22

2.2: Solutions and reagents for the extraction of DNA ..................................................... 24

2.3: List of primers used for the rapd analysis. ................................................................. 27

2.4. Sources of essential oils ............................................................................................. 31

CHAPTER 3: RESULTS

3.1: Morphological characteristics of different storage fungi isolated from sampled

mandarin fruits. ......................................................................................................... 40

3.2: Frequency of occurrence of different fungal genera isolated from sampled fruits. ... 43

3.3: Frequency of occurrence of storage fungi isolated from sampled fruits. ................... 43

3.4: Qualitative and quantitative spectrophotometric analysis of extracted DNA ............ 44

3.5: Random twenty decamers used in initial screening, their accessions, sequences,

number of polymorphic bands, percentage of polymorphic products and size of

bands produced by each primer. ............................................................................... 45

3.6: Chemical composition of essential oil extracted from A. indica. .............................. 55

3.7: Chemical composition of essential oil extracted from C. zylanicum. ........................ 60

3.8: Chemical composition of essential oil extracted from C. cyminum. .......................... 65

3.9: Chemical composition of essential oil extracted from C. citratus. ............................ 71

3.10: Chemical composition of essential oil extracted from E. globulus. ......................... 76

3.11: Chemical composition of essential oil extracted from E. caryophyllata. ................ 83

3.12: Chemical composition of essential oil extracted from F. vulgare. ......................... 88

3.13: Chemical composition of essential oil extracted from O. basilicum. ...................... 93

3.14: Chemical composition of essential oil extracted from O. sanctum. ......................... 99

3.15: Chemical composition of essential oil extracted from T. captivum. ...................... 104

xvii

LIST OF PLATES

TITLE PAGE #

3.1: Trial showing potential of eight isolates of P. italicum to cause postharvest

decay in mandarin fruits. 50

3.2: In Vivo antifungal effect of different concentrations of essential oil extracted from E.

caryophyllta. Pictures were taken after 15 days of incubation at 25ºC. 111

3.3: In Vivo antifungal effect of different concentrations of essential oil extracted from F.

vulgare. Pictures were taken after 15 days of incubation at 25ºC. 112

3.4: In Vivo antifungal effect of different concentration of essential oil extracted from T.

captivum. Pictures were taken after 15 days of incubation at 25ºC. 113

3.5: Mandarin fruits treated with three most effective essential oils with concentration of

200µL L-1

for 10 minutes, stored at 4ºC for 8 weeks. 132


200µL L-1



400µL L-1



400µL L-1


xviii

LIST OF ABBREVIATION

ANOVA Anaysis of variance

JECFA Joint expert committee on Food Additives

CTAB Cetyltrimethylammonium Bromide

Conc. Concentration

oC Degree centigrade

dNTPs Deoxynucleotide triphosphate

DNA Deoxyribonucleic acid

DMRt Duncan’s Multiple Range test

FAO Food and Agriculture Organization

LSD Least significant difference

g Gram

ha Hectare

GC-MS Gass chromatography mass spectrometery

bp base pairs

MgCl2 Magnesium chloride

TSS Total soluble solids

MEA Malt Extract Agar

m Micro meter

TA Titratable acidity

L Micro liters

RAPD Random amplified polymorphic DNA

mL Milliliter

mm Millimeter

WHO World health organization

ng Neon gram

μL-1

Per micro liter

mL–1

Per milliliter

% Percent

PCR Polymerase Chain Reaction

GOP Governement of Pakistans

pH Power of Hydrogen

rpm Revolutions Per Minute

1

Chapter 1

INTRODUCTION

Citrus constitutes an important group of fruits in the world. Its global demand is

attributed to its high content of vitamin “C” and its antioxidant potential (Gorinstein et

al., 2001). Citrus are evergreen that produce fruits of different forms and sizes. Citrus

fruits are remarkable for its typical fragrance, flavor and juice. The genus citrus is in the

family Rutaceae, which is composed of 1300 species belonging to 140 genera throughout

the world (Singh and Rajam, 2009). Citrus is divided into two subgenera: one is Citrus

and the other is Papeda. Commercially grown citrus fruits were obtained from subgenus

Citrus. It is very litigious to classify the various citrus plants into species. In early days,

just four Citrus species were recognized (Hooker, 1875). Later Sixteen to 162 different

species of citrus were identified by using different systems or criteria to distinguish one

species from another (Swingle and Reece, 1967; Tanaka, 1977).

Citrus is native to Asia and most of the historians believed that citrus plants may have

come from Southeast Asia at least 4000 years ago (Webber et al., 1967). It is generally

viewed that the fruit has been introduced from Asia to North Africa, the Mediterranean,

and then to Southern Europe by the middle ages via trade (Scora, 1975). Christopher

Columbus then introduced these fruits into the New World in 1493 on his second voyage

(Samson, 1980). The planting material of the fruit reached to South Africa by Dutch

merchants in 1654 (Oberholzer, 1969). Worldwide trade in citrus fruits did not appear

until the 19th century and trade in orange juice developed as late as 1940 (Florida Citrus

Mutual, 2009; UNCTAD, 2009).

Citrus is unique among all other fruits having thick or loose rind enclosed fruit containing

several juicy pulped segments. The flesh of the citrus fruit is pulpy which contains many

small glands full of fragrant oil. All citrus trees have glossy green leaves and bear white

and pink flowers. Citrus trees grow well in warm region and semi dry climate. Citrus

family is classified by four major varieties: oranges, grapefruit, tangerines, lemon and

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2

limes, which are being grown commercially. Its cultivars are grown in tropical and

subtropical climate.

Citrus fruits have long been esteemed as part of a nutritious and tasty diet. Its flavors are

in the course of the most privileged in the world and it is increasingly evident that citrus

not only tastes good, but is also good for health of the people. Citrus fruit are famous for

the source of essential nutrients like vitamin A, B, C, minerals, sugars, fiber, calcium,

folate, potassium, sodium, phosphorus, magnesium, copper, riboflavin, pantothenic acid

and a variety of phytochemicals (Nawaz et al., 2011). They also have been used in

pharmaceutical industry for the synthesis of medicines and soaps, as fragrance in

perfumes and other cosmetics and aromatherapy.

Within citrus family, Citrus reticulum L. (mandarin) is the largest planted cultivar in

Pakistan and is aboriginal to this part of the world. It is a hybrid of Citrus nobilis L. and

Citrus deliciosa. In 1940’s it was first introduced in Punjab from California (Niaz et al.,

2004) and at present Punjab is the center of production and supply of citrus fruit of high

quality and grade. In Punjab (Pakistan), 187 different varieties of citrus are commercially

available including oranges, Malta, Fruiter, Red Blood Mausami, grapefruit, tangerines,

lemon and limes (Naseer, 2010).

Mandarin c.v. Kinnow is one of the most eminent varieties which are domestically and

globally savored due to its flavor. Mandarin has longest growing period, in Pakistan it

starts from December and last till April. Harvested fruits are immediately transferred to

the fruit markets. It is manifest that the rate of picking is much higher than that of

consumption and therefore large quantity of citrus requires adequate storage. The skin of

the fruit is delicate, due to which the chances of damage during harvesting and

transportation become high.

1.1. CITRUS PRODUCTION

1.1.1.Citrus Production Worldwide

Commercially, citrus is one of the most leading fruit crops which are grown in

over 100 countries on six continents, with a worldwide crop of about 70 billion kilo

3

grams in 2004 (Anon, 2005; Terol et al., 2007) that are widely consumed both as fresh

fruit or juice. Citrus is mostly cultivated in tropical and subtropical regions of the world

and its production in the world fruit market is around 105 billion US dollar per annum

(Ismail and Zhang, 2004). Brazil is the largest grower followed by the United States of

America (USA), China, Spain and Mexico. Spain, USA and South Africa are the largest

exporters followed by the Turkey and Morocco (Citrus Commodity Note, 2005).

1.1.2. Citrus Production In Pakistan

Pakistan produces about 12.0 million tons of fruits and vegetables annually and

exports a large variety of fruits including mango, apple, date, pine nuts, banana, citrus,

grapes and guava etc. (Naseer, 2010). Among all the fruits, citrus has got a supreme

position with respect to area, production and export contributing to around 50% of the

total fruit production and about 25% of the country’s fruit export (Government of

Pakistan, 2006).

Pakistan is generally ranked tenth in citrus production worldwide (Sabir et al., 2010) and

stand at 8th and 38th position in area and production wise among all the citrus producing

countries in the world (FAO, 2008). The production of citrus fruit in Pakistan has been

increased eventually. Annual production was 1898 thousand tons in 2001 that has been

increased to 1982 thousand tons in 2011 (Agricultural Statistics of Pakistan, 2011).

The Pakistan is a sixth largest producer country of Kinnow in the world with crop

growing in an area of 0.5 million acres producing 2.3 million tons during the year of

2005-2006 (PHDEB, 2006). According to an estimate approximately 95% of total

Kinnow produced all over the world is grown in this country (Naseer, 2010). Citrus fruit

is grown in all the provinces but more than 95% of the citrus is produced in the Punjab

province because of its favorable growing conditions and adequate water. In Punjab

province, total area under the cultivation and production of citrus fruit was 184 thousand

hecters and the production was 1912 thousand tons in 2010-2011 (Agricultural Statistics

of Pakistan, 2011). Area under different varieties indicates that about 86% of the citrus is

4

covered by mandarin variety followed by musambi 10%, feutral 4% and red blood 1%

(Naseer, 2010).

In Pakistan, Sargodha district has top ranking in area and production of mandarin (46%

and 54% respectively) (Government of Pakistan, 2006). Despite the fact that the stipulate

of Pakistani fruits is massive, yet our exporting potential is a measly eight per cent due to

a big chunk of 25 per cent going to waste in consequence of poor harvest management

practices, transportation, packaging and storage conditions (Yaseen and Ahmad, 2010).

Citrus fruits are not only important for domestic consumption but can also serve as a

source of foreign exchange. Pakistan exports about 90 percent of citrus fruit to different

countries (Sharif and Ahmed, 2005; PHDEB, 2006).

Fig.1.1: Export of Kinnow from Pakistan (Source TDAP, 2010)

5

Table 1.1: Citrus area under cultivation in Pakistan from 2001-2011.

Year Punjab

(‘000’Hceters)

Sindh

(‘000’Hceters)

Balochistan

(‘000’Hceters)

Khyber

Pakhtoonkhaw

(‘000’Hceters)

Total Area

(‘000’Hceters)

2001 187.6 3.9 2.4 4.8 198.7

2002 183.2 4.1 2.4 4.5 194.2

2003 170.8 4.1 2.4 4.3 181.16

2004 166.6 4.2 1.3 4.4 176.5

2005 173.9 4.2 1.3 4.4 183.8

2006 182.1 4.4 1.3 4.5 192.3

2007 183.3 4.5 1.3 4.1 193.2

2008 189.2 4.5 1.3 4.4 199.4

2009 189.8 4.5 1.4 4.2 199.9

2010 188.2 4.5 1.4 4.3 198.4

2011 184.2 4.9 1.4 4.0 194.5

Source: Agricultural Statistics of Pakistan (2011).

Table 1.2: Province wise citrus production in Pakistan from 2001-2011.

Years Punjab Sindh Balochistan Khyber

Pakhtoonkhaw

Total

Production

(‘000’ tons)

2001 1813 30.9 13.6 40.2 1897.7

2002 1751.04 28.08 13.7 37.5 1830.32

2003 1623.56 27.73 13.06 38 1702.35

2004 1688.66 28.56 5.8 37.23 1760.34

2005 1872.25 28.56 6.04 36.82 1943.67

2006 2385.15 29.47 6.12 37.65 2458.39

2007 1400.75 31.48 5.81 34.44 1472.48

2008 2219.32 30.86 8.42 35.87 2294.47

2009 2059.51 30.53 8.41 33.83 2132.28

2010 2077.5 30.5 6.9 35.1 2150

2011 1912.0 30.9 7.0 32.3 1982.2

Source: Agricultural Statistics of Pakistan (2011).

6

1.2. PHYSICOCHEMICAL CHANGES IN FRUIT DURING

COLD STORAGE

Citrus fruits constitute a rich source of sugar, vitamins, minerals and other

compounds which are the important component of our daily diet. Fruits are taken either

as dessert or processed. The nutritional value of these fruits depends mainly on the

quality and concentration of their essential components.

Citrus fruits are harvested during November to December and then stored in cold storage

houses. The demand for the fruit increases from January onward but the quality of the

fruit can’t be retained. Improper storage results in rapid loss of sugars, ascorbic acid and

increased weight loss (Rab et al., 2010). Storage at low temperature is generally used to

slow down the deterioration of citrus fruits (Purvis, 1985). Since most of the prevailing

cold storage in Pakistan operate at 2-4ᴼC to increase the shelf life and to sustain the

quality of the citrus fruit especially Kinnow for a stipulated period.

Changes in sugar, ascorbic acid, tartaric acid, vitamin C and total soluble solids of the

juice depend on the storage conditions of the fruit. Higher temperature results in rapid

loss of water from the rind of the fruits (Kaushal and Thakur, 1996). The weight loss

varies from 1.5% to 34% depending on storage conditions and fruit coatings. Water loss

causes increase in levels of total soluble solids and acidity of the Kinnow (Chattopadhyay

et al., 1992; Angadi and Shantha Krishnamurthy, 1992). Kinnow stored under

refrigerated conditions is reported with a decrease in acids and percentage of the juice

content (Mukherjee and Singh, 1983; Ladaniya and Sonkar, 1996). If the coating is

applied on the surface of the fruit to protect the postharvest losses, then minimum weight

loss is about 1.5% although without coating it is 33.23% (Alam and Paul, 2001; Thakur et

al,. 2002).

Vitamin C is a water soluble acid which rapidly oxidized through light, heat and ascorbic

acid oxidase. Prolonged storage of citrus results in loss of vitamin C. under cold storage

conditions these changes are less as compared to those stored at room temperature (Pal et

al., 1997; Thakur, 2002).

7

1.3. CITRUS POST-HARVEST LOSSES

Unfortunately, fruits and vegetables are attacked by several pre and post harvest

plant pathogens that affects their quality. Up to 25 percent of the total fruit production is

subjected to fungal attack in both industrialized and developing countries and destruction

is frequently higher, greater than 50 percent (Spadaro and Gullino, 2004). More than 30%

of total crop yield is destroyed in developing countries where the protection and proper

handling of postharvest techniques are insufficient (Kader, 2002; Agriose, 2005).

As with other fruits and vegetables, fresh citrus fruits are susceptible to post-harvest

diseases which limit their storage period and marketing life (Schirra et al., 2000).

Moreover, huge economic losses occurred throughout the world due to post harvest

diseases. In Pakistan, about 40% of total citrus is lost during postharvest processes due to

poor disease management, weather extremes, harvest delay, poor harvest practices, cold

storage facilities and transportation (Iqbal, 1996; PHDEB, 2004; Naseer, 2010). These

factors may result in decay of the commodities and the microorganisms become activated

because of the physiological changes in the fruit (Wilson et al., 1991).

The quality of the citrus fruit starts deteriorating right after harvesting if not properly

managed. In consequence of poor handling conditions the citrus fruit meets

environmental stress and quality deteriorates promptly. These conditions more aggravate

to the development of physiological disorders such as, weight loss during marketing and

storage, high incidence of mold attack and fruit rots etc. Globally, numerous postharvest

technologies have been investigated to manage fruit disorders, maintain optimum fruit

quality and reduce the losses (Krochta, 1997: Hagenmaier, 2002; Bajwa and Anjum,

2007). The most ordinary technologies are low temperature storage, polyethylene packing

and emulsion applications as wax coatings (Perez et al., 2002; Thakur et al., 2002).

Citrus fruits are very susceptible to many diseases caused by different types of pathogens

such as fungi, bacteria, viruses and nematodes (Mukhopadhayay, 2004). In particular,

major postharvest decays and losses in harvested fruit are associated with the wide range

of fungal pathogens (Davis and Albrigo, 1994). Blue and green mould caused by

Penicillium italicum Wehmer and Penicillium digitatum Sacc. are generally the most

8

important postharvest diseases of citrus fruit in all production areas which occurs on the

surface of the fruit during storage and results in huge economic losses (Eckert and Eaks,

1989). Similarly grey mold caused by Botrytis cinerea Pers ex Fr. (Agriose, 1997), sour

rot caused by Geotrichum citri-autantii Link ex Pers (Howard, 1936; Hershenhorn et al.,

1992; Dewa et al., 1997), anthracnose caused by Colletotrichum gloeosporioides Penz

(Davis and Albrigo, 1994) and rots caused by various pathogens such as Alternaria citri

Ellis and Pierce, Trichoderma viridi Pos ex Gray (Whiteside et al., 1988), Phomopsis

citri Faw, Diplodia natalensis are also reported as a cause of postharvest decay in citrus

(Salunkhe and Desai, 1984; El-Ghaouth et al., 2002).

1.3.1. Blue Mold Disease In Citrus

Among the citrus fruits, Kinnow is highly perishable therefore, after harvest they

have to be marketed immediately. Penicillium is a versatile and opportunistic fungus.

More than ninety nine different Penicillium species have been identified (Carlos, 1982).

Penicillium species are one of the most common causes of fungal spoilage in stored

fruits. As among the various wound pathogens, Penicillium italicum causes a blue mold

disease which costs the loss up to 25% of the total worldwide citrus production (Palou et

al., 2007; Montesinos-Herrero et al., 2009).

Blue mold disease is most prevalent in cold storage fruits and easily identified due to the

presence of its filaments on the surface of the rotted fruits (Zhang et al., 2004). Infection

occurred though wounds which are caused by the poor harvest practices where the

nutrients are accessible to stimulate the spore germination. The initial symptoms of the

blue mold disease appeared as water soaked lesion. As the lesion enlarges to 1 to 2 inches

in diameter, white mycelium developed on the surface of the fruit followed by the

accumulation of blue spores and then it sporulates in the flesh of the fruit.

Colonies of Penicillium italicum on artificial media are more or less similar to the mould

that develops on surface of the infected fruit. Penicillium italicum is terverticillate,

metulae in verticils of 2-4, smooth 10-11 x 3.2-4.2 μm in size, phialides in verticils of 4-

9, smooth, cylindrical in shape and size is 8-12 x 2-3.3 μm. The conidia are produced on

9

phialides in chains and may vary in size (4.0-5.1 × 2.4-3.2 μm), smooth and thick walled

and ellipsoidal to cylindrical in shape (Samson and Varga, 2007).

Fig. 1.2: Penicillium italicum

1.4. CONTROL MEASURES

Post-harvest citrus spoilage by Penicillium italicum and Penicillium digitatum are

responsible for remarkable economic losses worldwide (Spadaro and Gullino, 2004;

Sharma et al., 2009). Fungal spoilage of fruits depends upon cultivation, harvesting,

handling, transport, post-harvest storage and marketing conditions (Effiuvwevwere,

2000). It is essential to control postharvest diseases in order to maintain the quality of the

fruit and improve its shelf life. However, different practices such as careful culling,

storage at low temperature or under controlled atmosphere and application of fungicides

have been used (Beuchat, 1987; Eckert and Ogawa, 1988). For the control of post-harvest

decay, chemical fungicides have also been applied on the surface of the fruits to prevent

the fungal growth. There are two crucial complications in using fungicides that is the

increasing public concern regarding contamination of perishables with fungicidal

residues and the development of resistance in the pathogen population (Wilson and

Wisnieski, 1989; Tripathi and Dubey, 2004).

Traditionally, a number of synthetic fungicides such as benzimidazoles, thiabendazole,

imazalil, sodium ortho-phenyl phenate, aromatic hydrocarbons and sterol biosynthesis

inhibitors are used as the primary means to reduce the postharvest decay and extend the

10

shelf-life of the fruits (Fan et al., 2000; Poppe et al., 2003). Currently, to control the

fungal diseases, annually 23 million kg of fungicides are used throughout the world

(Tripathi and Dubey, 2004) in both field and postharvest management. However,

extensive use of synthetic fungicides are developing resistant strains of the pathogens as

reported in case of imazalil (Bus et al., 1991; Eckret et al., 1994), thiabendazole (Timmer

and Duncan, 1999) and benomyl (Bus et al., 1991). Also the toxic residues have been

reported in products that affect the human health (Norman, 1988; Daferera et al., 2000).

Development of resistance against various fungicides has also been reported in

Penicillium digitatum and P. italicum (Fogliata et al., 2001). For these reasons, the use of

synthetic chemical fungicides has been discouraged (Spadaro and Gullino, 2004; Droby

et al., 2009).

However, due to the problems related to the fungicides applications, research has been

focused on the use of alternative control measures specially based on plant derived

products such as essential oils (EOs) and plant extracts as disease control agents. These

natural products tends to have low mammalian toxicity, less environmental hazards and

wide public acceptance (Janisiewicz and Korsten, 2002; Obagwu and Koresten, 2003;

Soylu et al., 2005; Lee et al., 2007; Tripathi and shukla, 2008). These secondary

metabolites obtained from different plant organs acts as novel chemotherapeutics in plant

protection and possess the potential to be used in pest management (Abdolahi et al.,

2010). Consequently, numerous studies have acknowledged the use of natural substances

as potential antifungal agents, and plants have been generally known as the source of

fungicidal and fungistatic phytochemicals (Bajpai et al., 2009).

Recently, Many explorations have been directed in the field toward antifungal activity of

essential oils obtained from plants against postharvest fungal decay (Daferera et al.,

2000; Venturini et al., 2002; Shahi et al., 2003; Tripathi and Dubey, 2004; Guynot et al.,

2005; Mercier and Smilanick, 2005; Neri et al., 2006; Nikos and Costas, 2007; Irkin and

Korukluoglu, 2007; Feng and Zheng, 2007; Kumar et al., 2007; Omidbeygi et al., 2007;

Kaloustian et al., 2008; Amiri et al., 2008; Liu, et al., 2009; Abdolahi et al., 2010: Xing

et al., 2010; Wang et al., 2010). The essential oils have two key advantages; they are

organic natural substances which signify both public health safety and environment

11

friendliness and there is low risk of resistance development by postharvest pathogens due

to the mixture of oil components with actually different antifungal activity mechanisms.

Essential oils and its active ingredients possess a broad spectrum of biological activity,

which may be of great upshot in several fields, from food chemistry to pharmacology and

pharmaceutics (Cristani et al., 2007). Such antimicrobial activity is due to the presence of

bioactive substances such as flavonoids, terpenes, coumarines and carotenes (Tepe et al.,

2005).

1.5. ESSENTIAL OILS

Essential oil is the natural oil typically obtained by distillation having fragrant

substances characteristic of the plant or a mixture of fragrant and odorless substances

(Burt, 2004).

The essential oils are also known as volatile, ethereal oils or etherolea which is defined as

concentrated, hydrophobic liquid that contain a large number of aromatic compounds and

organic constituents. These oils are "essential" because they represent the characteristics

like distinctive scent or essence, of the plant. Essential oils contain highly volatile

components that are isolated by a physical method known as steam distillation process of

a single botanical plant species. These essential oils normally identified with the name of

the plant species from which they are derived.

The essential oils constitute about 17 percent in the market of flavor and fragrance

worldwide. The world production of essential oil ranges from 40,000 to 60,000 tonnes

per annum. The stipulate for spice oils is to be found at 2,000 tonnes per annum (Joy et

al., 2001).

Essential oils comprise a major group of industrial products. These oils form a primary

ingredient of the necessities in many fields of human activity. Nowadays the world of

consumer boom, the role of essential oils has increased many folds. In Japan and

European countries, the essential oils are becoming more popular in therapeutics. Some

of these oils are reported to be in many ways better than antibiotics due to their safety and

12

wide spectrum of activity. The applications of these essential oils in agriculture as

antifeedants, repellents, biopesticides and growth boosters are still open to charming

realm of research (Joy et al., 2001).

The techniques of obtaining essential oils from the plants were first time developed by

the Arabs (Saeed, 1989). In 10th century, Aviceena, devised the method to extract the

essential oil from the rose flower by distillation process (Poucher, 1974). Therefore, rose

water has been reported for the first time by an Arabian historian, Ibn-e-Khulduae

(Hussain, 2009).

1.5.1.Extraction Of Essential Oils

There are various methods for the extraction of essential oils based on its

stability. The first method used for oil extraction is cold pressing. The whiff of this oil is

natural but impure. Steam distillation and hydro-distillation techniques are therefore more

commonly used for the isolation of pure essential oils (Masango, 2004). At present,

essential oils are also extracted by using latest techniques such as solvent extraction,

supercritical carbon dioxide, low or high pressure distillation employing boiling water

and hot steam (Bousbia et al., 2009; Donelian et al., 2009). Lawrence in 2002

demonstrated the oil extracted by solvent and sub and supercritical carbon dioxide are not

genuine because of the removal of a few components.

Distillation is the process of distilling plant material with steam that generally referred to

as a boiler. The main principle behind steam distillation and hydrodistillation is that a

mixture of two immiscible liquids were heated to expose the surface of both liquids to

vapor phase, each constituent exert a vapor pressure as if each liquid were pure

(Houghton and Raman, 1998). For two immiscible liquids the total vapor pressure of the

mixture is always equal to the sum of their partial pressures. As steam increases, it

ruptures the oil membranes of a plant and this releases its essential oil. The steam carries

the oil up through a condenser, where the mixture of oil and steam re-liquefies. As the

steam condenses back into water, the distilled essential oil gathers on top of it. This

process is therefore able to separate the volatile and non-volatile components with a

13

reduction in boiling point and thus avoiding extreme temperatures (Baker et al., 2000;

Donelian et al., 2009). However an obvious drawback of this process is the induction of

chemical changes by oxidation and hydrolysation reaction (Krell, 1982; Houghton and

Raman, 1998). Furthermore, the original composition of the essential oils is partially lost

due to their affinity of water (Baker et al., 2000; Masango, 2004). It is the most popular

widely used and cost effective method in use today for producing majority of the

essential oils throughout the world. About 93% of essential oil is produced by this

process on commercial scale but it is not preferred in research laboratories due to its

unavailability of steam generator and distillation vessels (Massango, 2004; Hussain et al.,

2008). So mostly in labs hydrodistillation process is used for the isolation of plants

essential oil through Clavenger type apparatus (Kulisic et al., 2004; Sokovic and

Grienseven, 2006; Hussain et al., 2008). In this process plant material is submerged in

water, which is heated to boiling point. In both distillation processes, the vapors are

allowed to condense and the oil is then separated from the aqueous phase (Houghton and

Raman, 1998).

Ackerman (2001) demonstrated that hydrodistillation process gave a better quality and

absolute product, as hot water is cooler than steam distillation. However Charles and

Simon (1990) evaluated the comparison of extraction methods for the rapid determination

of essential oil content and its composition. They found that steam distillation is more

efficient and gave higher yields of essential oil than hydrodistillation. They also approved

that hydrodistillation is simple and more rapid method for extracting essential oils. Eikani

et al. (2007) used subcritical water extraction (SCWE), hydrodistillation and Soxhlet

extraction and compared for the extraction of essential oil from coriander seed.

Hydrodistillation and Soxhlet extraction showed higher extraction efficiencies as

compared to SCWE.

In contrast, Khanavi et al. (2004) gave the comparison of essential oil composition

obtained by steam and hydrodistillation process. According to them better yield of

essential oil was obtained by hydrodistillation in comparison to steam distillation.

Sefidkon et al. (2007) have worked on the Setureja rechingeri L. essential oil obtained

through steam and hydro distillation process and found that highest yield of essential oil

14

was obtained in hydrodistillation. Silva et al. (2004) reported the variation in chemical

composition of essential oils of Ocimum spp. obtained by steam distillation, microwave

oven distillation and supercritical extraction with CO2 and showed that the main

constituent of these essential oils were the same but in different relative amounts. They

found that higher percentage of chemical constituents was observed in oil obtained

through hydrodistillation process.

1.5.2. Biological Activities Of Essential Oils

The use of plant essential oils has long been identified as a traditional means to

control plant diseases (Cowan, 1999). On the other hand the actual use of these products

in disease control has become an important field of study (Obagwu, 2003). The plant

essential oils have been used successfully for the control of postharvest diseases of stored

fruits and vegetables (Skocibusic et al., 2006; Bozin et al., 2006; Van Vuuren et al.,

2007). As they are the secondary metabolites, these are also known as the protective

compounds. They have several bioactivities such as antioxidant and antimicrobial

activities (Lertsatitthanakorn et al., 2006). The antimicrobial activity of many plant

essential oils has been tested at different concentrations against various microbes

including bacteria, fungi and yeast (Antunes and Cavaco, 2010). Ozcan and Erkmen in

2001 stated that essential oils provide an effective control by the inactivation of the

pathogen and enhance the shelf life of the food.

Essential oils and their constituents have been effective against numerous plant

pathogenic fungi, including Alternaria alternata F., Botrytis cinerea, Phytophthora

infestans, Phytophthors capsici B., Fusarium verticillioides, Rhizoctonia solani and

Seclerotium sclerotiorum (Muller-Riebau et al., 1995; Wilson et al., 1997; Soliman and

Badeaa, 2002; Feng and Zheng, 2005; Soylu et al., 2006). Similarly essential oils

extracted from rosemary, thyme, organo, lavender, sage, basil, cinnamon and marigold

have shown antifungal activity against postharvest pathogens (Daferere et al., 2000;

Soliman and Badeaa, 2002; Fandohan et el., 2004).

15

Essential oils exhibited different levels of antifungal activity depending on the type of the

oil, its concentration and the fungal species (Wilson et al., 1997; Soliman and Badeaa,

2002). Essential oils are effective against fungi at different stages like mycelilal growth

and spore germination (Fandohan et al., 2004; Feng and Zheng, 2005). The inhibitory

effect of essential oil is varying by the oil concentration and the sensitivity of the

pathogen to the chemical constituents present in the essential oil (Soliman and Badeaa,

2002). The antifungal activity of plant essential oils and their components is frequently

studied, where the need for the protection of plants and stored crops against fungi is of

great importance. The utilization of essential oils for the fungus free storage of food has

also been focused by various researchers (Burt, 2004, Sacchetti, et al., 2005; Bozin, et al.,

2006)

Bouchra et al. (2003) evaluated In Vitro antifungal activity of seven Moroccan Labiataes

against Botrytis cinerea. They revealed that Origanum compactum B. and Thymus

glandulosus among them greatly inhibited the mycelial growth of B. cinerea. The main

component these oils were thymol and carvacrol that showed the strongest antifungal

activity with 100% of inhibition at 100ppm.

Tripathi and Dubey in 2004 revealed the potential of some natural products such as flavor

compounds, acetic acid, jasmonates, glucosinolates, propolis, fusapyrone and

deoxyfusapyrone, chitosan, essential oils and plant extracts as an alternative strategy to

control postharvest fungal rotting of fruits and vegetables and prolonging storage life.

These biologically active natural products showed the potential to replace synthetic

fungicides.

Soylu and his colleagues in 2005 determined the antifungal activities of the essential oils

obtained from oregano, fennel, artemisia, laurel and lavender against the green mould of

citrus. Effect of different concentrations of these essential oils on conidial germination

and germ tube elongation of P. digitatum were determined In Vitro. Their results

elucidated that essential oils of oregano and fennel strongly inhibit the conidial

germination of P. digitatum.

16

In 2006 Yadav et al., also worked on various antifungal properties of essential oils of

Mentha spicata. This essential oil showed fungicidal effect on the mycelial growth of

Aspergillus ochraceus, Penicillium digitatum, Pyricularia oryzae and Alternaria

alternata. This oil possessed quick fungicidal action on broad pH range and fungitoxic

spectrum. Similarly, the essential oils of Cymbopogon citrates S., Ocimum basilicum L.

and Origanum majorana B. were found to be highly effective against Aspergillus niger,

Aspergillus flavus and Saccharomyces cerevisiae (Helal et al., 2006). Singh with his

colleagues in 2006 also determined complete zone inhibition against Aspergillus niger,

Asperigillus flavus, Fusarium graminearum and Fusarium moniliforme at 6 µL dose in

inverted plate method. While, in food poison technique it was found to be effective for A.

niger even at lower concentration.

In the same period another research by Yahyazadeh et al. (2007) showed In Vitro

antifungal activity of fennel, thyme, clove and sage essential oils against mycelial growth

of Penicillium digitatum. They stated that clove and thyme essential oils exhibited the

strongest toxicity and can totally inhibit the mycelial growth of the test fungus. They

found that the essential oils of clove and thyme showed fungistatic fungicidal activity in

In Vitro mycelial growth assay.

The essential oils from the seeds of neem, mustard, black cumin and asafetida were

extracted by Sitara et al., in 2008 and evaluated their antifungal activity against eight

seed borne fungi viz., A. niger, A. flavus, F. oxysporum, F. moniliforme, F. nivale, F.

semitectum, Drechslera hawiinesis and Alternaria alternata. Ridomyl gold was used for

the comparison. All the oils extracted except mustard showed fungicidal activity of

varying degrees against tested fungal species. Of these oils, asafoetida oil most

significantly inhibited the growth of all tested fungi except A. flavus. Black cumin oil

was also found effective but showed little fungicidal activity against A. niger followed by

neem, Ridomyl gold and mustard oil. In another study, the eucalyptus oil showed a wide

spectrum of biological activity including anti-microbial, fungicidal, insecticidal,

herbicidal and nematicidal (Batish et al., 2008). Amiri et al., in 2008 determine the In

Vitro and In Vivo activity of clove oil against four apple pathogens namely Phlyctena

vagabunda, Penicillium expansum L., Botrytis cinerea and Monilinia fructigena.

17

Complete inhibition of mycelial growth of test fungi was obtained at concentration of

150mL L-1.

Another study by Camele et al. (2010) showed the work on twelve essential oils from

Mediterranean aromatic plants. They tested different doses of EOs against four fungi

known as causal agents of orange fruit rot in post-harvest conditions. Their In Vitro

experiments showed that only the oils from Verbena officinalis L., Thymus vulgaris L.,

and Origanum vulgare L. exhibited some fungistatic activity against tested fungi. These

three essential oils were used in successive In Vivo tests carried out to protect healthy

orange fruits from artificial infection by the same micromycetes and enhanced the shelf

life of the fruit. Marjanlo and his co-worker in 2009 checked on cumin essential oil

against postharvest decay of strawberry. They found that storage life of strawberry fruits

was significantly increased by the use of cumin essential oil. Siripornvisal et al. in 2009

studied the antifungal activity of clove, cinnamon and lemongrass oils against grey mold

caused by Botrytis cinerea. The results indicated that these oils exhibited fungicidal

effect on pathogen and might be used as alternative options for the control of grey mold.

In addition to this Abdolah et al. (2010) demonstrated the potential of ajwain, fennel and

caraway essential oils as an antifungal agents for tomato fruits that are susceptible to

postharvest decay caused by A. alternata and P. digitatum. The results showed promising

prospects for the utilization of these essential oils on tomato fruit. Alizadeh-Salteh et al.

in 2010 monitored the antifungal effect of sage, savory and zatraria essential oils to

control the R. stolonifer on peach fruit to increase the shelf life and storage ability of the

fruit.

In a recent study Abdollahi et al. (2010) screened antifungal properties of sweet basil,

fennel, summer savory and thyme essential oils against two postharvest phytopathogenic

fungi. e.g. P. digitatum and R. stolonifer by poison food medium and vapor phase

methods. The results showed that thyme essential oil in vapor phase method completely

inhibited the mycelial growth of both fungi as well as in poison food technique thyme oil

showed highest antifungal activity while fennel oil showed the least inhibitory effect

against the pathogens.

18

In another study Singh et al. (2011) determined that essential oil of O. basilicum has

strong fungitoxic components against some storage fungi. They revealed that eugenol is

the major constituent of O. basilicum essential oil and exhibited broad fungitoxic

spectrum against A. alternata, A. fumigatus, A. niger, A. flavus, C. lunata, F. nivale, F.

oxysporum and P. italicum. Marandi et al. in 2011 successfully controlled the Penicillium

expansum and Botrytis cinerea on pear fruit with essential oils of Thymus kotschyanus L.,

Ocimum basilicum and Rosmarinus officinalis L.

Ippolito et al. (2012) checked efficacy of eight major components of essential oils (citral,

thymol, carvacrol, camphene, β-fellandrene, β-pinene, ɣ-terpinene, o-cymene) extracted

from some Mediterranean aromatic plants against five postharvest decaying fungi

including P. italicum. Among these compounds thymol showed significant fungistatic

activity against P. italicum.

In a recent study Arici et al. (2013) used essential oils from seven different plants

including cumin, thymus, lavandula, eucalyptus, rosemary, nigella and dill to manage

Fusarium oxysporum, the causal agent of tomato wilt. Essential oil from cumin and

thymus showed maximum antifungal activity against the tested fungi.

Tatjana et al., in 2014 tested sixteen different essential oils against 21 fungal pathogens.

All essential oils showed antifungal activity against tested fungi with varying degrees.

Savory, thyme and oregano oils that contain phenols and rose oil that possesses

monoterpene alcohols proved as most effective sources of antifungal compounds.

1.6. ANTIFUNGAL MECHANISM OF ESSENTIAL OILS

The diversity of plant derived essential oil constituents is immense and

comprised of a wide range of chemical constituents. Some of the constituents of many

essential oils have great potential against the microbes including bacteria, yeast and

fungi. Yet, the number of chemical compounds present in essential oils has no specific

target sites in the cell (Carson et al., 2002). Essential oils have the ability to damage the

cell membrane which disrupt its structure and make it more permeable (Ultee et al., 2002;

19

Burt, 2004). Injury to the cell wall and cell membrane can leads to lysis and to the escape

of macromolecules (Oussalah et al., 2006). Cell membrane is an important site of action.

The permeability of cell membrane gets disturbed by essential oils resulting in

interactions of the chemical constituents thus affecting the cell integrity.

Schncider et al., (2010) demonstrated that if there is no change in cell membrane

functionality, then site of action of essential oil is intracellular. In this case the target of

the essential oil is mainly proteins and enzymes. Thymol is the major constituent of the

essential oil that interacts with cell membrane and affects the membrane permeability

which has been documented by loss of membrane potential (Shapira and Mimran, 2007).

1.7. CHARACTERIZATION OF ESSENTIAL OILS

Essential oils are the mixture of naturally occurring compounds in different

concentrations (Bakkali, 2008). Chemical identification and quantification of all essential

oils are usually done by using Gas Chromatography with Mass Spectrophotometer (GC-

MS) or Gas Chromatography equipped with Flame Ionization Detector (GC-FID)

(Delaquis et al., 2002; Hussain et al., 2008; Anwar et al., 2009).

GC-MS is a method that combines the features of both gas liquid chromatography and

mass spectrometry to identify the different substances present within the sample. The gas

chromatograph utilizes a capillary column which depends on the column’s dimensions

(length, diameter, film thickness) as well as the phase properties. The difference in the

chemical properties between different molecules in a mixture will separate the molecules

the sample travels the length of the column. The molecules take different amount of time

(called retention time) to elute from the gas chromatograph, and this allow the mass

spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized

molecules separately. The mass spectrometer does this by breaking each molecule into

ionized fragments and detecting these fragments using their mass to charge ratio.

The identification of individual compound is based on the comparison of their retention

index (RI), Kovate Index (KI) on polar and non-polar column and their mass spectra from

GC-MS (Juliani et al., 2002).

20

1.8. AIMS AND OBJECTIVES

This study was designed with a principal objective to explore a plant based antifungal

strategy to control postharvest decay in stored citrus. This goal was accomplished

through following steps:

1. A survey in various agro ecological zones was conducted to achieve diversity in

sampling of stored citrus.

2. Isolation and identification of storage fungi from collected samples.

3. Essential oils extracted from ten selected plants were tested for their antifungal

activity against Penicillim italicum, the fungus that was isolated from sampled

citrus in highest frequency.

4. Quantitative and qualitative analysis of extracted essential oils using Gas

Chromatographic techniques.

5. To enhance the shelf life of the fruit the efficacy of the top three selected essential

oils were checked In Vivo in cold storage conditions. The study was extended to

analyzed fruit quality by its physiochemical analysis.

21

Chapter 2

MATERIALS AND METHODS

The research work presented in this dissertation was conducted in the

laboratories of the Institute of Agricultural sciences (IAGS), University of the Punjab,

Lahore, Pakistan; University of Vetnary and Animal Sciences (UVAS), Lahore,

Pakistan, and Forman Cristian College (FC), Lahore, Pakistan.

2.1. SAMPLE COLLECTION

The citrus variety mandarin c.v. Kinnow was selected for the present study

on the basis of its economic importance. The fruits were sampled from 22 different

cold storage houses in Punjab, Pakistan (Table 2.1). These regions were selected on

the basis of their storage importance and capacity. Specific locations of places are

indicated by the arrows in the map (Fig. 2.1)

Fig. 2.1: Map of Punjab Pakistan (30o 00´N 70o 00´E) Arrows showing major sampling

cities. Source: http://www.findpk.com/yp/html/punjab_.html.

Faisalabad

31˚ 30′N 73˚ 05′E

Sahiwal

30˚45′N 73˚ 8′E

Multan

30˚ 15′N 71˚ 36′E

Sargodha

32˚ 10′N 72˚ 40′E

Jhang

31˚ 15′N 72˚ 22′E

Chiniot

31˚ 45′N 73˚ 00′E

Lahore

31˚ 32′N 74˚ 22′E

Bhakkar

31˚40′N 71˚ 05′E

http://www.findpk.com/yp/html/punjab_.html

22

Table 2.1: List of Cold Storage Houses and Sampling Date

Sr. No. Name of cold storage

house

Location City Date of

sampling

01 Mian Amir cold storage Thokar niaz baig Lahore 21-01-09

02 Bismillah cold storage Sabzi mandi, ravi link

road

// 25-01-09

03 Nadim cold storage Sabzi mandi, ravi link

road

// 25-01-09

04 Lyallpur cold storage Main sussan road, Madina

town

Faisalabad 11-02-09

05 Punjab cold storage P-698 madina gali new

Khalid abad

// 11-02-09

06 R-Tek-Zia Khalid

corporation

Sussan road, inside

Lyallpur cold storage,

near mobilink-skans

college

// 11-02-09

07 Madina cold storage Sillanwali road Sargodha 13-02-09

08 Ranjha cold storage Wan Miana Tehsil

Bhalwal

// 13-02-09

09 Ittefaq cold storage Factory area // 13-02-09

10 Mian cold storage Sebzi mandi, bhakkar Bhaker 27-03-09

11 Siyal cold storage Chauk azam, jhang road // 27-03-09

12 Rafiq cold storage Fareed town Sahiwal 16-02-09

13 Afraat cold storage Shahana road // 16-02-09

14 Abdullah cold storage 88/6R Sharqee road // 18-02-09

15 Al-Makkah cold storage Sargodha road, jhang

sadar

Jhang 29-03-09

16 Khalid cold storage Bhakar road near Daewoo

terminal

// 29-03-09

17 Khawaja cold storage Sebzi mandi // 29-03-09

18 Multan cold storage Opp. MCB, Chowk Shah

abbas , vehari road

Multan 12-01-09

19 Multan Industries Cold

Storage

Chowk Shah Abbas

Vehari Road Multan

// 12-01-09

20 Khalil cold storage Bahawalpur road // 13-01-09

21 Agility Pakistan Pvt. Ltd Chah Dumri Wala, Near

Thatta Ghalla Godam Old

Duiya Pur Road

// 13-01-09

22 Shakarganj food product

limited

Ahmad nagar Chiniot 31-03-09

These samples were labeled, placed in sterilized sampling bags and were

transferred to the laboratory to store at 4oC in refrigerator till fungal isolation.

23

2.2. ISOLATION OF STORAGE FUNGI

The storage fungi were isolated from the samples collected from different

storage houses. Malt Extract Agar (MEA) and Potato Dextrose Agar (PDA) media

were used for the isolation of storage fungi.

2.2.1. Media Composition

a. Potato Dextrose Agar

Potato starch 20gm

Dextrose 20gm

Agar 20gm

Distilled water 1000mL

b. Malt Extract Agar

Malt 20gm

Agar 20gm

Distilled water 1000mL

Above chemicals were dissolved in 1000mL of distilled water. Then pH of the

solution was adjusted to 5.6-5.8 and autoclaved at 121ºC at 15 pounds per squares

inch (psi) pressure for about 15-20 minutes. After autoclaving, the medium was

allowed to cool to a temperature of 40-45ºC and then Chloromycetin was added as

bacteriostatic agent and poured into sterilized petri plates under aseptic conditions.

For isolation of storage fungi from the sampled fruits, the small portion of the

infected part was cut and placed on the surface of the plate containing above

mentioned media and incubated at 25ºC for 7 days. The developing fungal colonies

were counted and identified morphologically. The frequency of individual fungus

isolated from each sampled fruit was therefore determined by using the following

formula described by Giridher and Ready (1970).

Percentage Frequency (%) = No. of observations in which species appeared × 100

Total no. of observations

24

2.3. MORPHOLOGICAL CHARACTERIZATION

Fungal species isolated from the stored fruits were characterized on

morphological basis by using the most documented keys and literature in fungal

identification (Klich, 2002; Samson and Varga, 2007). These fungal species were

identified according to their macroscopical and microscopical characteristics like

colony diameter, colony color, texture, pigments, and characteristics of mycelium,

conidiophore and conidia (Haggag et al., 2006; Okereke et al., 2007).

2.4. ANALYSIS OF GENETIC DIVERSITY AMONG

FUNGAL ISOLATES

According to the percentage of occurrence Penicillium italicum was found as

the most frequently occurring storage fungi so was selected for further genetic

analysis. Genetic variation of eight morphologically similar but geographically

distinct P. italicum isolates was carried out through Random Amplified Polymorphic

DNA (RAPD) analysis.

2.4.1. DNA Extraction

The total genomic DNA of different isolates of P. italicum was extracted by

CTAB method (Doyle, 1991). Fungal tissue was taken and grinded in pestle and

mortar using fermentas glass beads. All the solutions and reagents employed in this

process are shown in Table 2.2.

Table 2.2: Solutions and Reagents for the Extraction of DNA

Solution I

(Extraction Buffer)

Solution II Solution III

(DNA precipitating solution)

2% CTAB

20mM EDTA

100mM Tris HCl (pH 8.0)

1.4M NaCl

0.2% β-Mercaptoethanol

Phenol: Chlorpform: Isoamyl

alcohol

(25:24:1)

Isopropanol (2⁄3 volume)

70% Ethanol

The freshly prepared pre-warmed (65ºC) extraction solution (2.5mL) was

added to the powdered fungal mass in new, autoclaved eppendorf (1.5 mL) and

incubated at 65ºC in water bath for 30 minutes until the mixture turned into jelly like

25

material indicating cell lysis. The sample was cooled on ice for 3-5 minutes till the

mixture was brought to room temperature, then an equal volume (2.5 mL) of solution

II was added. Sample was vortexed for 2 minutes and centrifuged at 10,000g for 10

minutes to separate the organic phases. Aqueous supernatants were transferred to new

tubes and two third volume of cold isopropanol was added to each and then incubated

overnight at 4 ºC.

After 24 hours, the sample was centrifuged at 10,000g for 10 minutes and

transparent DNA pellets were obtained. Washing of pellets was done with 70%

ethanol further pellets were air dried and resuspended in 50µL of Tris HCl EDTA

(TE) buffer.

2.4.2. Estimation of Extracted DNA

After the isolation of DNA the quantification and quality analysis of isolated

DNA is needed to be determined. The approximate quantity of DNA obtained is

necessary for further downstream analysis of the sample which includes polymerase

chain reaction (PCR) optimization steps and random amplified polymorphic DNA

(RAPD) amplifications.

2.4.3. DNA Quantification and DNA Quality Analysis

Quantification of the total genomic DNA from the samples was done using

Techne Spec gene Spectrophotometer (140801-2, UK). The spectrophotometer was

calibrated by taking distilled water at 260nm as well as 280nm as a blank. Then 10µL

of each genomic DNA sample from different isolates was diluted with 900µL of

distilled water in a cuvette and mixed well. The absorbance was taken at 260nm for

the estimation of the concentration of DNA as described by Hoisington et al. (1994).

The concentration was calculated on assumption that absorbance of 1 at 260nm is

equivalent to 50mg mL-1 double stranded DNA or 40mg mL-1 single stranded DNA

(Sambrook et al., 1989). The DNA quantity was calculated by the following equation:

DNA Con. in µg mL-1 ═ Absorbance at 260nm ×Dilution factor ×50

26

The optical density (OD) the DNA sample was also taken at 280nm and the

ratio of 260/280 was determined for the DNA purity. The ratio of the pure DNA

ranges from 1.5-1.9.

2.4.4. Agarose Gel Elecetrophoresis

The quality of the DNA was estimated through 1% agarose gel

electrophoresis. By using this method the DNA fragments were separated by

electrophoresis on 1% agarose gel. An adequate volume of electrophoresis buffer i.e.

1.0 X TAE buffer was made to fill the tank of electrophoresis apparatus. Agarose (1g)

was put into a flask, containing 100mL 1.0X TAE electrophoresis buffer. It was

melted in microwave oven for 1-2 minutes and swirled regularly to make sure proper

mixing of agarose. Gel casting tray was wrapped by scotch tape on its both sides and

a comb having specific number of wells was adjusted on it, for loading samples. The

temperature of melted agarose was lowered to 60 -50ºC before adding 5µL ethidium

bromide (10mg mL-1). The gel was poured in the gel casting tray; bubbles were

removed, and then solidified at room temperature. After that the casting tray was

placed in electrophoresis tank, removed the comb by avoiding tearing of wells. The

gel in casting tray was kept horizontally in an electrophoresis tank, containing 500mL

1x TAE buffer. Gel loading dye 3µL of the 6X was added to 5µL of each sample of

DNA prior to loading the samples into the wells of the gel. The electrophoresis

apparatus was connected to power supply and the immersed electrophoretic gel was

run at 100V for 45 minutes until the dye travelled one-third of the distance in the gel.

DNA bands were visualized using UV transilluminator. The DNA bands were

compared with the 1Kb DNA markers for quality estimation showing the

concentrations of DNA in ng µL-1. Hence, the DNA concentration was maintained to

25ng µL-1 for polymerase chain reaction (PCR) amplifications.

2.4.5. Random Amplification of Polymorphic DNA Analysis

The random amplified polymorphic DNA (RAPD) analysis is an exclusive

marker for studying DNA polymorphism in different fungal isolates for exploring

phylogenetic relation among them. RAPD analysis was performed by following

method as depicted by Ranganath et al. (2002).

27

2.4.5.1. Random Primer Screening

A set of twenty primers procured from SBS Genetech co. Ltd-Beijing, China

were used in Random Primer Screening (RAPD) analysis for the initial screening

(Table 2.3). Majority of these primers produced clear, distinct and reproducible bands

in different isolates of P. italicum. The primers were diluted up to 100 picomole

concentration before use in RAPD analysis. All the isolates of P. italicum were tested

against these primers.

Table 2.3: List of Primers Used for the RAPD Analysis.

Name Sequence ( 5'- 3' ) Length MW nmol GC (%)

SBSA01 CAG GCC CTT C 10 2964 111.3 70%

SBSA02 TGC CGA GCT G 10 3044 108.4 70%

SBSA03 AGT CAG CCA G 10 2997 110.1 60%

SBSA04 AAT CGG GCT G 10 3068 107.6 60%

SBSA05 AGG GGT CTT G 10 3099.1 106.5 60%

SBSA06 GGT CCC TGA C 10 3004 109.9 70%

SBSA07 GAA ACG GGT G 10 3117.1 105.9 60%

SBSA08 GTG ACG TAG G 10 3108.1 106.2 60%

SBSA09 GGG TAA CGC C 10 3053 108.1 70%

SBSA10 GTG ATC GCA G 10 3.68 107.6 60%

SBSA11 CAA TCG CCG T 10 2988 110.4 60%

SBSA12 TCG GCG ATA G 10 3068 107.6 60%

SBSA13 CAG CAC CCA C 10 2941.9 112.2 70%

SBSA14 TCT GTG CTG G 10 3050 108.2 60%

SBSA15 TTC CGA ACC C 10 2948 111.9 60%

SBSA16 AGC CAG CGA A 10 3046 108.3 60%

SBSA17 GAC CGC TTG T 10 3019 109.3 60%

SBSA18 ACG TGA CCG T 10 3068 107.6 60%

SBSA19 CAA ACG TCG G 10 3037 108.7 60%

SBSA20 GTT GCG ATC C 10 3019 109.3 60%

28

2.4.5.2. RAPD Reaction Mixture

RAPD reaction was carried out by using the standard reagents. All the process

for the preparation of reaction mixture was carried out on ice under sterile conditions.

The following RAPD reagents with optimized concentrations were tested.

PCR Buffer {10X} {10X} 5.0µL

MgCl2 (25mM) (1.5-3.0mM) 5.0µL

dNTPs (2.0 mM) (0.2mM) 5.0µL

RAPD primers (100pMole⁄µL) 5.0µL

Template DNA (0.5-1µg) 5.0µL

Taq Polymerase (2.5U⁄ µL) 1U 0.5µL

Double distilled deionized water 28µL

Total volume 50µL

The prepared reaction mixture was supplied by the company Enzynomics,

Korea continuing all above mention reagents excluding primers and DNA. The

advantage of using the supplied reaction mixture was to avoid the errors during the

preparation of PCR reaction mixture along with the less consumption of time.

Reaction mixture (Enzynomics, Korea) 10µL

DNA 1µL

RAPD Primers 1µL

Double distilled deionized water 8µL

2.4.5.3. RAPD Temperature Cycling Conditions

RAPD amplifications were carried out in PCR machine (Eppendorf) with

initial denaturation at 94ºC for 5 minutes, followed by 40 cycles of denaturation at

94ºC for 1 minute, annealing at 35ºC for 1 minute and extension was set for 1 minute,

final extension was done at 72ºC for 15 minutes. The reaction was hold at 4.0ºC. The

five annealing temperature conditions including 31, 32, 33, 34 and 35ºC were checked

to find the optimized temperature for the primers used in the study. The subsequent

amplifications were done at 35ºC optimum annealing temperature.

29

Fig. 2.2: Three PCR heat steps were innolved in temperature optimization.

2.4.5.4. Analysis of Amplified DNA Fragments

The analysis of the RAPD amplified bands were performed on 1% agarose

gel. Before loading the RAPD amplified DNA in the wells of gel, 5µL of 6 gel

loading dye was added to the 50M RAPD sample. DNA ladder of 1Kb was also

loaded on the both sides of the gel to compare the size of different amplified

fragments in RAPD analysis. The gel was run at 100 volts for 45 minutes at room

temperature. The bands were examined under UV transilluminator and photographed

on the gel documentation system.

2.4.5.5. Data Analysis

RAPD profiles were recorded by visually comparing RAPD amplification

profiles and scoring the presence or absence of each band for each primer

(Halmschlager et al., 1994). The bands obtained from 1% agarose gel electrophoresis

were combined in a binary matrix in two - discrete - character - matrix (0 and 1 for

absence and presence of RAPD–markers, respectively). The bands were calculated

starting from top of the lanes to their bottom. The amplification profiles of different

isolates of the P. italicum were compared with each other. In order to assess the

overall distribution of genetic diversity, data was analyzed by using MINITAB

software.

94ºC

5 min

94ºC

1 min

1 min

72ºC

5 min

Final extension

72ºC

10 min 35ºC

30

2.5. SCREENING OF MOST PATHOGENIC ISOLATE OF

P. ITALICUM

This trial was carried out to screen most pathogenic isolate of P. italicum

among eight genetically diverse isolates from different regions of Punjab. A very

simple, rapid and quantitative method for the evaluation of radial growth was

followed as described by Baiyewu et al. (2007) and Chukwuka et al. (2010) with

slight modifications. Individual isolate of P. italicum was tested on fresh mandarin

fruits and fungal deterioration of fruit was observed.

Fresh mandarin c.v. Kinnow fruits were taken and washed with running tap

water and rinsed with distilled water. After washing the fruits were surface sterilized

with 75% ethanol. A sterile stainless steel scalpel was used to make a 2mm deep

wound in each of the fruit. Spore suspension (15µL) of each isolate was inoculated

into each wound using a micropipette under aseptic conditions. The concentration of

spore suspension was adjusted to 106 spores per milliliter by using a haemocytometer.

The inoculated fruits were placed in sterile petri plates in incubator for a week at

25±1oC. This experiment was replicated three times. After incubation period

percentage of fruit decay for each isolate was calculated. The isolate causing highest

fruit decay was selected for the further trails. The stock cultures were prepared for

further experimentations and were stored in refrigerator at 4oC.

2.6. COLLECTION OF PLANT MATERIAL

The following plant species were used as raw material for the extraction of

essential oils (Table 2.4). These plants were selected keeping in view their

documented antimicrobial properties and easily availability. These plants can also be

easily grown in the prevailing climatic conditions of Pakistan.

Spices which comprised the seeds of carom, cumin and fennel, bud of clove

and bark of cinnamon were purchased from the market as they are easily available at

cheap prices in the South Asian region. These spices exhibit strong aromas due to the

high molecular weight volatile compounds and are also the main constituent of

pickles and sauces to enhance their shelf life against microbial attacks. Other plants

like the mature leaves of lemon grass, eucalyptus, neem, sweet basil and holy basil

31

were collected from the surroundings of the Institute of Agricultural Sciences,

University of the Punjab during early afternoon, because maximum contents of

volatile oil occur at this time (Clark and Menary, 1980). The collection was made at

different times of the year depending upon the availability of the plant species and

plant parts selected for extraction purpose. The collected plant materials were cleaned

thoroughly.

Table 2.4. Sources of Essential Oils

Sr.

No. Botanical name English name Family Plant part used

Collection

period

1 Eugenia caryophyllata T. Clove Myrtaceae Bud March 2009

2 Cinnnamomum zylanicum J. Cinnamon Lauraceae Bark //

3 Cuminum cyminum L. Cumin Umbelliferae Seeds //

4 Foeniculum vulgare M. Fennel Umbelliferae Seeds //

5 Trachyspermum captivum L. Ajwain Umbelliferae Seeds //

6 Cymbopogon citratus S. Lemon grass Poaceae Leaves April 2009

7 Azadirachta indica L. Neem Meliaceae Leaves June 2009

8 Eucalyptus globulus L. Eucalyptus Myrtaceae Leaves //

9 Ocimum basilicum L. Sweet basil Lamiaceae Leaves July 2009

10 Ocimum sanctum L. Holy basil Lamiaceae Leaves //

2.7. EXTRACTION OF ESSENTIAL OILS

Extraction of essential oils from the raw material was conducted by the

hydrodistillation in a Clevenger apparatus (Clevenger, 1928) for 4-5 hours as

prolonged extraction normally increase the yield of essential oil (Gildemeister and

Hoffman, 1961). Distillates of essential oils were then dried over anhydrous sodium

sulphate (Kumar et al., 2007) and stored in brown glass bottle at 4oC until analyzed.

2.8. ANALYSIS OF ESSENTIAL OILS

2.8.1. Chemical Analysis

The chemical identification and quantification of constituents of pure

extracted essential oils were undertaken by gas chromatography-mass spectroscopy

32

(GC-MS) and gas chromatography- flame ionization detector (GC-FID) techniques

respectively.

2.8.1.1. Gas Chromatography/Mass Spectrometry (GC-MS) Analysis

The essential oils were subjected to GC-MS analysis using a Hewlett-Packard

mass detector (model, 7890) coupled with mass spectrometer selective detector 5975.

Analysis was carried out using a column HP5 mass-selective detector (MSD) (30m ×

0.25mm; 0.25µm film thickness), the working conditions were as follows: Helium

was the carrier gas at a flow rate of 1mL/min. Diluted sample (1:100 v/v, in methanol)

of 1µL were inserted by hand at temperature 250ºC. Oven temperature conditions

were: 12.5 minutes isothermal at 62ºC subsequently programmed heating from 62 to

92ºC at a rate of 3ºC/min and from 92 to 165ºC at a rate of 5ºC/min and after that

10ºC/minutes steps up to 310ºC and finally held isothermally for 2.5 minutes.

2.8.1.2. Gas Chromatography/Flame Ionization Detector (GC-FID)

Analysis

For quantification of the components of the essential oils GC-FID (Agilent)

equipped with FID detector. Column HP-5 (5% Phenyl Methyl Siloxane; 30mm x

0.32mm film thickness 0.25µm) was used. Sample of 1µL was injected in split mode

(50:1). Nitrogen was used as a carrier gas with flow rate of 30ml/min. oven

temperature was maintained at 70°C for 1 minute then programmed heating from 70-

250 at rate of 30°C/minute and from 250-290°C, at a rate of 20°C/minute and then

10°C/minute steps up to 300°C and finally held for 2 minutes. The relative percentage

peak area of the individual component of the essential oils was obtained from the FID

chromatograms.

The different components of the essential oils were recognized by matching

their mass spectra. NIST (National Institute of Standards and Technologies) Mass

Spectra Library was used as a reference (Adam, 2001; Mass Spectral Library, 2002).

As well as based on the comparison of their retention index (RI), kovats index (KI)

(Finar, 1978) and by comparison with literature data (Mimica-Dukic et al., 2003;

Vagionas et al., 2007).

33

2.9. ANTIFUNGAL ACTIVITY

2.9.1. Concentration of Essential Oils

Stock solution (0.05%) of Tween 80 was prepared in distilled sterilized

water. Different concentrations of plant essential oils were prepared by adding 3, 6,

12, 24 and 48µL mL-1 of pure essential oil in 1mL of Tween 80 stock solution (Soylu,

2005).

2.9.2. Preparation of Spore Suspension

Culture of P. italicum (Lahore isolate) was grown on MEA medium at 25ᵒC

for seven days prior to each experiment. Spores from the surface of the culture were

removed and then suspended in 5 mL of sterile distilled water. Spore suspension was

prepared in 0.05% (v/v) Tween 80 and the inoculum concentration was adjusted to the

recommended dose for P. italicum i.e. 106 spores per milliliter by using

haemocytometer (Eckert and Ogawa, 1985; Abdolahi et al., 2010).

2.9.3. In Vitro Antifungal Assay

2.9.3.1. Agar Dilution Assay

For antifungal assay of essential oils against P. italicum (Lahore isolate) was

performed by mixing essential oils with MEA medium. The medium was autoclaved

at 121oC for 20 minutes at 15 Psi then cooled to about 45oC. The essential oil

concentrations were prepared as described above and subsequently mixed with sterile

molten MEA medium. Thirty milliliters of media was decanted into each petri plate

which was then inoculated with test fungi by using a fine sterilized needle. After

inoculation the Petri plates were placed in incubator for seven days at 25±1°C. Three

replicates of each treatment were experimented.

2.9.3.2. Volatile Assay

The antifungal activity of essential oils against P. italicum was also checked

through volatile assays. In this method the MEA plates were first inoculated at one

34

point with test fungi and then pure extracted essential oils in quantities of 3, 6, 12, 24,

and 48µL mL-1 were applied on the surface of the sterilized filter paper, which was

placed in the lid of the petri plate. After inoculation the plates were incubated in

inverted position for seven days at 25±1°C. The zone of inhibition was measured in

two directions at 90 degree to each other (Ndukwe et al., 2006).

The colony diameter of test fungi was measured by calculating the average

radial growth. The percentage of mycelial growth inhibition of each essential oil

concentration was calculated from the mean colony diameter of control plate and from

the mean colony diameter (cm) on each essential oil amended plate. Percentage

inhibition of mycelial growth was revealed using this particular formula (Nwachukwu

and Umechuruba, 2001).

%MGI = (�̅�-Xi /�̅�) × 100

Where,

% MGI denotes: % of mycelial growth inhibition

�̅�=Mean diameter (cm) of control colony on non-amended medium

Xi=Mean diameter (cm) of tested colony replicates on a single plant essential oil

concentration amended plate (zone of growth).

2.9.4. In Vivo Antifungal Assay

2.9.4.1. Surface Sterilization of Mandarin Fruit

The preliminary washing of mandarin c.v. Kinnow fruit was done under

running tap water for 2 minutes. After this the fruit was washed with 6% sodium

hypochlorite solution by dipping in a bucket (Kanan and Al-Najar, 2009).

Consequently, this was followed by dipping the fruit in sterile distilled water for two

minutes and surface sterilization in 70% ethanol for another two minutes. Finally the

fruits were placed inside a laminar flow cabinet under aseptic conditions for

subsequent inoculation.

2.9.4.2. Fruit Inoculation with Fungal Spores and Plant Essential Oils

A wound was made at the equatorial side of the fruit with a sterile stainless

steel scalpel. The size of each wound was approximately 4 mm long and 2 mm deep

(Zhu et al., 2006). Spore suspension (as prepared in section 2.9.2.) (15µL) was

35

inoculated into each wound using a micropipette under aseptic conditions. After two

hours of incubation, the pre-determined concentration from each plant essential oil

was inoculated into each wound (Kanan and Al-Najar, 2009). The sterile distilled

water was used instead of essential oil as a control along with the other same

treatments. Three replicates were prepared for each treatment. The treated fruits were

labeled, placed in sterilized petri plates and incubated at 25±2°C for fifteen days to

assess the rate of fungal deterioration on daily basis.

2.10. IN VITRO ANTIFUNGAL ACTIVITY OF MAJOR

COMPONENTS OF ESSENTIAL OILS AGAINST P.

ITALICUM

The commercial volatile compounds purchased from company ChromaDex, Inc.

Irvine, California, USA with at least 95% purity screened In Vitro for their ability to

control the mycelial growth of P. italicum. For this experiment concentrations of

50µL L-1, 100µL L-1 and 200µL L-1 were prepared for each compound (Neri et al.,

2006). The vapor phase method was conducted for the assessment of antifungal

activity of each volatile component against P. italicum. In this method, petri plates

containing MEA medium were inoculated with the test fungus (P. italicum), after that,

different concentrations of volatile compounds were added to the filter paper

(Whatmann no. 04) through micropipette and fitted on the inner side of the lid of petri

plate. Then petri plates were incubated in inverted position for 7 days at 25oC. For the

control, filter paper was impregnated with sterile distilled water instead of volatile

component. The three replicates of each treatment were experimented. The percentage

inhibition of mycelial growth was determined using the formulae (Abdolahi et al.,

2010).

IMG% =(dc − dt)

dc× 100

Where,

IMG= inhibition of mycelial growth

dc= mycelial growth measurement in control

dt= mycelial growth measurement in treatment

36

2.11. EFFECT OF SELECTED ESSENTIAL OILS ON POST-

HARVEST PHYSICOCHEMICAL CHANGES IN CITRUS

FRUIT DURING COLD STORAGE

For the evaluation of the fruit quality, fresh mandarin c.v. Kinnow fruits were

harvested from commercial garden located in Bhalwal (Punjab, Pakistan) and

immediately transferred to the laboratory. Healthy fruits of uniform physiological age

and size were selected. Fruits were washed with running tap water and then surface

sterilized by dipping in 6% sodium hyopocholorite solution for one minute (Kanan

and Al-Najar, 2009). Surface sterilization was followed by two washings with water.

In this experiment, the concentrations of 200 and 400µL L-1 (Solaimani et al., 2009)

of selected essential oils including T. captivum, F. vulgare and E. caryophyllata were

used. These essential oils proved to have characteristic significant antifungal

properties in previous bioassays. The essential oils were applied on the surface of the

fruit by immersing fruits for 10 and 20 minutes in essential oil solution (Solaimani et

al., 2009). After that the fruits were kept in wooden crates lined with double layer of

newspaper and stored at 4ºC for 8 weeks. For the control, same treatments were

accomplished on the fruit using sterile water instead of essential oil. Each crate

representing one replicate contained sixteen fruits. The quality of the fruits was

analyzed every week and following analyses were done on three replicates of each

treatment.

2.11.1. Weight Loss

All fruits were weighed (Digital Electrical Balance, JA5003N) on day one

and numbered respectively. The marked fruits from each treatment were weighed

again after every one week of storage period. The difference between the two was

used to calculate percentage of weight loss by using following formula:

W1 =w0 − 𝑤𝑡

𝑤0× 100

Where,

w1= Percentage weight loss

37

w0 = Initial weight of the fruit on day one.

wt = Weight of the fruit after a storage period.

Similarly following parameters were measured using described protocols after

regular intervals of 7 days up to 8 weeks.

2.11.2. Juice Content

The juice content in citrus fruit was determined by extracting the juice from

fruits of each treatment by using electric juice extractor and the volume of the juice

was determined by measuring glass cylinder. Extracted juice was then filtered through

two layers of cheese cloth for further analysis.

2.11.3. pH

The pH of extracted juice samples was determined by using pH meter (Bante

Instrument, PHS-38w microprocessor) already standardized to pH 7.

2.11.4. Total Soluble Solids (TSS)

Total soluble solids were examined directly from the citrus juice by using

hand refractrometer (model PGH). A few drops of juice were placed in the

refractrometer and total soluble solids were measured in ºBrix. Three readings were

taken from each sample and then mean value was used.

2.11.5. Ascorbic Acid

For the quantification of ascorbic acid, 1mL of citrus juice was diluted in 0.4%

oxalic acid solution and volume was made up to 10mL. Then this solution was titrated

against the standard dye solution until light pink color appeared. A blank titration was

also carried out. Ascorbic acid was calculated at eight days interval according to the

following formula (Anon, 1990).

Ascobic acid =F × T × 10

D × S× 100

38

F = factor for standardization =𝑤ℎ𝑒𝑟𝑒 (𝑚𝐿 𝑜𝑓 𝐴𝑠𝑐𝑜𝑏𝑖𝑐 𝑎𝑐𝑖𝑑)

𝑚𝐿 𝑜𝑓 𝑑𝑦𝑒

Where,

T = mL of dye used for sample – mL of dye used for blank

D = mL of sample taken for dilution

S = mL of dilute sample taken for titration

2.11.6. Titratable Acidity (TA)

Titratable acidity of extracted juice was determined as amount of citric acid

in g 100mL-1. Extracted juice was strained, using 4 folds muslin cloth. Ten milliliters

of juice and 50mL of deionized water was added into a 250mL Erlenmeyer flask with

few drops of phenolphthalein as an indicator and titrated against 0.1 N NaOH solution

at pH 8.2 to a definite pink end point. The results were converted to percent citric acid

and expressed in terms of fresh weight (AOAC, 2005).

TA = (mL NaOH × 0.1𝑁 ×0.064

6.00𝑔 𝑜𝑓 𝑗𝑢𝑖𝑐𝑒) × 100

2.12. STATISTICAL ANALYSIS

Data obtained from all experiments were subjected to different statistical analysis

using Analysis of Variance (ANOVA) and significant relationship at p<0.05 with

Duncan multiple range tests (DMRT) and p=0.05 with Fisher’s Least Significant

Difference (LSD) methods by using DSSTAT software. Results obtained from

physicochemical changes in citrus fruit during storage were also analyzed using linear

regression in Microsoft excel software. All the experimental results were performed in

triplicate of various tested parameters and the results were expressed as means ±

standard errors. The antifungal activity of the essential oils against P. italicum on

citrus fruit were documented in the text was calculated as percentage (%).

39

Chapter 3

RESULTS

A survey of different cold storage houses of major citrus growing districts of

Punjab including Sargodha, Lahore, Faisalabad, Multan, Sahiwal, Jhang and Bhakar

was conducted from January to March 2009.

3.1. ISOLATION AND IDENTIFICATION OF STORAGE

FUNGI

3.1.1. Morphological Identification

A total of sixteen different fungal species were isolated from all sampled

mandarin fruits and were identified on the basis of their cultural and morphological

characteristics. Binocular microscope was used in the identification of various fungal

isolates because defining characteristics were used to distinguish among them. The

fungi identified were Aspergillus niger Van Tiegh, Aspergillus flavus Link,

Aspergillus fumigatus Fresenius, Aspergillus terreus Thom, Penicillium verrucosum

Dierckx, Rhizopus arrhizus Fischer, Rhizopus stolonifer (Ehrenb) Vuill, Aspergillus

parasiticus Speare, Fusarium oxysporum Schlecht, Penicillium citrinum Thom,

Aspergillus awamorii Nakaz, Alternaria alternate (Fries) Keissler, Fusarium solani

(Mart.) Sacc., Mucor sp. and Dreshlera sp. (Table 3.1).

3.1.2 ANALYSIS OF GENETIC DIVERSITY AMONG

FUNGAL ISOLATES

The frequency of occurrence of each specie of fungi found associated with the

deterioration of sampled mandarin fruits is shown in Table 3.2. The principal

mycoflora obtained belongs to seven different genera including Aspergillus,

Penicillium, Rhizopus, Fusarium, Alternaria, Dreschlera, and Mucor. The genus

Aspergillus was found as the most dominant genera with a frequency of occurrence of

42.97% followed by the Penicillium (38%), Rhizopus (9.08%), Fusarium (4.95%),

Alternaria (2.47%), Mucor (1.65%) and Dreschlera (0.82%).

40

Table 3.1: Morphological Characteristics of Different Storage Fungi Isolated from Sampled Mandarin Fruits.

Name of

fungus

Colony

diameter

Colony

Texture

Obverse Reverse Characters

of hyphae

Conidiophore Conidia

Penicillium

italicum

5-6 cm in

14 days

Less

fasciculate

Bluish

green

Uncolored

to yellow

brown

Septate,

hyaline

Smooth walled,

hyaline, matulae and

phailides present

Ellipsoidal to cylindrical,

smooth walled, greenish

4.0-5.0 x 2.5-3.5 µm.

Penicillium

verrucosum

2.5-3.5

cm in 14

days

Velvety or

almost

floccose

Grey

green

Yellowish

Septate,

hyaline

Two-stage branched,

rough walled

Globose to subglobose,

smooth walled, greenish,

3.0-4.0 µm in diameter.

Penicillium

citrinum

2-3 cm in

14 days

Velutinous to

floccose

Greyish to

turquoise.

Bright

yellow

Septate,

hyaline

Smooth walled,

matulae and phailides

present

Spherical to subspherical,

smooth-walled or finely

roughened, 2.23.0 µm in

diameter.

Aspergillus

niger

5-6 cm in

7 days

Powdery Black Off-white Septate,

conidial head

radiate and

biseriate

Broad, long, hyaline,

thick walled, brownish,


covered the entire

vesicle

Globose, irregularly

roughened, brown to

black, 6-7 µm in diameter.

Aspergillus

flavus

6-7 cm in

7 days

Powdery Yellow

green

Pale

brown

Conidial

head radiate,

biseriate,

Hyaline, long,

roughened, matulae and

phailides covered the

entire vesicle

Subspherical, pale green,

smooth to finely

roughened, echinulate, 3.5

to 6 µm in diameter.

Aspergillus

fumigatus

7 cm in 7

days

Velvety Bluish

green

Greyish Vesicle

pyriform,

uniseriate

Clavate vesicle, thick

walled

Globose to subglobose,

smooth, echinulate, green,

2.5-3 µm in diameter.

Aspergillus

terreus

6-7 cm in

7 days

Velvety or

almost

floccose

Orange

brown

Yellow

brown

Conidial

heads

columnar,

biseriate

Long, smooth, thick

walled, hyaline, vesicle

hemispherical

Globose to slightly

ellipsoidal, smooth

walled, 1.6-2.5 µm.

Aspergillus

awamorii

4.5-

5.5cm in

7 days

Velvety Green Off-white Biseriate, Smooth walled, vesicle

subglobose

Cylindrical to ellipsoidal,

coarsely roughened, 3.5-

4.5 µm in diameter.

Conti…..

41

Aspergillus

parasiticus

5-6 cm in

7 days

Velvety or

floccose

Dark

green

Yellow

Orange

Uniseriate, Roughened, vesicle

spherical

Roughened, echinulate

and coarsely spiny,

globose, 4.2-5µm in

diameter

Rhizopus

arrhizus

Full plate

(9cm) in

3-4 days

Cottony White Brown Hyphae

branched and

aseptate,

rhizoids

present

Short, nonseptate,

smooth walled, simple

and branched,

columella globose

Unicellular, ovoid to

more or less globose,

hyaline to brown in color,

4-11µm in diameter

Rhizopus

stolonifer

Full plate

(9cm) in

3-4 days

Cottony white yellow

brown

Mycelium

aerial,

aseptate

hyphae,

rhizoids well

developed

Dark, subglobose rigid,

sporangia pale to

brown, straight,

columella conical

Angular-globose to

ellipsoidal, thick walled,

13µm in length.

Mucor

mucedo

Full plate

(9cm) in

3-4 days

Cottony Grey Yellowish

brown

Columella

obovoid,

ellipsoidal,

with truncate

base

Short, unbranched,

recurved and encrusted

wall Sporangia

brownish to grey

Thick walled, cylindrical

to ellipsoidal,

subspherical, 11-13 x 6-

7µm.

Fusarium

oxysporum

4.6-6.5

cm in 4-5

days

Sparce to

floccose

Snow

white

purple Macro and

microconidia

are present

Short, single in aerial

mycelium,

clamydospores are also

present

Macroconidia septate,

fusiform, moderately

curved, pointed at both

ends 23-54 x 3-4.5 µm,

microconidia non-septate,

ellipsoidal to cylindrical,

straight or often curved, 5-

12 x 2.3 - 3.5 µm.

Fusarium

solani

4.5-6.5 in

7 days

Floccose White to

cream

Brown Macro and

micro

conidia

present

Short and multi

branched form

sporodochia, hyaline,

globose, smooth to

roughened

Macroconidia fusiform,

curved, short, with blunt

apical and pedicellate

basal cells 28-42 x 4-6

µm, Microconidia

Conti…

..

42

clamydospores are

present.

cylindrical to oval, one- to

two-celled , 8-16 x 2-4.5

µm.

Alternaria

alternata

6-7 cm in

7 days

Cottony Greyish

black with

white

margin

Creamy Hyphae

branched and

septate,

brown

Simple, straight,

brown.

Ovoid, obclavate and dark

brown in color, 25-30 × 5-

9 µm.

Dreshlera

hawaiiensis

4-5.5 cm

in 7 days

Effuse Blackish

to brown

Black Hyphae

branched and

septate,

hyaline and

smooth

walled

Unbranched, brown

and septate.

Straight, fusiform to

ellipsoidal, septate,

rounded at both ends,

smooth walled, pale

brown, 12-22 × 8-15 µm.

43

Table 3.2: Frequency of Occurrence of Different Fungal Genera Isolated from

Sampled Fruits.

Sr. No. Fungi isolated % Frequency of occurrence

1 Aspergillus 42.9

2 Penicillium 38

3 Rhizopus 9.1

4 Mucor 1.6

5 Fusarium 4.9

6 Alternaria 2.5

7 Dreschlera 0.8

Where; n= 78

Table 3.3: Frequency of Occurrence of Storage Fungi isolated from Sampled

Fruits.

Sr. No. Fungi isolated % Frequency of occurrence

1 Penicillium italicum 29.7

2 Penicillium verrucosum 5.8

3 Penicillium citrinum 2.5

4 Aspergillus niger 14.9

5 Aspergillus flavus 9.1

6 Aspergillus fumigatus 7.4

7 Aspergillus terreus 5.8

8 Aspergillus awamorii 2.5

9 Aspergillus parasiticus 3.3

10 Rhizopus arrhizus 4.9

11 Rhizopus stolonifer 4.1

12 Mucor mucedo 1.6

13 Fusarium oxysporum 3.3

14 Fusarium solani 1.6

15 Alternaria alternata 2.5

16 Dreshlera hawaiiensis 0.8

Where; n= 78

44

When frequency of occurrence of each fungal species isolated from mandarin

fruits was calculated, Penicillium italicum was found the most frequently isolated fungus

with the highest percentage of occurrence of 29.75% among all isolates (Table 3.2). This

was followed by the Aspergillus species where A. niger showed a percentage of

occurrence of 14.87%, which was followed by A. flavus with 9.09% and A. fumigatus

with 7.43% frequency of occurrence. Whereas, least occurring fungi Mucor sp. and

Dreshlera sp. were found with the percentage of 1.65% and 0.82% respectively. The

results revealed that P. italicum is widespread among all examined sampled fruits that is

why it was selected for further experimentation.

3.1.3. Genetic Analysis of P. italicum Isolates

Penicillium italicum showed highest frequency of occurrence in all mandarin

fruits sampled from eight different districts of citrus producing belt in Punjab, Pakistan.

Genetic analysis of different isolates was carried out to determine the genetic variation

and genetic diversity among these eight geographically distinct isolates by using RAPD-

PCR technique (Table 3.4).

Table 3.4: Qualitative and Quantitative Spectrophotometric Analysis of Extracted

DNA.

Sr.

No.

P.

italicum

isolates

Spectrophotometric reading of

Extracted DNA

Ratio

260/280

Conc. of Extracted

DNA(µg mL-1

)

at 260 nm at 280 nm

1 Pi Lhr 1.504 1.332 1.13 60

2 Pi Mul 0.812 0.866 0.94 29

3 Pi Fsd 0.046 0.056 0.82 1.0

4 Pi Chn 1.479 1.303 1.13 59

5 Pi Jhg 0.084 0.080 1.04 3.0

6 Pi Bhk 0.163 0.175 0.93 6.0

7 Pi Sh 0.001 0.001 1.00 4.5

8 Pi Sgd 0.123 0.117 1.06 5.0

45

Table 3.5: Random twenty decamers used in initial screening, their accessions,

sequences, number of polymorphic bands, percentage of polymorphic products and

size of bands produced by each primer.

Name Sequence ( 5'- 3' ) Nano mole Total no.

of Bands

No. of poly

morphic

bands

%age of

polymorphic

bands

Size of

bands in

base pairs

SBSA01 CAG GCC CTT C 111.3 40 11 28 250-1000

SBSA02 TGC CGA GCT G 108.4 39 7 17% 200-1500

SBSA03 AGT CAG CCA G 110.1 39 15 38% 200-700

SBSA04 AAT CGG GCT G 107.6 45 10 7% 300-1000

SBSA05 AGG GGT CTT G 106.5 29 5 17% 300-900

SBSA06 GGT CCC TGA C 109.9 37 5 14% 200-1200

SBSA07 GAA ACG GGT G 105.9 34 13 38% 400-1800

SBSA09 GGG TAA CGC C 108.1 46 6 13% 200-1400

SBSA10 GTG ATC GCA G 107.6 39 15 38% 270-1500

SBSA11 CAA TCG CCG T 110.4 41 9 22% 200-1600

SBSA12 TCG GCG ATA G 107.6 42 18 43% 400-1900

SBSA13 CAG CAC CCA C 112.2 63 7 11% 340-2000

SBSA14 TCT GTG CTG G 108.2 14 6 43% 350-800

SBSA16 AGC CAG CGA A 108.3 10 2 20% 300-1500

SBSA17 GAC CGC TTG T 109.3 11 3 27% 900-1500

SBSA18 ACG TGA CCG T 107.6 50 2 4% 200-1400

SBSA19 CAA ACG TCG G 108.7 47 7 15% 250-1100

SBSA20 GTT GCG ATC C 109.3 45 5 11% 200-1100

A total of 20 RAPD decamers (Table 3.5) were used for testing the genetic

variability among eight isolates of P. italicum of which two primers (Primer SBSA08 and

SBSA15) did not show any amplification and hence dropped in the initial screening of

the random primers. Remaining 18 random primers produced easily scorable and

reproducible banding patterns, which were designated in genetic coefficient matrix in

Minitab 16 software for further analysis. Total amplified polymorphic bands ranged from

approximately 200 base pairs to 2000 base pairs in RAPD profile. There was a low

46

percentage of polymorphism in different isolates of P. italicum. The average number of

polymorphic bands perceived per primer was 8.22. The RAPD profiles produced with the

primers (SBSA06, SBSA07, SBSA11, SBSA12, SBSA13, and SBSA18) and banding

pattern are shown in Fig. 3.1. The ladder 100 base pairs was run as markers on both sides

along with the negative control in which only water was used instead of DNA that

indicates the credibility of the reaction mixture having no amplified band.

A dendogram based on UPGMA analysis indicated that the eight isolates formed three

major groups GI, G2 and G3 confirming some level of genetic variation among the

isolates of P. italicum (Fig. 3.2). The similarity coefficient ranged from 0 to 0.23

indicating that no two isolates are 100% similar and the differences are present among all

of the isolates. Group 1 (G1) was further divided in to three subgroups containing five

isolates naming Pi Lhr, Pi Mul, Pi Jhg, Pi Bhk and Pi Sdg. Group 2 (G2) consisted of two

isolates, Pi Fsd and Pi Chn while group 3 (G3) consisted of only one isolate, Pi Sh.

The first subgroup consisted of two isolates collected from Lahore and Multan, which

were 3% distant from each other while this subgroup is 9% distant from the second

subgroup containing two isolates collected from Jhang and Bhakar regions of Punjab.

There is much similarity seen in isolates collected from Jhang and Bhakar, which are 1%

distant from each other. This subgroup was 5% distant from the third subgroup which

contained only one isolate collected from Sargodha cold storage house. G2 is 2% distant

from G1 while G3 was 4% and 6% distant from G1 and G2, respectively.

47

-ve M 1 2 3 4 5 6 7 8 M -ve M 1 2 3 4 5 6 7 8 M

M 1 2 3 4 5 6 7 8 -ve M -ve M 1 2 3 4 5 6 7 8 M

-ve M 1 2 3 4 5 6 7 8 M -ve M 1 2 3 4 5 6 7 8 M

Fig. 3.1: RAPD profile of eight isolates (1-8 shown in Table 3) of P. italicum produced

by random primers (SBSA06, SBSA07, SBSA11, SBSA12, SBSA13, and

SBSA18. Molecular weight markers (M in base pairs) are indicated on the both

sides (100 base pair DNA ladder, cat # DM001), -ve sign shows the well with

negative control in PCR reaction.

48

Fig. 3.2: UPGMA cluster analysis based dendogram depicting the genetic relationship among

selected P. italicum isolates.

49

3.1.4. Screening of Most pathogenic Isolate of Penicillium

italicum

To select the most pathogenic strain of Penicillium italicum, all the eight

strains of the fungus were checked through their inoculations on mandarin fruits. The

results showed that all the eight isolates are pathogenic and have an ability to cause

postharvest infection in selected fruit. However, their pathogenic ability showed slight

variation (Fig. 3.3). The Lahore (Lhr) isolate was found most pathogenic among all as it

produces 93% decay in treated fruits. This was followed by Faisalabad (Fsd) isolate with

88% deterioration of inoculated fruits in seven days. Sargodha (Sdg) and Multan (Mul)

isolates were found equally pathogenic and they caused 85% decay in selected fruits.

Rest of the four isolates i.e., Sahiwal (Shw), Jhang (Jhg), Chiniot (Chn) and Bhakar

(Bhk) isolates found statistically equally significant (P=0.05) in causing deterioration of

treated fruits and the percentage of decay was between 60-70% (Fig. 3.3).

Fig 3.3: Screening of most pathogenic isolate of Penicillium italicum. Values are mean

of three replicates analyzed individually; Vertical bars show standard error of

means of three replicates. Values with different alphabetic letters indicate

significant differences (P=0.05) as determined by Duncan’s Multiple Range

(DMR) test.

0

20

40

60

80

100

120

Lhrisolate

Fsdisolate

Sdgisolate

Mulisolate

Shwisolate

Jhgisolate

Chnisolate

Bhkisolate

Per

cen

tage

of

fru

it d

eter

iora

tion

a ab

c c

c c

b b

50

PLATE: 3.1: Trial showing potential of eight isolates of P. italicum to cause postharvest

decay in mandarin fruits. Where: A= Lhr Isolate; B = Fsd Isolate; C = Sdg

Isolate; D = Mul Isolate; E = Chn Isolate; F = Shw Isolate; G = Jhg Isolate; H

= Bhk Isolate.

B C

E

A

D F

G H

51

3.2. ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS

The study was designed to investigate a plant based shelf life enhancer against

fungal deterioration in citrus during storage. In early In Vitro antifungal assays, potential

of essential oils from ten different sources were checked against Penicillium italicum the

causal agent of blue mold in citrus. All the selected essential oils showed variable

capacities of restricting fungal growth. However, the dosage effect was found

pronounced in every case. Dilution and volatile methods used for In Vitro studies showed

insignificant variation in their toxicity effects in most of the selected essential oils.

Similar trend was observed when selected essential oils were checked for their In Vivo

antifungal activities.

3.2.1. AZADIRACHTA INDICA L. (Neem)

3.2.1.1. In Vitro Antifungal Efficacy

The In Vitro antifungal activity of essential oil extracted from leaves of

Azadirachta indica L. found to be varied in both dilution and volatile assays. In dilution

assay the selected essential oil failed to produce any significant reduction in biomass

production of tested fungi. However, volatile assay showed that essential oil of A. indica

have a capacity to inhibit growth of P. italicum. The essential oil in concentration of 3

and 6μL mL-1

produced insignificant reduction in fungal biomass. However, this

antifungal activity increased with increase in concentration and highest tested

concentration of 48μL mL-1

resulted in 37.6% growth inhibition (Fig. 3.4).

3.2.1.2. In Vivo Antifungal Efficacy

In Vivo studies showed significant antifungal potential of essential oil extracted

from A. indica against selected fungi. Dosage effect was also found obvious thus

supporting results of In Vitro volatile assay. Lowest tested concentration of 3μL mL-1

produced 44.6% inhibition in colony growth that increased to 89.8% in samples treated

with essential oil in concentration of 24μL mL-1

. Whereas, the highest tested


resulted in 94.3% highly significant inhibition of fungal

52

growth. All tested concentrations delayed fungal growth to 3rd

day of inoculation (Fig.

3.5).

3.2.1.3. Chemical Composition

The GC-MS and GC-FID analysis of essential oil of A. indica showed the

presence of ten different compounds (Table 3.6), which represented 88.27% of the total

oil. This volatile oil contained oleic acid with relative percentage of 38.5% followed by

palmitic acid (21.45%), caryoplyllene (9.7%), limonene (7%), linalool (4.3%) and citral

(4%) as major components. Besides, this oil also had significant amount of minor

components (Fig 3.6).

53

Fig. 3.4: In Vitro antifungal activity of A. indica essential oil, used at various

concentrations on mycelial growth of P. italicum. (A) Dilution assay (B)

Volatile assay. Values are mean of three replicates analyzed individually.

Vertical bars show standard error of means of three replicates. Values with

different alphabetic letters indicate significant differences (P=0.05) as

determined by Duncan’s Multiple Range (DMR) test.

Fig. 3.5: In Vivo antifungal activity of A. indica essential oil, used in various

concentrations, on mycelial growth of P. italicum. Values are mean of three

replicates analyzed individually.

0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)

Conc. of essential oils (µL mL-1)

A

a a a a a a

0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)

Conc. of essential oils (µL mL-1)

B

a ab

abc bcd

cd d

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P.

itali

cum

(cm

)

Incubation Time (Day)

Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

54

Fig. 3.6: GC-MS chromatogram of essential oil extracted from leaves of A. indica.

S Oleic acid

Limonene

Palmitic acid

Citral

55

Table 3.6: Chemical Composition of Essential Oil Extracted from A. indica.

Components RI

%

Composition

β-pinene 943 0.70 ± 0.1f

Limonene 1018 6.9 ± 0.05d

Linalool 1082 4.3 ± 0.12e

Terpinene-4-ol 1137 1.02± 0.01f

Citral 1174 4 ± 0.49e

Caryophyllene 1494 9.7 ± 0.18c

Acetyl eugenol 1552 t

Palmitic acid 1968 21.45 ± 0.28b

Stearic acid 2167 1.5 ± 0.00f

Oleic acid 2175 38.5 ± 0.86a

TOTAL

88.07

Values are the mean of three replicates analyzed individually. Different alphabetic letters

in superscript indicate significant differences (P=0.05) as determined by Duncan’s

Multiple Range (DMR) test.

t = trace element, less than 0.05%.

Compounds listed in order of increasing RI value from a HP-5MS column.

Retention indices relative to C9-C24 n-alkanes on the HP-5MS column.

Mode of Identification is RT,RI and MS where;

RT = Identification based on retention time.

RI = Identification based on retention index.

MS = Identification based on comparison of mass spectra.

56

3.2.1.4. In Vitro Antifungal Efficacy of Major Components of

A. indica Essential Oil

Top five major components identified in tested essential oil of A. indica were

checked against P. italicum in concentrations 50, 100 and 200µL L-1

. The inhibitory

effect of all these compounds was found dosage dependent. Limonene found to be the

most active component of this essential oil as its highest tested concentration resulted in

96.55% growth reduction in terms of colony diameter in tested fungi. This was followed

by caryophyllene that restricted fungal growth by 87.36% at 200µL L-1

. After this,

linalool and citral were found equally significant with a percentage of inhibition around

74% at maximum tested concentration. However, at lower concentrations linalool

regulated fungal growth better than citral. Least antifungal activity was shown by

palmitic acid that resulted in 68.1% growth reduction at its highest tested concentration

that was found close to the lowest tested concentration of limonene (Fig. 3.7).

Fig. 3.7: Mycelial growth of P. italicum measured after 7 days of incubation on MEA

medium, treated with different concentrations of major components of A. indica

essential oil. Values are mean of three replicates analyzed individually. Vertical

bars show standard error of means of three replicates. Values with different

alphabetic letters indicate significant differences (P=0.05) as determined by

Duncan’s Multiple Range (DMR) test.

0

20

40

60

80

100

120

Per

cen

tage o

f in

hib

itio

n (

%)

50µL/L 100µL/L 200µL/L

gh

a

c d

c

g

h

b

d e

d

f

g

c

f

57

3.2.2.CINNAMOMUM ZYLANICUM J. (Cinnamon)


Cinnamomum zylanicum J. bark essential oil failed to inhibit tested fungi

significantly. The efficacy varied when tested through dilution and volatile methods.

Volatile method restricted fungal growth more effectively as compared to the dilution

method. Lowest tested concentration of 3μL mL-1

resulted in a reduction of 20.49% in

fungal growth when tested through volatile assay. However, the same concentration

proved inefficient when checked through dilution method. Insignificant reduction in

fungal growth was recorded in dilution method when concentration increased to 6, 12 and

24μL mL-1

. Highest tested concentration of 48μL mL-1

resulted in 32.43% fungal growth

reduction in dilution assay. In case of volatile assay the higher concentrations of 6 to

48μL mL-1

proved equally significant in controlling the tested fungi. The Highest tested


caused 11.74% more reduction in fungal growth when

checked through volatile assay in comparison to the dilution method (Fig. 3.8).


The data obtained from In Vivo antifungal assay shows that C. zylanicum bark

essential oil delayed fungal growth till third day of inoculation in comparison to the

control in which fungal growth appeared at second day of inoculation. No significant

difference (P=0.05) was recorded among the efficacy of various tested concentrations of

selected essential oil. After 15th

day of inoculation selected essential oil concentrations

caused a reduction in fungal growth that was between 83-90% (Fig. 3.9).


Table 3.7 shows the individual quantities of essential oil components estimated

in the C. zylanicum essential oil. Seventeen components were identified in examined oil

that constituted 95.34% of the oil. It contained cinnamaldehyde (41.47%) as the main

component, followed by the eugenol (21.5%) isoeugenol (11.2%) caryophyllene (5.1%)

and β-pinene (2.1%).

58

Fig. 3.8: In Vitro antifungal activity of C. zylanicum essential oil, used at various






Fig. 3.9: In Vivo antifungal activity of C. zylanicum essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)

Conc. of essential oil (µL mL-1)

A

ab

a a a a a

0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)

Conc. of essentail oil (µL mL-1)

B

a

ab

b b b b

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

59

Fig. 3.10: GC-MS chromatogram of essential oil extracted from bark of C. zylanicum.

Cinnamaldehyde

Eugenol

Iso eugenol

Caryophyllene

60

Table 3.7: Chemical Composition of Essential Oil Extracted from C. zylanicum.

Components RI

%

Composition

β-Pinene 943 2.1 ± 0.2f

ɣ-Terpinene 998 0.93 ± 0.03h

Limonene 1018 1.2 ± 0.17gh

p-Cymene 1042 1.5 ± 0.1f-h

Cinnamaldehyde 1136 41.7 ± 0.31a

Terpinen-4-ol 1137 1.4 ± 0.03gh

1,3-Cyclohexadien-1-menthanol,4-

(1-Methyletthyl 1240 t

Carvacrol 1267 1.14 ± 0.02gh

Eugenol 1392 21.5 ± 0.0b

1,8-Cineole 1398 3.1 ± 0.0e

Isoeugenol 1410 11.2 ± 0.05c

2-Aminotridecane 1492 t

Caryophyllene 1494 5.1 ± 0.05d

Aceteugenol 1552 1.3 ± 0.0gh

α-Humulene 1579 1.5 ± 0.16f-h

Palmitic acid 1968 1.7 ± 0.11fg

Stearic acid 2167 t

95.34




t= trace, less than 0.05%.







61


C. zylanicum Essential Oil

First five prime components identified in essential oil of Cinnamomum

zylanicum J. showed strong antifungal activity against selected fungi when tested

individually. Eugenol was found most significant in reducing the colony diameter of P.

italicum as its highest tested concentration completely restricted its growth. This was

followed by caryophyllene that resulted in a reduction of 87.36% at 200µL L-1

.

Statistically this activity was found in accordance with that of 100µL L-1

of eugenol. Rest

of the three compounds i.e., cinnamaldehyde, isoeugenol and β-pinene showed

insignificant difference (P=0.05) among their antifungal activity at 200µL L-1

. Lower

concentrations however showed slight differences among their antifungal potential (Fig.

3.11).


medium, treated with different concentrations of major components of C.

zylanicum essential oil. Values are mean of three replicates analyzed

individually. Vertical bars show standard error of means of three replicates.

Values with different alphabetic letters indicate significant differences

(P=0.05) as determined by Duncan’s Multiple Range (DMR) test.

0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

)


c

f

h

ef g

a

b

c c

e

g

b

d c C

62

3.2.3. CUMINUM CYMINUM L. (Cumin)


Essential oils extracted from Cuminum cyminum L. seeds depicted highly

significant In Vitro antifungal activity against P. italicum. However, the results obtained

in dilution and volatile assays varied in lower concentrations of 3 and 6μL mL-1

. On these

concentrations dilution assay showed highly significant reduction of 96 and 98.5% in

fungal growth. Whereas, above mentioned same concentrations could only produce 32

and 68.9% growth inhibition. Further increase in essential oil concentration highly

significantly controlled fungal growth in both assays. No growth was recorded in dilution

assay while traces of fungal mycelium were recorded in volatile assay (Fig. 3.12).


In Vivo assays illustrated a highly significant antifungal potential of C. cyminum

essential oil. All tested concentrations restricted growth till 5th day of inoculation. A

highly significant reduction of 88 – 91% was recorded in samples treated with

concentrations between 3 - 24μL mL-1

. The highest tested concentration of 48μL mL-1

restricted growth of selected fungi completely (Fig. 3.13).


The chemical composition of C. cyminum essential oil was obtained from GC-FID

and GC-MS analysis and is computed in Table 3.8. Thirty three compounds were

originated in essential oil of C. cyminum accounting for 98.61% of the total amount. As

the table 3.8 revealed that C. cyminum oil contained cuminaldehyde (36.4%), carvacrol

(24.46%), terpinen-4-ol (11.5%), citronellal (5.43%), gamma-terpinene (4.1%), cumic

acid (2.9), 4-carvomenthenol (2.12%) and thymol (0.13%) as the major components. In

addition, the tested cumin essential oil showed a substantial amount of numerous minor

constituents (Fig. 3.14).

63

Fig. 3.12: In Vitro antifungal activity of C. cyminum essential oil, used at various






Fig. 3.13: In Vivo antifungal activity of C. cyminum essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


A a

b b b b b 0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)

Conc. of essentia oil (µL mL-1)

B

c c c

bc

ab

a

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

64

Fig. 3.14: GC-MS chromatogram of essential oil extracted from seeds of C. cyminium.

Cumin aldehyde ɣ-terpinene Carvacrol

Citronellal Terpinene-4-ol

65

Table 3.8: Chemical Composition of Essential Oil Extracted from C. cyminum.

Components RI

%

Composition

β-Pinene 943 1.2 ± 0.005g-j

ɣ-Terpinene 998 4.13 ± 0.09e

Limonene 1018 0.1 ± 0.00j

p-Cymene 1042 0.09 ± 0.002j

Butyl-1-cyclohexene 1089 0.4 ± 0.006ij

2,6-Dimethyl-3,5,7-octatriene-2-ol,E,E- 1090 t

Ethanone, 1-(6-methyl-7-

oxabicyclo[4.1.0]hept-1-yl)- 1113 t

Silane, (4-ethylphenyl) trimethyl 1115 t

Citronellal 1125 5.43 ± 0.3d

Terpinen-4-ol 1137 11.5 ± 0.17c

Australol 1149 1.1 ± 0.00g-j

Cyclopentanepropanol, 2-methylene- 1159 t

Cuminaldehyde 1169 36.4 ± 0.52a

Estragol 1172 1.41 ± 0.00g-i

Phellandral 1175 0.05 ± 0.06j

4- Isopropyl-5-methyl-hexa-2,4-dienoic

acid, methyl ester 1188 t

4-Carvomenthenol 1189 2.12 ± 0.15fg

2-Caren-10-al 1230 1.43 ± 0.008g-i

1,3-Cyclohexadien-1-menthanol,4-(1-

methyletthyl 1240 0.7 ± 0.00h-j

Thymol 1262 0.13 ± 0.003j

Carvacrol 1267 24.46 ± 0.33b

7-Hydroxy-2-methyl-octa-3,5-dienoic

acid, methyl ester 1297 t

Bicyc[3.1.0]hexane-6-menthanol, 2- 1322 t

66











hydroxy-1,4,4-trimethyl-

β-Terpinyl acetate 1348 0.5 ± 0.005ij

Propane,1,2,3-trimethoxy-1-phenyl 1390 t

Isoeugenol 1410 0.3 ± 0.00ij

α-Amorphene 1440 1.76 ± 0.04gh

Acordiene 1474 0.7 ± 0.05h-j

9-Undecenol, 2,10- dimethyl- 1477 0.13 ± 0.03j

Caryophyllene 1494 0.1 ± 0.00j

4-Isopropylphenylacetic acid 1497 1.03 ± 0.08g-j

4-(1,5-Dihydroxy-2,6,6-

Trimethylcyclohex-2-enyl)but-3-en-2-one 1767 0.14 ± 0.01j

Cumic acid 2011 2.9 ± 0.38f

98.61

67


C. cyminium Essential Oil

All the seven major components of Cuminum cyminum L. significantly

inhibited tested fungi when checked individually. Data analyses proved thymol was the

most effective antifungal component of this essential oil as its lowest tested concentration

resulted in the highest inhibition of 93.10% in fungal growth. Further increase in

concentration completely inhibited fungal growth (Fig. 3.15). After this the highest tested

concentration of citronellal, cuminaldehyde and terpinen-4-ol also totally restricted

fungal growth. However, the lower concentrations of these components showed slight

variation among their antifungal activity. Following this, γ-terpinene resulted in 94.25%

reduction in colony diameter when compared to the control. Carvacrol and β-pinene were

the last two components with a percentage of reduction of 83.33 and 69.55% in tested

fungi when exposed to their maximum concentration (Fig. 3.15).

Fig. 3.15: Mycelial growth of P. italicum measured after 7 days of incubation on

MEA medium, treated with different concentrations of major components of C.

cyminium essential oil. Values are mean of three replicates analyzed individually.


different alphabetic letters indicate significant differences (P=0.05) as determined

by Duncan’s Multiple Range (DMR) test.

0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

)


a a b

g bc

de

b

cde

f

a b

de

a

e

bcd

e

f

b

de

f f

68

3.2.4. CYMBOPOGON CITRATUS S. (Lemon Grass)


Cymbopogon citratus S. essential oil was also found effective in controlling

growth of selected fungi in artificial media. Both dilution and volatile assays showed

equally effective antifungal activity. Concentration effect was also found slightly

significant. Lowest tested concentration of 3μL mL-1

caused a reduction of 70-71% in

both assays. This reduction in biomass increased to 94.5% in dilution assay and 97.1% in

volatile assay at 24μL mL-1

. The highest tested concentration of 48μL mL-1

reduced

growth of P. italicum up to 97% in dilution assay whereas negligible growth was

recorded in volatile assay on this concentration (Fig. 3.16).


Essential oils extracted from C. citratus significantly controlled In Vivo growth

of P. italicum. Increase in antifungal activity with increase in essential oil concentration

was not found very significant. Concentrations of 3, 6 and 12μL mL-1

showed almost

equally significant reduction in colony growth of P. italicum that was around 90%, and

the growth started after 4th

to 5th

day of inoculation. Essential oil in 24μL mL-1

restricted

fungal growth initiation till 8th

day of inoculation and the percentage of colony growth

suppression was recorded 93%. Highest tested concentration of 48μL mL-1

completely

inhibited fungal growth as can be seen in Fig. 3.17.


Chemical composition of C. citratus essential oil was analyzed by GC-MS

and GC-FID and is given in Table 3.9 with their percentages (Fig. 3.18). A total of twelve

compounds were identified in the oil, making up to 97.91% of the extracted oil. As seen

in Table 3.9, the major compound in the C. citratus oil was citral (46.64%). This was

followed by neral (29.73%), geraniol (7.64%), limonene (5.54%), caryophyllene (4.2%)

and geranil acetate (3.4%). Few minor compounds also existed in small quantities.

69

Fig. 3.16: In Vitro antifungal activity of C. citratus essential oil, used at various






Fig. 3.17: In Vivo antifungal activity of C. citratus essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


A

a

b bc

bc c c

0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


B

a

b bc

cd d d

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

70

Fig. 3.18: GC-MS chromatogram of essential oil extracted from leaves of C. citratus.

Neral

Geranil acetate

Citral

Limonene

71

Table 3.9: Chemical Composition of Essential oil Extracted from C. citratus.

Components RI

%

Composition

β-Myrcene 958 1.75 ± 0.14g

ɣ.-terpinene 998 t

Limonene 1018 5.54 ± 0.26d

Linalool 1082 1.5 ± 0.05gh

citronellal 1125 0.45 ± 0.02i

Neral 1174 29.73 ± 0.01b

Citral 1174 46.6 ± 0.00a

Geraniol 1228 7.64± 0.2c

Geraniol formate 1349 1.1 ± 0.05h

Geranil acetate 1352 3.4 ± 0.03f

Caryophyllene 1494 4.2 ± 0.1e

α-Cadinol 1580 t

TOTAL

97.91











72


C. citratus Essential Oil

Four major components identified in Cymbopogon citratus S.were checked

individually against P. italicum. Highest antifungal activity was recorded in neral and

limonene. Their highest tested concentration restricted the mycelial growth of P. italicum

up to 97%. All the three tested concentrations of these two components showed

insignificant differences among their antifungal potential. Caryophyllene followed this

with a reduction of 87.36% when checked in a concentration of 200µL L-1

. Least activity

was recorded in citral that in its lowest tested concentration could only cause a reduction

of 31.03% in tested fungi. This inhibition percentage increased with increase in

concentration and highest tested concentration showed a reduction of 73.57% in colony

growth (Fig. 3.19).


medium, treated with different concentrations of major components of C.

citratus essential oil. Values are mean of three replicates analyzed




0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

) 50µL/L 100µL/L 200µL/L

e d

b

g f

c

d

c

a

d

c

a

73

3.2.5. EUCALYPTUS GLOBULUS L. (Eucalyptus)


Essential oils extracted from Eucalyptus globulus L. leaves also significantly

inhibited selected fungi in In Vitro assays. Volatile assay showed a little higher inhibition

of fungal growth when compared to the dilution assay. However, in both assays the

increase in fungal inhibition with increase in essential oil concentration was not highly

significant (Fig. 3.20). In dilution assay lowest tested concentration of 3μL mL-1

reduced

fungal growth up to 56% that increased to 77% when essential oil was checked in a


. In contrast, Volatile assay has reduced the Penicillium

italicum growth up to 58 and 87% at concentrations of 3 and 48μL mL-1

respectively.


Results obtained from In Vivo antifungal assays also followed similar trend as

was recorded in In Vitro experiments. In comparison to the control, fungal growth started

two days later on fruits treated with E. globulus essential oil in its lowest tested


. The fungal growth inhibition was 80.79% that increased to

88.71% when concentration was made double. Further increase in tested oil concentration

restricted the fungal growth till 6th day after inoculation however it failed to increase the

percentage of inhibition significantly (Fig. 3.21).


The results of GC-MS and GC-FID analysis are reported in Table 3.10

revealing qualitative and quantitative analysis of E. globulus essential oil. As seen in

table, thirty nine various components were obtained amounting 97.07% of the total of the

extracted oil. E. globulus essential oil was found to be rich in eucalyptol (15.12%), E-

citral (12.16%), terpinene-4-ol, (11.5%), p-cymene (8.04%), isopulegol (5.3%),

phellandral (4.8%), globulol (4.6%), (-)-spathulenol (4.01%), linalool (3.94%), carvacrol

74

Fig. 3.20: In Vitro antifungal activity of E. globulus essential oil, used at various






Fig. 3.21: In Vivo antifungal activity of E. globulus essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


A a

b bc

bc bc c

0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


B

a

b bc

bcd cd

d

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16Colo

ny D

iam

eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

75

Fig. 3.22: GC-MS chromatogram of essential oil extracted from leaves of E. golobulus.

ɣ-terpinene

Linalool

Isopulegol

α-Gurjunene

Eucalyptol

Epiglobulol

Globulol

E-citral

76

Table 3.10: Chemical Composition of Essential Oil Extracted from E. globulus.

Components RI

%

Composition

α-Thujene 927 1.17 ± 0.19i-k

2,6-Octadiene,2,6-dimethyl- 985 t

ɣ- Terpinene 998 1.6 ± 0.05i

Limonene 1018 2.41 ± 0.05h

Eucalyptol 10.33 15.12 ± 0.03a

p-Cymene 1042 8.04 ± 0.01c

Terpinolen 1052 1.26 ± 0.00i-k

Linalool 1082 3.94 ± 0.05ef

4(10)-Thujen-3-ol 1085 1.08 ± 0.1i-l

Citronellal 1125 0.1 ± 0.00m

cis-p-Menth-2-en-1-ol 1126 t

Terpinene-4-ol 1137 11.5 ± 0.28b

р-Menth-1-en-8-ol 1143 t

Piperitone 1158 1.09 ± 0.17i-l

Estragol 1172 1.29 ± 0.11i-k

Phellandral 1175 4.8 ± 0.17d

Cephrol 1179 0.17 ± 0.01m

Isopulegol 1196 5.3 ± 0.17d

trans -Geraniol 1247 3.04 ± 0.01gh

Carvacrol 1262 3.4 ± 0.05fg

E-Citral 1267 12.16 ± 0.07b

2,3-Pinanediol 1276 1.27 ± 0.01i-k

Isoaromadendrene epoxide 1281 0.7 ± 0.05k-m

p-Cymene-8-ol 1284 2.9 ± 0.15gh

Lendene oxide-(III) 1293 0.1 ± 0.00m

Aromadendrene 1386 1.6 ± 0.08i

α-Gurjunene 1419 0.2 ± 0.03m

77

ɣ-Elemene 1431 t

&-Cadinene 1469 0.3 ± 0.14lm

Eremophilene 1474 0.1 ± 0.05m

B-Vatirenene 1489 0.3 ± 0.14lm

Caryophyllene 1494 0.76 ± 0.04j-m

Caryophyllene oxide 1507 t

Viridiflorol 1530 1.6 ± 0.03ij

Epiglobulol 1530 1.04 ± 0.02i-l

Globulol 1530 4.6 ± 0.12de

(-)-Spathulenol 1536 4.01 ± 0.01ef

Longipinocarvone 1569 t

α-Cadinol 1580 0.12± 0.01m

97.07










MS = Identification based on comparison of mass spectra

78

(3.4%), trans-geraniol (3.04%), P-cymene-8-ol (2.9%) and limonene (2.4%).

Furthermore, numerous minor components of this oil have also been characterized (Fig.

3.22).


E. globulus Essential Oil

Six major identified components in essential oil of Eucalyptus globulus L.

were tested independently against selected fungi. Terpinene-4-ol stood as the most

effective antifungal component of this essential oil as its highest tested concentration did

not allowed any growth in the media. Following this p-cymene resulted in 90.23%

growth reduction. Lowest tested concentration of the rest of the four components viz.,

carvacrol, citral, eucalyptol and linalool showed insignificant variation among their

antifungal activities. However, at highest tested concentration carvacrol and eucalyptol

was found equally efficient in controlling the fungi followed by citral and linalool with

significantly similar antifungal potentials (Fig. 3.23).


medium, treated with different concentrations of major components of E. globulus

essential oil. Values are mean of three replicates analyzed individually. Vertical bars

show standard error of means of three replicates. Values with different alphabetic letters

indicate significant differences (P=0.05) as determined by Duncan’s Multiple Range

(DMR) test.

0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

)


a b

e

b

f

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de

g

i

c

g

hi

cd

hi j

c

f

hi

79

3.2.6. EUGENIA CARYOPHYLLATA T. (Clove)


Essential oil of E. caryophyllata T. buds inhibited selected fungi highly

significantly. Insignificant variation was recorded in results of dilution and volatile

method. In dilution method the lowest tested concentration of 3μL mL-1

caused 84%

reduction in fungal growth when compared to the control. Increase in concentration

resulted in insignificant increment of antifungal proficiency and 24μL mL-1

restricted

fungal growth up to 99% (Fig. 3.24).

In case of volatile assay, lowest tested concentration reduced fungal growth up to 97%

that was 13% higher than recorded in dilution assay with same essential oil concentration.

Higher concentrations of 6-24μL mL-1

were also found equally effective as that of lowest

tested concentration.

The highest tested concentration of 48μL mL-1

completely inhibited P. italicum in both

volatile and dilution assays (Fig. 3.24).


In Vivo studies also supported significant antifungal proficiency of E.

caryophyllata bud essential oil. Selected essential oil in its lowest concentration caused

62.7% growth restriction in selected fungi. This percentage increased with increase in

concentration and 6 and 12μL mL-1

showed 79.6 and 93.2% fungal inhibition

respectively. In control the fungal growth started at 2nd

day after inoculation, whereas

essential oil treatment significantly delayed fungal growth. Fungal growth initiated after

4th

, 7th

and 10th

day of inoculation when fruits were treated with E. caryophyllata

essential oil in 3, 6 and 12μL mL-1

. Highest tested concentrations of 24 and 48μL mL-1

restricted initiation of fungal growth till 15th

day of inoculation (Fig. 3.25).

80


The chemical composition data of the E. caryophyllata essential oil is listed in

Table 3.11. Twenty one components of E. caryophyllata essential oil were identified by

GC-MS analysis accounting 97.33% of the oil. The results showed the presence of

eugenol (62.47%), acetyleugenol (12.9%), caryophyllene (10.02%) and Iso-eugenol

(1.92%) as its major components. Besides, the E. caryophyllata essential oil also

contained considerable amount of various minor components (Fig. 3.26).

81

Fig. 3.24: In Vitro antifungal activity of E. caryophyllata essential oil, used at various






Fig. 3.25: In Vivo antifungal activity of E. caryophyllata essential oil, used in various



-1

0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

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eter

(cm

)


A a

b b b b b

0

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Co

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(cm

)


B a

b b b b b

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5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P.

Itali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

82

Fig. 3.26: GC-MS chromatogram of essential oil extracted from buds of E. caryophyllata.

Iso-eugenol

Caryophyllene

Acetyl eugenol Eugenol

Eucalyptol

Cinnamaldehyde

83

Table 3.11: Chemical Composition of Essential Oil Extracted from E. caryophyllata.

Components RI % Composition

β- Terpinen 993 0.5 ± 0.01i

2- Nonanone 1053 t

Eucalyptol 1059 1.5 ± 0.0ef

4- Terpineol 1137 0.4 ± 0.005i-k

α-Terpinol 1143 0.3 ± 0.0i-l

Plastolin 1160 t

Cinnamaldehyde 1189 0.912 ± 0.05h

Anisole 1190 0.08 ± 0.0l

Chavicol 1203 0.09 ± 0.005l

Cuminaldehyde 1230 1.31± 0.02fg

Exagien 1281 0.15 ± 0.01kl

Shikimole 1327 0.17 ± 0.1j-l

Methyl Eugenol 1361 0.45 ± 0.05ij

Eugenol 1392 62.47 ± 0.3a

Isoeugenol 1410 1.92 ± 0.3d

α- Amorphene 1440 1.76 ± 0.01de

Caryophyllene 1494 10.02 ± 0.0c

Epoxycaryophyllene 1507 t

Acetlyeugenol 1552 12.909 ± 0.10b

Humulene 1579 1.3 ± 0.13fg

Caryophyllenyl alcohol 1677 1.07 ± 0.0gh

Total 97.331











84


E. caryophyllata Essential Oil

In case of Eugenia caryophyllata five top most components were checked individually

against selected fungi. Highest tested concentration of eugenol and cuminaldehyde

restricted any growth of tested fungi in selected media thus making them as the most

potent antifungal components. However, lower concentrations showed a little

discrepancy. Lowest tested concentration of 50µL L-1

caused a reduction of 78.74% in

fungal growth treated with cuminaldehyde and 68.96% in fungi exposed to eugenol (Fig.

3.27). Similarly when concentration of these essential oils was increased to 100µL L-1

cuminaldehyde showed slightly higher percentage of growth inhibition (94.26%) than

eugenol (82.19%). Caryophyllene and acetyl eugenol followed this and showed

significantly very close antifungal potential when checked in concentration of 100 and

200µL L-1

. Whereas, at lowest tested concentration of 50µL L-1

caryophyllene showed

significantly higher antifungal activity than was recorded against acetyl eugenol. Least

affectivity was depicted by isoeugenol (Fig. 3.27).


medium, treated with different concentrations of major components of E.

caryophyllata essential oil. Values are mean of three replicates analyzed




0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

) 50µL/L 100µL/L 200µL/L

e

h i

a

d ef

a ab

e

cd

ef g

bc

fg

i

85

3.2.7. FOENICULUM VULGARE M. (Fennel)


Essential oil extracted from F. vulgare M. seeds restricted growth of tested

fungi on malt extract media highly significantly. Statistically both dilution and volatile

assays were found equally significant. Essential oil in its lowest tested concentration of

3μL mL-1

reduced fungal biomass to 98-99% in both assays. Further increase in oil

concentration completely inhibited growth of selected fungi, except that of 6μL mL-1

in

dilution assay that inhibited fungi to 99% (Fig. 3.28).


In Vivo analysis followed similar trend as was recorded in In Vitro

experiments. Lowest tested concentration of 3μL mL-1

and that of 6μL mL-1

restricted

fungal growth till 9th

and 11th

day of inoculation. At 15th

day of inoculation above

mentioned concentrations showed a reduction of 93.2 - 96.6% respectively in growth of

tested fungi. Further increase in essential oil concentration did not allowed any growth of

fungi till 15th

day of inoculation (Fig. 3.29).


A detailed chemical composition of tested F. vulgare essential oil characterized

by GC-MS and GC-FID is listed in Table 3.12. A total of 21 components were identified

which accounts for 98.33% of the extracted oil. Major components were anethole

(61.41%), fenchol (13.7%), methyl chavicol (6.79%), eugenol (3.6%), aceteugenol

(2.5%), terpinene-4-ol (1.96%), limonene (1.9%), Apiole (1.7%), linalool (1.3%),

isoeugenol (1.2%) and caryophyllene (1.02%). Many other components were also present

in quantities less than 1% (Fig. 3.30).

86

Fig. 3.28: In Vitro antifungal activity of F. vulgare essential oil, used at various






Fig. 3.29: In Vivo antifungal activity of F. vulgare essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


A

a

b b b b b 0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


B

a

b b b b b

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

iam

eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

87

Fig. 3.30: GC-MS chromatogram of essential oil extracted from leaves of F. vulgare.

ɣ-terpinene

Methyl chavicol

Anethole

Fenchon

88

Table 3.12: Chemical Composition of Essential Oil Extracted from F. vulgare.

Components RI % Composition

ɣ-Terpinene 998 0.25 ± 0.04gh

Limonene 1018 1.9 ± 0.02ef

P-Cymene 1042 0.1 ± 0.00gh

Linalool 1082 1.3 ± 0.08fg

Fenchon 1121 13.7 ± 0.66b

Terpinene-4-ol 1137 1.95 ± 0.2ef

Mmethyl chavicol 1172 6.79 ± 0.12c

Anethole 1190 61.41 ± 0.26a

Thymol 1262 t

Carvacrol 1267 0.3 ± 0.00gh

Anisketone / Anisic acid 1318 0.5 ± 0.05gh

4-Methpxypropiophenone 1318 t

Benzenmethanol, a-ethyl-4-methoxy 1344 t

Eugenol 1392 3.6 ± 0.05d

Isoeugenol 1410 1.2 ± 0.11fgh

Caryophyllene 1494 1.02 ± 0.00fgh

Aceteugenol 1552 2.5 ± 0.00e

p-Methoxymandelic acid 1601 t

Apiole 1705 1.7 ± 0.15ef

Palmitic acid 1968 0.1 ± 0.00h

Oleic acid 2175 t

TOTAL 98.33



Multiple Range (DMR) test. t = trace element, less than 0.05%.






MS = Identification based on comparison of mass spectra

89


F. vulgare Essential Oil

Six major components identified in Foeniculum vulgare M. were checked

independently against selected fungi. Among these six compounds anethole stood as the

most potent inhibitory component of this essential oil. Its lowest tested concentration of

50µL L-1

resulted in an inhibition of 83.91%. Further increase in concentration totally

stopped fungal growth. This was followed by terpinen-4-ol and eugenol that in their

maximum concentration restricted any fungal growth but lower concentrations failed to

inhibit the selected fungi completely. After this, limonene resulted in 96.55% fungal

inhibition when checked in a concentration of 200µL L-1

. Fechon followed limonene with

90.23% of reduction in fungal colony growth. Whereas, least effective component of this

essential oil was methyl chavicol (Fig. 3.31).


medium, treated with different concentrations of major components of F.

vulgare essential oil. Values are mean of three replicates analyzed individually.




0

20

40

60

80

100

120

Per

cen

tag

e o

f in

hib

itio

n (

%)


a b

d

c

f

h

a

c

de

b

e

g

a

c

d

a a

c

90

3.2.8. OCIMUM BASILICUM L. (Sweet Basil)


In Vitro studies of antifungal potential of essential oil extracted from Ocimum

basilicum L. leaves also showed variation among dilution and volatile assay. Lower

concentrations of 3 and 6μL mL-1

failed to produce any reduction in fungal biomass when

tested through dilution assay. However, the volatile assay showed a reduction of 9 and

23.4% in fungal biomass tested against same concentrations (Fig. 3.32). Further increase

in concentration of essential oil to 12 and 24μL mL-1

generated insignificant reduction in

fungal growth in dilution assay, but volatile assay caused a significant decrease in fungal

biomass of 33.5 and 40.6% against these concentrations. Highest tested concentration of

48μL mL-1

significantly reduced fungal growth and a percentage reduction of 24 and 43%

was recorded in dilution and volatile assays (Fig. 3.32).


Supporting the results of In Vitro volatile assay, In Vivo studies also showed

significant antifungal activities. Lowest tested concentration of 3μL mL-1

generated

54.2% reduction in colony growth of tested fungi. This antifungal activity increased with

increase in concentration of essential oil. A highly significant reduction of 91.5 and

94.9% was recorded in samples treated with essential oil in concentration of 24 and 48μL

mL-1

. Increase in concentration also delayed initiation of fungal growth. Growth recorded

after 4th

day of inoculation in samples treated with 3μL mL-1

concentration. With further

increase in essential oil concentration the growth instigated late and in samples treated

with highest tested concentration of 48μL mL-1

growth started after 8th

day of inoculation

(Fig. 3.33).


The chemical composition determined in the essential oil of O. basilicum is

given in Table 3.13. A total of 29 compounds in this essential oil representing 97.52% of

the total amount, were identified by GC-MS and GC-FID (Fig. 3.34). The dominant

91

Fig. 3.32: In Vitro antifungal activity of O. basilicum essential oil, used at various






Fig. 3.33: In Vivo antifungal activity of O. basilicum essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

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eter

(cm

)


A

a a a ab

ab b

0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

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eter

(cm

)


B

a ab

bc c

c c

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny

Dia

met

er o

f P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

92

Fig. 3.34: GC-MS chromatogram of essential oil extracted from leaves of O. basilicum.

Eucalyptol Linalool

ɣ-terpinene

Β-cubebene

Carvacrol

Eugenol

93

Table 3.13: Chemical Composition of Essential Oil Extracted from O. basilicum.

Components RI

%

Composition

β-cis-Ocimene 976 2.37 ± 0.09fg

P- Cymene 1042 0.09± 0.00k

Eucalyptol 1059 1.9± 0.23g-i

Linalool 1082 31.5± 0.31a

Camphor, (+)- 1121 0.43± 0.01jk

Terpinene-4-ol 1137 2.12± 0.08f-h

Isoborneol 1138 t

Fenchol, exo 1138 0.3± 0.00jk

4-Allyanisole 1172 3.1± 0.08e

Citral 1174 15.1± 0.46b

Carvacrol 1262 9.1± 0.27d

Fenchyl acetate 1277 1.26± 0.01h-j

β-Cubebene 1339 3.97± 0.15e

Methyl Eugenol 1361 11.4± 0.28c

Eugenol 1392 4.05± 0.01e

β-Elemen 1398 t

Isoeugenol 1410 1.23± 0.08h-j

α-Bergamotene 1430 0.2± 0.05jk

ɣ-Elemene 1431 0.4± 0.03jk

ɣ-Cadinene 1435 2.01± 0.00g-i

ɣ-Muurolene 1435 0.17± 0.01jk

β-Sesquiphellandrene 1446 t

α-Bulnesene 1490 t

Caryophyllene 1494 1.98± 0.01g-i

Cis-α-Bisabolene 1518 0.1± 0.00k

Acetyl eugenol 1552 2.16± 0.08f-h

94

Cubenol 1580 0.46± 0.01jk

tau.-Cadinol 1580 1.02± 0.01i-k

Phytol 2045 1.1± 0.08h-k

TOTAL 97.52











95

compounds were Linalool (31.5%), citral (15.1%), methyl eugenol (11.4%), carvacrol

(9.1%), eugenol (4.05%), β-cubebene (3.97%), and 4-allyanisole (3.1%). Moreover, the

tested oil also contained a considerable amount of numerous minor compounds (Table

3.13).


O. basilicum Essential Oil

Among the five individually tested components of Ocimum basilicum, eugenol stood as

the most effective antifungal component. Its highest tested concentration did not allowed

any growth in the media. This was followed by methyl eugenol that gave a maximum of

91.96% control and carvacrol that restricted the selected fungi to 83.33% when checked

in a concentration of 200µL L-1

(Fig. 3.35). Citral and linalool showed significantly equal

antifungal potential at maximum tested concentration. However, slight variation was

recorded in lower concentrations (Fig. 3.35).

Fig. 3.35: Mycelial growth of P. italicum was measured after 7 days of incubation on

MEA medium, treated with different concentrations of major components of O.

basilicum essential oil. Values are mean of three replicates analyzed




0

20

40

60

80

100

120

Carvacrol Citral Eugenol Linalool Methyleugenol

Per

cen

tage

of

inh

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)


i

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c

ef cd

j

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c

fh

j

b

efg

96

3.2.9. OCIMUM SANCTUM L. (Holy Basil)


Ocimum sanctum L. leaf essential oil also controlled In Vitro growth of tested

fungi highly significantly in all tested concentrations. Lowest tested concentration of 3μL

mL-1

resulted in 80% growth inhibition in dilution assay. With increase in essential oil

concentration this assay showed a gentle rise in fungal growth reduction. A 98% decline

in fungal biomass was recorded at 24μL mL-1

(Fig. 3.36). In case of volatile assay the

upturn in antifungal activity was insignificant when concentration of the essential oil

increased from 3μL mL-1

to 24μL mL-1

and the percentage of inhibition was recorded

between 90 to 98.8%. Highest tested concentration of 48μL mL-1

completely restricted

fungal growth on selected media in both dilution and volatile assays (Fig. 3.36).


The In Vivo antifungal activity of O. sanctum essential oil was also found

equally effective as was shown by In Vitro assays. Lowest tested concentration of 3μL

mL-1

restricted fungal growth up to 4th

day of inoculation, whereas the reduction in

colony growth was 57.6% at 15th

day of inoculation (Fig. 3.37). Increase in essential oil

concentration increased this antifungal activity. No fungal growth was recorded till 8th

and 10th

day of inoculation in samples treated with basil essential oil in concentrations of

6 and 12μL mL-1

. In addition, these concentrations caused a significant reduction of 77.9

and 92.4% in colony growth at 15th

day of inoculation. Further increase in essential oil

concentration completely inhibited growth in tested fungi (Fig. 3.37).


The chemical composition of O. sanctum essential oil was analyzed by the

GC-MS and GC-FID (Fig. 3.38). A list of twenty identified compounds has been

recorded in Table 3.14 making 97.4% of the total oil. This oil was mainly composed of

linalool, caryophyllene, 1,8-cineole, ɣ-gurjuene, β-caryophyllene, citral, thymol,

carvacrol, B- ocimene and eugenol components with relative percentages of 49.36, 12.01,

6.01, 5.1, 4.37, 3.8, 3.19, 2.1, 2.07, 2.03% respectively. Rest of the components listed in

Table 3.14 were found in minor quantities (Fig. 3.38).

97

Fig. 3.36: In Vitro antifungal activity of O. sanctum essential oil, used at various






Fig. 3.37: In Vivo antifungal activity of O. sanctum essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

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(cm

)


A

a

b bc bc bc c

-1

0

1

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3

4

5

6

7

0 3 6 12 24 48

Co

lon

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)


B

a

b b b b

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-1

0

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4

5

6

7

0 2 4 6 8 10 12 14 16

Colo

ny D

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eter

of

P. It

ali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

98

Fig. 3.38: GC-MS chromatogram of essential oil extracted from leaves of O. sanctum.

Linalool

Thymol

Caryophyllene

Carvacrol

99

Table 3.14: Chemical Composition of Essential oil Extracted from O. sanctum.

Components RI

%

Composition

(-)-. Beta.-Pinene 943 1.01 ± 0.00h

B-Myrecene 958 t

Limonene 1018 1.02 ± 0.01h

β ocimene 1048 2.07 ± 0.01g

1,8-Cineole 1059 6.01 ± 0.00c

Linalool 1082 49.36 ± 0.37a

Terpinene-4-ol 1137 1.07 ± 0.00h

Citral 1174 3.8± 0.15f

Thymol 1262 3.19 ± 0.11f

Carvacrol 1263 2.1 ± 0.07g

Eugenol 1392 2.03 ± 0.02g

β-Caryophyllene 1438 4.37 ± 0.31e

ɣ-Gurjuene 1461 5.1 ± 0.09d

Caryophyllene 1494 12.01 ± 0.00b

Caryophyllene oxide 1507 0.7 ± 0.00hi

Germacrene D 1515 1.32 ± 0.17h

Viridiflorol 1530 t

α-Caryophyllene 1579 1.14 ± 0.02h

α-Cadinol 1580 0.05 ± 0.02h

B-Eudesmol 1593 t

96.35











100


O. sanctum Essential Oil

Seven major components of Ocimum sanctum identified in this study were checked

independently against P. italicum. Among these seven components thymol controlled

selected fungi highly significantly. Its lowest tested concentration restricted fungal

growth up to 93.1%. Further increase in concentration did not allowed any growth in the

media. Eugenol and 1,8-cineole were next in their antifungal potential. Their highest

tested concentration resulted in 100% fungal inhibition. At lower concentrations slight

variation was recorded among their significance to control the selected fungi. After this,

caryophyllene caused 87.36% growth inhibition at its maximum tested concentration. The

effect of carvacrol was found pretty close to that of caryophyllene at 200µL L-1

.

However, its lower concentrations produced less percentage of inhibition than

caryophyllene. Least affectivity was recorded by linalool and citral (Fig. 3.39.).


medium, treated with different concentrations of major components of O.

sanctum essential oil. Values are mean of three replicates analyzed




0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

)


b

e

i

jk

a

cd f

de

j k

c

f gh

cd

g

j

d

h

a a a

101

3.2.10. TRACHYSPERMUM CAPTIVUM L. (Carom)


Trachyspermum captivum L. essential oil extracted from its seeds proved as

a very potent source for restricting growth of Penicillium italicum as its lowest tested


did not allow any fungal growth (Fig. 3.40). The antifungal

activity was found equally efficient when checked through dilution and volatile assays.


In In Vivo studies the T. captivum seed essential oil followed similar trend as

was shown in In Vitro assays. No fungal growth was recorded on fruits treated with

essential oil concentration of 12μL mL-1

and higher till 15th

day of inoculation. Lower

concentrations of 3 and 6μL mL-1

reduced fungal growth up to 95.74 and 94.96%

respectively (Fig. 3.41).


The GC-MS and GC-FID analysis of T. captivum seed essential oil showed

the presence of thirty four identified compounds (Table 3.15) which represented 97.9% of

the total oil. The dominant compounds of the tested essential oil were thymol (42.9%)

followed by P-cymene (21.4%), ɣ-terpinene (13.5%), isothymol (2.5%), IR-alpha-pinene

(2.1%), eugenol (1.9%), (-)-Beta-pinene (1.7%), myrcene (1.63%), cuminal (1.5%) and

1,8-cineole (1.3%). Other compounds were present in small percentages (Fig. 3.42).

102

Fig. 3.40: In Vitro antifungal activity of T. captivum essential oil, used at various






Fig. 3.41: In Vivo antifungal activity of T. captivum essential oil, used in various



0

1

2

3

4

5

6

7

8

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


A a

b b b b b 0

1

2

3

4

5

6

7

0 3 6 12 24 48

Co

lon

y d

iam

eter

(cm

)


B

a

b b b b b

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Co

lon

y D

iam

eter

of

P.

Itali

cum

(cm

)


Control

Conc. 3µL/mL

Conc. 6µL/mL

Conc. 12µL/mL

Conc. 24µL/mL

Conc. 48µL/mL

103

Fig. 3.42: GC-MS chromatogram of essential oil extracted from seeds of T. captivum

Thymol

Iso thymol

P- cymene

104

Table 3.15: Chemical Composition of Essential Oil Extracted from T. captivum.

Components RI

%

Composition

6-Methyl-cyclodec-5-enol 809 t

Bicyclo[3.1.0]hexane, 4-methyl-1-(1-

methylethyl)-, didehydroderiv 873 t

α-Thujene 927 0.13 ± 0.005jk

(-)-. Beta.-Pinene 943 1.7 ± 0.08d-g

1R-alpha-pinene 948 2.1 ± 0.03de

B-Myrecene / Myrcene 958 1.63 ± 0.08d-g

Sabinene 967 0.31 ± 0.005i-k

ɣ-Terpinen 998 13.5 ± 0.28c

α-Tepinene 1015 1.15 ± 0.07e-j

Limonene 1018 0.51 ± 0.003h-k

B- phallederene 1030 0.77 ± 0.00g-k

P-Cymene 1042 21.4 ± 0.84b

1,8-Cineole 1059 1.3 ± 0.00e-i

Linalool 1082 0.2 ± 0.05jk

Terpinolene 1089 0.05 ± 0.01k

Cyclohexene, 2-ethenyl-1,3,3-

trimethyl 1105 t

Berbenone 1119 t

Camphor, (+)- 1121 0.64 ± 0.08g-k

Terpinene-4-ol 1137 0.45 ± 0.02h-k

Estragol 1172 0.98 ± 0.02f-k

Naphthalene, 1,2,3,4,4a, 5,6,7-

octahydro-4a-methyl- 1185 t

α-Terpineol 1189 t

Anisole 1190 0.56 ± 0.03h-k

Cuminal 1242 1.5 ± 0.03e-h

Thymol 1262 42.9 ± 0.00a

105

Isothymol 1267 2.5 ± 0.17d

Eugenol 1392 1.9 ± 0.03d-f

ɣ-Elemene 1431 0.34 ± 0.00i-k

Caryophyllene oxide 1507 0.09 ± 0.00k

Aceteugenol

α-Humulene

Palmitic acid

1552

1579

1968

1.01 ± 0.00f-k

0.23 ± 0.00jk

0.05 ± 0.0k

Oleic acid 2125 t

Gibberellic acid 2653 t

TOTAL 97.9











106


T. captivum Essential Oil

Among the six selected major components of Trachyspermum captivum

essential oil thymol proved as highly significant component to reduce fungal growth.

Only 7% growth was recorded against its minimum tested concentration. Doubling the

concentration to 100µL L-1

did not allow any growth (Fig. 3.43). Eugenol and 1,8-cineole

also completely restricted fungal growth at maximum tested concentration. This was

followed by γ-terpinene and p-cymene. Least potent antifungal component was β-pinene

whose highest tested concentration could only cause 69.54% reduction in growth of P.

italicum.


medium, treated with different concentrations of major components of T.

captivum essential oil. Values are mean of three replicates analyzed




0

20

40

60

80

100

120

Per

cen

tage

of

inh

ibit

ion

(%

)


f

j k

c

gh

j

ac

e

hi

d fg

b

de

i

a a a a

107

3.2.11. SELECTION OF MOST EFFECTIVE ESSENTIAL

OIL

3.2.11.1. In Vitro Antifungal Assay

In Vitro antifungal activity of ten essential oils was checked against P.

italicum using dilution and volatile assays. All the essential oils with an exception of

Cumin cyminium showed similar antifungal activity in both assays. The Trachyspermum

captivum essential oil found to be the most effective in inhibiting the growth of selected

fungi as it did not allow any growth even at its lowest tested concentration in both

dilution and volatile assays. This was followed by Foeniculum vulgare essential oil that

showed equally significant antifungal activity to T. captivum essential oil when checked

through volatile assay. However, in dilution assay the complete inhibition of tested fungi

was achieved at a bit higher concentration of 12μL mL-1

(Fig. 3.44).

This was followed by Eugenia caryophyllata and Ocimum sanctum essential oils as all

the tested concentrations of these oils showed highly significant antifungal activity

ranging between 90 – 100% against selected fungus in both assays. In case of C.

cyminium essential oil a little variation was recorded in results of both dilution and

volatile assays. Results from dilution assays proved C. cyminium essential oils equally

effective to E. caryophyllata and O. sanctum. However, lower concentrations of 3 and

6μL mL-1

could not prove as affective in volatile assay and the inhibition percentages

were 32 and 69% respectively. However, higher concentrations of 12 - 48μL mL-1

hardly

allowed any growth in the cultures of volatile assay (Fig 3.44B).

Essential oils of Cymbopogon citratus stood next in their potential to control In Vitro

growth of selected fungus. Both assays produced similar results. Lowest concentration of

3μL mL-1

produced 70-71% growth inhibition that increased with increase in

concentration and highest tested concentration restricted fungal growth up to 97% in

dilution and 99.4% in volatile assays.

108

This was followed by Eucalyptus globulus essential oil that produced 56-58% growth

reduction in P. italicum at lowest concentration of 3μL mL-1

. With increase in

concentration the percentage of growth inhibition reached to 77 and 87% in dilution and

volatile assays respectively.

Least significant activity was recorded in Cinnnamomum zylanicum, Ocimum basilicum

and Azadirachta indica essential oils. All tested concentrations of these three sources

produced insignificant antifungal activity in dilution assay. However, volatile assay

showed slightly significant increase in percentage of growth inhibition with increase in

concentration. Highest tested concentration of 48μL mL-1

could only produce 37.6, 43

and 11.7% growth inhibition in cultures treated with A. indica, O. basilicum and C.

zylanicum essential oils respectively (Fig. 3.44)

3.2.11.2. In Vivo Antifungal Assay

The results from In Vivo antifungal assay found comparable to In Vitro

studies with few variations. Similar to In Vitro studies, T. captivum and F. vulgare

essential oils found as most significant sources of antifungal activity as no fungal growth

was recorded in samples treated with these oils in concentration of 12μL mL-1

. Eugenia

caryophyllata and O. sanctum essential oils followed these results and they completely

inhibited fungal growth at a concentration of 24μL mL-1

(Fig. 3.45). Antifungal activity

of C. cyminium and C. citratus was found next to this as complete fungal inhibition was

recorded only at their highest tested concentration of 48μL mL-1

. Other essential oils

including A. indica, O. basilicum, E. globulus and C. zylanicum although failed to restrict

fungal growth completely even at their highest tested concentration but they also

produced a significant control on tested fungi with a reduction of fungal growth ranging

between 90 – 95% at their highest tested concentration (Fig. 3.45).

Keeping in view these results T. captivum, F. vulgare and E. caryophyllata were selected

as top three sources of antifungal activity among the ten tested essential oils. Further

studies were done with these essential oils.

109

Fig.3.44: In Vitro antifungal efficacy of various essential oils against P. italicum at

various concentrations. (A) Agar dilution method (B) Volatile method. Values

are mean of three replicates analyzed individually.

0

20

40

60

80

100

120

0 10 20 30 40

Per

cen

tage

of

inh

ibit

ion

(%

)

Concentrations (µL mL -1)

T. captivum E. caryophyllata C. zylanicum C. cyminiumF. vulgare C. citratus A. indica O. basilicumE. globulus O. sanctum

A

0

20

40

60

80

100

120

0 10 20 30 40

Per

cen

tage

of

inh

ibit

ion

(%

)

Concentrations (µL mL-1)

T. captivum E. caryophyllata C. zylanicum C. cyminium

F. vulgare C. citratus A. indica O. basilicum

E. globulus O. sanctum

B

110

Fig. 3.45: In Vivo antifungal efficacy of various essential oils against P. italicum at

various concentrations. Values are mean of three replicates analyzed

individually.

0

20

40

60

80

100

120

0 10 20 30 40

Per

cen

tage

of

inh

ibit

ion

(%

)

Concentrations (µL mL-1)

T. captivum E. caryophyllata C. zylanicum C. cyminium

F. vulgare C. citratus A. indica O. basilicum

E. globulus O. sanctum

111

At zero day

After 15 days

Control 3µL mL-1

6µL mL-1

12µL mL-1

24µL mL-1

48µL mL-1

PLATE 3.2: In Vivo antifungal effect of different concentrations of essential oil extracted from buds of E. caryophyllta.

Pictures were taken after 15 days of incubation at 25ºC.

112

At zero day

After 15 days

Control 3µL mL-1

6µL mL-1

12µL mL-1

24µL mL-1

48µL mL-1

PLATE 3.3: In Vivo antifungal effect of different concentrations of essential oil extracted from seeds of F. vulgare. Pictures

were taken after 15 days of incubation at 25ºC.

113

At zero day

At fifteenth day

Control 3µL mL-1

6µL mL-1

12µL mL-1

24µL mL-1

48µL mL-1

PLATE 3.4: In Vivo antifungal effect of different concentration of essential oil extracted from seeds of T. captivum. Pictures

were taken after 15 days of incubation at 25ºC.

114

3.2.12. EFFECT OF ESSENTIAL OILS ON POST-

HARVEST PHYSICOCHEMICAL CHANGES IN

CITRUS FRUIT DURING COLD STORAGE

Among the ten tested essential oils, three essential oils i.e.,

Trachyspermum captivum, Eugenia caryophyllata and Foeniculum vulgare were

selected on the basis of their significant potential to control post-harvest decay in

citrus fruit (mandarin) during In Vitro and In Vivo studies. The effect of these oils

were checked on physiological properties of the treated fruits including weight loss,

juice content, pH, ascorbic acid content, total soluble solids and titratable acidity.

3.2.12.1. Weight loss of fruit

A constant trend of weight loss was recorded in tested fruits during the

storage period of eight weeks as is clear by the regression line. The percentage of

weight loss was found significantly higher in control when compared to the treated

fruits. The weight loss significantly decreased with increase in essential oil

concentration and time of dipping.

Among the three selected essential oils T. captivum essential oil proved as the most

potent source for controlling the post-harvest decay in storage as the fruits treated

with this essential oil showed least decrease in their weights at the end of the

experimental period. A weight loss of 12.47% was recorded in fruits treated with

400μL L-1

for 20 minutes in this essential oil (Fig. 3.46). This was 46% lesser than the

losses recorded in control fruits. The reduction in weight loss percentages in fruits

treated with 200μL L-1

for 20 minutes and 400μL L-1

for 10 minutes was insignificant

(P=0.05).

Essential oil of F. vulgare followed these results as 16.56% weight loss was recorded

in fruits treated with this essential oil in a concentration of 400μL L-1

for 20 minutes.

As recorded previously, the effect of 400μL L-1

for 10 minutes and 200μL L-1

for 20

minutes was found insignificantly different with a percentage loss of 25.21 and 26.5%

respectively. Fruits dipped in 200μL L-1

for 10 minutes showed a weight loss of

35.3%

115

Fig. 3.46: Linear regression fitted for the effect of different concentrations of essential

oils on weight loss (%) of mandarin fruit during cold storage at 4ºC for 8

weeks. (A) 200µL L-1

for 10 minutes (B) 200µL L-1

for 20 minutes.

y = 7.3096x - 2.0347 R² = 0.9871

y = 3.7068x - 2.8777 R² = 0.966

y = 4.9372x - 2.293 R² = 0.9908

y = 4.3851x - 2.3368 R² = 0.968

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9

Wei

gh

t lo

ss (

%)

Storage period (weeks)

ControlT. captivumE. caryophyllataF. vulgare

A

y = 7.3096x - 2.0347 R² = 0.9871

y = 2.9327x - 2.4818 R² = 0.9536

y = 3.9716x - 1.8005 R² = 0.9894

y = 3.4228x - 1.0557 R² = 0.992

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9

Wei

gh

t L

oss

(%

)

Storage Period (weeks)


B

116


oils on weight loss (%) of mandarin fruit during cold storage at 4ºC for 8

weeks. (A) 400µL L-1 for 10 minutes (B) 400µL L-1 for 20 minutes.

y = 7.3096x - 2.0347 R² = 0.9871

y = 2.5888x - 2.2831 R² = 0.9686

y = 3.3257x - 2.9386 R² = 0.9543

y = 3.2561x - 2.2911 R² = 0.9774

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9

Wei

gh

t lo

ss (

%)



A

y = 7.3096x - 2.0347 R² = 0.9871

y = 1.5998x - 1.6327 R² = 0.9379

y = 2.5763x - 1.9972 R² = 0.9666 y = 2.0921x - 1.4296

R² = 0.9592

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9

Wei

gh

t lo

ss (

%)



B

117

In case of E. caryophyllata, the essential oil concentration of 200μL L-1

for 10

minutes was found equally significant as that of the same concentration of F. vulgare

(Fig 3.46). Doubling the dipping time in this concentration could only increase 6.68%

in reduction in weight losses in comparison to the fruits dipped in same concentration

for 10 minutes. This oil in 400μL L-1

resulted in 25.65% weight loss in fruits dipped

for 10 minutes. This weight loss decreased to 20.47% on the increase of dipping time

to 20 minutes. Thus fruits treated with 400μL L-1

for 20 minutes showed a reduction

of 38.34% in weight losses over the control (Fig. 3.47).

3.2.12.2. Juice content

The linear regression line showed a continuous increasing trend in % of

juice loss in treated and untreated fruits during the storage period of eight weeks. On

the first day the average juice content was around 157.7mL. In control fruits this

decreased to 45.3mL (71.25%) at the end of eight weeks. Essential oil of T. captivum

highly significantly controlled these losses in juice content. Samples treated with its

lowest tested concentration of 200μL L-1

for 10 and 20 minutes gave an average of

92.33 and 96.66mL juice per sample (Fig. 3.48). Hence the percentage of losses in

juice content was 41.44 and 38.69% respectively. The efficiency of this oil increased

with increase in concentration to 400μL L-1

that resulted in an average juice content of

112.33 and 132mL in samples dipped for 10 and 20 minutes respectively. When

compared to the control the highest tested concentration of this oil decreased

percentage losses in juice by 54.98% (Fig. 3. 49).

Essential oil of E. caryophyllata was found next to this. With an average production

of 49.3ml juice per sample, the lowest tested concentration of 200μL L-1

for 10

minutes showed a decrease of 68.71% in juice content (Fig. 3.48A). Doubling the

time of dipping in this concentration highly significantly increased juice content to

81.66mL. Highest tested concentration of 400μL L-1

for 20 minutes decreased these

losses in juice content to 40.8% with an average production of 93.3mL per sample

(Fig. 3.49B).

118


oils on juice content of mandarin fruit during cold storage at 4 ºC for 8



for 20 minutes.

y = 8.3887x + 1.0596 R² = 0.9922

y = 3.7333x - 2.5711 R² = 0.9703

y = 6.7484x - 2.2987 R² = 0.9884

y = 4.9241x - 1.2614 R² = 0.9914

-10

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

% L

oss

of

juic

e

Storage period (weeks )


A

y = 8.3887x + 1.0596 R² = 0.9922

y = 2.1791x - 1.4196 R² = 0.9504

y = 3.5095x + 2.0901 R² = 0.8771

y = 5.4369x + 0.577 R² = 0.9526

-10

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

% L

oss

of

juic

e



B

119

Fig. 3.49: Linear regression fitted for the effect of different concentrations of

essential oils on juice content of mandarin fruit during cold storage at 4ºC

for 8 weeks. (A) 400µL L-1


for 20 minutes.

y = 8.3887x + 1.0596 R² = 0.9922

y = 4.1283x + 2.1047 R² = 0.9572

y = 5.6742x + 3.5498 R² = 0.9817

y = 6.5507x - 1.8375 R² = 0.9858

-10

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

% L

oss

of

hu

ice


Control

T. captivum

E. caryophyllata

F. vulgare

A

y = 8.3887x + 1.0596 R² = 0.9922

y = 2.2099x - 1.8757 R² = 0.9063

y = 5.2798x + 1.4064 R² = 0.9921

y = 3.4574x - 2.1716 R² = 0.975

-10

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

% L

oss

of

juic

e



B

120

Essential oil of F. vulgare was found least effective in decreasing percentage losses in

juice content. Its efficacy was found pretty close to that of essential oil of E.

caryophyllata. In fact the samples dipped in this essential oil concentration of 200μL

L-1

for 10 minutes produced 15% more juice in average (Fig. 3.48A). Doubling the

concentration of this essential oil failed to produce a significant (P=0.05) increase in

the juice content. An average of 62.33mL juice was extracted from samples treated

with 400μL L-1

for 10 minutes (Fig. 3.49A). Increasing the time of dipping in this

highest tested concentration increased this juice content to 88mL per sample and thus

the percentage losses were recorded as 44.18% when compared to the control.

Significantly the essential oils of E. caryophyllata and F. vulgare in their highest

tested concentration of 400μL L-1

for 20 minutes were found equally effective as that

of lowest tested concentration of 200μL L-1

for 10 minutes of T. captivum essential

oil.

3.2.12.3. pH

The results acquired showed a linear trend among the residual and

expected values of pH of treated and untreated juice. The pH value of fresh mandarin

juice was 3.4 at the start of the experiment, while at the end of the experiment after

eight weeks of storage the value reached at 3.85 in control. Essential oil of T.

captivum highly significantly maintained its pH up to 3.5 in fruits treated with highest


for 20 minutes (Fig. 3.50B). Whereas, at lowest


for 10 minutes the pH value obtained was 3.91(Fig.

3.50A). Essential oil of E. caryophyllata followed this as its maximum tested

concentration of 400µL L-1

for 20 minutes resulted in a pH of 3.87. Efficiency of F.

vulgare essential oil found close to that of E. caryophyllata as the fruits dipped in

same above concentration showed a pH of 3.89 (Fig. 3.50 and 3.51).

121


oils on pH of mandarin fruit during cold storage at 4ºC for 8 weeks. (A)

200µL L-1


for 20 minutes.

y = 0.0537x + 3.4481 R² = 0.9698

y = 0.0625x + 3.4585 R² = 0.961

y = 0.0632x + 3.491 R² = 0.9335

y = 0.0627x + 3.4819 R² = 0.9649

3.4

3.6

3.8

4

4.2

0 1 2 3 4 5 6 7 8 9

pH


ControlT. captivumE. caryophyallataF. vulgare

A

y = 0.0508x + 3.4644 R² = 0.9705

y = 0.0446x + 3.4912 R² = 0.9758

y = 0.0469x + 3.5228 R² = 0.8488

y = 0.057x + 3.4824 R² = 0.9853

3.4

3.6

3.8

4

4.2

0 1 2 3 4 5 6 7 8 9

pH



B

122


oils on pH of mandarin fruit during cold storage at 4ºC for 8 weeks. (A)

400µL L-1


for 20 minutes.

y = 0.0537x + 3.4481 R² = 0.9698

y = 0.0377x + 3.4521 R² = 0.9363

y = 0.0546x + 3.4251 R² = 0.9542

y = 0.0676x + 3.4457 R² = 0.9823

3.4

3.6

3.8

4

4.2

0 2 4 6 8 10

pH



A

y = 0.0537x + 3.4481 R² = 0.9698

y = 0.0138x + 3.4302 R² = 0.9343

y = 0.0404x + 3.5373 R² = 0.7705

y = 0.0532x + 3.4179 R² = 0.9596

3.4

3.6

3.8

4

4.2

0 1 2 3 4 5 6 7 8 9

pH



B

123

3.2.12.4. Total soluble solids

Assessment of total soluble solids in fruits like citrus is an important

indicator of fruit quality. In the present study the regression line showed a gradual

increase in total soluble solids in samples stored over a period of 8 weeks. At the start

of the experiment the quantity of the total soluble solids was recorded as 10 oBrix.

This gradually increased and was found 15oBrix in control samples after 8

th weeks of

storage. Percentage increase in total soluble solids in control samples was recorded as

50%. This increase in total soluble solids was significantly checked by using essential

oils from three selected sources. Essential oil of T. captivum found most effective in

restricting any change in total soluble solids. The samples treated with its highest


for 20 minutes showed total soluble solids at 10.63

oBrix (Fig. 3.53B). Hence the percentage of increase in total soluble solids was found

only 6.3%. This was followed by E. caryophyllata. The samples treated with this

essential oil in the same above concentration showed quantity of total soluble solids at

11.9 oBrix. This increase in terms of percentage was 19%. Whereas essential oil of F.

vulgare caused highest increase of 27.6% in the total soluble solids and was found

equally significant (P=0.05) as that of the T. captivum in a concentration of 400μL L-1

for 10 minutes (Fig. 3.53A).

124


oils on total soluble solids (TSS) of mandarin fruit during cold storage at

4ºC for 8 weeks. (A) 200µL L-1


for 20

minutes.

y = 0.5311x + 10.331 R² = 0.9599

y = 0.33x + 9.7874 R² = 0.887

y = 0.4472x + 10.037 R² = 0.99

y = 0.5106x + 10.161 R² = 0.977

10

11

12

13

14

15

16

0 1 2 3 4 5 6 7 8 9

Ch

an

ge

in T

SS

(⁰B

rix)



A

y = 0.5311x + 10.331 R² = 0.9599

y = 0.1789x + 10.299 R² = 0.921

y = 0.3256x + 10.279 R² = 0.9723

y = 0.375x + 10.222 R² = 0.9736

10

11

12

13

14

15

16

0 1 2 3 4 5 6 7 8 9

Ch

an

ge

in T

SS

(⁰B

rix)


Control

T. captivum

E. caryophyllata

F. vulgare

B

125


oils on total soluble solids (TSS) of mandarin fruit during cold storage at

4ºC for 8 weeks. (A) 400µL L-1


for 20

minutes.

y = 0.5311x + 10.331 R² = 0.9599

y = 0.1689x + 10.069 R² = 0.7513

y = 0.2811x + 10.076 R² = 0.9672

y = 0.365x + 9.8881 R² = 0.9822

10

11

12

13

14

15

16

0 1 2 3 4 5 6 7 8 9

Ch

an

ge

in T

SS

(⁰B

rix

)



A

y = 0.5311x + 10.331 R² = 0.9599

y = 0.0672x + 10.001 R² = 0.7511

y = 0.2189x + 10.139 R² = 0.9596

y = 0.3594x + 9.8474 R² = 0.9586

10

11

12

13

14

15

16

0 1 2 3 4 5 6 7 8 9

Ch

an

ge

in T

SS

(⁰B

rix)


Control

T. captivum

E. caryophyllata

F. vulgare

B

126

3.2.12.5. Ascorbic acid content

The ascorbic acid content measured in terms of mg 100g-1

in fresh juice

of stored mandarin fruits was found to be decreased due to its degradation during the

storage period of eight weeks. The regression line showed a continuous decreasing

trend of ascorbic acid content during storge period in all treatments. In control

samples the ascorbic acid was quantified as 73.84 mg 100g-1

of fresh weight that

decreased to 42.15 mg 100g-1

after 8 weeks of storage. The percentage of ascorbic

acid degradation was as high as 42.9%. The selected essential oil treatments decreased

this degradation of ascorbic acid significantly. Essential oil of T. captivum was found

most effective in controlling ascorbic acid degradation. Lowest tested concentration

of 200μL L-1

reduced ascorbic acid degradation to 18.08 and 12.98% in samples

dipped for 10 and 20 minutes respectively (Fig. 3.54). Increasing the concentration to

400μL L-1

further restricted ascorbic acid degradation to 11.17 and 7.94% in samples

treated for 10 and 20 minutes respectively (Fig. 3.55). Hence the degradation recorded

in samples treated with this essential oil in a concentration of 400μL L-1

for 20

minutes was 34.96% lower than the control samples (Fig. 3.55B).

This was followed by the essential oil of E. caryophyllata that caused 31.5%

degradation of ascorbic acid in samples treated with a concentration of 200μL L-1

for

10 minutes. Highest tested concentration of 400μL L-1

for 20 minutes reduced

ascorbic acid degradation to 15.79% and hence was found equally effective as that of

200μL L-1

of T. captivum. In comparison to the control the degradation percentage in

samples treated with 400μL L-1

of E. caryophyllata essential oil for 20 minutes was

27% lower (Fig. 3.55B).

Essential oil of F. vulgare controlled ascorbic acid degradation least effectively. Its

lowest tested concentration of 200μL L-1

caused an ascorbic acid degradation of

35.4% in samples dipped for 10 minutes (Fig. 3.54A). Doubling the time of dipping in

same concentration lowered this percentage to 30.21%. As recorded previously the

affectivity of this essential oil also increased with increase in concentration and time

of dipping. Highest tested concentration of 400μL L-1

for 20 minutes caused a

degradation of 19.31% in ascorbic acid content that was 23.59% lower when

compared to the control.

127


oils on ascorbic acid of mandarin fruit during cold storage at 4ºC for 8



for 20 minutes.

y = -3.9142x + 71.877 R² = 0.9788

y = -1.6338x + 73.566 R² = 0.9992

y = -2.9972x + 74.828 R² = 0.9927

y = -3.2356x + 72.986 R² = 0.9969

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

Asc

orb

ic a

cid

(m

g/1

00g)



A

y = -3.9142x + 71.877 R² = 0.9788

y = -1.1371x + 73.691 R² = 0.9706

y = -2.2788x + 73.304 R² = 0.9955

y = -2.7753x + 73.345 R² = 0.9986

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

Asc

orb

ic a

cid

(m

g/1

00g)



B

128


oils on ascorbic acid of mandarin fruit during cold storage at 4ºC for 8



for 20 minutes.

y = -3.9142x + 71.877 R² = 0.9788

y = -1.0416x + 73.747 R² = 0.9936

y = -2.0279x + 73.56 R² = 0.9986

y = -2.5398x + 73.128 R² = 0.993

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

Asc

rbic

aci

d (

mg

/10

0g)



A

y = -3.9142x + 71.877 R² = 0.9788

y = -0.7417x + 74.105 R² = 0.9818

y = -1.4501x + 74.082 R² = 0.994

y = -1.7337x + 73.592 R² = 0.9972

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9

Asc

orb

ic a

cid

(m

g/1

00g)



B

129

3.2.12.6. Titratable acidity

The regression line of titratable acidity also showed significantly

drecreasing trend during eight weeks of the storage period. On the onset of the

experiment the average titratable acidity was measure as 1.6g 100g-1

fresh weight in

control samples that decreased 65.8% in the period of eight weeks and samples of last

harvest showed titratable acidity at 0.546g 100g-1

fresh weight. The three selected

essential oils helped significantly to overcome this reduction. The essential oil of T.

captivum was found most potent in this activity. Samples treated with 400μL L-1

of

this essential oil for 20 minutes showed titratable acidity at an average of 1.243g

100g-1

fresh weight. Hence the percentage loss in titratable acidity decreased to 22.3%

that was 43.48% less than the control (Fig. 3.57B).

Results showed by the essential oil of E. caryophyllata were found quite close to that

of essential oil of T. captivum. Titratable acidity was recorded at 1.026g 100g-1

fresh

weight in fruits treated with 400μL L-1

of E. caryophyllata essential oil for 20

minutes. In terms of percentage losses the titratable acidity decreased to 35.87% that

was 29.9% less in comparison to the control (Fig. 3.57B).

Similarly essential oil of F. vulgare decreased titratable acidity to 39.6% as the fruits

treated with its highest tested concentration for 20 minutes showed an average of

0.966 % titratable acidity. When compared this was 26.17% lower than the control. In

all the essential oils, increase in concentration and time of dipping increased their

efficacy.

130

Fig. 3.5:6 Linear regression fitted for the effect of different concentrations of essential

oils on titratable acidity of mandarin fruit during cold storage at 4ºC for 8



for 20 minutes.

y = -0.164x + 1.8479 R² = 0.8959

y = -0.0951x + 1.6023 R² = 0.993

y = -0.1188x + 1.6466 R² = 0.9911

y = -0.112x + 1.6599 R² = 0.9906

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8 9

TA

(g/1

00g f

resh

wei

gh

t)

Storage Periods (weeks)


A

y = -0.164x + 1.8479 R² = 0.8959

y = -0.0784x + 1.6134 R² = 0.9925

y = -0.0952x + 1.6327 R² = 0.9922

y = -0.0974x + 1.642 R² = 0.9944

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8 9

TA

g/1

00g f

resh

wei

gh

t)

Storage period (Weeks)


B

131

Fig. 3.57: Linear regression fitted for the effect of different concentration of essential

oils on titratable acidity of mandarin fruit during cold storage at 4ºC for 8



for 20 minutes.

y = -0.164x + 1.8479 R² = 0.8959

y = -0.0691x + 1.5957 R² = 0.9939

y = -0.0914x + 1.643 R² = 0.9869

y = -0.0914x + 1.643 R² = 0.9869

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8 9

TA

(g/1

00g f

resh

wei

gh

t)



A

y = -0.164x + 1.8479 R² = 0.8959

y = -0.0461x + 1.6042 R² = 0.9912

y = -0.0739x + 1.6158 R² = 0.9997

y = -0.0916x + 1.6199 R² = 0.9762

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8 9

TA

(g/1

00g f

resh

wei

gh

t)


Control

T. captivum

B

132

At 0 day After 8 weeks

Control fruit

E. caryophyllata

F. vulgare

T. captivum

Plate 3.5: Mandarin fruits treated with three most effective essential oils with


for 10 minutes, stored at 4ºC for 8 weeks.

133


Control fruit

E. caryophyllata

F. vulgare

T. captivum




134


Control fruit

E. caryophyllata

F. vulgare

T. captivum




135


Control fruit

E. caryophyllata

F. vulgare

T. captivum




136

Chapter 4

DISCUSSION

The blue mold disease caused by Penicillium italicum Wehmer, is most important

problem in all citrus producing regions of the world (Eckert and Eaks, 1989). A recent study

states that more than 90% of citrus fruit decay is under fungal attack that is responsible for high

economic losses annually (Al-Hindi et al., 2011). At present different postharvest fungicides

including benzimidazoles, thiabendazole and imazalil are used to control citrus fruit decay (Fan

et al., 2000). Although, the increasing reports of pathogen resistance to conventional pesticides is

partially the result of a continual and haphazard use of the same fungicide against similar

pathogens for long term. Moreover the consumer is becoming more health conscious and is

highly concern about the hazardous residues of pesticides. All these features are of great concern

in the international trade market and therefore have forced researchers in the field to focus on

development of a new environment safe strategy to control postharvest fungal decay and

improve fruit quality during storage. The reports on citrus fruit losses have increased drastically

in the last decade, as no control measures has proved effective against storage fungi on this fruit

for longer duration (Zhu et al., 2006). Part of the solution to this problem can be the use of plant

based chemicals including plant extracts and essential oils (Prasad et al., 2004; Chuang et al.,

2007; du Plooy et al., 2009; Askarne et al., 2012).

Consequently, present study was designed to investigate a plant based ecologically safe strategy

against postharvest decaying fungi of citrus. Ameziane et al., (2007) also reported that

application of essential oils can be a very attractive and environment safe method for controlling

postharvest diseases and thus can enhance the shelf life of the fruits. Antimicrobial activity of

essential oils is also proven by earlier studies (Shimoni et al., 1993; Mishra and Dubey, 1994;

Cox et al., 1998; Ozcan, 1998; Cosentino et al., 1999; Aligiannis et al., 2001; Elgayyar et al.,

2001; Arras and Usai, 2001).

137

A survey conducted during this study in major citrus growing areas of Punjab, Pakistan revealed

Aspergillus, Penicillium and Rhizopus as the top most fungal genera associated with the stored

mandarin fruits. The most abundant fungal specie found in the collected samples was P. italicum.

In 2005, Tournas and Katsoudas isolated a number of Aspergillus species including A. niger, A.

fumigatus, A. nidulans, A. varsicolor and A. candidus along with some Fusarium species from

stored citrus fruits. Similarly Bukar et al. (2009) reported association of six fungal genera

including Penicillium, Aspergillus, Fusarium, Rhizopus, Alternaria and Mucor with massive

deterioration of citrus fruits. In an earlier study, Niji et al. (1997) reported the presence of

Aspergillus niger in decaying citrus fruits. Penicillium digitatum, Rhizopus stolonifer and

Aspergillus niger have also been isolated from stored oranges by Akintobi et al., in 2011.

The study was extended to evaluate any genetic variation among various isolates of P. italicum

that appeared as most dominating storage fungi in this study. The morphological characteristics

of all the geographically distinct isolates of P. italicum when cultured on malt extract ager were

found closely related. As morphological study is not sufficient in examining the diversity among

microbial isolates, these isolates were further confirmed for their variability by the molecular and

genetic marker studies. Randomly Amplified Polymorphic DNA (RAPD) fingerprinting

technique was used to fulfill this purpose. Dupont et al. (1999) revealed comparatively

significant level of similarities among twelve Penicillium species using molecular markers when

morphological characterization exhibited moderate level of similarities. Tiwari et al., in 2011

also stated that variations in genetic material cannot be studied morphologically, while RAPD

technique may overcome such type of problems and is very effective for microbial specie

characterizations. In present study the genetic relationship among isolates of P. italicum shown

by dendogram was found completely compatible with the geographical locations and belts from

where the isolates have been collected. Lahore and Multan are distant but fall in the same belt in

citrus producing areas of the Punjab. Lahore, Multan, Bhakar, Jhang and Sargodha fall in the

same geographical belt whereas Chiniot and Faisalabad render a separate geographic region.

Consequently the collected isolates from different regions form distinct groups showing a range

of variability as seen by UPGMA results. Sahiwal being more close to southern Punjab showed

more variation as compared to the other isolates. Here another point of concern cannot be

negated that the mutation frequencies fluctuate considerably along the genomic nucleotide

138

sequences such that mutations focus at certain positions called hotspots. Mutation hotspots in

DNA reflect the effect of the environmental factors in which the organism prevail (Rozin and

Pavlov, 2003). These mutations are largely due to the point mutations, insersions, deletions,

inversions and translocations in the chromosomes, which cause the variability in the genome that

is easily detected through the fingerprinting process using various molecular marker tools such

as RAPD.

As all the eight isolates of P. italicum were found genetically variable they were then checked

for their pathogenicity potentials by artificial inoculation on mandarin fruits. All the isolates

infect the mandarin fruit at different levels. The Lhr isolate stand as the most pathogenic isolate

as it caused maximum deterioration in tested fruits in 7 days followed by the Fsd and Sdg

isolates. Therefore, Lhr isolate was selected for further investigations and trials.

Naturally plants have special chemical compounds in the cell compartments of their seeds, roots,

shoots, fruits and flowers which are high molecular weight phenolic and aromatic compounds

and have antimicrobial and antifungal activities. The Essential oil composition of plants varies

considerably in different families, genera and other small taxonomic groups. These essential oils

are ethno-medicinally important and used in different medicinal drugs to cure microbial

infections (Singh et al., 2011). Antifungal mechanism of essential oils and their components also

varies from restricting hyphal growth rendering lack of cytoplasm and cell organelles along with

the loss of integrity of the cell wall to the halted conidial and germ tube development (Obagwa,

2003). This antimicrobial activity is due to their hydrophobicity which allows the permeability of

essential oils through lipid bilayer of cell membranes causing the leakage of cell cytoplasm and

its contents (Lambert et al., 2001).

In the present study essential oils from various parts of ten selected plants were employed for the

control of test microbe and these oils were then further characterized for the presence of

antimicrobial compounds playing an active role. Essential oils extracted from ten plants namely

Eugenia caryophyllata T., Cinnnamomum zylanicum J., Cuminum cyminum L., Foeniculum

vulgare M., Trachyspermum captivum L., Cymbopogon citratus S., Azadirachta indica L.,

Eucalyptus globulus L., Ocimum basilicum L. and Ocimum sanctum L. were screened for their

antifungal properties against P. italicum. These plant sources were selected because they are well

139

known for their antimicrobial properties (Partra et al., 2002; Dhaliwal et al., 2004; Rasooli and

Owlia, 2005; Soylu et al., 2006; Rasooli and Lee at al., 2007; Yahyazadeh et al., 2008; Mossini

et al., 2009; Camele et al., 2010; Singh et al., 2011; Marandi et al., 2011; ). Recently some

workers have also reported few of these essential oils against blue mold disease in citrus,

however their investigations are restricted to In Vitro assays (Szczerbanik et al. 2007;

Yahyazadeh et al. 2008; Combrinck et al. 2011). The novelty of this study lies in its extensive

scale of work where the studies have been extended to In Vivo assays. The essential oils were

tested in 3, 6, 12, 24 and 48µL mL-1

concentrations against selected fungus. All tested essential

oils exhibited diverse degrees of antifungal activity against P. italicum at different

concentrations. The maximum antifungal activity was shown by T. captivum followed by F.

vulgare, E. caryophyllata, O. sanctum, C. cyminium and C. citratus.

In Vitro and In Vivo studies showed the efficacy of T. captivum essential oil that completely

inhibited fungal growth even at its lowest tested concentration of 3µL mL-1

. Results of this study

are in line with Singh et al. (2004) who reported complete inhibition of selected Penicillium

species against the essential oil of T. captivum even at a low dose of 2µL mL-1

. Different

researchers have previously shown a strong antifungal effect of T. captivum essential oil at

concentrations of 100, 200 and 300ppm (Meera and Sethi, 1994; Diwivedi and Singh, 1999). On

chemical analyses of the extracted essential oil of T. captivum, thymol was found the major

component of the essential oil. This was followed by P-cymene and ɣ-terpinene. This was in

close accordance with the results of Saatchi et al. (2014) who reported thymol as major

component of T. captivum essential oil along with p-cymene, r-terpinen and b-pinene. Waghmare

et al. (2014) also reported the chemical composition of the T. captivum essential oil fairly similar

to this study. When six major identified components of this essential oil were compared for their

antifungal activities, thymol stand as the most effective antifungal component among all. As the

major constituent of the T. captivum essential oil i.e., thymol was also the most effective

antifungal component among all the identified and tested compounds this can rightly be a reason

behind the highest antifungal activity of this oil. Thymol also stands as the most effective

antifungal component among all the twenty four tested components of the ten essential oils.

Hence in this study thymol appeared as the utmost effective chemical compound whose

concentration actually determined the efficiency of different essential oils. In a recent study, the

140

In Vitro antifungal efficacies of terpinen-4-ol, eugenol, carvone, eucalyptol and thymol were

tested against Fusarium sp. Aspergillus sp., Penicillium sp. and Alternaria alternata and found

thymol as the most efficient antifungal component (Morcia et al., 2012). Other similar studies

also reported the higher efficacy of thymol as compared to carvacrol and eugenol when checked

against A. niger, P. italicum and P. digitatum (Bouddine et al., 2012; Perrez-Alfanso et al.,

2012). No comprehensive study using T. captivum essential oil before this has been documented

against blue mold decay of citrus. Previous studies documented that antifungal activity of thymol

is due to its potential to alter the hyphal morphology resulting in lyses of hyphal wall, reduced

hyphal diameter or formation of hyphal aggregates (Numpaque et al., 2011). More over this

compound is also known to alter cell membrane permeability thus causing leakage of

macromolecules (Segvic et al., 2007).

The second most effective essential oil found in this study against P. italicum was the seed

essential oil of F. vulgare which was assessed through In Vitro and In Vivo antifungal assays.

Soylu et al. (2005) reported the F. vulgare oil as the most effective oil among Origanum

syriacum, Artimisia annua, Laurel nobilis and Lavender stoechas against green mold i.e., P.

digitatum on citrus fruit during storage. They also exposed the microscopic degenerative

mechanisms of F. vulgare and Origanum syriacum essential oils on fungal hyphal morphology.

They reported that mycelial cells become devoid of cytoplasm and cytoplasmic organelles which

showed overall suppressed growth of the fungus. Essential oil of F. vulgare is also known to

inhibit various other storage fungi including A. alternata, F. oxysporum, A. flavus and A.

parasiticus (Aggarwal et al., 2000). The most prominent chemical constituent found in F.

vulgare essential oil by using recent techniques of GC-MS and GC-FID was anethole followed

by fenchol, methyl chavicol and eugenol. Many previous investigations have reported similar

chemistry of this essential oil and have mentioned anethole as its major component (Fernandez-

Ocana et al., 2004; Singh et al., 2006; Abdolahi et al., 2010). Similar to the thymol, anethole

also completely inhibited tested fungi at 100µL L-1

but at lower concentration it was found

slightly less effective than thymol. Parallel to the results of T. captivum the most potent

antifungal component of this essential oil appeared as the major component, constituting 61.4%

of the oil. Hence this can be a cause of high antifungal activity of this oil. When compared

141

among all the twenty four tested essential oil components anethole stood second after thymol in

its ability to control fungi.

Eugenia caryophyllata and Ocimum sanctum essential oils showed approximately similar results

at higher concentrations of 24 and 48µL mL-1

during In Vitro assay. But at lowest tested

concentration of 3µL mL-1

, E. caryophyllata showed slightly higher inhibition over O. sanctum.

In Vivo studies also revealed E. caryophyllata as the next most effective essential oil against blue

mold pathogen. Deng et al. (2013) evidenced E. caryophyllata oil as the most effective and

remarkably fungistatic against P. italicum and Fusarium sp. Their experiment demonstrated that

F. vulgare oil is less effective in contrast to E. caryophyllata oil which is not in accordance with

the results obtained in the present study. The essential oil of E. caryophyllata was also reported

to have fungicidal activity on several food borne fungal species (Lopez et al., 2005). Eugenia

caryophyllata essential oil significantly suppressed the microbial growth of A. fumigatus and A.

acculeatus and displayed greater pathogenicity. According to Pawar and Thaker (2006), E.

caryophyllata essential oil can be also effective against the growth of A. niger. The results of

Sunita and Mahendra (2008) proved the efficacy of O. sanctum essential oil against several

pathogenic fungi including A. fumigatus and A. niger. When chemistry of both the essential oils

was compared, Eugenol was found as the major component of the E. caryophyllata essential oil,

whereas in O. sanctum, linalool was recorded with maximum percentage. The antifungal

effectivity of eugenol was significantly higher than linalool. In case of E. caryophyllata,

cuminaldehyde was also found equally potent as that of eugenol. However quantity of

cuminaldehyde was much lower than eugenol. In contrast to this major component of O. sanctum

i.e., linalool was found as least effective compound against tested fungi. The effective

components of the essential oil of O. sanctum were thymol, eugenol and 1,8-cineole that were

present in very low percentages. This may be the cause of its lower antifungal potential than E.

caryophyllata. The same findings were evidenced by Eugenia et al., in 2009 who investigated

the antifungal activity of E. caryophyllata essential oil and eugenol against Candida and

Aspergillus species. In this study, eugenol was evidenced to be the third most effective chemical

component after thymol and anethole thus making its essential oil as the third most effective

source of antifungal activity. Previous studies have also confirmed efficacy of E. caryophyllata

essential oil against P. italicum. Hall and Fernandez (2004); Yahyazadeh et al. (2008) and

142

Combrinck et al. (2011) also showed In Vitro efficacy of E. caryophyllata essential oil against

blue mold fungus. Plaza et al., in 2004, documented significant In Vitro antifungal potential of E.

caryophyllata essential oil against blue mold fungi. However when they extended their studies to

In Vivo assays they found it ineffective against P. italicum when tested directly on inoculated

wounds. When they used this oil in combination with wax or incorporated in packaging by

soaking pads, severe rind damage was observed making this technique impracticable. No such

rind damage or bad odour was recorded in present study.

Essential oils rich in phenolic compounds are reported to possess high level of antifungal

activities. Ozcan and Boyraz (2000) and Tepe et al. (2005) documented that phenolics usually

show strongest antifungal activities followed by aldehyde, ketones, alcohols and other

hydrocarbons. In present study thymol that is a phenol which showed highest inhibition of tested

fungi among twenty four tested essential oil components. It was followed by anethole that is a

derivative of phenylpropene and eugenol that is a phenylpropene.

The essential oils of C. cyminium and C. citratus stood as the next potent source of antifungal

activity. Considerable antifungal activity of C. cyminium essential oil has been reported by a lot

of researchers against A. flavus, P. expansum, A. ochraceus, P. verrucosum, Salmonella

typhimurium, Escherisia coli and various other microbes (Hammer et al., 2003; Burt, 2004;

Iacobellis et al., 2005; Nguefack et al., 2012). In another study Lee et al. (2007) demonstrated

strong growth inhibitory activities of C. cyminium and C. citratus essential oils against the

pathogen causing gray mold of apple. The essential oil of C. citratus has also been documented

exhibiting various degrees of antifungal activity against phytopathogenic fungi (Sun et al., 2007;

Sunita and Mahendra, 2008). The chemical analyses of the tested essential oil of C. cyminium

showed cuminaldehyde as its prime component. This was followed by carvacrol, terpinen-4-ol

and citronella. Kawther (2007) have also reported the presence of γ-terpinene, β-pinene and

cuminaldehyde in this essential oil. In the present study, when identified components were

compared for their effectivity, cuminaldehyde and terpinen-4-ol was found as most potent

components in restricting the fungal growth after thymol that was found in traces in this essential

oil. Carvacrol also established as a major component of this oil but its antifungal activity was

recorded significantly lower than other constituents. On the other hand C. citratus illustrated

citral as its major component followed by neral, geraniol, limonene and caryophyllene. The

143

component with highest percentage of occurrence i.e., citral showed least antifungal activity.

However, the second most abundant component neral stood as the most effective antifungal

component of this oil along with limonene. Adegok et al. (2000) also revealed limonene as the

most potent antifungal constituent of C. citratus essential oil against A. flavus and A. parasiticus.

They also confirmed that limonene is actually involved in the membrane injury of fungal

pathogen. In another study citral, was reported as prime antifungal component of this essential

oil (Silva et al., 2009). Oliveira et al. (2011) confirmed that the antimicrobial efficiency of C.

citratus oil is due to the presence of neral and geranial.

Rest of the four essential oils including C. zeylanicum, O. basilicum, A. indica and E. globulus

could not prove as effective as the above mentioned sources against selected fungi. Paul and

Sharma (2002) stated that the extract of A. indica did not effectively inhibit the fungal growth of

Drechslera graminea and Alternaria alternata. In this study the effectivity of these four essential

oils varied when checked In Vivo and In Vitro. When their components were identified and

checked individually it was found that their prime components were weak antifungal compounds

and the constituents showed higher antifungal activity were found in lower percentages in these

components. In case of C. zeylanicum essential oil the prime constituent is the cinnamaldehyde,

followed by the eugenol, isoeugenol, caryophyllene and β-Pinene as also reported by previous

workers (Schmidt et al., 2006; Kamaliroosta et al., 2012) whereas, the most powerful antifungal

component is eugenol followed by caryophyllene and cinnamaldehyde. Ozcan and Boyraz

(2000) also confirmed eugenol as most effective antifungal constituent of cinnamon essential oil.

Similarly in O. basilicum linalool constituted the major part of the essential oil but its antifungal

activity was positioned after eugenol, methyl eugenol and carvacrol. Oxenham et al. (2005) and

Sunita and Mahendra (2008) also reported similar composition of this essential oil. In contrast to

our studies, these four essential oils have been reported effective against other fungi by various

workers. Ocimum basilicum essential oil is acknowledged for its wound healing properties and

reported as fungitoxicant and antimycotoxicant (Rios and Recio, 2005; Singh et al., 2011). The

significant inhibitory effect of A. indica, E. globulus and O. sanctum plant extracts has been

reported against Fusarium by sexena and Mathela (1996). However, in other studies different

scientists noted that essential oil of A. indica showed low antimicrobial activity (Martinez, 2002;

Carneiro, 2008). Eucalyptus globulus essential oil and extract has been clinically approved as

144

food hyperglycemic and antioxidant activities (Takahashi et al., 2000). Eucalyptus globulus oil

showed strong antifungal activity against Fusarium solani and Fusarium oxysporum which

inhibited mycelial growth and spore production in the pathogenic fungi (Fiori et al., 2000;

Oluma and Garba, 2004; Liu et al., 2008). Dhaliwal et al. (2004) reported that the essential oil of

Eucalyptus camaldulensis is highly effective against P. digitatum causing green mold disease in

mandarin c.v. kinoo under both In Vitro and In Vivo conditions. Our results of C. zeylanicum

essential oil efficacy are in agreement with Simic et al. (2004), according to which this essential

oil is significantly effective against filamentous fungi such as A. niger, A. flavus, A. tereus and P.

versicolor. According to Xing et al. (2010) this oil is active against postharvest pathogens of

citrus fruit like Rizopus nigricans, A. flavus and P. expansum. This essential oil also exhibited

strong inhibitory effects on the growth of Botrytis cinerea (Sirirat et al., 2009; Sessou et al.,

2012).

In Vitro studies were performed using both dilution and volatile assays. The antifungal effects of

most of the essential oils were found more pronounced in volatile assay than in dilution assay.

The reason behind this is that the active chemical compounds can act in a more concentrated

form rather when they get diluted in a medium. These results were in close agreement with the

study of Bouddine et al. (2012) who used both dilution and volatile methods for determining the

efficacy of various essential oils. Their study demonstrated that the volatile method was more

effective due to the high concentration of major components in the oil that could inhibit fungal

growth in a more pronounced way. Other studies in literature also supported the similar findings

(Suhr and Nielsen, 2003; Inouye et al., 2000). However, in case of C. cyminium essential oil the

antifungal effect was higher when checked through dilution method in comparison to the volatile

method.

Top three essential oils found active in controlling selected postharvest fungi were then checked

for their effect on physicochemical changes that occurs in a fruit during storage. Mandarin fruits

treated with different concentrations of T. captivum, E. caryophyllata and F. vulgare essential

oils for 10 and 20 minutes were stored at 4oC for eight weeks. Readings for various parameters

were taken after every seven days. As was expected the fruit quality in untreated fruits

diminished with passage of time. Fruits treated with selected essential oils showed significantly

lower rates of deterioration. Essential oil of T. captivum found most effective in reducing

145

postharvest decay followed by F. vulgare and E. caryophyllata. Earlier reports also confirm that

different physicochemical changes occur in perishables during storage that results in its

deterioration (Aquino, 2003). It is therefore that storage of fruits at low temperature is most

desired practice to maintain the fruit quality that also extends the shelf life of fruit (Sala and

Lafuente, 1999). Recent research reports the use of wax that reduce losses in firmness and

strength of fruits like citrus (Hagenmaier, 2000; Rojas et al., 2002; Perez et al., 2003).

First sign of quality deterioration is the weight loss in the fruits that is also visually noticeable as

wilted fruits. Results of this study were in accordance with the findings of Roongruangsri et al.

(2013) who demonstrated that the percentage of weight loss increased and the moisture content

of citrus peel decreased at higher temperature and longer duration. However, low temperature

storage reduces the losses of fruit weight and moisture content of the peel and preserved the

external quality better than storage at room temperature. Wills et al. (2007) demonstrated that

weight loss was caused by fruit transpiration in which water evaporated and resulted in the

wilted fruit rind and a shriveled appearance. In Mandarin fruit, 5-6% water loss resulted in some

changes in appearance and firmness of the fruit that could be detrimental to its marketability

(Ladaniya, 2008). It is evident from present study that essential oils can be a safe alternative for

commercial coatings and waxes to reduce weight losses during storage. Chafer et al. (2012)

reported that coating of chitosan; bergamot thyme and tea tree essential oils significantly reduce

the physicochemical changes in citrus fruit throughout the cold storage. Many earlier reports

confirmed that the effect of coatings of edible composites significantly control the weight loss of

citrus fruit (Valencia-Chamorro et al., 2008; Silvia et al., 2010). Weight loss was not only due to

the moisture loss from the peel but the juice content was also found to be reduced when checked

periodically. Essential oil of T. captivum reduced these losses to minimum level thus increased

its shelf life.

The pH of stored fruits slightly increased with storage time probably due to the breakdown of

acids during respiration (Pesis et al., 1999). However, it remained within acceptable limits even

after eight weeks of storage. Earlier workers have also documented similar changes in pH in

stored citrus juice (Martin et al., 1995; Esteve et al., 2005; Asghari- Marjanlo et al., 2009; Ali et

al., 2011; Chafer et al. 2012). When compared with the changes in the acidity, the variations in

the pH were found less pronounced during eight weeks of the storage period at temperature 4oC.

146

Esteve et al., in 2005 also concluded similar changes in pH and acidity of stored citrus juice.

According to them this may be because of the natural buffer medium including potassium

citrates and malates in orange juices.

Titratable acidity is a measure of the total acid present in juice. Citric acid is the major acid in

orange juice but there are also small amounts of malic acid and tartaric acid. The total acid may

not be measured by pH because the acids concerned are “weak acids” and not completely

ionized. The acid content was therefore measured using a titration with sodium hydroxide.

Untreated fruits showed a decline in TA that was significantly overcome by the coatings of

selected essential oils. Minimum change in TA with respect to the control was observed in

samples treated with T. captivum essential oil, followed by F. vulgare and E. caryophyllata. Our

results are in line with the findings of Elshiekh and Abu-Goukh (2008), who found that TA

increased slightly during the first fifteen days of the storage and then progressively decreased in

all fruits. Similar findings were recorded in a study conducted by Ali et al. (2011) who revealed

that the TA decreased at a slow rate with the chitosan coatings, regardless of storage time.

Decrease in TA with time is due to the utilization of acids as respiratory material or their

conversion into sugars that can be further utilized in metabolic process of fruits (Faasema et al.,

2011). In another study of Tzortzakis (2007) no significant change in titratable acidity and pH

was recorded in tomato and strawberry fruits treated with the essential oils of E. globulus and C.

zeylanicum.

Total soluble solids content is the amount of soluble solids such as sugar, salts, proteins, acids,

etc. present in an aqueous solution. In the current study, it was noted that total soluble solids

(TSS) increased with storage period. Control fruits showed higher TSS values than treated ones.

Least amount was recorded in fruits treated with essential oil of T. captivum, followed by E.

caryophyllata and F. vulgare essential oils. Previous workers have also reported similar increase

in TSS in citrus fruits during storage. Various treatments adopted by these workers decreased

TSS value significantly when compared to the control (Attia, 1995; Elshiekh and Abu-Goukh,

2008; Aborisade and Ajibade, 2010). During storage of citrus fruits, increase in TSS level is due

to the loss of moisture content which increases the concentration of TSS (Elshiekh and Abu-

Goukh, 2008). In contrast, Asghari-Marjanlo et al. (2009) reported no significant change in TSS

in treated fruits as compared to that in the control samples.

147

Citrus is famous for its high ascorbic acid content. This is also a parameter to monitor fruit

freshness and postharvest fruit spoilage has also been found associated with losses in this

antioxidant compound (Sanusi et al., 2008). However, ascorbic acid is quite unstable and it

decomposes easily through both aerobic and anaerobic pathways (Fennema, 1977; Johnson et al.,

1995; Lee and Coates, 1999). Factors responsible for this degradation involves oxygen, heat,

light (Robertson and Samaniego, 1986), storage temperature and storage time (Fellers,

1988; Gordon and Samaniego-Esguerra, 1990). Present study also confirmed gradual losses in

ascorbic acid during storage. These losses were overcome by treating fruits with selected

essential oils. Minimum loss of ascorbic acid content was noticed in fruits treated with T.

captivum essential oil followed by E. caryophyllata and F. vulgare at highest tested

concentration and time i.e. 400µL L-1

for 20 minutes. During storage anaerobic degradation of

ascorbic acid occurs (Johnson et al., 1995; Solomon et al., 1995). This means that the essential oil

coating acted as an affective barrier against environmental factors like light, oxygen and

temperature thus it helped in reducing ascorbic acid losses due to these factors (Gordon, 2006).

The findings of Zamani-Zadeh et al. (2013) support these results, as they revealed that the T.

captivum and C. zeylanicum essential oil reduce the gray mold disease in strawberry fruit during

storage along with least losses in ascorbic acid content. They found that the loss of ascorbic acid

is due to the changes in the storage conditions including mold growth, changes in the storage

temperature and moisture, and other factors that can deteriorate the ascorbic acid content. It was

established that ascorbic acid destruction may be due to the cell wall break down by fungal

infection (Zamani-Zadeh et al., 2013).

This study concludes the possible use of T. captivum, E. caryophyllata and F. vulgare essential

oils and their constituents against postharvest decay especially caused by P. italicum and quality

preservation in citrus during storage. However, one should keep in view their effect on human

health and environment. As far as top most effective essential oil component found in this study

i.e., thymol is concerned, US environmental protection agency EPA in 2009 have confirmed that

thymol has minimal potential toxicity. Also it has been documented that thymol naturally

degrades very quickly thus has low ecological risks (Hu and Coats, 2008). On the other hand,

eugenol that stood second in its potential to inhibit tested fungi has been reported as a

hepatotoxic compound and is also known to cause some allergies (Fujisawa et al., 2002).

Conversely it is being used in perfumeries, flavoring and in medicines as a local antiseptic and

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148

anaesthetic (Jadhav et al., 2004). Similarly anethole that significantly helped in quality

preservation and control against fungal decay in stored mandarins also has few reports for its

carcinogenic activity and liver toxicity (Newberne et al., 1989). However, currently the data is

not sufficient to support these findings and Joint FAO/WHO Expert Committee on Food

Additives (JECFA) has confirmed that anethole is safe to be used as flavoring agents (JECFA,

2001). Keeping in view the antifungal potential and safety concerns about these essential oil

components, the present study recommends use of T. captivum essential oil and its active

components as thymol in controlling postharvest decay in citrus.

149

CONCLUSIONS AND FUTURE PROSPECTS

CONCLUSION

This study concludes the possible use of essential oils from selected sources in reducing

postharvest losses caused by fungal decay in perishables especially fruits like citrus. Among the

ten selected essential oils T. captivum essential oil proved as most effective source of antifungal

components as thymol that should be exploited in future for development of an ecologically safe,

shelf life enhancer against fungal deterioration in citrus during storage.

FUTURE PROSPECTS

Despite all documented advantages, much work remains to be done on the detailed

examination of the biological activity, mode of action and dispersion of essential oils and

their components in fruit tissues to develop a formulation that maintains fungicidal

activity without causing undesirable effects to the product, human health and

environment.

More studies are required to establish the safety and toxicity levels of the essential oils

found effective against targeted microbes. The minimum inhibitory concentrations have

also to be determined for each specific essential oil and its components and commodity.

Combination of essential oils from different sources can provide a better control over

their individual use that needs an extensive study.

As essential oils do not fill such a broad spectrum as synthetic pesticides, there is a need

to improve their effectiveness by using them in combination with effective preharvest,

harvest and postharvest techniques, carefully designed packaging and low temperature

storage.

150

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY

ISSN Print: 1560–8530; ISSN Online: 1814–9596

13S–004/2013/15–6–1283–1288

http://www.fspublishers.org

Full Length Article

To cite this paper: Akhtar, N., T. Anjum and R. Jabeen, 2013. Isolation and identification of storage fungi from citrus sampled from major growing areas of Punjab, Pakistan. Int. J. Agric. Biol., 15: 1283‒1288

Isolation and Identification of Storage Fungi from Citrus Sampled from

Major Growing areas of Punjab, Pakistan

Nosheen Akhtar1, Tehmina Anjum

1* and Rasheda Jabeen

1

1Institute of Agricultural Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore-54590, Pakistan

*For Correspondence: [email protected]

Abstract

Stored citrus fruits were collected from cold storage houses of major citrus growing areas of the Punjab, Pakistan and storage

fungi were isolated from the sampled fruits. The fungal isolates were identified as Aspergillus niger, A. flavus, A. fumigatus, A.

terreus, Penicillium verrucosum, Rhizopus arrhizus, R. stolonifer, A. parasiticum, Fusarium oxysporum, P. citrinum, A.

awamorii, Alternaria alternata, F. solani and Mucor sp. based on morphological characteristics. The frequency of occurrence

of each fungal species was determined; P. italicum was found the most frequent with the highest occurrence of 29.75%

among all isolates, followed by the A. niger (14.87%), while Dreshlera sp. having the least occurrence (0.82%). Hence, P.

italicum was chosen for the molecular characterization among its different geographically distinct isolates through random

amplified polymorphic DNA (RAPD) technique. The UPGMA based dendrogram showed three distinct groups, which

coincide with the geographical locations from where the isolates were collected. The total variation of 23% among the isolates

confirmed that the isolates are of the same species and the source of variation can be due to the environmental factors. The

variation can also be contributed by switching off and switching on of some genes under certain environmental conditions. ©

2013 Friends Science Publishers

Keywords: Citrus; Fungi; Penicillium italicum; RAPD; Random primers

Introduction

Citrus is one of the most important winter fruit crops of the

world and has grown commercially in more than 100

countries across six continents (Terol et al., 2007). They

contain several phytochemicals including vitamin C with

disease preventing and life sustaining functions (Dillard and

German, 2000). Pakistan is ranked 10th in citrus production

worldwide (Sabir et al., 2010). Moreover, huge economic

losses occur throughout the world due to post harvest

diseases. In Pakistan, about 40% of total citrus produced is

wasted during storage in post-harvest process (Naseer,

2010), and is frequently reduced by fungal pathogens attack

(Liu et al., 2007). Storage fungi can reduce the shelf life and

acceptability of fresh produce.

Blue mold, caused by Penicillium italicum, is

generally severe postharvest disease of citrus, which costs

the loss up to 25% of the total production worldwide (Palou

et al., 2007; Montesinos-Herrero et al., 2009). Mold growth

in citrus fruit leads to the production of the hazardous

mycotoxins (Moss, 2008). Taxonomic relationships of

fungal species have been clarified by applying many

phenotypic and genotypic approaches (Varga et al., 2000).

The molecular markers including biochemical protein

markers and DNA markers are extremely valuable tools for

weighing genetic similarity and determining species

individualities. Among the DNA molecular markers,

random amplified polymorphic DNA (RAPD) typically are

more eminent due to high variability and reproducibility

with an added advantage of no prior knowledge of DNA

sequence for the fingerprinting of any genomic DNA.

Analysis of genetic diversity among closely related species

is crucial step towards understanding the fungal populations.

The objectives of this investigation were to

characterize storage fungi isolated from stored citrus fruit of

different storage houses in Punjab using morphological

characteristics and to find the most occurring storage fungi

among the storage fungi and to assess the diversity among

these isolates through RAPD-PCR technique to show

genetic variability within and between these isolates.

Materials and Methods

Sample Collection

Samples of citrus fruits were collected from different cold

storage houses in eight major citrus growing areas of

Province Punjab, Pakistan. These samples were placed in

separate sterile plastic bags; transferred to the laboratory and

kept in refrigerator at 4oC till further analysis. These regions

were selected on the basis of their fruit production

importance.

mailto:[email protected]

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Isolation and Identification of Storage Fungi

Isolation of storage fungi from each of the collected fruit

sample was carried out by using the technique of Baiyewu

et al. (2007). A small portion of disease tissue were cut with

sterile scalpel and placed on previously prepared Malt

extract agar and Potato dextrose agar medium plates and

incubated at 25±1oC for 7 days. The developing fungal

colonies were counted to calculate percentage frequency

and further characterized on morphological basis by using

the most documented keys and literature for fungal

identification (Samson and Varga, 2007). Percentage

frequency of individual isolated fungal species was

determined by using the method described by Giridher and

Ready (1997) as:

( )

Finding Genetic variation in all Isolates

According to the percentage of occurrence, P. italicum was

selected for genetic analysis as a most abundant blue mold

fungus on citrus during storage. Genetic variation of eight

morphologically similar but geographically distinct P.

italicum isolates was carried out using RAPD analysis.

DNA Extraction

The total genomic DNA was extracted by using CTAB

method (Doyle and Doyle, 1991) form the fungal mat and

ground to fine powder with Fermentas glass beads. The

freshly prepared pre-warmed (65ºC) extraction solution was

added to the powdered fungal mass in new autoclaved

Eppendorf (1.5 mL) tubes and incubated at 65ºC. An equal

volume of solution Choloform isoamyl alcohol (24:1) was

added and centrifuged at 10,000 × g to separate the phases.

Aqueous supernatants were transferred to new tubes and 2⁄3

volume of cold isopropanol was added. The sample was

centrifuged and transparent DNA pellets were obtained.

After washing, the pellets were air dried and resuspended in

50 µL of Tris HCl EDTA (TE) buffer.

Estimation of Quantity and Quality of DNA

The quantity and quality of extracted DNA was determined

by Techne Spec gene Spectrophotometer (140801-2, UK).

The concentration was calculated on assumption that

absorbance of 1 at 260 nm is equivalent to 50 mg⁄mL double

stranded DNA or 40 mg⁄mL single stranded DNA

(Sambrook et al., 1989). The quantity of DNA was

calculated by the following equation:

DNA Con (µg/mL) ═ Absorbance at 260 nm ×Dilution factor ×50

The quality of the DNA was also estimated through

1% agarose gel electrophoresis in which DNA bands were

compared with the 1kb DNA markers showing the

concentrations of DNA in ng/µL. Hence, the DNA

concentration was maintained to 25 ng/µL for polymerase

chain reaction (PCR) amplifications.

Random Amplification of Polymorphic DNA Analysis

The RAPD analysis is a valuable tool for studying DNA

polymorphism in different fungal isolates for exploring

phylogenetic relationships among them. RAPD analysis was

carried out by following the method described by

Ranganath et al. (2002).

Random Primer Screening

A set of 20 primers procured from School of Biological

Sciences (SBS) Genetech Co. Ltd-Beijing, China were used

in RAPD analysis for the initial screening (Table 1).

Majority of these primers produced clear, distinct and

reproducible polymorphic bands in different isolates of P.

italicum. The primers were diluted up to 100 picomole

concentration before use in RAPD analysis. PCR reactions

were carried out in 25 µL volume containing PCR Buffer

(10X), 2.5 mM MgCl2, 0.2 mM of each dNTP, and 0.6 U

DNA polymerase (Enzynomics, Korea). PCR conditions

and separation of RAPD-PCR fragments were done

according to Messner et al. (1994). The amplifications were

carried out in Techne-412 thermal cycler with temperature

profile as initial denaturation at 94ºC for 5 min and then

primers were subjected for denaturation at 94ºC for 1 min,

annealing at 25ºC for 1 min and final extension at 72ºC for 5

min to a total of 40 cycles.

Statistical Analysis

RAPD profiles were recorded by visually comparing RAPD

amplification profiles and scoring the presence or absence

of each band for each primer (Halmschlager et al., 1994).

The bands obtained from 2% agarose gel electrophoresis

were combined in a binary matrix in two - discrete -

characters - matrix (0 and 1 for absence and presence of

RAPD – markers, respectively). In order to assess over all

distribution of genetic diversity, data was analyzed by using

MINITAB software (MINITAB, 2004).

Results

In the present study, different fungi associated with the

deterioration of citrus fruit during cold storage were isolated

from the collected fruit samples. A total of sixteen fungal

species belonging to seven different genera included were

isolated and identified on the basis of their cultural and

morphological characteristics which are revealed in detail in

Table 1. The frequency of occurrence of each isolate of

fungi which showed that P. italicum was the most

frequently isolated fungus with the highest occurrence of

29.75% among all isolates, followed by the Aspergillus

niger (14.87%), A. flavus (9.09%), A. fumigatus (7.43%),

A. terreus and P. verrucosum (5.78%), Rhizo pus arrhizus

(4.95%), R. stolonifer (4.13%), A. parasiticus and Fusarium

Identification of Storage Fungi Isolated from Stored Citrus Fruit / Int. J. Agric. Biol., Vol. 15, No. 6, 2013

1285

oxysporum (3.3%), P. citrinum, A. awamorii and Alternaria

alternata (2.47%), F. solani and Mucor sp. (1.65%) with

Dreshlera sp. having the least occurrence of 0.82% (Table

2).

The morphological characteristics of P. italicum such as colonies on Malt Extract Agar (MEA) medium were 5-6

cm in diameter after 7 days. The colonies were bluish green in color with velvety and floccose and reverse side of the

colony is yellow to brown. The microscopic characteristics

such as septate and hyaline hyphae; conidiophores smooth

walled and conidia ellipsoidal to cylindrical in shape smooth walled and blue in color were observed. The

cultural and microscopic characteristics of P. italicum seen

in the present study were correlated to those as described by

Singh et al. (1991).

Eight geographically distinct isolates of P. italicum

were collected during this study and subjected to further

characterization by using RAPD-PCR technique (Table 3). A

total of 20 RAPD decamers (Table 4) were used for testing

the genetic variability among eight isolates of P. italicum of

which two primers (Primer SBSA08 and SBSA15) did not

show any amplification and hence dropped in the initial

screening of the random primers. Remaining 18 random

primers produced easily scorable and reproducible banding

patterns, which were designated in genetic coefficient matrix

in Minitab 16 software for further analysis. Total amplified

polymorphic bands ranged from approximately 200 bp to

2000 bp sizes in the RAPD profile. There was a low

percentage of polymorphism in different isolates of P.

italicum. The most probable reason is that the isolates are of

single species, which was correspondingly seen by their similar morphology. The average number of polymorphic bands perceived per primer was 8.22. The RAPD profiles

produced with the primers (SBSA06, SBSA07, SBSA11,

SBSA12, SBSA13, and SBSA18) and banding pattern are

shown in Fig. 1. The ladder 100 bp was run as markers on

Table 1: Morphological Characteristics of Different Storage Fungi Isolated from Stored Citrus Fruit

Name of fungus Colony diameter Colony Texture Obverse Reverse Character of hyphae conidiophore conidia

Penicillium italicum

5-6 cm bluish green

yellow brown

septate, hyaline smooth walled ellipsoidal to cylindrical

Penicillium

verrucosum

2.5-3.5 cm velvety or

almost floccose

grey green yellowish

brown

septate, hyaline two-stage branched,

rough walled

globose to subglobose

Penicillium

citrinum

2-3 cm blue green bright

yellow

septate, hyaline smooth walled,


present

globose to subglobose,

sooth walled

Aspergillus

niger

5-6 cm powdery black Off-white conidial head radiate,

conidia matulae and

phailides present

broad, long, thick

walled, brownish

globose , irregularly

roughened,

Aspergillus

flavus

6-7 cm powdery yellow

green

Pale brown conidial head radiate, hyaline, long, rough

walled,

globose to subglobose,

finely roughened to echinulate

Aspergillus

fumigatus

7 cm bluish

green

Greyish Vesicle pyriform,

uniseriate

clavate vesicle, thick

walled

Smooth, globose to

subglobose Aspergillus

terreus

6-7 cm velvety or

almost floccose

orange

brown to

brown

uncolored matulae present, conidial

heads columnar,

smooth walled,

hyaline, with

hemispherical vesicle

globose to slightly

ellipsoidal, smooth

walled Aspergillus

awamorii

5-6cm green green matulae and phailides

present

smooth walled,

vesicle subglobose

cylindrical to ellipsoidal,

coarsely roughened

Aspergillus parasiticus

5-6 cm green colorless Uniseriate, vesicle spherical

roughened, matulae absent

echinulate and coarsely spiny

Rhizopus

arrhizus

cottony white brown Hyphae branched and

aseptate, rhizoids present

sporangiophore short,

columella globose

spoangiospores ovoid to

more or less globose, dark in color

Rhizopus

stolonifer

fluffy greyish yellow

brown

columella subglobose to

oval

sporangiophore

subglobose rigid,

sporangia pale to brown,

straight, sporangiospore

thick walled

Mucor sp. full plate cottony white to

yellow

Brownish Columella obovoid,

ellipsoidal, with truncate base

Sporangiophore short

branches, recurved and encrusted wall

Sporangia brownish to

grey, spores ellipsoidal to subglobose

Fusarium

oxysporum

4.6-6.5 cm sparce to

floccose

white purple Macroconidia present septate, fusiform,

moderately curved, pointed at both ends

Fusarium

solani

4.5-6.5 floccose whitish cream macroconidia present curved, short, with blunt

apical and pedicellate basal cells

Alternaria

alternata

6-7 cm dark brown

blackish,

Hyphae branched and

septate, brown in color

conidiophore simple,

straight and curved

conidia formed in long

chain, ovoid, obclavate and dark brown in color

Dreshlera sp. 4-5.5 cm dark brown

to black

yellow to

brown

conidiophore , conidia simple and branched Cylindrical or ellipsoidal,

septate with round ends.


1286

both sides along with the negative control in which only

water was used instead of DNA that indicates the credibility

of the reaction mixture having no amplified band.

A dendogram based on UPGMA analysis indicated

that the eight isolates formed three major groups GI, G2 and

G3 confirming some level of genetic variation among the

isolates of P. italicum (Fig. 2). The similarity coefficient

ranged from 0 to 0.23 indicating that there is no 100%

similarity occurs among any two isolates. Group 1 was

further divided in to three subgroups containing five isolates

naming Pi Lhr, Pi Mul, Pi Jhg, Pi Bhk and Pi Sdg. G2

consisted of two isolates, Pi Fsd and Pi Chn while G3

consisted of only one isolate, Pi Sh.

The first subgroup consisted of two isolates collected

from Lahore and Multan, which were 3% distant from each

other while this subgroup is 9% distant from the second

subgroup containing two isolates collected from Jhang and

Bhakar regions of Punjab. There is much similarity seen in

isolates collected from Jhang and Bhakar, which are 1%

distant from each other. This subgroup was 5% distant from

the third subgroup which contained only one isolate

collected from Sargodha cold storage house. G2 is 2%

distant from G1, while G3 was 4% and 6% distant from G1

and G2, respectively.

Discussion

This study showed that a number of storage fungi of genera

Aspergillus, Penicillium, Rhizopus, Fusarium, Alternaria,

Dreschlera, and Mucor are associated with the spoilage and

loss of citrus fruit during storage in Pakistan. The most

abundant of all these fungi was P. italicum. Several

Penicillium species were frequently established on citrus

fruits and cause storage rot commonly referred as blue and

green mold (Filtenborg et al., 1996). Bukar et al. (2009)

who reported that six genera of fungi namely Penicillium

sp., Aspergillus sp., Fusarium sp., Rhizopus sp., Alternaria

sp. and Mucor sp. were associated with massive

deterioration of citrus fruit. Similarly, Niji et al. (1997)

reported that A. niger was associated with the decay of

citrus fruit. P. digitatum, R. stolonifer and A. niger have also

Table 4: Random twenty decamers used in initial screening, their accessions, sequences, number of polymorphic bands,

percentage of polymorphic products and size of bands produced by each primer

Name Sequence ( 5'- 3' ) nmol Total no. of Bands No. of poly morphic bands %age of polymorphic bands Size of bands (bp)

SBSA01 CAG GCC CTT C 111.3 40 11 28% 250-1000

SBSA02 TGC CGA GCT G 108.4 39 7 17% 200-1500 SBSA03 AGT CAG CCA G 110.1 39 15 38% 200-700

SBSA04 AAT CGG GCT G 107.6 45 10 7% 300-1000

SBSA05 AGG GGT CTT G 106.5 29 5 17% 300-900 SBSA06 GGT CCC TGA C 109.9 37 5 14% 200-1200

SBSA07 GAA ACG GGT G 105.9 34 13 38% 400-1800

SBSA09 GGG TAA CGC C 108.1 46 6 13% 200-1400 SBSA10 GTG ATC GCA G 107.6 39 15 38% 270-1500

SBSA11 CAA TCG CCG T 110.4 41 9 22% 200-1600 SBSA12 TCG GCG ATA G 107.6 42 18 43% 400-1900

SBSA13 CAG CAC CCA C 112.2 63 7 11% 340-2000

SBSA14 TCT GTG CTG G 108.2 14 6 43% 350-800 SBSA16 AGC CAG CGA A 108.3 10 2 20% 300-1500

SBSA17 GAC CGC TTG T 109.3 11 3 27% 900-1500

SBSA18 ACG TGA CCG T 107.6 50 2 4% 200-1400 SBSA19 CAA ACG TCG G 108.7 47 7 15% 250-1100

SBSA20 GTT GCG ATC C 109.3 45 5 11% 200-1100

Table 2: Frequency of occurrence of fungi isolated from

stored citrus fruit

Fungi isolated % Frequency of occurrence

Penicillium italicum 29.75

P. verrucosum 5.78 P. citrinum 2.47

Aspergillus niger 14.87

A. flavus 9.09 A. fumigatus 7.43

A. terreus 5.78

A. awamorii 2.47 A. parasiticus 3.3

Rhizopus arrhizus 4.95

R. stolonifer 4.13 Mucor sp. 1.65

Fusarium oxysporum 3.3

F. solani 1.65 Alternaria alternata 2.47

Dreshlera sp. 0.82

Table 3: Qualitative and quantitative spectrophotometric

analysis of extracted DNA

P. italicum isolates

Spectrophotometric reading of Extracted DNA

Ratio 260/280

Conc. of Extracted DNA (µg/ml)

at 260 nm at 280 nm

Pi. Lhr 1.504 1.332 1.13 60 Pi Mul 0.812 0.866 0.94 29

Pi Fsd 0.046 0.056 0.82 1.0

Pi Chn 1.479 1.303 1.13 59 Pi Jhg 0.084 0.080 1.04 3.0

Pi Bhk 0.163 0.175 0.93 6.0

Pi Sh 0.001 0.001 1.00 4.5 Pi Sgd 0.123 0.117 1.06 5.0

Identification of Storage Fungi Isolated from Stored Citrus Fruit / Int. J. Agric. Biol., Vol. 15, No. 6, 2013

1287

been isolated from orange fruits (Akintobi et al., 2011). Al-

Hindi et al. (2011), showed that more than 90% of citrus

fruit were under fungal decay. Numbers of Aspergillus species have been reported such as A. niger, A. fumigatus,

A. nidulans, A. variecolour and A. candidus. Fusarium spp

were also the most commonly associated fungi in citrus fruits

(Tournas and Katsoudas, 2005).

The morphological characteristics of P. italicum of the

lab cultured isolates collected from different citrus

producing districts of Punjab in Pakistan were closely

related. This morphological study was not sufficient in

examining the diversity among these isolates. Therefore,

with the advent of modern approaches including molecular

and genetic marker studies, these isolates were further

characterized by the RAPD fingerprinting technique.

Dupont et al. (1999) reported that the molecular markers

reveal comparatively significant level of similarities among

twelve Penicillium species whereas morphological

characterization exhibited moderate level of similarities.

Variations on genetic material cannot be studied

morphologically, while RAPD technique may overcome

such type of problems and is very effective for microbial

species characterizations (Tiwari et al., 2011). The results

shown in dendogram are completely compatible with the

geographical locations and belts from where the isolates

have been collected. Lahore and Multan are distant but

fall in the same belt in citrus producing areas of the

Punjab. Lahore, Multan, Bhakar, Jhang and Sargodha fall

in the same geographical belt, whereas Chiniot and

Faisalabad render a separate region. Consequently the

collected isolates from different regions form distinct

groups showing a range of variability as seen by

UPGMA results. Sahiwal being more close to southern

Punjab showed more variation i.e., 23% compared to

other isolates. Here another point of concern cannot be

negated that the mutation frequencies fluctuate

considerably along the genomic nucleotide sequences such

that mutations focus at certain positions called hotspots.

Mutation hotspots in DNA reflect the effect of the

environmental factors in which the organism prevail

(Rozin and Pavlov, 2003). These mutations are largely due

to the point mutations, insertions, deletions, inversions

and translocations in the chromosomes, which cause the

variability in the genome that is easily detected through

the fingerprinting process using various molecular

marker tools such as RAPD.

The citrus producing belt is in extreme danger from P.

italicum due to its most occurrences in storage conditions.

Therefore, suitable integrated control measure should be

taken to reduce this threat. Most probably the bio-control

measures are more efficient, environmental safe and cost

effective in a country like Pakistan. Accordingly, the future

studies should be focused on the use of environmentally

safer control measures.

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(Received 10 March 2013; Accepted 24 August 2013)

International Conference on Applied Life Sciences (ICALS2012)Turkey, September 10-12, 2012

321ISALS

© 2012 Anjum and Akhtar.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Antifungal Activity of Essential Oils Extracted From Clove, Cumin and Cinnamon Against Blue Mold Disease on Citrus FruitTehmina Anjum* and Nosheen Akhtar

Institute of Agricultural Sciences, Quaid-e-Azam Campus, University of the Punjab, Lahore, Pakistan * Corresponding author, Tel.: +92-42-99231846; fax: +92-42-99231187

Email: [email protected]

Abstract

Essential oils obtained from Cumin seeds, Clove buds and Cinnamon bark was checked for their antifungal potentials against Penicillium italicum, causal agent of blue mold disease in citrus fruit. Selected essential oils were checked in different concentrations of 3, 6, 12, 24 and 48µl/ml for their ability to inhibit the mycelial growth of the test fungi. The in vitro study revealed that the essen-tial oils of cumin and clove have the potential to inhibit mycelial growth of test fungi completely at concentrations of 12 and 48µl/ml, respectively. Essential oil of cinnamon, however failed to completely inhibit the mycelial growth even at maximum used concentration of 48µl/ml. In vivo assays also supported these results. Clove and cumin oils showed complete fungal inhibition at concentration of 24 and 48µl/ml, respectively when applied on citrus fruits. Whereas, cinnamon essential oil could not stop fungal infection even at its highest tested concentration. The study was extended to chemical identification of tested essential oils through GC-MS.

Keywords: essential oils, citrus, Penicillium itallicum, antifungal activity

1. IntroductionFungi cause significant losses in almost all perishables due to post harvest rots. One of these fungi is Penicillium italicum Whemer (blue mold) that results in a universal post-harvest disease of almost all kinds of citrus fruit[1]. Chemicals imazalil, sodium ortho-phenyl phenate, and thia-bendazole, have been widely used to control this problem [2]. However increase in public concern regarding contamination of perishables with fungicidal residues and proliferation of resistance in the pathogenic population [3] has forced the community to search for environment safe strate-gies. In the past few years, there has been a huge increase in the search of natural substances such as essential oils and plant extracts as potential antifungal agents [4]. The use of essential oils to control post-harvest fruit diseases have been deeply investigated and is well documented since the volatile compounds may have better applicability as fumigants for control [5]. In the present work, it has been therefore thought desirable to discover the antifungal potencies of essential oils extracted from three different plants against P. italicum, a dominant mycotoxin producing fungi during citrus storage.

322

2. Methodology

2.1. Test Fungi

The strain of Penicillium italicum used in this study was isolated from the decaying citrus fruit (variety: Mandarin). The fungal culture was maintained on Malt Extract Agar (MEA) medium at 4±1oC. A 7-14 days old culture of the isolate was used as the source of inoculum and for the preparation of spore suspension for various studies. The spores were removed from the surface of the culture, suspended in 5 ml of sterile distilled water containing 0.05% (v/v) Tween 80 and its concentration was adjusted to 106sp/ml using a haemocytometer for further in vivo studies.

2.2. Extraction of Essential Oils

Seeds of cumin (Cuminum cyminum L.), clove buds [Syzygium aromaticum (L.) Merrill & Per-ry], and cinnamon bark (Cinnamomum verum J. Presl) were imperiled to hydrodistillation in a modified Clevenger apparatus for 3-4 hours. Isolated EOs were stored in glass after dehydrating with anhydrous sodium sulphate and were kept in the refrigerator at 4±1oC before use. Differ-ent concentrations of plant essential oils (Eos) were prepared by adding 3, 6, 12, 24 and 48 µl of pure Eos in 1 ml of 0.05% (v/v) Tween 80 in case of in vivo and 0.5% (v/v) Tween 80 in case of in vitro experiments.

2.3. In Vitro Antifungal Assay by Agar Dilution Method

Prepared concentrations of EOs were mixed with sterile molten MEA medium. Thirty milliliters of media containing different concentrations of EOs and 0.5% Tween 80 was poured into each petri plate which was then inoculated with test fungi and incubated for 7 days at 25±1°C.

2.4. In Vitro Volatile Assay to Check Antifungal Activity

In this method the MEA plates were first inoculated with test fungi and then pure extracted EOs in quantities of 3, 6, 12, 24, and 48 µl were applied on the surface of the sterilized filter paper, which was placed in the lid of the petri plate. After inoculation the plates were incubated in in-verted position for seven days at 25±1°C. The zone of inhibition was measured in two directions at right angles to each other. The percentage of mycelial growth inhibition by each essential oil concentration was calculated from the mean colony diameter (cm) on medium without essential oil amendment (control) and from the mean colony diameter (cm) on each essential oil amended plate (zone of growth).

2.5. In vivo antifungal assay

Fruits were sterilized with 6% sodium hypochlorite solution followed by immersion in sterile distilled water for two minutes and surface sterilization in 70% ethanol for another two minutes. Fruit was wounded (2-wounds per fruit) at the equatorial side with a sterile stainless steel scalpel where each wound was about 4 mm long and 2 mm deep. 15 µl of spore suspension was in-oculated into each wound using a micropipette under aseptic conditions. Two hours later, each wound was inoculated with a pre-determined concentration from each plant essential oil. Con-

http://en.wikipedia.org/wiki/Carolus_Linnaeus

http://en.wikipedia.org/wiki/Jan_Svatopluk_Presl


323ISALS

trol fruit was subjected to the same treatments except that sterile distilled water was used instead of essential oil. The treated fruits were labeled, placed in sterilized petri plates and incubated at 23±2 °C for two weeks to assess decay and fungal growth symptoms on daily basis.

2.6. Chemical identification of essential oils by GC-MS

Qualitative analysis of the tested essential oils was undertaken by gas chromatography-mass spectroscopy (GC-MS) using a Hewlett-Packard mass detector (model 7890) coupled with mass spectrometer selective detector 5975. Analysis was carried out using a column HP5 mass-selec-tive detector (MSD) (30 m x 0.25 mm; 0.25 µm film thickness), the operating conditions were as follows: Helium was the carrier gas at a flow rate of 1 ml/min. diluted samples (1:100 v/v, in methanol) of 1 µl were injected manually at temperature 250°C. NIST (National Institute of Stan-dards and Technologies) Mass Spectra Library was also used as a reference.

3. Results and Discussion

3.1. In vitro antifungal efficacy of essential oil

In vitro antifungal activity of selected oils were checked through two methods i.e., dilution method and the volatile method (Table 1). A little variation was observed in results of both the assays. In dilution method cumin oil gave best control on the mycelial growth of P. itali-cum as its lowest concentration of 3µl/ml gave 96% inhibition. This increased with increase in oil concentration and 12µl/ml completely inhibited the fungal growth. The results of cumin oil was followed by clove oil, whose lowest tested concentration i.e., 3µl/ml showed 84% mycelial growth inhibition. However, the complete inhibition of fungal growth was recorded at maximum tested concentration i.e., 48 µl/ml. Cinnamon gave the lowest effect among the three tested EOs.

In volatile method clove oil gave best control of P. italicum instead of cumin that showed highest inhibition of mycelial growth in dilution method perhaps because of high volatility of phenols that are abundantly present in clove essential oil. Clove oil in 3, 6 and 12µl/ml con-centrations gave 97% control on tested fungi. Increase in oil concentration of clove oil gave similar results as were recorded when the same oil was checked through dilution method. Complete inhibition of mycelial growth was observed at maximum tested concentration. In case of cumin the lowest tested concentration of 3µl/ml could only inhibit P. italicum up to 32% in contrast to its significant control of 96% recorded in dilution method. Concentration increased the inhibition percentage up to 98-99%. Romagnoli et al.,[6] reported a strong anti-fungal activity against dermatophytes and phytopathogens including fungi and yeast. They also found cumin aldehyde, pinenes, and p-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides as the most active ingredients of cumin essential oil. Cinnamon oil also depicted better control on tested fungi in volatile method when compared to the dilu-tion method perhaps because it is also rich in volatile phenols live clove essential oil. How-ever, still the inhibition effect of cinnamon essential oil failed to match with the controlling capacity of other two tested oils.

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Essential

Oils

Concen-

trations

(µl/ml)

Average colony diameter of P. italicum

(cm)

Inhibition of mycelial growth

of P. italicum (%)

Dilution Method Volatile Method Dilution MethodVolatile

Method

Control 0 6.666+1.7a 5.66+0.83a 0.0+0.0g 0.0+0.0g

Cumin 3 0.266+0.03de 3.83+0.09bc 96+8.3a 32.33+7.1e

6 0.1+0.07e 1.76+0.03e 98.49+13a 68.9+9.4b

12 0.0+0.0g 0.066+0.02g 100+0a 98.83+13a

24 0.0+0.0g 0.1+0.07f 100+0a 98.23+7.5a

48 0.0+0.0g 0.066+0.03g 100+0a 98.83+3.9a

Clove 3 1.066+0.05c 0.166+0.08f 84+6.2b 97.06+19a

6 0.733+0.09d 0.166+0.05f 89+11b 97.06+3.7a

12 0.333+0.04d 0.166+0.09f 95+8.6ab 97.06+6.7a

24 0.066+0.03f 0.1+0.06f 99+14a 98.23+9.1a

48 0.0+0.0g 0.0+0.0h 100+0a 100+0.0a

Cinnamon 3 6.666+.74a 4.83+0.59b 0.0+0.0g 14.66+1.5f

6 6.333+1.04a 3.33+0.37cd 4.995+0.03f 41.16+9.5cd

12 6.166+0.07ab 3.5+0.83c 7.5+0.14e 38.16+6.2d

24 5.833+0.43b 3.66+0.51bc 12.496+0.37d 35.33+6.8de

48 4.5+0.21bc 3.166+0.09d 32.49+2.9c 44.06+5.7c

* Values are means (n=3). Mean values followed by different letters within the column are significantly dif-ferent according to Duncan Multiple Range Test (P<0.05).

Table 1. In vitro antifungal activity of plant essential oils, used at various concentrations, on mycelial growth

of Penicillium italicum*

3.2. In Vivo Antifungal Efficacy of selected Essential OilsThe results of in vivo antifungal efficacy indicated that all the three EOs had a good inhibitory effect on mycelial growth of P. italicum when tested on the surface of the citrus fruit. Yahyazadeh et al.,[7] revealed that essential oil can result in loss of pigmentation in fungal conidia as they became hyaline that may affect virulence of the pathogen; hence a decrease in the incidence of the infection.

Figure 1A, shows that clove essential oil exhibited most pronounced antifungal potentials against P. italicum as it completely inhibited the mycelial growth at concentration of 24µl/ml after 15 days of incubation. At concentration of 3µl/ml the inhibition of P. italicum was 62% and growth started at 4th day of inoculation. Increase in concentration to 6 and 12µl/ml increased fungal inhibition up to 79 and 93% respectively, and also the fungal growth on citrus fruit was delayed till 7th and 11th day.


325ISALS

Fig 1. In vivo antifungal activity of A: Clove; B: Cumin; C: Cinnamon essential oils, used at various concentra-

tions, on mycelial growth of Penicillium italicum

Cumin oil showed complete inhibitory effect at concentration of 48µl/ml while cinnamon oil did not completely inhibit fungal progression even at its highest concentration when applied on citrus fruit. The minimal inhibitory effect of cumin and cinnamon essential oils at lowest tested concentration (3µl/ml) was 88.27 and 83.05 % respectively. Whereas, no significant difference was recorded in inhibitory effects caused by both the cumin and cinnamon essential oils used in higher concentrations of 6 – 48µl/ml.

The data presented in Fig. 1B indicated that cumin oil in concentrations of 3 and 6µl/ml resulted in the inhibition of 88.71 and 89.27% respectively and growth started from the 5th day of the inoculation. However the concentrations of 12 and 24µl/ml delayed the mycelial growth in citrus fruit till 6th and 7th day. While the data presented in Fig. 1C shows that the mycelial develop-ment of tested fungus was started from 3rd day of inoculation at all tested cinnamon oil concen-trations (3-48µl/ml) except the concentration of 24µl/ml that delayed the growth up to 5 days. These results depicted that the efficacy of clove essential oil during in vivo assays followed by that of cumin, whereas cinnamon oil showed least inhibitory effect.

In general, the results obtained from GC-MS analysis of the essential oils used were in accor-dance to the previous literature. Clove oil shows the presence of eugenol, alpha-terpineol, Iso-eugenol and beta-terpinene as its major components. Eugenol has been reported by different

326

workers to be the most effective component of the clove and cinnamon EOs against various pathogens[8]. Vazquez et al.,[9] reported complete inhibition of P. citrinum by 2000 ppm of eu-genol in a liquid medium.

The major components found in cinnamon oil were eugenol and cinnamaldehyde, whereas cum-in oil revealed the presence of gamma-terpinen, cuminaldehyde and 4-carvomenthenol. Singh and Upadhayay[10] showed antifungal activity of Cuminaldehyde against Aspergillus flavus and Aspergillus niger. In a recent study Romagnoli et al.,[6] found cumin aldehyde, pinenes, and p-cymene, and a fraction of oxygenate compounds such as alcohol and epoxides as the most ac-tive ingredients of cumin essential oil.

4. References[1] D. Prusky, J.L. McEvovy, R. Saftner, W.S. Conway, and R. Jones. Relationship between host

acidification and virulence of Penicillium spp. on apple and citrus fruit. Biotech. Cell. Biol. 2004. 94: 44-51.

[2] Eckert, J. W.; Eaks, I. L. Postharvest disorders and diseases of citrus fruits. In The Citrus Industry; Reuter, W., Calavan, E. C., Carman, G. E., Eds.; University of California Press:Berkeley, CA, 1989; pp 179− 260.

[3] P. Tripathi, and N.K. Dubey, Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables. Post-harvest Biol. Technol. 2004. 32: 235-245.

[4] V.K. Bajpai, T.J. Lee, and S.C. Kang. Chemical composition and in vitro control of agricultural plat pathogens by the essential oil and various extracts of Nandina domestica Thunb. J. Sci. Food Agric. 2009. 89: 109-116.

[5] J. Kaloustian, J. Chevalier, C. Mikail, M. Martino, L. Abou and M.F. Vergnes. Etude de six huiles essentielles: composition chinique et activite antibacterienne. Phytotherapie. 2008. 6: 60-164.

[6] C. Romagnoli, E. Andreotti, S. Maietti, R. Mahendra and D. Mares. Antifungal activity of essential oil from fruits of Indian Cuminum cyminum. Pharm. Biol. 2010. 48(7): 834-838.

[7] M. Yahyazadeh, R. Omidbaigi, R. Zare, and H. Taheri. Effect of some essential oils on mycelial growth of Penicillium digitatum Sacc. J. Microbiol. Biotechnol. 2008. 24: 1445-1450.

[8] R. Montes-Belmont, and M. Carvajal. Control of Aspergillus flavus in maize with plant essential oils and their components. J. Food Protect. 1998. 61: 616-619.

[9] B.I. Vazquez, C. Fente, C.M. Franco, M.J. Vazquez, and A. Cepeda. Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese. Int. J. Food Microbiol. 2001. 67: 157- 163.

[10] G. Singh, and R.K. Upadhyay. Fungitoxic activity of cuminaldehyde, main constituent of Cuminum cyminum oil. Fitoterapia. 1991. 62: 86.

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