INTEGRATED MANAGEMENT OF MYCOTOXINS IN RED CHILLI

SHAISTA AKHUND

10-Arid-1985

Department of Botany

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2016

ii

INTEGRATED MANAGEMENT OF MYCOTOXINS IN RED CHILLI

by

SHAISTA AKHUND

(10-Arid-1985)

A thesis submitted in partial fulfilment of

the requirements of the degree of

Doctor of Philosophy

in

Botany

Department of Botany

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2016

iii

CERTIFICATION

I hereby undertake that this research is an original one and no part of this

thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible

for the consequences.

Student’s Name: Shaista Akhund Signature: ________________

Registration No: 10-arid-1985 Date: ____________________

Certified that the content and form of thesis entitled “Integrated

Management of Mycotoxins in Red Chilli” submitted by Miss Shaista Akhund

have been found satisfactory for the requirement of the degree.

Supervisor: __________________________

(Dr. Abida Akram)

Member: ____________________________

(Dr. Rahmatullah Qureshi)

Member: ____________________________

(Dr. Farah Naz)

Member: ____________________________

(Dr. Nafeesa Qudsia Hanif)

Chairman: ___________________________

Dean: _______________________________

Director, Advanced Studies: ____________

iv

DEDICATION

This Dissertation is dedicated to

My Beloved Parents

For their endless love, support and encouragement

v

vi

CONTENTS

Page

List of Tables ix

List of Figures xi

List of Abbreviations xii

Acknowledgements xiv

ABSTRACT 1

1 INTRODUCTION 3

2 REVIEW OF LITERATURE 10

2.1 MYCOTOXINS IN RED CHILLI 10

2.2 MYCOFLORA IN RED CHILLI 16

2.3 MANAGEMENT OF MYCOTOXINS AND

MYCOFLORA IN LABORATORY TRIALS

22

2.4 MANAGEMENT OF MYCOTOXINS AND

MYCOFLORA IN GREENHOUSE TRIALS

37

3 MATERIALS AND METHODS 43

3.1

EVALUATION OF RED CHILLI FOR

ASSOCIATED MYCOFLORA AND

MYCOTOXINS

43

3.1.1 Collection of Samples 43

3.1.2 Isolation and Identification of Fungi 43

3.1.2.1 Dilution method 44

3.1.2.2 Agar plate method 44

3.1.2.3 Blotter paper method 44

3.1.2.4 Deep freezing method 44

3.1.3 Detection and Quantification of Mycotoxins 45

3.1.3.1 Chemicals and standards 45

3.1.3.2 Analytical method performance/ Optimization 46

vii

3.1.3.3 Sample preparation and aflatoxins analysis by TLC 46

3.1.3.4 Sample preparation/extraction and clean up by HPLC 48

3.1.3.5 Analytical method performance 48

3.2 IN-VITRO INHIBITION OF MYCOFLORA AND

REDUCTION OF MYCOTOXINS 49

3.2.1 Preparation of Plant Diffusates 49

3.2.2 Inhibition of A. flavus Growth by Poisoned Food

Technique 51

3.2.3 Reduction of Aflatoxins. 51

3.2.4 Inhibition of A. flavus and Reduction of Aflatoxins

by Bio agents 52

3.2.4.1 Collection and preparation of bio agents 52

3.2.4.2 Growth media and culture preparation 52

3.2.4.3 Inhibition of A. flavus by poisoned food technique 53

3.2.4.4 Reduction of aflatoxins 53

3.2.4.5 Nutritional and quality profile of red chilli 53

3.2.4.5.1 Proximate analysis 53

3.2.4.5.2 Sample extraction 54

3.2.4.5.3 Determination of total phenols 54

3.2.4.5.4 DPPH radical scavenging activity 55

3.2.4.5.5 Determination of vitamin C contents 55

3.2.4.5.6 Data analysis 55

3.3 MANAGEMENT TRIALS IN GREENHOUSE 56

3.3.1 Management by Manipulating the Irrigation Regimes 56

3.3.1.1 Preparation and sterilization of potting mixture 56

3.3.1.2 Sowing and transplantation of Nagina seeds 56

3.3.1.3 Inoculum preparation and addition in clay pots 56

3.3.1.4 Treatments design 57

viii

3.3.1.5 Parameters studied 57

3.3.2 Management of A. flavus and Aflatoxin by Yeast

Application 57

3.3.2.1 Preparation and application of yeast inoculum 58

3.3.2.2. Data analysis 58

3.4 INTEGRATED MANAGEMENT OF

MYCOTOXINS IN RED CHILLI 58

3.4.1 Pre-Harvest Management 59

3.4.2 Post-Harvest Management 59

3.4.3 Seed Viability Test 59

3.4.4 Data Analysis 60

4 RESULTS AND DISCUSSIONS 61

4.1 EVALUATION OF RED CHILLI FOR

ASSOCIATED MYCOFLORA AND

MYCOTOXINS

61

4.1.1 Dilution Method 61

4.1.2 Agar Plate Method 61

4.1.3 Blotter Paper Method 63

4.1.4 Deep Freezing Method 63

4.1.5 Detection and Quantification of Mycotoxins 78

4.2 IN-VITRO INHIBITION OF MYCOFLORA AND

REDUCTION OF MYCOTOXINS 84

4.2.1 Inhibition by Plant Diffusates 84

4.2.1.1 Inhibition of A. flavus growth 84

4.2.1.2 Reduction of aflatoxin B1 85

4.2.2 Inhibition by Bio agents 88

4.2.2.1 Inhibition of A. flavus growth 88

4.2.2.2 Reduction of aflatoxin B1 91

ix

4.2.3 Nutritional and Quality Profile of Red Chilli 97

4.2.3.1 Proximate analysis 97

4.2.3.2 Determination of total phenols 97

4.2.3.3 DPPH radical scavenging activity 97

4.2.3.4 Determination of vitamin C contents 100

4.2.3.5 Determination of capsaicin 100

4.3 MANAGEMENT TRIALS IN GREENHOUSE 100

4.3.1 Management by Manipulating the Irrigation Regimes 100

4.3.2 Management by Yeast Application 101

4.4 INTEGRATED MANAGEMENT OF

MYCOTOXINS IN RED CHILLI 105

4.4.1 Pre-Harvest Management 105

4.4.2 Post-Harvest Management 106

SUMMARY 111

CONCLUSIONS AND RECOMMENDATIONS 114

LITERATURE CITED 115

x

LIST OF TABLES

Table No. Page

3.1 Spike recoveries of total aflatoxins in red chillies. 47

4.1 Occurrence (%) of mycoflora by dilution plate method from the

fruits of six red chilli cultivars.

62

4.2 Occurrence (%) of mycoflora by agar plate method from surface

disinfected pericarp of six red chilli cultivars.

64

4.3 Occurrence (%) of mycoflora by agar plate method from surface

disinfected seeds of six red chilli cultivars.

65

4.4 Occurrence (%) of mycoflora by agar plate method from surface

non disinfected pericarp of six red chilli cultivars.

66

4.5 Occurrence (%) of mycoflora by agar plate method from surface

non disinfected seeds of six red chilli cultivars.

67

4.6 Occurrence (%) of mycoflora by blotter paper method from

surface disinfected pericarp of six red chilli cultivars.

68

4.7 Occurrence (%) of mycoflora by blotter paper method from

surface disinfected seeds of six red chilli cultivars.

69

4.8 Occurrence (%) of mycoflora by blotter paper method from

surface non disinfected pericarp of six red chilli cultivars.

70

4.9 Occurrence (%) of mycoflora by blotter paper method from

surface non disinfected seeds of six red chilli cultivars.

71

4.10 Occurrence (%) of mycoflora by deep freezing method from

surface disinfected pericarp of six red chilli cultivars.

72

4.11 Occurrence (%) of mycoflora by deep freezing method from 74

xi

surface disinfected seeds of six red chilli cultivars.

4.12 Occurrence (%) of mycoflora by deep freezing method from

surface non disinfected pericarp of six red chilli cultivars.

75

4.13 Occurrence (%) of mycoflora by deep freezing method from

surface non disinfected seeds of six red chilli cultivars.

76

4.14 Incidence (%) and frequency (%) of A. flavus detected from six

red chilli cultivars by using dilution plate, agar plate, blotter

paper and deep freezing methods.

79

4.15 Aflatoxin contamination (mean) levels (µg/kg) and range of

aflatoxin concentration in different cultivars.

82

4.16 In-vitro effect of plants and bio agents on nutritional profile of

red chilli.

98

4.17 In-vitro effect of plants and bio agents on quality profile of red

chilli.

99

4.18 Effect of irrigation levels on the management of A. flavus and

aflatoxin B1 production in greenhouse trial.

102

4.19 Effect of yeast application on the management of A. flavus and

aflatoxin B1 production in greenhouse trial

104

4.20 Integrated management of aflatoxin content and A. flavus by the

most effective treatments selected from the greenhouse trial with

the most effective dose of yeast and optimum irrigation level.

107

4.21 Integrated management of aflatoxin production and A. flavus

growth in red chilli by best treatments selected from laboratory

trial with the most effective bacterial cultures

108

xii

LIST OF FIGURES

Figure No. Page

3.1 Chromatogram of standard mixture with pre column derivatization 50

4.1 Incidence (%) of aflatoxins in different cultivars of red chillies 80

4.2 Efficacy of plant diffusates on inhibition of radial mycelial growth

of A. flavus

86

4.3 Efficacy of plant diffusates on reduction of aflatoxin B1. 87

4.4 Effect of bacterial culture filtrates on radial mycelial growth of A.

flavus

90

4.5 Effect of culture filtrates of bacteria on reduction AFB1 in red chilli. 92

4.6 Effect of culture filtrates of yeast on reduction of AFB1 in red chilli

by culture

93

4.7 Effect of plant diffusates and bio agents on reduction of aflatoxin

B1 in red chilli

96

4.8 Reduction of aflatoxin B1 in red chilli by post-harvest treatments 109

4.9 Combined effect of pre and post-harvest treatments on seed

germination of red chilli

110

xiii

LIST OF ABBREVIATIONS

AF Aflatoxins

AFB1 Aflatoxin B1

AFB2 Aflatoxin B2

AFG1 Aflatoxin G1

AFG2 Aflatoxin G2

AFT Total Aflatoxins

ANOVA Analysis of Variance

AOAC Association of Official Analytical Chemist

AWC Available Water Content

CFU Colony Forming Unit

DMRT Duncan's Multiple Range Test

EU European Union

FAO Food and Agriculture Organization

FASFC Federal Agency for the Safety of the Food Chain

FCBP Fungal Culture Bank of Pakistan

HPLC High Performance Liquid Chromatography

ISTA International Seed Testing Association

LAB Lactic Acid Bacteria

LOD Limit Of Detection

LOQ Limit Of quantification

MEA Malt Extract Agar

MIC Minimum Inhibitory Concentration

NCYC National Collection of Yeast Cultures

xiv

OTA Ochratoxin A

PBS Phosphate Buffered Saline

PDA Potato Dextrose Agar

PPB Parts Per Billion

SE Standard Error

TFA Trifluoroacetic Acid

TLC Thin Layer Chromatography

USDA United States Department of Agriculture

YM Yeast Malt

xv

ACKNOWLEDGEMENTS

I am thankful to Almighty Allah for blessing me good health, strength and

perseverance needed to complete this study. I offer my humble gratitude from the

core my heart to the Holy Prophet Hazrat Muhammad (Peace be upon him) who

is forever a torch of guidance for the humanity as a whole.

I wish to thank my supervisor & advisor, Dr. Abida Akram, Associate

Professor, Department of Botany; PMAS-Arid Agriculture University Rawalpindi.

She has been supportive since the days I began working on my PhD project. She

has supported me not only by providing a research assistantship but also

academically and emotionally through the rough road to finish this thesis.

A special thanks to Prof. Dr. Mohammad Arshad, Chairman, Department

of Botany; PMAS-Arid Agriculture University Rawalpindi for providing me the

opportunity and making the department facilities available for research work. I

wish to acknowledge my supervisory committee members Dr. Rahmatullah

Qureshi, Associate Professor, Department of Botany; PMAS-Arid Agriculture

University Rawalpindi for his comments and suggestions. I whole heartedly thank

other members of supervisory committee Dr. Farah Naz (Assistant Professor,

Department of Plant Pathology; PMAS-Arid Agriculture University Rawalpindi)

and Dr. Nafeesa Qudsia Hanif (Principle Scientist, Romer Labs, Pakistan) for

their help, professionalism and valuable guidance throughout the entire program of

study.

My sincere thanks also goes to Dr. Irfan-ul-Haq, Professor, Department of

Plant Pathology PMAS-Arid Agriculture University Rawalpindi who motivated me

to join this university. His polite, humble and kind behaviour helped me to start

new journey with full passion

I do not have enough words to express my deep and sincere appreciation to

my lab fellow Mr. Brian Gagosh Nayyar for his endless support and tolerance as

he always stood beside me at every difficult step and made it easy for me. Without

his assistance, the completion of this work would have been immeasurably more

difficult.

xvi

This study was made possible through the financial support of Higher

Education Commission of Pakistan (HEC) under HEC Indigenous Ph.D. 5000

Fellowship Program Phase-VII (PIN No. 117-7663-Bm7-221). I highly

acknowledge Kunri Research Station, Mirpur Khas, Sindh for sample collection

and facilitating me to survey their fields for my research. I would also like to thank

USDA-ARS Culture Collection (NRRL) for providing me microbial cultures for

my research.

I do thank all non-teaching staff of Department of Botany; PMAS-Arid

Agriculture University Rawalpindi, especially Mr. Tariq Mahmood (lab

attendant) for all the help he rendered me during my research. Heartiest thanks to

fellow researchers and students of my department especially Miss Wajiha Seerat

for her pleasant association and help in various forms. I record my strong

appreciation to entire staff of Romer labs, Pakistan but Senior Scientist, Miss Iffat

Tahira deserves my wholehearted thanks.

I must express my profound gratitude to my parents and siblings for

providing me with unfailing support and continuous encouragement throughout my

years of study. Their prayers for me was what sustained me thus far. Dr. Imran

Akhund, my elder brother is highly acknowledge at this juncture, without his

efforts PhD studies would have undoubtedly been more difficult. I also thank all of

my nieces and nephews as well.

Finally, I thank all those who have helped me directly or indirectly in the

successful completion of my thesis. Anyone missed in this acknowledgement are

also thanked.

Shaista Akhund

1

ABSTRACT

Red chilli (Capsicum annuum L.) is a major crop of Pakistan. Sindh

contributes 85% of its production and a small town “Kunri” is one of the largest

centres for red chilli production in Asia. Red chilli is a major food ingredient and

is utilized for the production of essence, pungency and red color. It is an excellent

source of vitamin C and has several medicinal uses. The overall production of red

chilli has decreased during the years 2006-2007. One of the main reasons for this

decline is mycotoxin contamination. Contamination by mycotoxins in the red chilli

crop drastically reduces its quality, due to which Pakistani red chilli is unable to

enter in the world market and has been banned by European Union Food

Authorities, which led to the decrease in export and production. Mycotoxins are a

chemically diverse group of fungal metabolites that have a wide variety of toxic

effects. The most serious and toxic example are the Aflatoxins (B1, B2, G1, G2)

produced by Aspergillus flavus and A. parasiticus. Aflatoxin B1 is considered a

major cause of liver cancer. The present study was planned to develop an integrated

approach for the management of mycotoxin contamination in red chilli. For this

purpose, available germplasm was evaluated to determine the resistance level of

red chilli varieties against mycoflora and production of mycotoxins. The mycoflora

were isolated by employing standard techniques; associated mycotoxins were

analyzed by chromatographic techniques; different antagonistic bioagents and plant

diffusates were tested in-vitro for their effectiveness in managing the mycotoxins

and mycotoxin producing fungi. The highly susceptible variety „Nagina‟ was

subjected to crop management trials in the greenhouse. Firstly, different irrigation

levels were evaluated; which have been reported to induce significant reductions in

1

2

toxins. Secondly, different species of yeast like Saccharomyces cerevisae, S.

bayanus, S. postoranus and their doses were applied at flowering stage of crop.

Finally, the best treatment from these individual trials was incorporated in an

integrated mycotoxin management experiment. The significance of each treatment

in trials was evaluated by detection and quantification of mycotoxins, estimation of

yield components (fresh weight, dry weight, number of pods), nutritional profile

and seed viability of produce. At this stage, the formulation of plant diffusates and

bio agents with highest proficiency in the in-vitro management trial were applied to

chilli pods and pre and post-application status of mycotoxins were recorded. This

study provides a record of the mycoflora and mycotoxins associated with various

varieties of red chilli. Most importantly, it provides the resistant/tolerant locally

available red chilli cultivars (Kunri & Drooping type) which were less

contaminated. In addition, the use of Saccharomyces species during pre-harvest and

Lactobacillus rhamnosus at the post-harvest stage were some major findings of this

study. Finally, the integration of best pre and post-harvest treatments was the most

useful strategy for the management of mycotoxins in red chilli. This work provides

better and more cost effective technology for farmers to produce high quality toxin

free chillis which will not only increase the demand for Pakistani products in

international markets but will also become the source of enhanced foreign

exchange and farmer income.

3

Chapter 1

INTRODUCTION

Pakistan is an agricultural country, as most (65%) of its population depends

upon agriculture livelihood. The agriculture sector contributes 23% of the country‟s

GDP and retains about forty percent of its labour. Several crops such as Chilli,

Citrus, Mango, Rice, Wheat, Cotton, various fruits and vegetables are produced in

bulk and are exported to several countries. Among them red chilli is known as both

vegetable and spice crop of economically significant and valuable cash crop of

Pakistan.

Red chilli is in the genus Capsicum, an important member of the

Solanaceae. It is a perennial herb with entire leaves. Flowers are in clusters of up

to three. The calyx is shallowly 5-toothed and campanulate. Corolla is rotate and 5-

lobed. There are 5 stamens with bluish, dehiscence longitudinal anthers. Ovary is

usually bilocular. Fruit posseses many seeds and in the form of dry indehiscent

inflated berry, shorter than the pedicel and variously coloured. Seeds are

compressed and usually smooth (Nasir, 1985). There are 27 species of which 5

species, Capsicum annuum L., C. frutescens L., C. chinense Jacq., C. baccatum L.

and C. pubescens Ruiz and Pavon are cultivated throughout the world (Csillery,

2006). The most common species for dried spice production is C. annuum.

Worldwide production of chilli is about 24 million tons per anum. The production

areas for chilli are situated in Asia and it contributed 16 million tons (Anonymous,

2005).

3

4

Chilli crops are cultivated around the globe, as it is a main ingredient of

many dishes. It is used for its spiciness (heat level/pungency) and also for the red

colour and essence production in sausages, pickles and salads. The compound

“capsaicin” is responsible for the pungency of red chilli and is present in the septa

and skin of the chilli fruit. Pungency of red chilli depends on the environment and

cultivar (Klieber, 2000). There are several factors responsible for the colour of red

chilli, including ripeness of the fruit at harvest, processing methods, storage

conditions and finally the spice powder. Red chillies are used as a pain killer and

antibiotic and are an excellent source of vitamins A, B, C, D and P (Flavonoids). In

Ayurvedic herbal medicines, red chillies are used to avert several diseases

including heart attacks, lung diseases and even cancers (Kloss, 2009).

Red chilli is the biggest spice traded world wide with 50% merchandized by

Asia (FAO, 1987). India is the leading exporter followed by China (24%), Spain

(17%), Mexcio (8%), Pakistan (7.2%), Morocco (7%) and Turkey (4.5%) (Iqbal et

al., 2010a). Pakistan exports chillies to Sri Lanka, Gulf States, Canada, USA, and

European Union nations. It is traded in various structures: chilli powder, curry

powder, fresh chillies, stalkless chillies and also in the form of paprika oleoresin.

Chilli crop is a very important cash crop of Pakistan cultivated as

approximately 38,000 hectares with production of 85,000 to 90,000 tons annually.

The normal production rate is 1.7 tons/hectare and contributes 1.5% to Pakistan‟s

GDP (SBI, 2010). Sindh is the major chilli producing province cultivating forty

seven thousands hectares and producing 85% of Pakistan‟s chillies followed by

Punjab (about 7000 hectares), Baluchistan (2000 hectares) and Khyber

5

Pukhtoonkhawa (400 hectares). Kunri a small town in Umar Kot (Sindh) is a hub

of red chilli. It is one of the largest markets of red chilli in Asia. Yield per hectare

of this highly valuable cash crop is decreasing in spite of having massive areas of

cultivation. Chilli producing areas and their production levels have decreased by

9.2% and 14.2% respectively; average yield has declined by 5.5 percent, during

2006-2007. Several factors are responsible for this reducing yield but the main

problem is mycotoxin contamination.

Mycotoxins are secondary metabolites produced by fungi which have an

extensive range of damaging impacts. It has been estimated (CAST, 2003) that

mycotoxins affect twenty five percent of the world‟s food crops, which include

many basic food items, animal feed, as well as crops. The fungi responsible for

mycotoxin production are in the genera like Aspergillus, Penicillium and Fusarium.

Mycotoxins are harmful to human and animal health and are associated with many

chronic health risks, including immune suppression, induction of cancer and

digestive, blood and nerve defects (CAST, 2003; Shephard, 2006). They occur

more frequently under tropical conditions. In many developing countries, diets are

based on crops vulnerable to mycotoxins which results in high levels of chronic

health problems. In the early 1960s the discovery of aflatoxins (Blount, 1961) led

researchers worldwide to make significant progress in identifying and

understanding the major classes of mycotoxins and the fungi that produce them.

Numerous mycotoxins are stable and survive during processing. The economic

consequences of mycotoxin contamination are profound, as crops contaminated

with high levels of a mycotoxin often must be destroyed.

6

With respect to chilli and other spices, large numbers of fungi are

connected. The most well-known pathogens are Aspergillus flavus, A. parasiticus,

A. ochraceus, Fusarium verticillioides, F. graminearum and Penicillium spp.

(Kumar et al., 2008). The most widely recognized mycotoxins are aflatoxins (B1,

B2, G1 and G2) and ochratoxin A. Aflatoxins are dangerous to both humans and

animals because of their cancer-causing, teratogenic, genotoxic, immunotoxic and

mutagenic properties (Creppy, 2002). Although several Aspergillus spp. can

produce aflatoxins, A. flavus is most commonly found in red chilli. This fungus is

saprophytic and survives in the soil as mycelium, conidia and sclerotia (Mehan et

al., 1991b; Cole et al., 1995). About 40% of human productivity lost to diseases in

developing countries may be due to diseases aggravated by aflatoxin contamination

(Miller, 1996). In 2004, an outbreak of aflatoxicosis occurred in Kenya causing 125

deaths (Azziz-Baumgartner et al., 2005), and the problem reoccurred in 2005. In a

number of developing nations, where high amounts of aflatoxins are found in some

staple food stuffs, the toxins are considere to a cause of liver cancer in people.

Hepatitis is a dangerous type of aflatoxicosis and in serious cases, prompts jaundice

or even death. Every year, roughly 25,200 to 155,000 new HCC (hepatocelluler

carcinoma) cases are reported around the world (Iqbal et al., 2011).

The fungal and aflatoxin contamination can occur at any phase of food

production from pre to post-harvest, at whatever point ideal conditions prevail. In a

moist climate, red chilli gets contaminated by A. flavus and seeds are infected by

the fungi. Other than aflatoxins, ochratoxin A is found as a coexisting contaminant

(Kumar et al., 2008). The detection of various fungi like A. flavus, A. nidulans, A.

ochraceus, A. niger, A. sydowii, Penicillium and Rhizopus spp. has been reported

7

(Ath-Har et al., 1988) from C. frutescens and other spices. Dry and semi-dry to

moist conditions are associated with contamination, and the poor population of

nations subsists on unhygienic staples in developing nations including, Pakistan. In

such areas, infected foods are thought to be connected with liver cancer (Qazi and

Fayyaz, 2006). High temperature and high moisture support the growth and

sporulation of Aspergillus spp. In fields the soil becomes dry during drought stress,

which promotes fungal growth and dispersal of Aspergillus condia through wind.

This increase in spore load results in an increase in contamination levels in the

whole crop. Moreover, 85% relative humidity and a temperature of 27–38 °C are

ideal for the development of Aspergillus (Cassel et al., 2001), conditions which are

quite common in Pakistan.

Contamination of food commodities by these toxins results in reduction in

quality and market value, with significant economic losses for farmers and food

processors, and serious health implications for consumers (Cardwell and Miller,

1996). The significant amount of mycotoxins in red chilli has affected Pakistan‟s

export when red chilli consignment was rejected by European Union Food

Authorities thus Europe has banned the import of red chillies from Pakistan. This is

the reason of decline in the trade and production of Pakistani red chilli (Ziaf et al.,

2006). Due to this rational the present study was designed to develop strategies

which can eliminate/reduce the mycotoxin contamination from red chillies so that

Pakistani red chilli can regain its status in international markets. Samples of

different red chilli varieties were collected from Kunri town which is a hub of red

chilli production and export. These varieties were analysed to determine the most

8

susceptible and resistant/tolerant varieties. The most susceptible variety was

subjected to management trials.

Mycotoxin occurrence in field crops is to a great extent a matter of

uncontrollable biological events. The complete removal of toxins is practically

difficult to accomplish, as it is an unavoidable regular contaminant, therefore

management strategies from pre to post-harvest will be valuable to decrease

mycotoxin levels. According to some previous field tests and appropriate

agronomic practices such as manipulating the irrigation system can be useful

strategies in controlling aflatoxin contamination in various crops, particularly corn

and peanuts (Payne et al., 1986; Craufurd et al., 2006). Spray of specific strain of

yeast in fields has reduced incidence of A. flavus in pistachio gardens (Hossein et

al., 2007). At post-harvest level various microbes, including fungi, bacteria and

yeast have been used for their capacity in decreasing mycotoxigenic fungi and

resultant mycotoxin contamination in food stuff. Many bacteria such as Bacillus,

Burkholderia, Lactobacillus, Pseudomonas and Ralstonia spp., have demonstrated

the capacity to reduce fungal development and production of aflatoxins by

Aspergillus spp. in-vitro (Reddy et al., 2010b). Other than bio control antagonists,

plant extracts have ability to minimize aflatoxin B1. For example, Ocimum

sanctum, Curcuma longa, Allium sativum and Sizigium aromaticum has shown

complete inhibition of A. flavus development and aflatoxin B1 production in

laboratory trials (Reddy et al., 2009). The essential oils of different plants can have

great antifungal properties. The inhibitory effects of plant extracts on aflatoxin

production have also been investigated. The extract of Azadirachta indica was seen

9

to be great inhibitor of both development of A. flavus and A. parasiticus, and toxin

production in laboratory (Bhatnagar et al., 1990).

In viewpoint of previously reported management of mycotoxins and

mycotoxigenic fungi in different plant species; application of yeast, irrigation,

antagonists and plant diffusates might play a critical role in management of

mycotoxins in red chilli. Thus, for the first time in Pakistan, the present study was

designed to explore these management strategies independently and afterward

consolidating them to create an integrated approach for the control of mycotoxin

contamination in red chilli.

The objectives of this study are to

1. Determine the resistance of various red chilli cultivars/varieties to

mycoflora associated with mycotoxins.

2. Manage the mycoflora and related mycotoxins by investigating different

laboratory and greenhouse trials to find out the best treatments.

3. Develop an integrated mycotoxin management approach by the combining

the best treatments from the management trials in solarized and

nonsolarized soil.

10

Chapter 2

REVIEW OF LITERATURE

Red chilli is one of the most important crops in Pakistan, but this is one of

the most susceptible host crops to fungal invasion and ultimately mycotoxin

production.

2.1 MYCOTOXINS IN RED CHILLI

Red chilli contamination has been reported from all over the world

including Pakistan. A survey was conducted from various stores in Karachi,

Pakistan for aflatoxin contamination in red chilli. Whole, crushed and powdered

red chilli samples were collected. Aflatoxins were detected in 176 red chilli

samples by using a modified Romer method followed by 2-directional thin layer

chromatography (TLC). As a result, 66% aflatoxin B1 contamination was observed

(Shamsuddin et al., 1995). Another report from Pakistan showed generally low AF

levels, but 255µg/kg AFB1 levels in seven samples of red pepper (Ahmad &

Ahmad (1995). Paterson (2007) highlighted the chilli samples contaminated with

aflatoxin from Pakistan. High levels of aflatoxin B1 were observed from all ground

samples. Aflatoxin B1 and B2 concentrations showed direct relation. But no relation

was observed between aflatoxin and A. flavus. Aflatoxin B1 were determined (Iqbal

et al., 2010a) in chillies by using HPLC from Pakistan. Nineteen (86.4%) and 16

(73.0%) whole and ground samples of chillies were observed for infection,

respectively. The concentrations exceeded the legislative limit set by the European

Union. Iqbal et al. (2010b) in another study assessed 43 and 42 chillies samples for

total aflatoxins collected during winter and summer, respectively to observe the

10

11

effect of season on infection. Samples percentage of AFB1 and total aflatoxins,

greater than the European Union legislative boundary for whole chillies in the

winter season were 48 and 36% compared with ground chillies with percentage of

50 and 45%, respectively. While in summer season, percentage of AFB1 and total

aflatoxins in whole chillies were 52 and 38% as compared to ground chillies with

percentage of 54 and 49%, respectively. Iqbal et al. (2011) determined total

aflatoxins by using reverse phase HPLC with fluorescence detector in 156 samples

of chilli from Punjab, Pakistan. They tested 78 samples of whole and ground chilli

each, most of the samples were contaminated with high concentrations of total

aflatoxins in both whole and powdered red chilli. Thirty three percent samples of

whole chillies were contaminated by aflatoxins ranged from 0.00 to 81.5 µg kg-1.

However, in case of ground chillies 40% samples were positive for aflatoxins in a

range of 0.00 to 84.6 µg kg-1. The level of contamination exceeded the limit of

European Union legislative boundary in 26 and 19% samples of whole and ground

chilli, respectively.

Seventy five percent red chilli samples obtained from USA were

contaminated with AFB1 with concentration of 30 µg/kg (Wood, 1989). The

highest level of aflatoxin i.e. 250 to 525 µg/kg was reported by Fufa and Urga

(1996) from 64 pepper samples in Ethiopia. MacDonald and Castle (1996)

determined the highest concentration of the total aflatoxin content in chilli (48

µg/kg) among spices of United Kngdom. Yildirim et al. (1997) reported aflatoxin

in 8 out of 34 samples of red pepper (23.5%) collected from Northwestern

provinces of Turkey ranging between 1.6 and 15 µg/kg. Aflatoxin B1 were found in

46 out of 141 red pepper samples collected from various regions (Hazir and

12

Coksoyler, 1998). Coksoyler (1999) described a vulnerable product as red pepper

for the formation of aflatoxin depending on unsuitable processing conditions.

Surveys were conducted in Turkey and he reported the occurrence of aflatoxin B1

(80 µg/kg) in 1 out of 9 samples of pepper that were dried openly on soil and in 4

pepper samples (20-80 µg/kg) which were dried as whole on soil, concrete

grounds. In another study, Erdogan (2004) reported total aflatoxins (AFT) level of

contamination ranged from 1.1 to 97.5 µg/kg in 18.2% of ground red pepper (GRP)

samples in Turkey. Abdulkadar et al. (2000) found total aflatoxin (AFT)

concentration ranging from 5.60 to 69.28 µg/kg in chilli pepper powder in Qatar.

Dokuzlu (2001) reported AFB1 contamination from 5 to 25 µg/kg in red pepper

samples in Bursa. Seventy nine powdered samples of twelve numerous kinds of

ground spice for aflatoxin contamination in Portugal were screened (Martins et al.,

2001). Aflatoxin level of 69.3 µg/kg and 5.1 µg/kg was reported in chilli powder

and mixed spices powder, respectively from Qatar (Abdulkadar et al., 2004).

In India, various chilli pods were collected from cold stores at monthly

intervals to detect the presence of mycotoxins by HPLC. Natural occurrence of

aflatoxin B1 in chilli pods which were kept in cold storage was tested and results

showed that samples were infected by aflatoxin B1 upto 5.5 µg/kg (Ravikiran et al.,

2009). Aflatoxin B1 was detected from 43% samples. The concentration of

aflatoxin in powdered red pepper ranged between 1 and 20 µg/kg. Reddy et al.

(2001) studied aflatoxins B1 in different grades of chillies by indirect competitive

ELISA in India. Various samples of chilli pods were collected from cold store

houses and markets of Andhra Pradesh (AP), India. Among them, 182 samples of

chillies were investigated, 59% samples were found to be contaminated with AFB1

13

while 18% having the toxin above the permissible limits. Yerneni et al. (2012) also

reported the occurrence of AFB and AFG in red chillies of India.

Ninety one samples of different spices were contaminated with aflatoxins

(B1, B2, G1, G2) and ochratoxin A (OTA) in Hungary (Fazekas et al., 2005). They

worked on 70 samples of ground red pepper among them 18 samples (25.7%)

contained AFB1 and seven of them exceeded the pemissible limit of 5 µg/kg (mean

6.1-15.7 µg/kg). However, thirty two samples contained OTA in a range of 10.6-

66.2 µg/kg. Some workers evaluated that chillies are susceptible for aflatoxin

contamination produced by Aspergillus flavus (Romagnoli et al., 2007). They

tested 103 samples of spices, herbal teas, aromatic herbs and medicinal plants were

tested for aflatoxin contamination in Italy. They noted that among all samples, 7

samples of spices showed positive results: 5 chili-peppers, 1 cinnamon and 1

nutmeg. They observed aflatoxins contamination ranging 0.57-30.7 µg/kg in 45.5%

samples of red pepper

Report from Kayseri, Turkey showed AFB1 contamination from 1.48 to

70.05 µg/kg in red pepper samples (Kanbur et al., 2006). While, contamination of

AFB1 was at highest level in red paprika (9.68 µg/kg) amongst 60 cereals samples

and 55 samples of spices (14 of cumin, 12 of ginger, 14 of paprika and 15 of

pepper) bought from local markets of Morocco (Zinedine et al., 2006). Aydin et al.

(2007) investigated the AFB1 contamination level from 0.025 to 40.9 µg/kg in

ground red pepper samples in Istanbul. They tested 100 samples of powdered red

pepper for AFB1 by using microliter plate (ELISA). They observed unacceptable

contamination levels in 18 samples which were higher than the maximum

14

permissible level (5 µg/kg) of Turkish Food Codex and the European Commission.

Speijers and Speijers (2004) described interactions probability for example

synergistic or additive effects increases in paprika and chilli by the co-occurrence

of different mycotoxins that may increase the risk to human health. Seventy five

deep-red ground pepper samples obtained from markets in Sanliurfa (Turkey)

(Ardic et al., 2008) and assayed through microtitre plate Enzyme Linked

Immunosorbent Assay (ELISA) method by using immunoaffinity columns and

AFB1 contamination was observed in 96% samples ranged from 0.11-24.7 µg/kg.

Some samples (14.7%) were exceeding the regulatory limits used in the European

Union. Cho et al. (2008) evaluated 88 spices and processed spice products for total

aflatoxins (aflatoxins B1, B2, G1, and G2) commercialized in Korea. Twelve

samples showed total aflatoxins with incidence of 13.6% including two red pepper

pastes (Kochujang), seven red pepper powder, two curry and one ginger product.

Confirmation of aflatoxins in 12 samples after HPLC was done by LC–MS/MS.

Aflatoxins were tested in several foods marketed in Manama city, Bahrain.

Among them maximum level of aflatoxins exceeded in three red chilli samples

powder (69.2, 52.6 35.9 ug/kg), one sample of unshelled pistachio nuts (81.6 µg

/kg) and one sample of black pepper powder (27.7 µg/kg) (Musaiger et al., 2008).

Report by Santos et al. (2010) suggested aflatoxins as the second highest

mycotoxins in tested Capsicum samples obtained from Spanish market while

studying co-occurrence of aflatoxins with other mycotoxins. Set and Erkmen

(2010) detected aflatoxins in red chilli powder and nuts of pistachio from retailed

market of Turkey by using HPLC and observed total aflatoxins in 17.1% (14/82)

samples and AFB1 in 23.1% (19/82) unpacked red chilli powder exceeding legal

15

limits while only one packed sample was exceeding legal limits with AFB1 (89.99

µg/kg). Aflatoxin contamination was found in 79.2% market samples of red chilli

and 63.6% samples of red chilli powder collected from Turkey. AFB1 in four red

chilli flake and three samples of red chilli powder were above the EU regulatory

limit of 5 µg kg-1

(Ozbey and Kabak, 2012). Aflatoxin B1 was reported as

predominant mycotoxin in almost 77% of Sri Lankan chilli samples

(Yogendrarajah et al., 2014b). Remarkably, AFB1 exceeded EU maximum level

(ML) of 5 µg/kg in 67% of the Sri Lankan chilli samples and total aflatoxins

exceeded the EU ML of 10 µg/kg in 44% of the samples. They also observed that

AFB1 contamination in 9 of the 11 positive samples of chilli exceeded the EU ML

from Belgium. Hammami et al. (2014) reported exceeded AFB1 and total aflatoxins

levels in fourteen samples of spice collected from local markets in Doha, Qatar.

Other studies have demonstrated mycotoxin contamination in red chilli in

different countries, including the United Kingdom (MacDonald & Castle, 1996;

Patel et al., 1996 ), Portugal (Martins et al., 2001), Hungary (Fazekas et al., 2005),

Ireland (O‟Riordan & Wilkinson, 2008), Australia (Klieber, 2001), Japan (Tabata

et al., 1993), China (Hu and He, 2006), Moroco (Zinedine et al., 2006), Turkey

(Bircan, 2005; Colak et al., 2006), Korea (Cho et al., 2008). Van Egmond et al.

(2007) reported that most countries in trading commodities have established strict

monitoring conditions for mycotoxins level.

According to the Regulations of European Commission (Commission

Regulation No. 165/2010) the stipulated maximum level (ML) allowed in spices

for AFB1 is 5 µg kg−1

and 10 µg kg−1

for total AFs (sum of AFB1, B2, G1 and G2).

16

For spices, only OTA is currently regulated by the EU in addition to AFs. The ML

for OTA is 30 µg kg−1

in Capsicum spp. and 15 µg kg−1

for all other spices

(Commission Regulation No. 105/2010). As from 2015, a lower ML for Capsicum

spp. is foreseen (European Commission, SANCO-Ares, 2012). Meanwhile,

maximum AFs levels of 10–20 µg kg−1

are agreed for commercial transactions

within the international spice trade (Almela et al., 2007). In 2007, the Scientific

Committee of the Federal Agency for the Safety of the Food Chain (FASFC) in

Belgium decided the necessity for further research into “silent carriers” of

mycotoxins like spices, spice extracts and food supplements (Federal Agency for

the Safety of Food Chain, 2007). Within the European Union (EU), AFs in

Capsicum fruits are regulated with a maximum tolerable limit of 10 µg/kg for total

AFs (B1 + B2 + G1 + G2) and 5 µg/kg for aflatoxin B1 (Commission Regulation

EC No. 1881/2006). Legislation and levels for OTA in spices are currently being

considered (Commission Regulation No. 1881/2006).

2.2 MYCOFLORA IN RED CHILLI

Aspergillus species are shown as a dominant component of the mycoflora

of red chilli, usually occur in chilli fruits stored in humid regions (Flannigan and

Hui, 1976). Seenappa et al. (1980) described that with increasing relative humidity

(RH), A. flavus incidence increases, compared to other Aspergillus species. Various

species of fungi in two chilli varieties were identified (Wadia et al., 1983). These

were Alternaria alternata, Aspergillus flavus, A. niger, A. flavipes, Cladosporium

herborum, C. cladosporiodes, Curvularia lunata, Colletotrichum gloesporioides,

Dreshslera rostrata, D. hawaiiense, Fusarium semitectum, F. oxysporum,

17

Penicillium citrinum, Phoma sp., Pestalotiopsis theae, P. mangifera, Rhizopus

minutus, R. nigricans, Syncephalastrum racemosum, and Sclerotium oryzae. There

was a relationship between fungi associated with the fruit surface and storage

spoilage.

Seven genera and 11 species of fungi were isolated from stem, root, seeds

and leaves of infected plants of red pepper (C. frutescens) compared with 7 genera

and 10 species from bell pepper (C. annuum). Cephalosporium acremonium,

Alternaria alternata, Fusarium monliforme, F. proliferatum, F. oxysporum, F.

solani, F. anthophilum, Macrophomina phaseolina, Rhizoctonia solani were

observed prevalent in plants with wilting symptoms in District Mirpur Khas

(Sindh), Pakistan (Mushtaq and Hashmi, 1997). Sharfun-Nahar et al. (2004)

studied mycoflora associated with seeds and fruits of red chillies by using deep-

freezing technique and standard blotter method from India. From forty samples, 47

fungal species were isolated from seeds and pericarp of C. annuum i.e.

Acremonium fusidioides, Absidia corymbifera, Aspergillus tamarii, Cladosporium

accacicola, Blakeslea sp., Ulocladium tuberculatum, Cephaliophora irregularis,

Streptomyces sp., Tritirachium sp., and Scopulariopsis sp. Among them isolates of

Aspergillus flavus, A. niger, Rhizopus stolonifer and Alternata alternata were

predominant from both the seeds and fruits. Hussain et al. (2013) reported eleven

fungi contaminating fruits and leaves of chillies including Alternaria solani, Al.

alternata, Aspergillus niger, A. flavus, Botrytis cinerea, Colletotrichum capsici,

Cercospora capsici, Verticillium spp. and Leveillula taurica from local markets of

lower Sindh including Hyderabad, Mirpurkhas, Tando Allahyar, Kunri, Umerkot,

Samro, Digri and Kot Ghulam Muhammad.

18

Some fungi, A. flavus, A. terreus, A. niger, A. candidus, Fusarium

sporotrichioides, F. moniliforme, Penillicium corylophilum, Paccilomyces variotii

and Syncephalastrum racemosum were detected from decaying stored chilli fruits

in humid region (Prasad et al., 2000). Kiran et al. (2005) reported high incidence of

Aspergillus species in chillies (Capsicum annuum L.) as compared to Mucor spp.

Alternaria spp., and Fusarium spp. from cold stores of India. Chigoziri and Ekefan,

(2013) observed the incidence of 20 genera and 36 species from three classes of

fungi isolated and identified from 800 seed samples of pepper in Benue State of

Nigeria. Aspergillus flavus, A. niger and Colletothrichum capsici occurred

frequently at 29.75 % 44.00% and 54.75%, respectively.

Seventeen samples of spice and condiment imported from several parts of

the world mainly Pakistan, India, Iran and USA were collected and tested by using

standard dilution plate method on various media in Bahrain. Aspergillus,

Cladosporium, Penicillium, Trichoderma and Rhizopus were predominant genera.

Relative occurrence values of taxa were ranged between 36.4% for A. flavus and

0.6% for Absidia corymbifera and Aspergillus parasiticus (Mandeel, 2005).

Ten different kinds of spices (fifty samples) including black and red pepper

obtained from different places in Jeddah Governorate were assessed by using

dilution plate method (Bokhari, 2007). He reported 15 genera and 31 fungal species

in addition to one species variety. Aspergillus, Penicillium and Fusarium were the

most common genera. Highest incidence of A. flavus and A. niger among all

identified fungi was observed. The samples of dried red chilli were collected from

cold stores for one year at monthly intervals and mycoflora was observed

19

(Ravikiran et al., 2009). Aspergillus flavus and A. niger showed high prevalence

among all the samples followed by Alternaria alternata, Fusarium spp. and Mucor

spp. Due to these fungi. steady decrease in the levels of biochemical constituents,

such as carbohydrates, total phenols, ascorbic acid total protein and reducing sugars

in cold-stored red chillies was observed with increase in storage period

Numerous biochemical constituents like carotenes, capsaicin, ascorbic acid,

polyphenols, minerals, sugars (soluble and insoluble), fats and proteins were

quantitatively reduced in powdered red pepper after the successful growth of

Aspergillus flavus for 30 days. In all of dietary constituents, total carotenoids

showed the nutritional loss at maximum level (88.55%) followed by total sugars

(85.5%). The protein content of the contaminated sample was increased from

18.01% to 23%. The authors determined variations in biochemical contents

between infected and control (mould free) samples by indicating that mycoflora

colonization generate some biochemical changes in the food properties by the

colonization of mycoflora during its growth which needs to be considered intensely

(Tripathi and Mishra, 2009).

Different fungi were isolated by evaluating underground parts (roots and

stem base) of hot pepper (Capsicum annuum) plants cultivated near Lublin (south-

eastern Poland), Alternaria alternata, Colletotrichum coccoides, Fusarium solani,

F. equiseti, F. oxysporum, Gilmaniella roseum, G. humicola, Penicillium

janczewskii, P. cyclopium, Trichoderma harzianum and T. hamatum.

(Jamiolkowska, 2009). Red pepper samples collected from Razavi Khorasan

province and in Sabzevar (Iran) were analysed for the presence of different fungi.

20

High incidence of mold and yeast were observed ranged from 2.4×103 to 4.6×10

6

cfu g-1

and Aspergillus spp., Rhizopus spp. and Penicillium spp. were the most

predominant fungal genera (Salari et al., 2012).

Some workers characterized the fungal flora of 80 paprika, pepper and

cumin especially on Aspergillus section Flavi isolates marketed in Morocco. They

observed that Aspergilli section Flavi widely contaminated the spices. Among

them, 57% were observed to be toxigenic (El-Mahgubi et al., 2013). Jeswal and

Kumar (2013) stated that the most dominant species isolated from samples of red

chilli were Aspergillus flavus and A. niger. In another study, occurrence of A.

flavus was very high in red chilli followed by dry ginger. From red chilli, 56% of

A. flavus were toxigenic and produced aflatoxins. Three isolates of Colletotrichum

capsici, one isolate each of Fusarium oxysporum. F. moniliforme, F.

pallidoroseum, Aspergillus flavus and Alternaria alternata were associated with

the disease samples of chilli fruits collected from India including Vijaypur, Chatha,

Marh, Akhnoor, and Jammu areas (Parey et al., 2013).

In a survey, fourteen samples of spices were collected from local markets in

Qatar, Doha for the presence of mycoflora contamination. Among them, highest

presence of fungal propagules was shown by chilli powder, while samples of garlic

ginger and curry were not contaminated by fungi. Most were species of Penicillium

and Aspergillus genera. The dominant species were Aspergillus flavus, A. niger and

A. nomius. Screening of 37 Aspergillus isolates for aflatoxin production using three

primers focusing on aflP, aflM and aflR genes suggested that 9 A. flavus isolates

could produce aflatoxins (Hammami et al., 2014).Two species of Fusarium were

21

reported by Samyal and Sumbala (2014) and observed that F. verticillioides and F.

semitectum contaminated the samples of red chilli powder in Jammu and Kashmir

State, India.

Sri Lankan peppers (Piper nigrum L.) were assessed and found that

Aspergillus flavus, A. niger, A. parasiticus and Penicillium spp. were the most

dominant fungi (Yogendrarajah et al., 2014a) Moulds were characterized in malt

extract agar (MEA) and A. flavus and parasiticus agar (AFPA) in 77 black pepper

(BP) and 11 white pepper (WP) samples. In total, A. flavus and/or A. parasiticus

(AfAp) contaminated 73% of the BP and 64% of the WP samples. A BP sample

with water activity (aw) 0.70 recorded the highest count of AfAp (4.3*104 CFU/g).

Moreover, 75% of the BP samples exceeded the safe aw limit (0.65) set by the

European Spice Association (ESA). The frequency of occurrence of A. niger in BP

was 62% with counts up to 1.3x103 CFU/g. Penicillium spp. were found in 61%

and 55% of the BP and WP samples, respectively. In BP 94% of the samples had a

Penicillium contamination below 103 CFU/g. Other Aspergillus spp, found in

peppers included Aspergillus terreus, Aspergillus tamarii, Aspergillus candidus,

Aspergillus penicilloides, Aspergillus sydowii and Aspergillus fumigatus. Mould

counts in BP (102-10

4 CFU/g) were significantly higher than that of WP (<10

2

CFU/g).

From Taif city (Saudi Arabia), 60 samples of chili including ground chili

(20 samples), crushed chili (20 samples) and chili sauce (20 samples) were

obtained. High contamination of fungi was seen in crushed chili as compared to

two other chilli products tested, whereas highest occurrence of total aflatoxins was

22

observed in chili powder. The most common genera isolated from chilli samples

were Aspergillus, Eurotium and Penicillium (Gherbawy et al., 2015). In another

study from India, Jeswal and Kumar (2015) detected various fungi in 55 red chilli

samples collected from local markets of Bihar India including, Alternaria

alternata, Aspergillus flavus, A. ochraceus, A tamarii, A. versicolor, A. niger,

Penicillium verrucosum, P. citrinum, Mucor hiemalis Fusarium moniliforme,

Chaetomium globosum and Rhizopus oryzae.

This literature survey suggested that red chilli is a good substrate for

mycoflora and mycotoxin contamination and that there is a dire need to manage

this issue to reduce contamination levels below permissible limits of the EU. Trials

have been conducted throughout the world for the management of mycotoxins and

mycoflora under laboratory and in fields.

2.3 MANAGEMENT OF MYCOTOXINS AND MYCOFLORA IN

LABORATORY TRIALS

Biological control or biocontrol is the suppression of damaging activities of

one organism by one or more other organisms, often referred to as natural enemies.

The organism that suppresses the pest or pathogen is referred to as the biological

control agent (BCA). Biocontrol of fungal growth is a potential mean for toxin

control in the field. Various organisms including yeasts, bacteria, and nontoxigenic

(atoxigenic) strains of the causal organisms have been tested for biological control

of aflatoxin contamination among which only atoxigenic strains have attained

commercial use (Yin et al., 2008).

23

The US Environmental Protection Agency has approved the biological

control of aflatoxin production in crops in the US following testing of a

commercial product based on atoxigenic Aspergillus flavus strains (Afla-Guard®).

Two atoxigenic isolates of A. flavus have been detected in Nigeria as atoxigenic

strains for exclusion of toxigenic fungi in maize fields. In both laboratory and field

trials, these strains reduced toxin contamination by 70 to 99% (Atehnkeng et al.,

2008).

Antifungal activity of ethanolic extracts of Euphordia hirta against the

plant pathogens A. niger, Botryodiplodia theobromae, Fusarium pallidoroseum,

Colletotrichum capsici and Phomopsis caricae-papayae was assayed by disc

diffusion method. The extracts showed inhibition zones ranged 7 to 9 mm against

pathogens (Mohamed et al., 1996). Verma and Dubey (1999) observed that as

compared to synthetic pesticides plant metabolites and plant based pesticides are

better alternatives as they have minimal environmental impact and danger to

consumers. Antibacterial activity of ethanolic extract of E. hirta was tested against

Gram positive and Gram negative organisms and observed that the extract is more

active against Gram positive organisms than Gram negative bacteria (Nelofar et al.,

2006). Sudhakar et al. (2006) revealed antimicrobial effect against Escherichia coli

(enteropathogen), Staphylococcus aureus, Proteus vulgaris and Pseudomonas

aeruginosa. Inhibition zones diameters were 21, 19, 23, and 19 mm respectively

with the MIC values 0.189, 0.216 0.2 and 0.166 mg/ml. Ogbulie et al. (2007)

observed antibacterial activity of crude ethanolic extract of E. hirta against the

growth of Bacillus subtilis, E. coli, P. aeruginosa, and S. aureus. Suresh et al.

(2008) analyzed the ethanol extract from E. hirta leaves for antimicrobial potential

24

against Bacillus cereus, S. aureus, Salmonella typhi, P. aeruginosa, Klebsiella

pneumonia and fungal species Rhizopus oryzae, Aspergillus flavus, Aspergillus

niger and Aspergillus fumigatus. Antimicrobial activity was ascribed to alkaloids,

tannins, saponins, proteins, flavonoids, glycosides and sterols. Furthermore, leaves

collection from August to December showed more significant antimicrobial

activities. Abubakar (2009) analysed the antibacterial capability of the water,

hexane and methanolic extract of E. hirta against a group of Gram negative

bacteria that frequently cause enteric infections in humans including E. coli,

Proteus mirabilis, Shigella dysentriae, K. pneumonia, and S. typhi. More

antibacterial effectiveness was observed by water extracts than organic solvent

extracts. Phytochemical screening of the crude extracts showed the presence of

flavonoids, alkaloids, saponins, tannins, phenolics, cardiac glycosides and

anthroquinones. These bioactive constituents have been linked to antimicrobial

activity.

Under laboratories trials, plant extracts of many higher plants exhibited

antifungal, antibacterial and insecticidal properties (Satish et al., 2007). Due to

these reasons, the use of various plant extracts, spices and their constituents may

provide an alternative approach for the prevention of fungal growth and

mycotoxins formation (Vagi et al., 2005; Kumar et al., 2007; Lee et al., 2001,

2007). In-vitro antifungal activities were investigated (Bokhari, 2009) by extracting

the lemon grass (Cymbopogon citrates DC.) Stapf., olive leaves (Olea europaea

L.), lantana (Lantana camara L.), basil (Ocimum basilicum L.) and nerium

(Nerium oleander L.) by using water or different organic solvent. Highest activities

were shown by methanolic extract of lemon grass, nerium and lantana followed by

25

their ethyl acetate extracts against Trichophyton rubrum. This causes the growth

inhibition of T. rubrum at a concentration of 100 μg ml-1

by 85-90 and 80-85%,

respectively, while aqueous extracts causes growth inhibition of T. rubrum at the

same concentration by 32-77%. Methonolic extracts of five selected plants were

tested against strains of pathogenic fungi including T. mentagrophytes,

Microsporum canis and M. gypseum. Among them, lemon grass showed the

highest antifungal activity followed by lantana. Moderate activities showed by

Nerium and basil while olive extract showed lowest activity.

Volatile components extracted from the stems, flowers and leaves of

Hibiscus rosa-sinensis, Lantana camara and Malvaviscus arboreus, cv. white

flowers and red flowers were tested against the fungi Botrytis cinerea, Fusarium

solani f. sp. cucurbitae, F. oxysporum f. sp. niveum, Verticillium dahlia, Pythium

ultimum, Alternaria solani and Rhizoctonia solani. Extracted volatile components

from the flowers and stems showed the strongest inhibitory effect. Complete

inhibition was observed against V. dahlia. While P. ultimum showed the weakest

effect (Boughalleb et al., 2005).

The antimicrobial activity of nystatin and methanolic extract of the leaves

of E. hirta were assessed using the checkerboard method against Candida albicans.

(Jackson et al., 2009). It was observed that some nystatin combinations with extract

might be synergistic in activity for some ratio combinations and similar for some

others. The antimicrobial activity of ethanolic extracts of the aerial parts of E. hirta

was then investigated. Akinrinmade and Oyeleye (2010) examined the

effectiveness and tissue reaction of the crude ethanolic extract of E. hirta in canine

26

infected incised wounds. It was observed that the crude ethanolic extract of E. hirta

is not effective for the growth of S. aureus as well as tissue reaction in canine

wounds, therefore, it was recommended for usage in surgical site preparation.

Mohammad et al. (2010) evaluated the antimicrobial potential of E. hirta leaves,

root, stem and flower extracts using microbial samples including four Gram

negative bacteria (P. mirabilis, E. coli, S. typhi, and K. pneumoniae), four Gram

positive bacteria (Bacillus thuringensis, S. aureus, B. subtilis and Micrococcus sp.)

and one yeast (C. albicans). Zones of inhibition observed between 16 and 29 mm.

Leaf extract showed inhibition for the growth of all tested microorganisms with

large inhibition zones, followed by the flowers, which also inhibited all the bacteria

except S. aureus and Micrococcus sp., the most susceptible microbes to all extracts.

Root extract exhibited larger zones of inhibition against Gram positive bacteria

than Gram negative bacteria, and had larger zones of inhibition as compared to the

stem extract. E. coli and C. albicans showed lowest MIC values (3.12 mg/ml),

followed by S. aureus (12.50 mg/ml) and P. mirabilis (50.00 mg/ml). All other

bacteria had MIC values of 100.00 mg/ml. The results support the usage of E. hirta

in traditional medicine. Gayathri and Ramesh (2013) described that antifungal

activity exhibited by the ethyl acetate extract of the inflorescence of Euphorbia

hirta targeting the cell membrane which might be result in leakage of cellular

proteins.

Evaluation of aqueous leaf extracts of Croton sparsiflorus, Azadirachta

indica, Clerodendron spp., Calotropic procera, seed and leaf of Lantana camara,

seed and leaf of Putranjiva roxburghii, leaf of Luffa cylidrica, Moringa oleifera,

leaves of Senna alata, Salvadora persica, Trichosanthes dioica, and Trema

27

orientales showed some potentiality for the inhibition of some seed borne fungi i.e.

Aspergillus flavus, A. niger, Phomopsis vexans, Penicillium spp., Fusarium

oxysporum and Curvularia lunata improved the seed germination of brinjal (Kuri,

2011). In another study, Mostafa et al. (2011) examined that Punica granatum

extract showed the highest antiaflatoxigenic and antifungal activities followed by

Zingiber officinalis. Extract of P. granatum prevented production of aflatoxin B1 at

5 mg/ml and inhibited the mycelial growth of A. flavus 100% at 10 mg/ml. Extracts

of Olea europaea and Zingiber officinalis showed a moderate antifungal activities

and exhibited a significant antiaflatoxigenic effectiveness as they completely

inhibited production of aflatoxin B1 at 15 mg/ml. Weak antifungal activity was

showed by Lantana camara extract while Allium sativum L. showed no effect. The

GC/MS analysis of plant extracts showed that P. granatum extract was mainly

composed by ellagic acid (37.01%) followed by pedunculagin (6.40%),

punicalugin (5.64%) and polyphenol as lumicolchicine (4.68%) while components

of Z. officinalis were gingerol (46.85%), cedrene (8.39%), zingiberene (7.40%) and

α-curcumene (7.32%).

Maximum percentage growth inhibition was observed against Alternaria

alternata at 1 mg/ml concentration by the fungitoxic spectrum of the extracts of

tested plant‟s leaf & stem (Saraf et al., 2011). Medeiros et al. (2012) detected 68

putative compounds from Lantana camara oil. Cytotoxic effect of essential oils

from leaves of L. camara were observed against V79 mammalian cells and Artemia

salina by showing 50% lethal concentration (LC50) values from 0.00023 mg/ml.

These essential oils significantly inhibited the growth of two tested fungi Candida

krusei and Candida albicans. Assessment and comparison of antimicrobial activity

28

among plants, Bougainvillea glabra choicy, Lantana camara L. and Delonix regia

(Hook) Raf, leaves by using agar well diffusion method was carried out against

Aspergillus niger, Streptococcus mitis, Lactobacillus sp. and Candida albicans

(Rani et al., 2012). It was observed that excellent antimicrobial activity showed by

Delonix regia (Hook) Raf against all the test organisms followed by Bougainvillea

glabra choicy considered moderately active against all the test organisms while

Lantana camara L. having high antibacterial activity and no antifungal activity.

Among extracts of all three plants, high antibacterial activity showed by Lantana

camara while highest antifungal activity was observed in Delonix regia. In another

study, Naz and Bano (2013) assessed the antimicrobial activity of Lantana camara

and determined that maximum antibacterial activity was shown by methanolic leaf

extract of L. camara against Pseudomonas aeruginosa and Staphylococcus aureus

and was also effective against other bacterial strains as compared with ethanol and

aqueous extracts of leaves. The significant inhibition was shown by methanolic leaf

extract of L. camara against Aspergillus flavus (66%) and Aspergillus fumigatus

(71%). Antifungal activity of Lantana camara was evaluated against Aspergillus

niger, Aspergillus flavus, Aspergillus fumigatus and the dermatophyte

Microsporum gypseum isolated from the soil. Good inhibition was observed. (Rizvi

et al., 2013). However, thirteen test bacteria and eight test fungal strains were

tested against crude acetone and methanolic extracts of Lantana camara. Inhibition

of the growth of Staphylococcus aureus was observed by both the solvent extracts

to the maximum.

Assessment of antifungal potential of Trianthema portulacastrum Kavitha

et al. (2014) was carried out against the clinical isolates of Aspergillus flavus, A.

29

fumigatus, A. niger, Candida albicans, Mucor indicus and Rhizopus oryzae. Less

than 100% inhibition was shown by the methanol and chloroform extracts against

Aspergillus fumigatus, A. niger, Candida albicans and Rhizopus. Aqueous extracts

of nine weeds Parthenium hysterophorus L., Chenopodium album L., Solanum

nigrum L., Trianthema portulacastrum L., Malvestrum coromandelianum (L.)

Garcke, Nicotiana plumbaginifolia Viv., Coronopus didymus (L.) Sm., Digera

muricata (L.) Mart., and Sphaeranthus indicus L. were applied against

Myrothecium roridum Tode strain by poison food technique. They observed that

the extract of N. plumbaginifolia exhibited growth inhibition of 88%, P.

hysterophorus (71%) and S. nigrum, C. didymus, S. indicus and T. portulacastrum

L. restrained the colony growth up to 66, 65, 64 and 60%, respectively. Digera

muricata was observed as least effective with 11% of colony growth (Naz et al.,

2015).

Antimicrobial activity of Amaranthus viridis ethanolic extracts was

evaluated against two strains of Gram positive bacteria, Bacillus subtilis and

Staphylococcus aureus and four strains of Gram negative bacteria via; Escherichia

coli, Proteus vulgaris, Peudomonas picketii and Klebsiella pneumonia.

Antibacterial assay showed that Amaranthus viridis had inhibitory activity against

Escherichia coli, Bacillus subtilis, Peudomonas picketii and Proteus vulgaris.

Antifungal activity of ethanolic extract of Amaranthus viridis was tested against

five different strains of fungal species, including; Alternaria species, Aspergillus

flavus, A. niger, A. fumigatus and Fusarium solani. It was determined that

moderate antifungal activity was observed in Amaranthus viridis (41-51%) against

Alternaria species while low activity (below 40%) against Aspergillus flavus, A.

30

niger and A. fumigatus (Sarwar et al., 2016). Islam et al. (2010) investigated the

antimicrobial and irritant activities of the Amaranthus viridis L., Malvastrum

coromandelianum L. and Malva parviflora L. extracts. Malvastrum

coromandelianum L. and Malva parviflora L. extracts against E. coli exhibited

similar patterns of antibacterial activity with some variations in the antibacterial

response against Staphylococcus aureus, Bacillus subtilis and Proteus vulgaris. All

of extracts showed almost same antifungal activities on Aspergillus oryzae and A.

niger. The antibacterial action of ethanolic Amaranthus viridis L. extracts was

more prominent than that of its polar mass and aqueous extract. Though, plants

extracts prepared in hexane exhibited a greater antibacterial potential against Gram

negative and Gram positive microorganisms as compared to extracts of chloroform.

Apart from plant sources many microorganisms like bacteria and yeasts

have been considered as a good source of mycoflora and mycotoxin management.

It was concluded that uptake of aflatoxins by the cells greatly affected by viability

and population of bacteria (Line and Brackett, 1995) Lactic acid bacterial (LAB)

strains inhibited aflatoxin biosynthesis but not effective for the removal of aflatoxin

from contaminated media (Coallier- Ascah and Idziak, 1985; Thyagaraja and

Hosono, 1994). El-Nezami et al. (1998a) studied the ability of selected dairy strains

of lactic acid bacteria for the removal of AFB1 from liquid media. Removal of

AFB1 approximately 80% at 0 hr by Lactobacillus rhamnosus strain GG (LBGG)

and L. rhamnosus strain LC-705 (LC705) was observed.

Without production of toxic by-products, aflatoxin can be removed from

liquid medium and food products by the microorganism Flavobacterium

31

aurantiacum. Then mechanism for aflatoxin detoxification by this organism is not

known (Ciegler et al., 1966; Hao and Brackett, 1988; Line and Brackett, 1995).

The binding of AFB1 with bacteria that were treated previously with heat or acid

was observed (Haskard et al., 1998). The binding depends on the original

concentration of aflatoxin of B1. Haskard et al. (2000) revealed the mode of action

of bacteria L. rhamnosus by which aflatoxins binds the surface of bacterial cell wall

through protein or carbohydrate components present in the cell wall of bacteria.

Presence of hydrophobic interactions in binding indicated by the occurrence of

decrease in binding while treating with urea. Like treatment with NaCl and CaCl2,

electrostatic interactions also play a minor role. pH has not affected aflatoxin B1,

but had considerable influence on aflatoxin B2a binding, indicating that with

respect to binding mechanisms, different metabolites of the same mycotoxins may

show considerable differences.

Microorganisms like yeast and LAB cells are known to bind different

molecules such as killer toxins and metal ions on complex binding structures on the

cell wall (Brady et al., 1994; Bolognani et al., 1997; Santos et al., 2000). Raju and

Devegowda (2000) reported the mechanism of binding of mannans with other

mycotoxins such as ochratoxin A toxin and T-2. Yeast like Saccharomyces

cerevisae, candida krusi has been considered as a best source of amino acids,

cytokinins, enzymes and vitamins which help in binding of aflatoxins to the surface

of bacterial and yeasts cell and help in the mycotoxin management (Mahmoud,

2001). Huwig et al. (2001) stated that replacing of yeast cells with physically

extracted cell walls enhanced the binding of ochratoxins. By physical binding

(Haskard et al., 2001), bacterial strains of lactic acid remove toxins from liquid

32

media. Aflatoxin B1 complexes stability formed with 12 bacterial strains in viable

and nonviable forms was evaluated by repetitive aqueous extraction. Up to 71% of

the total aflatoxin B1 remained bound by the fifth extraction. Highest amount of

aflatoxin B1 retained by nonviable bacteria. It was observed that from solution, L.

rhamnosus strain LC-705 (DSM 7061) and Lactobacillus rhamnosus strain GG

(ATCC 53103) removed aflatoxin B1 efficiently. The accessibility of bound

aflatoxin B1 to an antibody in an indirect competitive inhibition ELISA suggests

that in binding, surface components of these bacteria are involved. By solvent

extraction, 90% of the bound aflatoxin can be recovered from the bacteria.

Sonication and autoclaving did not release any detectable AFB1. Variation in pH (2

to 10) and temperature (4 to 37°C) did not show any significant influence on the

amount of aflatoxin B1 released. Aflatoxin B1 binding appears to be predominantly

extracellular for viable and heat-treated bacteria. Celyk et al. (2003) investigated

the effects of adding chlortetracycline (CTC) at 2.5 ng/g, baker yeast (BY) at 2.0 %

and both BY + CTC to a control diet containing 200 ng/g of aflatoxin B1 (C +

AFB1) on performance, serum parameters and pathology alterations of 100 broilers.

Body weight, feed efficiency and feed efficiency were recorded weekly. Increase in

serum glutamic oxalacetic transaminase (GOT) was observed in C+AFB1 birds.

Significantly reduction in body weight and feed consumption in group AFB1 was

observed while higher body weight observed in birds receiving BY + AFB1, CTC +

AFB1 and BY + CTC + AFB1, CTC + AFB1 showed better feed efficiency than the

others.

The binding of AFM1

was also assessed in contaminated phosphate buffered

saline (PBS). Streptococcus thermophilus ST-36 and Lactobacillus delbrueckii

33

subsp. bulgaricus CH-2 were used. By using contaminated reconstituted milk and

contaminated yoghurt, removal activities of these strains were also assessed.

ELISA procedure was used. Results showed high binding ability of Streptococcus

thermophilus ST-36 (bound in PBS at 29.42% and in milk at 39.16%) than

Lactobacillus delbrueckii subsp. bulgaricus CH-2 (bound in PBS at 18.70% and in

milk at 27.56%). The rate of binding of both of micro-organisms was higher in

milk as compared to PBS. AFM1

was bound at 14.82% level in yogurt

(Sarimehmetoglu and Kuplulu, 2004). Authors reported that mycotoxins removed

by the adhesion of components of cell wall rather than by metabolism or covalent

binding as dead cells kept remain their binding ability (Raju and Devegowda, 2000;

Celyk et al. 2003; Santin et al., 2003 Baptista et al., 2004). Yiannikouris et al.

(2004a,b) stated the binding of zearalenone to β-D-glucans. Bejaouii et al. (2004)

stated that adsorption physical nature of ochratoxin binding indicated by heat

treated cells and rapid nature of removal of toxin from liquid medium. Interactions

between zearalenone and β-D-glucans, based on NMR and X-ray diffraction

studies were reported (Yiannikouris et al., 2004c). Lahtinen et al. (2004) showed

that peptidoglycan or the structures closely related with peptidoglycan might be the

most likely carbohydrate that is involved in binding process of aflatoxin B1. A

stable intra-helical association with zearalenone supported by β-1,3-D-glucan

chains and stabilized by β-1,6-D-glucans side chains has been reported by Jouany

et al. (2005). In an investigation the removal of aflatoxin B1 (AFB1) by selecting

bacterial strains of lactic acid isolated from traditional sourdough ferments were

conducted. (Zinedine et al., 2005). MRS broth comprising a known concentration

of AFB1 used for the growth of isolates for 48 h at 30°C. Determination of AFB1

34

was done by HPLC. It was observed that Lactobacillus strains removed more AFB1

than Leuconostoc and Pediococcus strains. For all the strains studied, reduction of

the initial amount of AFB1 ranged from 1.80 to 44.89% was observed. Five strains

of Lactobacillus rhamnosus, one strain of a L. casei and one strain of L. lactis

reduced AFB1 by more than 20%. Fourty five percent AFB1 reduction observed in

L. rhamnosus strain Lb50.

The Inhibitory effects of four bacteria (Bacillus subtilis, Lactobacillus

plantarum, Leuconostoc mesenteroides and Lactobacillus casei) were examined on

the growth and aflatoxin production of A. parasiticus, which are found in

fermented foods (Kim, 2007). Modified APT broth used for the growth of

microorganisms. High performance liquid chromatography (HPLC) was used for

the determination of aflatoxin B1. Co-inoculation of the four bacteria resulted in the

reduction of mycelial growth of A. parasiticus ranged between 20.9 to 86.2% and

aflatoxin production ranged from 21.6 to 70.4%. High reduction was observed by

mold inoculation with B. subtilis followed by Leuconostoc mesenteroides,

Lactobacillus casei and L. plantarum.

The removal of aflatoxin B1 was investigated from contaminated media by

using dairy strains of lactic acid bacteria (Shahin, 2007). Twelve isolates out of

forty two from raw milk, yogurt and karisk cheese showed different levels for

aflatoxin B1 binding. For binding AFB1, highest isolates were identified as

Sterptococcus thermophiles and Lactococcus lactis. Dead cells (by boiling) of S.

thermophiles and L. lactis bind 100% and 86.1 of the AFB1 (2 μg) added to the

phosphate buffer solution, respectively while the viable cells of both strains bind

35

only 81.0% and 54.35, respectively. Hundred percent removal of AFB1 (2 μg)

contaminating maize, sunflower and soybean oils was observed by dead cells pellet

of L. lactis while dead cells of S. thermophilus pellet removed 81.0, 96 and 96.8%

of the toxin contaminating sunflower, soybean and maize oils, respectively.

The effect of different fermenting microorganisms on growth of Aspergillus

nomius was assayed (Munoz et al., 2010). Two lactic acid bacteria, Lactobacillus

rhamnosus and L. fermentum and baker‟s yeast Saccharomyces cerevisiae, were

tested for fungus inhibitory properties. Assays were carried out by subsequent

inoculation of one of the microorganisms followed by the fungus. Growth

inhibition of the mycotoxin producing Aspergillus strain was shown by all the three

microorganisms. They evaluated that highest fungal inhibition of the

microorganisms assayed was shown by L. rhamnosus which was isolated from

sheep milk. The capacity of Lactobacillus rhamnosus for the removal of AFB1

from contaminated medium was also assessed (Bovo et al., 2014). Culturing of L.

rhamnosus was done in MRS broth. Quantification of AFB1 was carried out by

high performance liquid chromatography followed by performing Scanning

electron microscope. No significant differences was observed between AFB1

binding efficiency for pH 3.0 and 6.0 by L. rhamnosus cells in freeze-dried (36.6 ±

7.1% and 27.2 ± 4.0%, respectively) or in solution (45.9 ± 8.8% and 35.8 ± 7.7%,

respectively). Though, during atomization, AFB1 binding ability completely lost by

the spray-dried cells. AFB1 binding capacity of L. rhamnosus retained only when

its cell wall remained intact. Haggag et al. (2014) tested various organisms

including fungi, yeast and bacteria for controlling aflatoxin contamination. It was

observed that Pichia anomala, Streptomyces aureofaciens, Bacillus pumilus,

36

Bacillus subtilis and Pseudomonas putida showed the ability to inhibit fungal

growth and production of aflatoxin by Aspergillus flavus and A. parasiticus in

laboratory experiments by giving good efficacies to protect corn grain crops.

A clear picture of binding capacity of L. casei Shirota to AFB1 into the cell

envelope of bacteria was revealed for the first time (Hernandez-Mendoza et al.,

2009). The images showed that aflatoxin binding develop some changes in

structure which cause modification in the cell surface of bacteria. Oluwafemi et al.

(2010) tested biological detoxification strategy by using lactic acid bacteria in

Nigeria and artificially inoculated maize grains by using toxigenic and atoxigenic

A. flavus i.e. LA 32G_28 and A. flavus (LA32_79) respectively. Aflatoxin B1

concentrations of 50, 100, 200, and 500 ng/g were prepared in four samples of bulk

maize grains. Significant reductions in aflatoxin B1 were observed in all treatments,

pH of the medium decreased from 5.0 to 4.0 by lactic acid bacteria. Highest

reduction in aflatoxin B1 was observed in maize contaminated at 50 ng/g (44.5%)

while least reduction was observed in maize contaminated at 500ng/g (29.9%). For

degradation of aflatoxin B1, L. plantarum was the most effective organism.

The ability of four strains of lactic acid bacteria to remove aflatoxin B1 was

studied (Kasmani et al., 2012). Three indigenous isolates (Pedioccus pentosaceus

TMU457, Lactobacillus rhamnosus TMU094 and Lactobacillus fermentum

TMU121) and a non-indigenous isolate (Labacillus rhamnosus PTCC1637) were

studied. Incubation of strains with AFB1 was done at different time. All species

showed reduction in toxin quantity. Aflatoxin binding by LAB varied from 19.41

to 75.06%. L. rhamnosus PTCC1637 bound 19.41 to 35%, P. pentosaceus bound

37

24.86 to 63.21%, Lactobacillus rhamnosus TMU094 bound 25.64 to 75.06% and L.

fermentum bound 38.63 to 72.15% of AFB1.

2.4 MANAGEMENT OF MYCOTOXINS AND MYCOFLORA IN

GREENHOUSE TRIALS

Biofertilizers and biological agents were evaluated for the management of

seed and seedling diseases of Sesamum indicum L. (sesame) and observed that the

untreated seeds were found to have maximum percent incidence of mycoflora and

minimum population was recorded in the treatment of Trichoderma +

Pseudomonas fluorescence, Azatobacter + Trichoderma, Rhizobium +

Trichoderma, Azatobacter, Trichoderma and Pseudomonas fluorescence in

decreasing order of effectiveness (Bharathi et al., 2013). A cell wall constituent

„mannan‟ in yeast possess binding capacity to sterols from the medium (Thompson

et al., 1973). A stimulatory effect of yeast on cell division and enlargement,

chlorophyll formation and protein and nucleic acid synthesis was reported (Kraig

and Haber, 1980 and Castelfranco and Beale, 1983). Coallier-Ascah and Idziak

(1985) suggested the inhibition of aflatoxins related to heat stable, low-molecular-

weight inhibitory compound by Lactobacillus cell free supernatants. Due to its

cytokinins content, during stress it participates in a positive way (Barnett et al.,

1990). Luchese and Harrigan (1990) repoted that Lactobacillus spp. was observed

to delay aflatoxin biosynthesis while other lactic strains like L. lactis stimulate

aflatoxin accumulation. Occurrence of “anti-mycotoxinogenic” metabolites that

inhibits aflatoxin accumulation in Lactobacillus cell-free extracts produced during

LAB growth was indicated by using a dialysis assay (Gourama, 1991). Karunaratne

38

et al. (1990) proposed that inhibition of aflatoxin biosynthesis was not the

consequence of a pH decrease or a hydrogen peroxide production.

During pre-harvest aflatoxin contamination management of cottonseed, 800

beneficial bacterial isolates were tested, recovered from cotton field soils, leaves

and stems and immature as well as opened bolls, for inhibition potential of A.

flavus growth on cottonseed. As a result, inhibition of fungus by six isolates

partially or totally was observed. All of these effective isolates prevented the

fungus fioin infecting simulated pink bollworm exit holes in immature bolls in the

field (Misaghi et al., 1993). Thyagaraja and Hosono (1994) stated that numerous

papers have focused on the inhibition of mycotoxin biosynthesis by Lactic Acid

Bacteria. Earlier studies considered LAB as inefficient binders of aflatoxin B1

(Thyagaraja and Hosono, 1994; Coallier-Ascah and Idziak, 1985).

There is possibility during cell lysis that LAB releases molecules that

potentially cause inhibition in mould growth and hence accumulated lower rate of

their mycotoxins (Gourama and Bullerman, 1995). Kamilova et al. (2009)

described that species of the genus Trichoderma is most promising and effective

bio control agent. Chet et al. (1997) tested Trichoderma as antagonist controlling

widely the microbes while they have much more complex mechanism of

mycoparasitism, involves hyperparasitism, nutrient competition, antibiosis, space

and cell wall degrading enzymes.

Binding ability is highly strain specific indicated by different strains of

LAB with respect to aflatoxin binding (Turbic et al., 2002). Tehrani and Ramezani

(2003) reported that six Bacillus spp. produced volatile metabolites isolated from

39

the rhizosphere inhibiting mycelial growth of Fusarium oxysporum in-vitro. They

added the isolates in soil treatment; the isolates had the highest effect in reducing

the Fusarium wilt of onion. Freimund et al. (2003) demonstrated the excellent

binding of modified yeast β-1, 3-glucan with T-2 toxins in addition to zearalenone

mycotoxins. This indicates that there are the possibilities for mycotoxin binding on

cell wall on more than one target.

Bio-Organic Farming effect on potato plants was assessed on their growth,

yield and Pests Infestation in Egypt (Gomaa et al., 2005). Reduction in the

infestation numbers of thrips and white fly was observed by foliar application of

yeast with the percentages for white fly and thrips ranged from 0.2 to 17.9 and 33

to 64, respectively. On potato yield, organic fertilization and the foliar application

of yeast showed synergistic effect, whereas significant variations was observed in

the combined application of organic fertilizers and yeast. Potato yield increased

with the percentage from 21 to 73. Kamilova et al. (2009) reported that the bio

control strains P. fluorescens PCL1751 and P. putida PCL1760 under soil and

hydroponic cultivation conditions, were effectively suppress Tomato foot and root

rot.

Foliar spray on kidney bean by using active yeast extract (AYE) was

applied with concentrations of 50 ml, 100 and 150 ml AYE/L (Nassar et al., 2011).

Significant promotive effects were observed at concentrations of 100 and 150 ml

AYE/L on all investigated morphological characters, number of pods/plant, seed

yield/plant, yield of green pods/plant, number of seeds/plant, and the percentage of

crude protein in seeds of Kidney bean 'Giza 6' in both studied seasons. Maximum

40

significant increase was showed in sprayed Kidney bean plants with active yeast

extract at100 ml/L concentration of 14.7 and 8.6% for seed protein, 22.5 and 25.2%

for yield of green pods/plant and 34.0 and 36.6% for yield of seeds/plant in the first

and second season, respectively.

Positive significant difference was observed in shoot characteristics and in

all yield traits by spraying bread yeast or seaweed extract. All the detected traits

were enhanced by the interaction between yeast and seaweed extract. Since

cucumber plant received 6 g.l-1

bread yeast and sprayed with a mixture of 0.33ml.l-1

Alga 600 +2.5 ml.l-1

Sea force 2 were characterized by the highest values of all

shoot and yield characteristics (Sarhan et al., 2011). Abdel-Kader et al. (2012)

worked for the control of foliar diseases of vegetables under greenhouse conditions,

by testing various bio agents including Bacillus subtilis, Saccharomyces serevisiae,

Trichoderma Viride, T. harzianum and Pseudomonas flourescens as alternatives.

Significant reduction was observed in diseases incidence by the application of B.

subtilis and T. harzianum as compared with the other applied bio agents. Combined

treatments of chemical inducers and S. serevisiae were recorded, i.e. (Chitosan +

Thyme oil), (Chitosan + Calcium chloride + S. serevisiae), (Chitosan + Saccharin),

(Saccharin + Potassium monohydrogen phosphate), (Chitosan + Potassium

monohydrogen phosphate), (Chitosan + S. serevisiae) and (Humic & folic + Thyme

oil) compared with other applied treatments and untreated control.

Studies on foliar application by EM “Effective Microorganisms”, Amino

acids and yeast on growth, yield and quality of two cultivars of onion plants under

Newly Reclaimed Soil were carried out (Fawzy et al., 2012). Positive promoting

41

effects were observed by providing supplemental doses of EM, amino acids and

yeast on growth, yield and its quality. Though, highest yield and quality of onion

plants was observed by using EM at rates of 3 cm/L. Mostly, highest amount of

growth, yield and quality of onion plants were examined by using Super X cv. of

onion with foliar spraying of EM. Sukorini et al. (2013) carried out a study in

which as an alternative to synthetic fungicide, plant crude extracts and yeasts

combination was used to control green mould on citrus fruit caused by Penicillium

digitatum. Combination of Candica utilis and Eugenia caryophylata crude extracts

showed best results with 90.3% incidence reduction and 96.26% disease severity in

green mould. Moreover, this combination had effective antifungal activity than

imazalil. Marzauk et al. (2014) conducted an experiment to study the influence of

combined foliar application of yeast extract (0, 3 and 6 ml/L) and vitamin E (0, 500

and 1000 ppm) on broad bean (Vicia faba L.) growth, yield and some biochemical

constituents. Spraying with high level of vitamin E (1000ppm) and yeast extract (6

ml/L) showed highest values of plant growth such as number of leaves and

branches, branches and whole plant, plant length, highest values of total pods yield

and different organs, fresh and dry weight of leaves and the content of nitrogen and

protein % in seeds tissues.

By application of active yeast extract, the growth and productivity of

vegetable crops like eggplant (Fathy and Farid, 1996; Hewedy et al., 1996; El-

Tohamy et al., 2008), tomatoes (El-Ghamriny et al., 1999; Fathy et al., 2000), pea

(Tartoura, 2001; Amer, 2004; El-Desuki and El-Greadly, 2006), beans (El-Tohamy

and El-Greadly, 2007) were improved. Similarly, such enhancement influence of

active yeast extract on growth and fruiting of horticultural plants was recorded by

42

Ahmed et al. (1997), Atawia and El-Desouky (1997). Hegab et al. (1997) on citrus,

El-Mogy et al. (1998), Abd El-Ghany et al. (2001) and Ismaeil et al. (2003) on

vines.

43

Chapter 3

MATERIALS AND METHODS

3.1 EVALUATION OF RED CHILLI FOR ASSOCIATED MYCOFLORA

AND MYCOTOXINS

The sampling was done from Kunri (Sindh province) which is a hub in Asia

for red chilli production and marketing. Therefore, a survey was conducted in the

fields of Kunri to obtain the fruits of red chilli.

3.1.1 Collection of Samples

Sixty nine (69) samples of red chillies from six different cultivars, namely

Kunri, Nagina, Tall Round (T.R), Tall Pointed (T.P), Drooping Type (D.T) and

Maxi, were collected from the field of Kunri Research Station. The chillies were

harvested during August to December 2012 by hand picking at monthly intervals,

when the pods change colour from green to red. These samples were placed in air

tight polyethylene bags and brought in laboratory within 48 hours, where they were

sundried for 10-15 days. The samples were ground in a grinder, homogenized and

stored at 4 ºC for further analysis.

3.1.2 Isolation and Identification of Fungi

The samples were analysed for the presence of fungi from all parts (seed &

pericarp) of fruit in order to investigate external and internal mycoflora. A variety

of methods was applied including the standard protocols of International Seed

Testing Association (ISTA, 2001) viz. Agar Plate, Blotter Paper, Deep Freezing.

43

44

Moreover, the samples were also analysed using a dilution plate method (Sharfun

Nahar, 2004). The seeds and pericarp were surface disinfected with 2 % sodium

hypochlorite (NaOCl) for 2 minutes followed by rinsing three times in sterile

distilled water. Surface non-disinfected seeds and pericarp were also analysed.

3.1.2.1 Dilution method

Samples (10 g) were ground and added to 100 ml of distilled water to make

a stock suspension from which tenfold dilutions were made. One ml of each

dilution was spread on Potato Dextrose Agar (PDA) and incubated at 28 ºC

(Sharfun Nahar, 2004).

3.1.2.2 Agar plate method

Surface disinfected and non-disinfected seeds and pericarp were placed at

the rate of 25 seeds and 15 pieces per Petri dish, respectively on sterilized PDA.

Dishes were incubated for seven days at 28 ºC (ISTA, 2001).

3.1.2.3 Blotter paper method

Three filter (Whatman No 1, 9cm) papers were moistened with sterile

distilled water and transferred to sterilized Petri dishes. Surface disinfected and

non-disinfected seeds (25/plate) and pericarp (15/plate) were placed on filter papers

and incubated at 28 ºC for 7 days (ISTA, 2001).

3.1.2.4 Deep freezing method

In this method plating of seeds and pericarp were same as described for the

45

blotter paper method, but the cultures were incubated for 1 day at 28 ºC followed

by -20 ºC for 24 hours and then at 28 ºC for 7 days (ISTA, 2001).

After incubation, the fungi were isolated, purified and maintained on PDA

slants. Fungal colonies were observed for colony and conidial characteristics and

identified by reference to standard manuals Domsch et al., 1980; Ellis, 1971, 1976;

Booth, 1971; Raper and Thom, 1949; Raper and Fennel, 1965. The colonies were

counted and frequencies and incidence were calculated using the formulae

(Marasas, 1988):

Frequency (%) = (Ns/N) x 100

Incidence (%) = (ng/Ng) x 100

Where, Ns is the number of fungi in samples, N is the number of samples,

ng is number of infected seeds or pericarp and Ng is the total number of seeds or

pericarp. Colony forming units (cfu) were also calculated using the dilution plate

method.

3.1.3 Detection and Quantification of Mycotoxins

Thin layer chromatography (TLC) and high performance liquid

chromatography (HPLC) were performed for the detection and quantification of

mycotoxins.

3.1.3.1 Chemicals and standards

The standard mixture of AFB1, AFB2, AFG1, AFG2 and AflastarTM

Immunoaffinity columns (3ml) were purchased from Romer Labs, Austria. The

standard mixture comprised 2.02µg AFB1, 0.508 µg AFB2, 2.01 µg AFG1 and

46

0.508 µg AFG2 in 5 ml acetonitrile. Acetonitrile (Fisher-USA), methanol (Fisher-

USA) and water (Applichem- Germany) were of HPLC grade. Trifluoroacetic acid

(TFA) (Acros-USA) was used for derivatization.

3.1.3.2 Analytical method performance/ optimization

Method optimization in terms of precision, linearity, accuracy, limits of

detection (LOD) and limit of quantification (LOQ) were determined by spiking of

chilli blank samples with six concentrations 1, 2, 4, 8, 16 and 32 µg/kg of AFB1

and AFG1, 0.25, 0.5, 1, 2, 4 and 8 µg/kg for AFB2 and AFG2. One set of unspiked

chillies was used as a blank (Table 3.1). Calibration curves were drawn by

calculating the peak area (y) versus concentration (x) for each compound. Linear

regression analysis was used to determine the linearity which was expressed as the

square of correlation coefficient (R2).

3.1.3.3 Sample preparation and aflatoxin analysis by TLC

Aflatoxin analysis was carried out according to the standard protocol of

Association of Official Analytical Chemists (2000). Red chilli powder (25 g) was

macerated in 125 ml methanol-water (55:45; v/v), after that 50ml of n-hexane and 1

g sodium chloride were added and the mixture shaken for 30 minutes. Extracts

were filtered, shaken vigorously in separating funnel and two layers separated. The

lower layer (12.5ml) was collected and transferred into another separating funnel in

which chloroform (12.5ml) was added and shaken for 30-60 seconds. Again, the

lower layer was collected and evaporated over night (16 h) at room temperature.

Residual contents were redissolved in 400µl of toluene: acetonitrile (98:2; v/v).

47

Table 3.1: Spike recoveries of total aflatoxins in red chilies

Limit of Detection (LOD) – 0.1 µg/kg; Limit of Quantification (LOQ) – 0.5 µg/kg

Aflatoxin B1/G1 Aflatoxin B2/G2

Spiked

Levels

(µg/kg)

Observed

Levels

(µg/kg)

Spike

Recovery

(%)

Spike

Levels

(µg/kg)

Observed

Levels

(µg/kg)

Spike

Recovery

(%)

1 0.92 92.0 0.25 0.20 80.0

2 1.91 95.50 0.5 0.47 94.0

4 3.62 90.20 1 0.97 97.0

8 7.89 98.60 2 1.85 97.0

16 15.52 97.0 4 3.45 86.25

32 33.60 105 8 7.65 95.62

48

Extracts (40 µl) along with aflatoxin standards were loaded on to silica gel

aluminium TLC plates (20x20 cm) with 200 µm gel thickness.

Chloroform:Acetone (9:1 v/v) was used as developing solvent. The developed TLC

plates were visualized under UV light at the wavelength (λ) of 365 nm. The

standard distance migrated i.e. Retention factor (Rf) for AFB1, AFB2, AFG1 and

AFG2 were 0.45, 0.40, 0.35 and 0.30, respectively.

3.1.3.4 Sample preparation/ extraction and clean up by HPLC

Twenty five grams of red chilli powder was macerated in 100 ml of

methanol water in a ratio of 60:40 v/v. The mixture was shaken on orbital shaker

for one hour at maximum speed and filtered through whatman no. 1 filter paper.

Four millilitre of filtrate was mixed into 8 ml phosphate buffer saline (PBS) and the

pH adjusted to 7. The sample was passed through an immunoafinity column of 3ml

capacity purchased from Romer Lab, Austria. The flow rate was 0.5ml min-1

. After

washing the column with 20ml of distilled water, methanol (3ml) was passed and

collected. The sample was dreid at 55ºC and derivatized by adding 100 µl of TFA

(Akiyama et al., 2001). The sample was kept in darkness for 15 minutes and

redissolved in 400 µl acetonitrile and water (1:9 v/v). The sample was vortexed and

injected (20 µl) into the HPLC (Shimadzu SPD-20A, Japan). It was equipped by

UV detector 365 nm and C18 column, 4.6x150 mm x 5µm. Mobile phase was

water, methanol and acetonitrile in the ratio of 60:20:20v/v/v. The flow rate and

column temperature was adjusted at 1 ml per minute

and 30 ºC, respectively (Feizy

et al., 2012).

3.1.3.5 Analytical method performance

49

The HPLC chromatogram of an aflatoxins standard is shown in Figure 3.1.

AFG1 eluted at 6.958 mins followed by AFB1 (8.935), AFG2 (12.751) and AFB2

(17.593). According to linear regression analysis, the calibration curves for all four

aflatoxins indicated good linearity with a correlation of approximately 0.99.

3.2 IN-VITRO INHIBITION OF MYCOFLORA AND REDUCTION OF

MYCOTOXINS

In-vitro efficacy of botanicals and bio agents was evaluated for the

inhibition of A. flavus and reduction of aflatoxins.

3.2.1 Preparation of Plant Diffusates

Four plants: Lantana camara L. (Chandan), Euphorbia hirta L. (Bathu),

Amaranthus viridis L. (Chulai) and Trianthema portulacastrum L. (It-sit) were

collected from the Arid Agriculture University, Rawalpindi, in September 2014.

The plant specimen were identified, a voucher number allotted and submitted in the

Department of Botany, PMAS-AAUR. The plants were immediately moved to the

laboratory and carefully washed in running tap water to remove dust particles and

debris then rinsed in distilled water and shade dried at room temperature. Dried

leaves were ground into a fine powder using a home grinder (Muschietti et al.,

2005). Ten g of powder was macerated in 100 ml of 80% methanol and shaken on

orbital shaker at 150 rpm for 48 hours. The extracts were filtered through 4 layers

of muslin cloth and evaporated to dryness in a 40°C water bath. Approximately,

12mg of methanolic extract of each plant was redissolved in 1 ml of extraction

solvent to make the stock solution. Concentrations of 25, 50, 75 % were prepared

by diluting stock solution with distilled water.

50

B1

B2

G1

Figure.3.1 Chromatogram of standard mixture with pre column derivatization

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

0.0

2.5

5.0

mVDetector A:365nm

2.4

19

2.6

23

3.0

76

6.9

58

8.9

35

10.2

20

11.4

82

12.7

51

17.5

93

G2

51

3.2.2. Inhibition of A. flavus Growth by Poisoned Food Technique

In this technique, A. flavus was inoculated onto PDA, in triplicate and

incubated at 28±1°C for three days, to obtain young, actively growing colonies. For

treatment set 0.5 ml of different concentrations of plant extracts were mixed into 20

ml PDA, poured into Petri dishes and allowed to solidify at room temperature for

thirty minutes. A mycelial disc, 6 mm in diameter, cut from margin of three days

old culture, was aseptically inoculated onto the agar containing the plant diffusate.

PDA with 0.5 ml of solvent and the antifungal drug, terbinafine were used as

negative and positive controls, respectively (McCutcheon et al., 1994). The

inoculated cultures were incubated at 28±1 °C and colony diameter recorded after

seven days. Percent mycelial growth inhibition was calculated as given below:

I = C – T x 100

C

Where, I = % inhibition

C = Colony diameter in control

T = Colony diameter in treatment

The experiment was repeated twice with three replicates.

3.2.3. Reduction of Aflatoxins

The stock solution and dilutions (25, 50 and 75%) of each plant were also

tested for the reduction of aflatoxins. Briefly 5 g chilli powder was placed in 100

ml conical flask and 300 µl of AFB1 standard was added. The flask was kept in a

cool and dark place for two hours for the penetration of toxin. After that 2 ml of

each plant extract was added to the sample and mixed well. The flask was then

incubated at 28±1 °C for 16 hours. Aflatoxins were detected using the Best Food

52

(BF) method described in 3.1.3. Controls containing AFB1 but no plant diffusates

along with blank (without toxin & diffusates) were also prepared.

3.2.4. Inhibition of A. flavus and Reduction of Aflatoxins by Bio agents

Four species of bacteria and three species of Saccharomyces were evaluated

for the inhibition of A. flavus and reduction of aflatoxins.

3.2.4.1 Collection and preparation of bio agents

Bacillus subtilis (FCBP-189) and Pseudomonas fluorescens (FCBP-188)

were obtained from the Fungal Culture Bank of Punjab, Lactobacillus rhamnosus

(NRRL B-442) and Flavobacterium aurantiacum (NRRL B-184) were obtained

from United States Department of Agriculture (USDA), while Saccharomyces

bayanus (NCYC-2578), Saccharomyces cerevisiae (NCYC-505) and

Saccharomyces postorianus (NCYC-392) were from the National Collection of

Yeast Cultures (NCYC), UK.

3.2.4.2 Growth media and culture preparation

Bacterial cultures were grown in Luria-Bertan (LB) liquid medium

containing (g/L) Tryptone 10, NaCl 10 and yeast extract (Oxoid) 5. The

Saccharomyces species were grown in yeast malt (YM) broth containing (g/L)

yeast extract 3, malt extract 3, dextrose10 and peptone 5, purchased from Oxoid.

The bacterial cells were activated by transferring a loopful of culture taken from

slants to LB liquid media. The freeze-dried Saccharomyces were first activated in

10 ml of YM broth. After incubation all cultures were streaked in YM agar to

confirm the growth patterns. Finally all organisms were cultured in 100 ml YM

broth and incubated at 30 °C for 48 hours.

53

3.2.4.3. Inhibition of A. flavus by poisoned food technique

All culture suspensions were tested for inhibition of A. flavus using the

poisoned food technique as described in 3.2.2. Five hundred µl of each suspension

was mixed with 20 ml PDA and poured into Petri dishes. After solidification of the

medium, A. flavus was inoculated. Cultures were incubated for 7 days at 28±1 °C

and colony diameter measured. Percent inhibition of growth was calculated.

3.2.4.4. Reduction of Aflatoxins

Five g of ground red chillies were added to a 100 ml conical flask. 300 µl of

aflatoxin standard (1000 ng) was added and left for 2 hours for the toxin to

penetrate the sample. Two ml of culture broth was added and the mixture incubated

in the darkness at room temperature for 16 hours. Controls containing AFB1

without yeast and bacterial cells were also prepared along with a blank without

toxin and cells. The aflatoxins were detected by the BF method described in 3.1.3.

All assays were performed in triplicate and repeated twice.

3.2.4.5 Nutritional and quality profile of red chilli

The effects of the treatments, including plant extracts and bio agents on red

chilli pods were also evaluated to observe changes in quality and nutrients, if any.

3.2.4.5.1 Proximate analysis

The proximate composition of red chilli powder was determined according

to the Association of Official Analytical Chemists (AOAC, 2012) standard

techniques. Moisture was determined by drying the samples overnight at 105 °C.

54

The ash content was determined by ashing the samples overnight at 550 °C. The

crude protein content was determined by following the method of Kjeldahl and the

fat content was determined by the Soxhlet method. The carbohydrate content was

calculated by difference (total mass of moisture, total fat, ash and crude protein)

subtracted from the total mass of spices.

3.2.4.5.2 Sample extraction

A sample was extracted following the protocol of Nuutila et al. (2003). A

powdered sample of 0.1 g was extracted with 10 ml methanol by agitating for one

hour at room temperature followed by sonicating for 20 minutes in an ultrasonic

bath. The mixture was centrifuged at 3000 rpm for 20 minutes, the supernatant

decanted and the extraction repeated with an additional 10 ml of methanol. The

combined supernatant from the two extractions was used for analysis, as described

below.

3.2.4.5.3 Determination of total phenols

Methanolic extract (0.5 ml; 10 mg/mL) of Capsicum annuum was mixed

with 0.5 mL Folin-Ciocalteu reagent and 1.50 mL 7.5% Na2CO3 solution and

placed at room temperature for 1 h. After incubation, absorbance was measured at

760 nm using UV-Vis-Spectrophotometer (SP-1920, Spectrum, Germany)

(Kumazawa et al., 2002). The calibration curve was prepared using gallic acid in a

concentration range of 2-20 mg/L. The total phenolic content was calculated and

reported as gallic acid equivalent (GEA, mg of gallic acid per g dried fruit).

55

3.2.4.5.4 DPPH radical scavenging activity

Free radical scavenging activity was determined using the stable 1,1-

diphenyl-2-picrylhydrazyl (DPPH) free radical. Extract (0.5 ml) was added to 3.0

ml of a 0.004% methanolic solution of DPPH. After 30 minutes, absorbance was

measured at 517 nm (Banerjee et al., 2005). Inhibition was calculated as [1 –

Aextract /Ablank] x 100.

3.2.4.5.5 Determination of vitamin C contents

The titration method is based on the reduction of the blue dye 2, 6-

dichlorophenolindophenol (DCP) by ascorbic acid (AOAC, 2012). The end point

of the titration is indicated by the appearance of the pink acid form of the dye. This

official method is simple and was used in the determination of vitamin C content of

the chilli extract. Five ml of each sample was treated with 10 ml, 3% meta

phosphoric acid and filtered to remove possible protein interference. The filtrate

was then titrated against freshly standardized 2, 6-dichloroindophenol (0.0012%).

Standardization was with 10 ml of standard ascorbic acid. Triplicate titration was

conducted for all samples. L-ascorbic acid concentration was calculated by

comparison with the standard and expressed as mg/g dry weight.

3.2.4.5.6 Data analysis

Experiments were performed in triplicate, and data were subjected to

Analysis of Variance (ANOVA) using a Completely Randomized Factorial Design.

Means were separated by Duncan's multiple range test (DMRT), when ANOVA

was significant (P<0.05) by using SPSS 16.0 (Chicago, IL, USA).

56

3.3. MANAGEMENT TRIALS IN THE GREENHOUSE

The susceptible chilli variety Nagina was subjected to greenhouse trials for

the management of mycotoxin and mycoflora.

3.3.1 Management by Manipulating the Irrigation Regimes

In this trial different water levels were applied to the plants at the preharvest

stage to reduce A.flavus and aflatoxin contamination in red chillies.

3.3.1.1 Preparation and sterilization of potting mixture

The potting mixture (clay, farmyard manure 3:1) was sterilized with 37%

commercial formalin. One part of formalin (Saudi Formaldehyde Chemical Co.

Ltd.) was diluted with nine parts of water. The potting mixture was placed over a

cemented path in layers. The mixture was then moistened with formalin solution

and covered with a polyethylene sheet for 48 hours. Later, the mixture was exposed

to air until the formalin smell vanished.

3.3.1.2 Sowing and transplanting of Nagina

The potting mixture was placed in plastic bags (6x4 inches). The seeds were

sterilized in 15 % sodium hypochlorite for 30 minutes and 10 sterilized seeds were

sown in each bag. Plants were raised for 45 days and healthy plants were

transplanted to clay pots (9 inches diameter).

3.3.1.3 Inoculum preparation and addition to clay pots

Inoculum of A. flavus was prepared by mixing scraped mycelia of 3 days

old culture in 0.05 % Tritone X-100. The spore numbers (5x106) were adjusted by

57

haemocytometer and sprayed at the start of flowering stage. Three pots were

prepared, as three replicates for each treatment.

3.3.1.4 Treatment design

Four irrigation levels were selected by measuring Field Capacity (FC),

Wilting Point (WP) and Available Water Content (AWC) of soil. Field capacity was

tested using a pressure membrane apparatus (LEESON®, USA; Model#

C6C14DB6F) in the Department of soil sciences, PMAS-AAUR. Six treatments

were employed including control.

Treatments were as follows:

T1 = Control (inoculated with A.flavus @ 5x106

spores ml-1

)

T2 = Control (uninoculated)

T3 = No stress (100% FC + 5x106

spores ml-1

of A.flavus)

T4 = Low stress (45% AWC + 5x106 spores ml

-1 of A.flavus)

T5 = Moderate stress (30% AWC + 5x106 spores ml

-1 of A.flavus)

T6 = Severe stress (15% AWC + 5x106

spores ml-1

of A.flavus)

3.3.1.5 Parameters studied

Fruits were collected at intervals, fresh weight recorded and then chillies

were dried after which the dry weight was also noted. After the collection of whole

produce, the number of pods per plant was counted. Then from each sample, the

mycoflora was isolated and identified and aflatoxins were also assayed.

3.3.2 Management of A. flavus and Aflatoxin by Yeast Application

The trial was conducted in the glasshouse of the Botany Department in

58

PMAS-AAUR. Three types of yeasts, Saccharomyces bayanus (NCYC-2578),

Saccharomyces cerevisiae (NCYC-505) and Saccharomyces postorianus (NCYC-

392) were used.

3.3.2.1 Preparation and application of yeast inoculum

Yeast strains were subcultured to 100ml YM broth and shaken for 5 days.

Suspensions were centrifuged at 6000 x g for 15 minutes. From each strain, five

inoculum concentrations 105

,

106

, 107

, 108

, 109

cfu /ml were prepared by adjusting

cell numbers using a haemocytometer. At the start of flowering stage, yeast

suspensions were sprayed twice at15 days interval by hand-held sprayer, with 25

ml per plant. Control plants were sprayed with tap water. Each treatment was

replicated three times.

3.3.2.2. Data analysis

Data for the five parameters were recorded and analysed using SPSS with

completely randomized factorial design; means were separated by Duncans

multiple range test (DMRT).

3.4 INTEGRATED MANAGEMENT OF MYCOTOXINS IN RED

CHILLI

The best treatments selected from pre-harvest and post-harvest experiments

were incorporated to develop an integrated management strategy for mycoflora and

mycotoxin contamination. The most susceptible variety of chilli Nagina, found

during the screening trial (3.1) was inoculated with yeast strain S. bayanus selected

59

from trial 3.3.2 and treated with the optimum irrigation level chosen from trial

3.3.1. These findings were incorporated into a final integrated management trial, in

the greenhouse to achieve mycotoxin control at a pre-harvest level. This trial was

carried out in solarized and nonsolarized soil.

3.4.1 Pre-Harvest Management

Four treatments were employed in the green house:

T1 = Control (inoculated with A.flavu,s 5x106

spores ml-1

)

T2 = Control (uninoculated )

T3 = S. postorianus 109

cfu/ml +Moderate stress (45% AWC) in solarized soil

T4 = S. postorianus 109

cfu/ml+ Moderate stress (45% AWC) in non-solarized soil

3.4.2 Post-Harvest Management

After picking, the chillies were dried in the laboratory. The yield parameters

were recorded and the chillies ground. Samples were divided and subjected to the

post-harvest treatments. Each set was treated with culture filtrates of F.

aurantiacum and L. rhamnosus. Controls were not treated with filtrates. A.flavus

and aflatoxins were analysed.

3.4.3 Seed Viabilty Test

In this trial, a seed viability test was also conducted to check any harmful

effects of the combined treatments on the viability of seeds. Seeds treated with

culture filtrates were placed in 2% water agar at a rate of 25 seeds per Petri dish.

60

The dishes were incubated at 28 ºC for 7 days and the number of germinated seeds

counted, germination percent was determined. The test was performed in triplicate.

3.4.4 Data Analysis

Experiments were performed in triplicate, and data were subjected to

ANOVA using Completely Randomized Factorial Design. Means were separated

by LSD, when ANOVA was significant (P<0.05) by using SPSS 16.0 (Chicago, IL,

USA).

61

Chapter 4

RESULTS AND DISCUSSION

4.1 EVALUATION OF RED CHILLI FOR ASSOCIATED MYCOFLORA

AND MYCOTOXINS

Out of 69 samples, 47 species viz. Aspergillus candidus, A. flavus, A.

fumigatus, A. nidulans, A. niger, A. ochraceus, A. penicilloides, A. tamarii,

Alternaria destruens, Al. tomaticola, Al. alternata, Al. brassicicola, Al.

chlamydospora, Al. citri, Al. dianthicola, Al. godetiae, Al. infectoria, Al. longipes,

Al. subulata, Al. tangelonis, Al. triticina, Al. vaccariae, Bipolaris sorokiniana,

Cercospora sp., Cladosporium uridinicola, Curvularia ovoidae, Cu. brachyspora,

Cu. lunata, Cu. pallesence, Cu. tuberculata, Cu. trifolii, Drechslera sp., Fusarium

anthophilum, F. oxysporum, F. semitectum, F. solani, F. sporotricioides, F.

tabacinum, Helicorhoidion botryoideum, Penicillium corylophilum, P. expansum,

P. rubrum, P. rugulosum, Rhizomucor sp., Rhizopus oryzae, Scolecobasidium sp.,

Syncephalstrum racemosum in 14 genera were isolated using various techniques.

4.1.1 Dilution Plate Method

A total of 15 species were isolated using the dilution plating technique

(Table 4.1). The most common fungi found A. flavus with incidence of 67.80 from

cv Nagina, although it was in 0.26% samples from cv. Kunri.

4.1.2 Agar Plate Method

This method was applied to detect fungi from both pericarp as well as seeds

61

62

Table 4. 1. Occurrence (%) of mycoflora by dilution plate method from the fruits of

six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus candidus 0.66

2 Aspergillus flavus 0.26 16.62 52.22 15.46 42.38 67.80

3 A.spergillus niger 12.23 2.26 1.42 1.48 20.15

4 Alternaria alternata 56.09 4.25 12.12

5 Alternaria brassicicola 7.43 16.95

6 Alternaria citri 1.06

7 Alternaria longipes 34.59

8 Alternaria subulata 47.98 8.48

9 Curvularia pallesence 27.03

10 Drechslera sp. 1.59 1.13

11 Fusarium oxysporum 27.91 54.06 0.48 5.65 7.11

12 Fusarium solani 0.33

13 Fusarium tabacinum 27.25 17.56

14 Helicorhoidion botryoideum 1.91

15 Penicillium rugulosum 6.37

*Tall Round, ** Tall Pointed, ***Drooping type

63

of chilli. A total of 21 species in 11 genera were identified from surface disinfected

samples of pericarp (Table 4.2). A. flavus was the most common fungus (25.5%) in

cv.Nagina, but was not detected from cv. Kunri and the Drooping type. However,

seeds yielded 21 species. Here, the most common species were A. niger (21%)

followed by A. flavus (19.7%) from cv Tall Round and Nagina, respectively (Table

4.3).

Non disinfected pericarp and seeds yielded greater numbers of fungi, 26 and

24 respectively than disinfected pericarp and seeds. A. flavus showed the highest

incidence from Nagina (Tables 4.4 & 4.5).

4.1.3 Blotter Paper Method

Sixteen species of fungi in 6 genera were detected (Table 4.6). Here also A.

flavus was the most common (25 %) from Nagina, followed by A. niger (24.6 %)

from Tall round. Seeds yielded 18 fungal species in 6 genera. A. flavus (11.7 %)

and A. niger (10.13 %) were detected mainly in Nagina (Table 4.7).

Twenty one species of fungi were found in non-disinfected pericarp and 20

species from seeds. A. flavus was found with high incidence in pericarp and seeds

(Tables 4.8 & 4.9).

4.1.4 Deep Freezing Method

Thirteen species of fungi in 5 genera were isolated (Table 4.10). Overall in

this method, fungi were found in low incidence as compared to Agar plate and

Blotter paper method from pericarp as well as from seeds, although A. flavus was

64

Table 4.2. Occurrence (%) of mycoflora by agar plate method from surface

disinfected pericarp of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 1.00 13.30 3.30 25.50

2 Aspergillus niger 1.86 25.00 6.67 19.70

3 Alternaria alternata 1.50 2.88 0.50

4 Alternaria brassicicola 2.33

5 Alternaria citri 0.67

6 Alternaria destruens 0.27

7 Alternaria subulata 2.00 3.33

8 Alternaria vaccariae 0.67

9 Bipolaris sorokiniana 1.00 0.13

11 Cercospora sp. 0.33

12 Cladosporium uridinicola 0.33 1.07

13 Curvularia brachyspora 0.38

10 Curvularia ovoidae 1.00

14 Curvularia tuberculata 3.00

15 Drechslera sp. 0.50

16 Fusarium oxysporum 2.50 3.50 3.75 5.50 0.33 0.27

17 Fusarium solani 0.53

18 Fusarium tabacinum 1.17 0.07

19 Penicillium corylophilum 3.10

20 Rhizopus oryzae 0.02

21 Syncephalstrum racemosum 0.13

*Tall Round, ** Tall Pointed, ***Drooping type

65

Table 4.3. Occurrence (%) of mycoflora by agar plate method from surface

disinfected seeds of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 11.00 19.70

2 Aspergillus niger 2.22 21.00 4.00 15.47

3 Alternaria dianthicola 4.50

4 Alternaria alternata 0.33 3.00 0.75 0.40

5 Alternaria citri 0.50

6 Alternaria destruens 0.80

7 Alternaria vaccariae 0.67

8 Bipolaris sorokiniana 1.17 0.25

9 Cercospora sp. 0.47

10 Cladosporium uridinicola 0.33 1.87

11 Curvularia brachyspora

0.25

12 Curvularia lunata 0.10

13 Curvularia ovoidae 0.33

14 Curvularia tuberculata 12.50

15 Drechslera sp. 0.50

16 Fusarium oxysporum 7.83 7.25 7.75 0.67 2.00

17 Fusarium semitectum 0.40

18 Fusarium solani 1.73

19 Fusarium tabacinum 0.16

20 Penicillium rubrum 2.11

21 Scolecobasidium sp. 6.34

*Tall Round, ** Tall Pointed, ***Drooping type

66

Table 4.4. Occurrence (%) of mycoflora by agar plate method from surface non

disinfected pericarp of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 11.10 10.00 1.70 3.30 2.20 23.20

2 Aspergillus nidulans 0.11 0.25

3 Aspergillus niger 3.71 2.23 1.67 8.90 4.44 22.81

4 Aspergillus ochraceus 0.06 0.47

5 Aspergillus penicilloides 0.13

6 Aspergillus tamarii 0.50

7 Alternaria alternata 2.00 3.00 0.73

8 Alternaria chlamydospora 1.67

9 Alternaria dianthicola 5.00

10 Alternaria infectoria 0.13

11 Alternaria subulata 5.00

12 Alternaria tomaticola 0.13

13 Alternaria vaccariae 1.00

14 Bipolaris sorokiniana 4.84

15 Cercospora sp. 0.47

16 Curvularia brachyspora 0.50 0.25

17 Curvularia lunata 0.07

18 Curvularia ovoidae 0.67

19 Curvularia tuberculata 7.50

20 Drechslera sp. 0.75

21 Fusarium oxysporum 3.83 6.00 7.00 7.75 1.00 1.33

22 Fusarium solani 0.40

23 Fusarium tabacinum 0.67 0.34 0.33 0.02

24 Helicorhoidion botryoideum 1.07

25 Rhizomucor sp. 0.67

26 Syncephalstrum racemosum 0.50

*Tall Round, ** Tall Pointed, ***Drooping type

67

Table 4.5. Occurrence (%) of mycoflora by agar plate method from surface non

disinfected seeds of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 13.50 3.00 25.10

2 Aspergillus niger 3.55 25.00 3.00 4.00 13.14

3 Aspergillus ochraceus 0.22

4 Aspergillus tamarii 0.50

5 Alternaria alternata 2.83 6.25 1.03

6 Alternaria citri 1.50

7 Alternaria destruens 1.00

8 Alternaria dianthicola 10.00

9 Alternaria subulata 2.67

10 Alternaria tomaticola 0.27

11 Alternaria vaccariae 1.07

12 Bipolaris sorokiniana 2.50 0.25

13 Cercospora sp. 1.00

14 Cladosporium uridinicola 3.00

15 Curvularia brachyspora

0.38

16 Curvularia lunata 0.25 0.27

17 Curvularia tuberculata 12.5

18 Drechslera sp. 0.33

19 Fusarium oxysporum 9.17 3.50 9.50 11.75 4.33 4.80

20 Fusarium semitectum 0.80

21 Fusarium solani 2.00

22 Fusarium tabacinum 4.00 1.13 0.98

23 Helicorhoidion botryoideum 0.07

24 Scolecobasidium sp. 8.50

*Tall Round, ** Tall Pointed, ***Drooping type

68

Table 4.6 Occurrence (%) of mycoflora by blotter paper method from surface

disinfected pericarp of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 21.70 25.00

2 Aspergillus niger 24.60 10.37

3 Alternaria alternata 0.33 0.33 0.87

4 Alternaria brassicicola 1.50 3.00

5 Alternaria citri 0.17

6 Alternaria destruens 0.40

7 Alternaria subulata 1.00

8 Alternaria vaccariae 0.40

9 Cercospora sp. 0.20

10 Curvularia brachyspora 0.50

11 Curvularia lunata 0.25

12 Curvularia trifolii 0.20

13 Curvularia tuberculata 2.50

14 Drechslera sp. 0.33 0.33

15 Fusarium oxysporum 1.33 3.50

16 Fusarium semitectum 0.13

*Tall Round, ** Tall Pointed, ***Drooping type

69

Table 4.7 Occurrence (%) of mycoflora by blotter paper method from surface

disinfected seeds of six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 7.00 11.70

2 Aspergillus niger 0.67 10.13

3 Alternaria alternata 1.00 0.33 1.43

4 Alternaria brassicicola 2.17 2.00 4.50

5 Alternaria destruens 0.60

6 Alternaria godetiae 1.33

7 Alternaria vaccariae 0.67

8 Bipolaris sorokiniana 0.50

9 Cercospora sp. 0.33

10 Curvularia brachyspora

0.50

11 Curvularia lunata 3.25

12 Curvularia trifolii 0.27

13 Curvularia tuberculata 11.50

14 Fusarium anthophilum 0.34

15 Fusarium oxysporum 2.5 4.50 0.73

16 Fusarium solani 1.20

17 Fusarium sporotrichioides 0.67

18 Fusarium tabacinum 0.62

*Tall Round, ** Tall Pointed, ***Drooping type

70

Table 4.8 Occurrence (%) of mycoflora by blotter paper method from surface non

disinfected pericarp of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 2.80 26.70 28.60

2 Aspergillus niger 25.00 6.96

3 Alternaria alternata 2.00 1.20

4 Alternaria brassicola 1.75

5 Alternaria chlamydospora 2.00

6 Alternaria citri 0.50

7 Alternaria destruens 0.40

8 Alternaria dianthicola 4.00

9 Alternaria subulata 1.00

10 Alternaria tangelonis 0.07

11 Alternaria vaccariae 0.40

12 Cercospora sp. 0.33

13 Curvularia lunata 2.25

14 Curvularia trifolii 0.47

15 Drechslera sp. 0.17

16 Fusarium oxysporum 2.33 1.20 3.75 0.27

17 Fusarium semitectum 0.20

18 Fusarium solani 0.53

19 Fusarium tabacinum 1.17 6.00 0.04

20 Scolecobasidium sp. 2.20

*Tall Round, ** Tall Pointed, ***Drooping type

71

Table 4.9 Occurrence (%) of mycoflora by blotter paper method from surface non

disinfected seeds of six red chilli cultivars

S.No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 6.10 2.60 2.00

27.50

2 Aspergillus fumigatus

0.16

3 Aspergillus niger 0.45

8.53

4 Alternaria alternata

7.00

1.00 2.07

5 Alternaria brassicicola

3.20 4.00 5.00

6 Alternaria destruens

0.07

7 Alternaria dianthicola

6.00

8 Alternaria subulata

2.00

9 Alternaria tangelonis

0.20

10 Alternaria triticina

2.00

11 Alternaria vaccariae

1.13

12 Cercospora sp.

1.33

13 Curvularia trifolii

0.60

14 Curvularia tuberculata 12.50

15 Fusarium anthophilum

1.67

16 Fusarium oxysporum 5.50

7.00 2.00

1.00

17 Fusarium semitectum

1.00

18 Fusarium solani

1.13

19 Fusarium sporotrichioides

1.67

20 Fusarium tabacinum 2.22

2.38

1.04

*Tall Round, ** Tall Pointed, ***Drooping type

72

Table 4.10. Occurrence (%) of mycoflora by deep freezing method from surface

disinfected pericarp of six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 1.70 5.60

2 Aspergillus niger 1.67 0.89

3 Alternaria alternata 0.33 0.67 0.20

4 Alternaria brassicicola 1.75

5 Alternaria chlamydospora 1.67

6 Alternaria destruens 0.20

7 Alternaria diathicola 4.50

8 Alternaria subulata 2.00

9 Alternaria vaccariae 0.33

10 Cercospora sp. 0.07

11 Curvularia lunata 1.13 0.25

12 Curvularia tuberculata 1.00

13 Fusarium oxysporum 1.83 1.25

*Tall Round, ** Tall Pointed, ***Drooping type

73

again most commont with an incidence of 5.6 %. The seeds yielded 15 species

(Table 4.11). A. flavus (12%) was the most common. This method yielded 18

species from non-disinfected pericarp and 19 species from seeds. In both cases A.

flavus was found at an incidence of 20 % in the Nagina cultivar (Table 4.12 &

4.13).

Overall the highest fungal numbers were obtained by the agar plate method

with 33 species from pericarp and 26 from seeds, followed by Blotter paper method

with 22 species from pericarp and 24 from seeds. Deep freezing yielded 21 species

from pericarp and 20 from seeds, while the dilution plating method gave the lowest

number of species (14). Results for the blotter paper method were similar to those

reported by Sharfun-Nahar et al. (2004), where seeds yielded higher number of

fungi than pericarp. However, these results differ in the case of deep freezing, as in

the present study pericarp yielded more fungi. The deep freezing method is suitable

for the detection of slow growing parasitic fungi because they draw nutrition from

the dead seed embryo. Furthermore the growth of fast growing saprophytic fungi is

checked due to the interrupting deep-freezing period of twenty four hours.

Such a high fungal diversity in red chilli detected in the present study was

also supported by earlier reports. Kobina and Ebenezer (2012) investigated the fruit

borne mycoflora of Capsicum annuum from Accra metropolis. Eighteen fungal

species in eight genera were found in surface sterilized and non-sterilized fruits.

The highest (2.79 log10 CFU/g) fungal load was recorded with A. flavus being the

most common species. Three isolates of Colletotrichum capsici, and single isolates

of Alternaria alternata, Fusarium pallidoroseum, F. moniliforme, F. oxysporum,

74

Table 4.11. Occurrence (%) of mycoflora by deep freezing method from surface

disinfected seeds of six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 1.00 12.00

2 Aspergillus niger 0.53

3 Alternaria alternata 0.17 1.00 0.40

4 Alternaria brassicicola 2.67 2.25

5 Alternaria destruens 0.67

6 Alternaria dianthicola 2.00

7 Alternaria vaccariae 0.40

8 Cercospora sp. 0.40

9 Curvularia lunata 1.38

10 Curvularia trifolii 0.27

11 Curvularia tuberculata 12.50

12 Fusarium oxysporum 1.00 6.00 1.50 0.07

13 Fusarium semitectum 0.40

14 Fusarium sporotrichioides 0.33

15 Fusarium tabacinum 0.73

*Tall Round, ** Tall Pointed, ***Drooping type

75

Table 4.12. Occurrence (%) of mycoflora by deep freezing method from surface

non disinfected pericarp of six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 20.00

2 Aspergillus niger 7.11

3 Alternaria alternate 1.67 0.80

4 Alternaria brassicicola 1.25 2.50

5 Alternaria destruens 0.60

6 Alternaria triticina 0.25

7 Alternaria vaccariae 0.47

8 Cercospora sp. 0.40

9 Curvularia brachyspora

0.38

10 Curvularia lunata 0.25

11 Curvularia trifolii 0.33

12 Curvularia tuberculata 3.50

13 Drechslera sp. 0.17

14 Fusarium anthophilum 0.65

15 Fusarium oxysporum 1.00 2.50 0.50

16 Fusarium tabacinum 1.28

17 Helicorhoidion botryoideum 0.27

18 Penicillium corylophilum 0.10

*Tall Round, ** Tall Pointed, ***Drooping type

76

Table 4.13. Occurrence (%) of mycoflora by deep freezing method from surface

non disinfected seeds of six red chilli cultivars

S. No Name of Fungi Kunri Maxi TR* TP** DT*** Nagina

1 Aspergillus flavus 3.10 20.10

2 Aspergillus niger 25.00 2.40

3 Alternaria alternate 2.0 0.33 1.33

4 Alternaria brassicicola 4.25 6.00

5 Alternaria citri 0.33

6 Alternaria destruens 0.47

7 Alternaria subulata 0.67

8 Alternaria vaccariae 1.13

9 Cercospora sp. 0.67

10 Cladosporium uridinicola 3.00

11 Curvularia lunata 0.75 1.75

12 Curvualria pallesence 0.22

13 Curvularia trifolii 0.67

14 Curvularia tuberculata 12.50

15 Fusarium oxysporum 1.60 7.25 1.34 0.27

16 Fusarium semitectum 0.53

17 Fusarium solani 0.53

18 Fusarium sporotrichioides 1.33

19 Fusarium tabacinum 3.62 0.88 1.20

*Tall Round, ** Tall Pointed, ***Drooping type

77

and Aspergillus flavus were found in diseased samples of chilli fruits in India

(Parey and Sofi, 2013). In contrast, Sharfun-Nahar et al. (2004) has reported 47

fungal species from an Indian consignment of red chilli. Among them, A. flavus, A.

niger, A. alternata, Chaetomium bostrychodes, F. moniliforme, Paecillomyces sp.

and R. stolonifer dominated in seeds and pericarp. A. flavus occurred in all pericarp

samples. Species of Alternaria, Colletotrichum, Fusarium and Phoma were

reported by Hashmi (1989) from samples of capsicum imported from India. Wadia

et al. (1983) reported that the fruit surface mycoflora of C. annuum included A.

niger, P. citrinum, and F. semitectum. However S. racemosum, P. theae, A.

flevipes, C. herbarum, Phoma sp. R. minutus, and S. oryzae were isolated less

frequently. Mushtaq and Hashmi (1997) found eleven species, including F.

anthophilum, A. alternata, Cephalosporium acremonium, F. moniliforme, F.

solani, F. oxysporum, F. proliferatum, Macrophomina phaseolina, Rhizoctonia

solani and Pythium aphanidermatum in red chillies in Mirpurkhas Sindh. Another

report from Sindh (Hussain et al., 2013) showed that, of five fungi found, A. flavus,

A. niger and Colletotrichum capsici dominated. A. solani and A. alternata were less

frequent. Jamiolkowska (2009) isolated A. alternata, Colletotrichum coccoides, F.

oxysporum, F. equiseti, F. solani, Gilmaniella humicola, P. janczewskii, P.

cyclopium, Gliocladium roseum, T. hamatum and T. harzianum from red chilli

plants in Poland.

Red chilli is clearly heavily contaminated with fungal flora at every stage

of production. Hence, pre and post-harvest losses of red chillies pose a major

challenge to developing countries like Pakistan. Due to this problem quality of both

seeds and fruits of this cash crop is being reduced as these fungi produce

78

mycotoxins which cause health hazards in humans and animals. Several

mycotoxins, like deoxynivalenol, zeralenone, fusarubin, bostrycoidin,

moniliformin, aflatoxins and ochratoxins are produced by the fungi isolated in this

study (Yogendrarajah et al., 2014b). A. flavus which is the most common fungus

of this genus found in Capsicum is known to produce aflatoxins which are

carcinogenic (IARC, 2002).

This fungus was the most common species isolated in the present study. A

summary of colony forming units (cfu), frequency and incidence of Aspergillus

flavus isolated using the different techniques is presented in Table 4.14. Nagina

was a highly contaminated variety, with a frequency of 93%, 2.14x104

cfu and high

incidence in the range of 5.6-28.6%. It yielded A. flavus with all techniques tested.

However, in the cv. Drooping Type, A. flavus was detected (2.2%) using the agar

plate technique from the pericarp of non-disinfected samples only. Frequency was

33%, however, cfu was higher in dilution technique. In contrast, the lowest cfu was

found in Kunri and the fungus was only restricted to non-disinfected samples of

seeds and pericarp. This report is the first to detect the mycoflora in six local

cultivers of chilli from Kunri, Sindh.

.

4.1.5 Detection and Quantification of Mycotoxins

Of 69 samples, 46 (67%) were positive for aflatoxins B1 and B2. However,

AFG1 was detected in only one sample and G2 was not detected in any sample. The

incidence of B1 and total aflatoxins was highest in Nagina (78.8%) while it was

lowest in cv Kunri, and Drooping Type, at 50% in each cultivar (Figure. 4.1).

79

Table 4.14. Incidence (%) and frequency (%) of A. flavus detected from six red chilli cultivars by using dilution plate, agar plate,

blotter paper and deep freezing methods

*SND=Surface non-disinfected, ** SD= Surface disinfected

T.R – Tall round, T.P – Tall Pointed, D.T – Drooping type

PERICARP SEED

Frequency

Dilution

Method

Agar

Plate

Blotter

Paper

Deep

Freezing

Agar

Plate

Blotter

Paper

Deep

Freezing

VARIETY %age Cfu SND* SD** SND* SD** SND* SD** SND* SD** SND* SD** SND* SD**

Kunri 66.7 5.5x10 11.1 --- 2.8 --- --- --- 13.5 --- 6.1 --- 3.1 ---

Maxi 50 2.5x103 10 1 --- --- --- --- --- --- 2.6 --- --- ---

TR 75 1.23x 10 4 1.7 13.3 26.7 21.7 --- 1.7 3 11 2 7 --- 1

TP 50 6.93x103 3.3 3.3 --- --- --- --- --- --- --- --- --- ---

DT 33 8x103 2.2 --- --- --- --- --- --- --- --- --- --- ---

Nagina 93 2.14x104 23.2 25.5 28.6 24.6 20.0 5.6 25.1 19.7 27.5 11.7 20.1 12.0

80

Figure 4.1 Incidence (%) of aflatoxins in different cultivars of red chillies

T.R= Tall Round, T.P= Tall Pointed, D.T= Drooping Type

81

Highest concentrations were recorded in Nagina, with AFB1 and total

aflatoxins contamination levels of 87.7 µg/kg and 121.9 µg/kg, respectively (Table

4.15). Lowest contamination levels were found in Kunri, with 5.5 µg /kg AFB1 and

7.9 µg/kg total Aflatoxins, and Drooping types with 6.3 µg/kg AFB1 only. This

result correlated with the presence of A. flavus in the chillies as incidence and level

was also high in the same variety. The correlation for aflatoxins and fungal

contamination was found with the drooping type variety also. Data for other

cultivars did not correlate with the aflatoxin contamination, agreeing with the

findings of Paterson (2007) who reported that occurrence of A. flavus was not

correlated with the production of aflatoxins. Month wise difference in

contamination was also noticed as the samples collected in the month of August

had high levels of contamination with an average of 284 µg/kg, compared with

14.7 and 5.4 µg/kg in October and December, respectively. The month of August

was the driest month with no rainfall as compared to October and December with

average rainfall of 6 mm in each (Anonymous, 2015). These data suggest

correlation between drought and production of aflatoxins. Qualitative variation in

aflatoxins was also observed. AFB2 was produced in August, while in October only

one sample showed the presence of AFB2, however the production of AFG2 was

recorded in the December samples.

Surveys and studies reported from Pakistan (Paterson, 2007) showed high

levels of aflatoxin contamination (0.1-96.2 µg/kg) in red chilli samples collected

from Karachi, Pakistan. Khan et al. (2013) found mean aflatoxin levels in

powdered, crushed and whole chillies of 27.8, 31.2 and

82

Table. 4.15 Aflatoxin contamination (mean) levels (µg/kg) and range of aflatoxin concentration in different cultivars.

Aflatoxin contamination levels (µg/kg)

Number of samples in range µg/kg

Varieties AFB1 AFB2 AFG1 AFG2 Total Aflatoxins Min max ˂1 1-5 5-50 50-100 100-600

Kunri 5.5 <LOD 2.4 <LOD 7.9 1.2 30.3 --- 3 2 --- ---

Maxi 22.4 <LOD <LOD <LOD 22.4 15.2 63.6 --- --- 2 1 ---

T.R 37.7 53.34 <LOD <LOD 91.1 3.6 272.7 --- 1 2 --- 1

T.P 45.2 49.95 <LOD <LOD 95.2 2.4 266.6 --- 2 --- --- 1

D.T 6.3 <LOD <LOD <LOD 6.3 2.4 27.3 1 2 2 --- ---

Nagina 87.7 34.17 <LOD <LOD 121.9 1.2 600.0 --- 4 14 2 6

T.R – Tall round, T.P – Tall Pointed, D.T – Drooping type

83

11.7µg kg-1

. In another study, samples collected from all over Pakistan were

contaminated with B1 in whole and powdered red chillies. In contrast, work in

Peshawar (Hussain et al., 2012) showed that powdered red pepper was

contaminated below the maximum allaowable level. Eighteen samples analysed, 12

samples were found contaminated with aflatoxin B1. Iqbal et al. (2010a) reported

that the number of positive samples for aflatoxin B1 was higher (73.0 %) in

powdered chillies than whole chillies (86.4 %). Hoowever, in powdered chillies,

the concentrations were 0.00-89.56 µg/kg, compared with 0.00-96.3 µg/kg in

whole chillies. In another study (Iqbal et al., 2010b), authors made comparison

between the samples of summer and winter collected fruit from Punjab to

determine aflatoxin contamination. In winter the ranges of aflatoxins were 0.00-

52.30 µg/kg and 0.00-74.60 µg/kg in whole and ground chillies, respectively. In

summer samples the concentrations were 0.00-61.50 µg/kg and 0.00-95.90 µg/kg in

whole and ground chillies, respectively. Iqbal et al. (2011) reported the effect of

climate change on aflatoxin contamination in chillies from Punjab. Whole chillies

and ground chillies were contaminated with aflatoxins in a range of 0.00 to 81.5 µg

kg-1

and 0.00 to 84.6 µg kg-1

, respectively. Khan et al. (2013), reported

contamination in a range of 6.62-148.75 µg/kg from whole red chillies collected

from Kunri-Sindh. The occurrence of aflatoxins above the maximum permitted

level in previous studies are in agreement with the present report of high

contamination levels in red chilli samples although they were collected from field.

The present study strongly suggested that aflatoxin contamination in red

chilli is not only a storage problem, but also prevails at the pre-harvest stage.

Health hazards and critical control point (HACCP) based management is required

84

to reduce aflatoxins in this crop. Selection of resistant varieties is the initial step

towards mycotoxin management strategies. In this work, two chilli varieties

Drooping Type and Kunri, were found to be less contaminated by aflatoxins as

compared to other varieties grown in Kunri, Sindh. Farmers could therefore,

benefited by cultivating these varieties which can boost Pakistan‟s economy. This

study carried out in Pakistan for evaluation of A. flavus, aflatoxin incidence and

contamination level in chilli cultivars to determine variations in susceptibility and

resistance. The most susceptible cultivar Nagina, was selected for further studies of

aflatoxins and A. flavus management.

4.2 IN-VITRO INHIBITION OF MYCOFLORA AND REDUCTION OF

MYCOTOXINS

Based on the results of objective number 1, the most common fungus, A.

flavus isolated from the highly susceptible cv. Nagina, was chosen for testing

management options in laboratory trials. Diffusates extracted from various plants

and culture filtrates of bioagents were used.

4.2.1 Inhibition by Plant Diffusates

Methanolic extracts from leaves of four plants, Lantana camara, Euphorbia

hirta, Amaranthus viridis and Trianthema portulacastrum were screened against A.

flavus isolated from Nagina cv. for inhibition of mycelial growth and aflatoxin

reduction in red chilli pods.

4.2.1.1 Inhibition of A. flavus growth

Poisoned food technique was used to test inhibition of the growth of A.

85

flavus. All plants were equally effective against A. flavus with maximum activity of

63.3% at the highest concentration (12mg/ml) showed by T. portulacastrum,

followed by L. camara (61.1%), E. hirta (58.5%) and A. viridis (56.3%) (Figure

4.2). There was no significant (P > 0.05) difference in antifungal activity between

the plants as indicated by the DMRT. The replicates/ dilutions of L. camara

showed consistency in their antifungal ability. Dabur et al. (2007) found no

antifungal activity of methanolic extracts of L. camara against A. flavus, but a

water extract was active. Mostafa et al. (2011) observed that methanolic extracts of

this plant had weak antifungal (19%) activity against A. flavus. Previous studies

(Rao et al., 2010; Singh & Kumar, 2013) reported that there was no inhibitory

effect of E. hirta on the growth of A. flavus. Weak antifungal activity of A. viridis

against A. flavus was detected (Sarwar et al., 2016). T. portulacastrum showed no

antifungal activity against A. flaus (Kavitha et al., 2014). However, chloroform

extracts of this plant were active against various other human and fungal plant

pathogens with moderate inhibition in comparison to standard drugs (Sharma et al.,

2011). Adjou et al. (2012) recommended the use of oil of L. camara leaves to

inhibit fungal growth and aflatoxin production. This may be due to presence of

fungitoxic components in the oil.

4.2.1.2 Reduction of Aflatoxin B1

Red chilli pods deliberately contaminated with 1000 ng/g of AFB1 were

treated with diffusates from four types of plants. AFB1 was only reduced by E.

hirta extract (Figure 4.3) while other treatments were inactive, even those with

86

Figure 4.2 Efficacy of plant diffusates on inhibition of radial mycelial growth of A. flavus

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

L. camara E. hirta A. viridis T. portulacastrum

48.5

34.4

40.0 37.8

52.6

34.8

48.9

38.5

55.2

47.8

51.9 51.5

61.1 58.5

56.3

63.3 p

erce

nta

ge

Plant species

25% dilution

50% dilution

75% dilution

100% stock

solution

87

Figure 4.3. Efficacy of plant diffusates on reduction of aflatoxin B1.

88

antifungal activity. The reason may lie behind the fact that some plant metabolites

are more active as aflatoxin inhibitors than as fungal growth suppressors (Kumar et

al., 2007; Prakash et al. 2010b). Hence it is concluded here that E. hirta possesses

both antifungal and antiaflatoxigenic activity. E. hirta leaves are rich in active

constituents like tannins, alkaloids, flavonoids, glycosides, sterols, saponins and

proteins (Rao et al., 2010). The inhibitory activity is may be due to one or more

groups of these constituents. Extracts of several other plants inhibited A. flavus and

aflatoxins production but literature is scarce on the activity of the plants selected in

present study.

Increasing concerns about food safety have recently led to the development

of natural antifungal agents to control food borne pathogens. Weeds are unwanted

plants grown seasonally in large quantities. In the present study this easily

accessible resource was utilized against pathogens and production of toxic

metabolites to provide an economical and ecofriendly solution against these

problems.

4.2.2 Inhibition by Bioagents

Four bacteria and three Saccharomyces species were screened for their

ability to inhibit the radial mycelial growth of A. flavus isolated from Nagina

cultivar, and to reduce aflatoxins in red chilli pods.

4.2.2.1 Inhibition of A. flavus growth

In-vitro inhibition of A. flavus was investigated by poisoned food technique.

There was significant (P < 0.05) inhibition of A. flavus growth by all biological

treatments but L. rhamnosus was the most effective microorganism in controlling

89

fungal growth (Figure 4.4). The lowest activity was observed in the P. flourescens

treatment (Figure 4.4). Differences might be due to the nature of the species itself,

substrate and/or culture condition (Kim, 2007). Several mechanisms have been

proposed for the inhibitory effects of bacteria on fungal growth, such as secondary

metabolites, nutritional competition, pH or combinations of these mechanisms (El-

Nezami et al., 2002). In this study, the low activity of B. subtilis is on line with

result of Haggag et al. (2014) who recorded its antifungal activity against A. flavus.

Previously, Kim (2007) observed growth inhibition of A. parasiticus in a

range of 20.9-86.2% with B. subtilis. In another study, a B. subtilis strain showed

antifungal properties against various fungi (Klich et al., 1991; Munoz et al. 2010).

L. rhamnosus was the most effective microorganism in controlling fungal growth.

Production of antifungal peptides by Bacillus cereus, Streptomyces setonii

Pseudomonas syringae was reported by Pohanka (2006). Inhibition of

mycotoxigenic fungi is necessary in order to avoid toxin formation in food and

animal feed. Priority should be given to the use of natural resources such as

beneficial microorganisms to control mycoflora. The use of antifungal

Lactobacillus (LAB) could possibly replace chemical preservatives used in the food

industry, to produce organic food without addition of chemicals. In addition to the

already known excellent properties of LAB these biological products could

enhance the nutritional value and prolong the shelf life of food.

90

Figure 4.4. Effect of bacterial culture filtrates on radial mycelial growth of A.

flavus

91

4.2.2.2 Reduction of Aflatoxin B1

In this assay significant (P < 0.05) inhibition of AFB1 occurred in

treatments with L. rhamnosus (74%) and F. aurantiacum (60%), P. fluorescens

showed lowest activity (35%). However, B. subtilis did not show any activity

(Figure 4.5). The performance of the Sacharomyces species was similar (Figure

4.6) as there were no significant (P > 0.05) differences between inhibition

activities. Earlier reports supported the present results, but almost all previous work

focused on the inhibition of AFB1 production in liquid media. According to these

reports LAB strains were the most promising bio-agents for the detoxification of

AFB1. The detoxifying ability of LAB was shown by many authors Zhang et al.

(1990), Abdella et al. (2005), Kasamani et al. (2012), Shetty and Jepersen (2006),

Hernandez-Mendoza et al. (2009), Dalie et al. (2010), and Haskard et al. (2001)

due to the presence of metabolites. Other investigations reported the transformation

of AFB1 by lactic acid bacteria into the nontoxic aflatoxins B2a in acidogenous

yogurt (Megalla and Hafez, 1982). In addition the amount of AFB1 also reduced

due to fermentation of yogurt and acidified milk (Rasic et al., 1991). Despite the

fact that the mode of action of AFB1 reduction by lactic acid bacteria is still

unknown, it was proposed that aflatoxins are bound to the cell wall components of

bacteria. In support, Haskard et al. (2001) suggested that AFB1 is bound to the

bacteria by weak non covalent interactions, such as associating with hydrophobic

pockets on the bacterial surface. Lactic acid bacteria have been shown to bind

mycotoxins to their components of cell wall. It is known that toxin binding in LAB

is strain dependent (Haskard et al., 2001) and many of them possess considerably

high potential of mycotoxin binding ability (Shetty and Jespersen, 2006). Lactic

92

Figure 4.5 Effect of culture filtrates of bacteria on reduction AFB1 in red chilli.

0.0

60.0

74.0

35.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

B. subtilis F. aurantiacum L. rhamnosus P. fluorescens

per

cen

tage

Bacteria

93

Figure 4.6. Effect of culture filtrates of yeast on reduction of AFB1 in red chilli.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

S. pastorianus S. cerevesiae S. bayanus

per

cen

tage

Yeast

94

acid bacteria have been previously reported to bind various dietary contaminants

such as aflatoxins, as well as suppression of mycotoxin-producing fungal growth

(Hernandez-Mendoza et al., 2009; Dalie et al., 2010).

Among LAB strains, L. rhamnosus was the most efficient at binding AFB1

(Kasamani et al., 2012). The destruction of specific components of the bacterial

cell wall, e.g., carbohydrates and proteins, resulted in reduction in AFB1 binding by

L. rhamnosus strain GG (Hernandez-Mendoza et al., 2009). It is likely, however,

that multiple components are involved in AFB1 binding. Differences in AFB1

binding between strains are probably due to different bacterial cell wall and cell

envelope structures (Peltonen et al., 2001). Even dead bacterial cells can

decontaminate AFB1. In the work of Bovo (2014) dead bacterial cells were selected

for decontamination of aflatoxin, because viable cells would probably ferment the

medium, which is undesirable in food items. This effect has been explained by

Lahtinen et al. (2004): binding of toxin with dead cells is due to the physical union

of cell wall components, particularly peptidoglycans and polysaccharides, instead

of covalent bonding or degradation by microbial metabolism. The impact of

inactivation treatments on the ability of four strains of Lactobacillus spp. to adsorb

AFB1 was assessed and recorded by Azab et al. (2005) and it was found that

following heat (33.5 - 71.9%) and acid (58.6 - 87.0%) treatments, binding

capability was increased as compared to the viable cells in buffer solution (16.3 -

56.6%). However, alkaline (8.3 - 27.4%) and ethanolic (15.9 - 46.5%) treatments

reduced the amount of adsorbed AFB1. This phenomenon was also observed by

others (Shahin, 2007; Peltonen et al., 2001; El-Nezami et al., 1998a,b;

Kankaanpaa et al., 2000 and Pierides et al., 2000) who all suggested that physical

95

binding was one mechanism of toxin removal. Binding is probably reversible, but

the stability of the complexes formed depends on strain, treatment and

environmental conditions. The effective removal of AFB1 by all dead bacteria

suggests that binding rather than metabolism is involved in all cases. Denaturation

by high temperatures did not cause the most strongly bound AFB1 to be released.

Moreover, AFB1 was not bound to loosely attached bacterial components.

Flavobacterium aurantiacum is the only micro-organism tested so far

which significantly remove aflatoxin from liquid medium and food products with-

out the production of toxic by-products (Ciegler et al., 1966; Hao and Brackett,

1988; Line and Brackett, 1995). So far, the mechanism by which this organism

detoxifies aflatoxins is not known. However, Smiley and Draughon (2000)

demonstrated that the degradation of AFB1 by F. aurantiacum was enzymatic.

Zorlugenc and Evliya (2011) obtained reductions of AFB1 (96.52%) in highly

contaminated red chillies (2000ng/g AFB1).

In present study, B. subtilis did not reduce AFB1 which is not in agreement

with previous reports in which moderate (Haggag et al., 2014) or remarkable

reduction of AFB1 (21.6 to 70.4%) by B. subtilis (Kim, 2007) was observed.

Moreover, there are few previous studies of aflatoxin degradation by S. postorianus

during fermentation of alcoholic beverages (Inoue et al., 2013). An overall

summary of the assays given in 4.2.1 and 4.2.2 is presented in Figure 4.7, from

which it can easily be concluded that the most effective bio agents for the inhibition

of AFB1 were L. rhamnosus and F. aurantiacum.

96

Figure 4.7. Effect of plant diffusates and bio agents on reduction of aflatoxin B1 in red chilli

0

45.7

0.0 0.0

41.0

48.7 48.7

0.0

60.0

74.0

35.0

0

10

20

30

40

50

60

70

80

per

cen

tage

Plants & Bio agents

97

4.2.3 Nutritional and Quality Profile of Red Chilli

Whenever food commodities are infected and spoiled with aflatoxins, only two

possible approaches are applicable; 1) the contaminated products can be destroyed, or 2)

the level of toxins can be decreased to less toxic or non-lethal concentrations.

Particularly in developing nations, the contaminated items are generally consumed by

people and animals, due to economical and health issues. Hence, cost effective, rapid

and safer methods for elimination of these contaminants are needed. However,

prevention of reduced nutritional value in detoxified commodities should be assured. In

this study, therefore, nutritional and quality profiles of treated red chillies were

examined. The data are presented in Table 4.16 & 4.17.

4.2.3.1 Proximate analysis

Moisture content increased in all treated samples due to addition of culture

filtrates. Ash and carbohydrates decreased. Fat, fiber and proteins increased. However

energy content remained the same in all samples except those treated with E. hirta.

4.2.3.2 Determination of total phenols

Polyphenol contents did not change significantly in samples treated with L.

rhamnosus and P. fluorescens, however they increased in other treatments except S.

bayanus

4.2.3.3 DPPH radical scavenging activity

Inhibition of DPPH radical scavenging activity in treatments with increased

98

Table 4.16 In-vitro effect of plants and bio agents on nutritional profile of red chilli

S. No Treatments Moisture (%) Ash (%) Fat (%) Fiber (%) Protein (%)

Carbohydrate

(%)

Energy

(Kcal/100g)

1 Control 11.55 d* 6.87 a* 8.76 d* 6.13 c* 12.89 d* 53.56 c* 344.56 abc*

2 Control+AFB1 12.54 d 6.73 a 9.48 c 6.82 b 8.60 g 56.72 a 346.50 abc

3 S. pastorianus 33.84 a 6.08 bc 8.81 d 7.26 ab 15.41 a 48.28 e 334.03 abc

4 S. cerevisiae 31.38 b 5.56 d 11.79 a 7.73 a 14.03 c 47.03 f 350.28 a

5 S. bayanus 33.03 a 6.65 a 8.72 d 7.72 a 15.16 b 47.69ef 329.81 cd

6 F. aurantiacum 33.66 a 5.67 cd 10.76 b 6.81 b 15.39 a 47.23 f 347.24 ab

7 L. rhamnosus 32.96 a 6.13 b 7.80 e 7.01 b 15.38 a 49.63 d 330.11 bc

8 P. fluorescens 33.98 a 6.00 bc 9.56 c 7.62 a 12.67 d 49.95 d 336.54 abc

9 E. hirta 14.04 c 6.94 a 7.10 f 7.61 a 11.67 f 54.84 b 312.62 d

*Means in column followed by the same letters are not significantly different according to DMRT

99

Table 4.17 In-vitro effect of plants and bio agents on quality profile of red chilli

S. No Treatments β Carotene (ppm) Polyphenol (mg

GAE/g)

% Inhibition

(DPPH)

Capsaicin

(ppm)

Vitamin C

(mg/g)

1 Control 43.20 e* 9.90 e* 36.84 e* 23.25fg* 1.23 c*

2 Control+AFB1 38.28 f 11.20 d 42.16 c 29.73 b 1.39 b

3 S. pastorianus 51.58 c 12.61 b 45.80 a 28.68 de 1.54 a

4 S. cerevisiae 31.97 g 12.11 c 39.77 d 26.36 ef 1.37 b

5 S. bayanus 47.28 d 9.58 f 27.36 h 46.49 a 1.10 e

6 F. aurantiacum 46.80 d 8.54 g 24.15 I 45.39 ab 0.98 f

7 L. rhamnosus 57.30 b 9.87 ef 31.71 f 42.98 bc 1.18 cd

8 P. fluorescens 46.74 d 9.72 ef 29.63 g 40.46 c 1.13 de

9 E. hirta 62.03 a 12.95 a 44.77 ab 20.83 g 1.49 a

*Means in column followed by the same letters are not significantly different according to DMRT

100

by S. pastorianus and S. cerevisiae and E. hirta, but decreased in the remaining

treatments (Table 4.17).

4.2.3.4 Determination of vitamin C contents

Vitamin C remained the same or increased in all treatments, except for

samples of P. fluorescens and S. bayanus (Table 4.17).

4.2.3.5 Determination of capsaicin

Capsaicin content increased in all treatments, with the exception of E.

hirta. (Table 4.17).

On the basis of these assays it was concluded that L. rhamnosus and F.

aurantiacum were the best bio agents, as few parameters changed in the treatments.

These treatments were selected for the integrated management trials for control of

aflatoxins and A. flavus on chilli plants.

4.3 MANAGEMENT TRIALS IN GREENHOUSE

The cultivar Nagina was subjected to greenhouse trial for the management of

aflatoxins and A. flavus. Yield parameters were also recorded to determine the

growth level of the crop.

4.3.1 Management by Manipulating the Irrigation Regimes

Four levels of irrigation were applied for management of aflatoxin and A.

flavus. The treatment with 45% available water content eliminated aflatoxin

101

contamination and reduced fungal growth, whereas yield parameters were not

significantly affected. However, in other treatments, there was significant (P <

0.05) decrease in yield as it was so low that it was not possible to investigate

aflatoxin content. Therefore the 45% AWC treatment was considered most useful

in controlling aflatoxins (Table 4.18).

The basic concept directing the design of this experiment was that

aflatoxins contamination increases during drought stress. It has been proposed that

drought stress contributes to increased airborne conidia, because the fungus

survives in drier soils and is more easily disseminated by wind blowing dry soil

containing the conidia (Payne et al., 1986). Because A. flavus is predominately

soilborne, plant debris in the soil is assumed to be the primary source of airborne

inoculum. The factors that influence survival of A. flavus in soil are not well

understood, but the fungus may not compete well with other fungi in moist soil.

The cooler and wetter season may reduce initial levels, as well as survival and

growth of the fungus. Ultimately this water stress appears to be involved in

aflatoxin contamination in crops. The results of the present study suggest that by

controlling irrigation levels, fungal growth can be reduced which ultimately leads

to mycotoxin reduction or elimination.

4.3.2 Management by Yeast Application

Following spraying spore suspensions of S. cerevisae, S. bayanus and S.

postorianus on to plants, there was a highly significant (P < 0.05) effect of S.

cerevisae and S. bayanus on inhibiton of aflatoxins contamination and occurrence

of A. flavus. S. postorianus did not reduce aflatoxin production except

102

Table 4.18 Effect of irrigation levels on the management of A. flavus and aflatoxin B1 production in a greenhouse trial.

Treatments No of pods Fresh Wt.

of pods (g) Dry Wt. of

pods (g) Aflatoxin A. flavus

% Incidence

T1- Control (inoculated with A.flavus @ 5x106 ml-

1) 15.00 a* 29.85 a* 9.84 a* 96.15 a* 80.12 c*

T2- Control (uninoculated ) 18.75 a 32.16 a 11.42 a 27.47 b 1.56 a

T3-No stress (100% FC+ A.flavus @ 5x106 ml-

1) 1.75 a 3.03 a 0.82 a --- 0.70 a

T4- Low stress (45% AWC+ A.flavus @ 5x106 ml-

1) 20.50 a 36.37 a 11.16 a ND c 6.17 a

T5- Moderate stress (30% AWC+ A.flavus @ 5x106

ml-1) 2.25 a 6.09 a 1.47 a --- 72.27 c

T6- Severe stress (15% AWC+ A.flavus @ 5x106 ml-

1) 5.50 a 10.55 a 3.08 a --- 34.93 b

*Means in column followed by the same letters are not significantly different according to DMRT

103

at the highest dose (Table 4.19). However, fungal contamination was significantly

decreased. Increase in yield parameters (number and weight of pods) were also

observed in almost all treatments but they were not significant (P > 0.05).

Foliar spray of bio-stimulants such as yeast and chitosan significantly

increase growth parameters such as number of leaves, fresh and dry weight of

leaves, stem and plant height (Shehata et al. 2012). Application of dry yeast has a

positive effect on plants like tomato and magnolia, possibly due to the natural

source of amino acids, enzymes, cytokinins, minerals and vitamins (Khedr & Farid,

2000; Mahmoud, 2001), stimulation of synthesis of chlorophyll, protein, nucleic

acids and of cell division and cell enlargement (Kraig and Haber, 1980;

Castelfranco & Beale, 1983). During stress conditions, yeast plays an important

role due to its content of cytokinins (Barnett et al., 1990). Several workers reported

the beneficial effects of active yeast extracts on growth and yield of vegetable

crops such as beans (Fathy & Farid, 1996; Amer, 2004; El-Tohamy & El-Greadly,

2007), eggplant (Hewedy et al., 1996 and El-Tohamy et al., 2008), pea (Tartoura,

2001; El-Desuki & El-Greadly, 2006), potatoes (Sarhan & Abdullah, 2010 and

Ahmed et al., 2011), snap bean (Fawzy et al., 2010), sweet pepper (Ghoname et

al., 2010) and on tomatoes (El-Ghamriny et al., 1999 and Fathy et al., 2000).

Chitosan and yeast application significantly increased quality parameters such as

fruit diameter, fruit weight, fruit length, total soluble solids (T.S.S) and over all

fruits yield. The beneficial outcomes of applying active dried yeast were

recognized to increase the contents of various nutrients, protein, vitamin B and also

growth regulators, for example cytokinins (Glick, 1995; Fathy & Farid, 1996),

104

Table 4.19 Effect of yeast application on the management of A. flavus and aflatoxin B1 production in greenhouse trial

Treatments Aflatoxins

(ppb) No of pods Fresh Wt. of

pods (g) Dry Wt. of

pods (g) A. flavus

% Incidence T1- Control (inoculated with A.flavus) 96.15 a* 11.25a* 29.85a* 9.84a* 80.12 h* T2- Control (uninoculated ) 27.473 abc 18.75a 32.16a 11.42a 1.56 a

T3- Saccharomyces bayanus 105 N.D c 41.00a 90.85a 30.06a 12.33 bc

T4- Saccharomyces bayanus 106 N.D c 34.25a 68.45a 20.01a 19.17 cde

T5- Saccharomyces bayanus 107 N.D c 13.25a 28.76a 7.82a 20.67 def

T6- Saccharomyces bayanus 108 N.D c 30.25a 55.37a 15.26a 28.50 g

T7- Saccharomyces bayanus 109 N.D c 12.25a 26.53a 7.31a 9.07 ab

T8- Saccharomyces cerevisiae 105 N.D c 33.50a 87.63a 24.28a 12.03 bc

T9- Saccharomyces cerevisiae 106 N.D c 15.50a 34.45a 10.15a 14.40 bcd

T10- Saccharomyces cerevisiae 107 N.D c 7.25a 16.59a 4.60a 25.47 efg

T11- Saccharomyces cerevisiae 108 N.D c 17.50a 31.27a 9.27a 13.33 bcd

T12- Saccharomyces cerevisiae 109 N.D c 3.25a 5.93a 1.27a 28.00 fg

T13- Saccharomyces postorianus 105 78.78 ab 37.75a 72.73a 21.93a 15.67 bcd

T14- Saccharomyces postorianus 106 94.94 a 9.50a 20.82a 5.97a 11.10 bc

T15- Saccharomyces postorianus 107 43.43 abc 9.75a 22.29a 5.83a 16.50 bcd

T16- Saccharomyces postorianus 108 39.39 abc 7.00a 17.17a 4.72a 1.67 a

T17- Saccharomyces postorianus 109 9.3 bc 7.75a 15.18a 5.92a 12.37 bc

*Means in column followed by the same letters are not significantly different according to DMRT

105

gibberlic acid and indole acetic acid (Sarhan & Abdullah, 2010). Similar results,

were reported by several scientists for several crops, e.g., tomato (El-Ghamriny et

al., 1999; Fathy et al., 2000; Abou El-Yazied and Mady, 2012).

Mekki and Ahmed (2005) reported that the increase in yield and yield

components of soybean plants as a result of yeast treatment could mainly be

attributed to a stimulating effect on the plant for building up dry matter (Heikal,

2005) and enhanced orientation and translocation of photoassimilates from leaves

to flowers and immature seeds (Hopkins, 1995). Furthermore, yeast extract may

play a beneficial role in improvement of flower formation and set for some plants

as well as enhancing the accumulation of carbohydrate due to high auxin and

cytokinin contents (Barnett et al., 1990).

4.4 INTEGRATED MANAGEMENT OF MYCOTOXINS IN RED CHILLI

Mycotoxin contamination can occur at every stage of crop production. Even if

it is reduced at pre-harvest stage, the produce is still under threat of mycotoxin

contamination because the source of contamination i.e. fungal inoculum cannot be

eliminated completely as demonstrated in this study in which aflatoxin was

eliminated but the fungus prevailed in produce. The final trial was conducted to

contribute all the most effective treatments from the previous trials and to observe

any integrated management effects from the pre-harvest to the postharvest stage.

4.4.1 Pre-Harvest Management

The cv. Nagina was grown in same conditions in the greenhouse as discussed

above and subjected to the pre-harvest treatment. The most effective dose (109) of

106

S. postorianus was sprayed onto the plants and most effective irrigation level (45 %

AWC) was maintained separately in solarized and non-solarized soils (Table 4.20).

The treatment with solarized soil significantly (P < 0.05) reduced aflatoxin

contamination and fungus infection to 1.13% while yield parameters were

unaffected. However, samples grown in non-solarized soil were highly

contaminated and therefore were brought to laboratory for post-harvest

management.

4.4.2. Post-Harvest Management

Contaminated samples obtained from pre-harvest management trial were

treated with culture filtrate of F. aurantiacum and L. rhamnosus. In samples treated

with L. rhamnosus, aflatoxin B1 was significantly reduced down to 8.08 µg/kg

(Table 4.21) closed to the permissible limit set by the European Union for AFB1 in

red chilli. Reduction percent of AFB1 was 87.5% (Figure 4.8). However, there was

no significant (P > 0.05) effect on seed germination in this sample, showing that

these treatments have no harmful effects on normal growth of plants (Figure 4.9). It

can be concluded that integration of treatments at every step from pre-harvest to

post-harvest is necessary in order to produce sound product.

Various discrete techniques for the management of mycotoxins have been

reported in different crops which can be investigated for their efficacy in managing

the mycotoxins and fungal contamination in chilli. Each technique can protect the

crop at any particular stage. However, by an amalgamation of these techniques at

various pre and post-harvest stages of chilli crop, the mycotoxin contamination can

be avoided.

107

Table 4.20. Integrated management of aflatoxin content and A. flavus by the most effective treatments selected from the

greenhouse trial with the most effective dose of yeast and optimum irrigation level.

Treatments No of pods Fresh wt. of

pods (g)

Dry wt. of

pods (g)

AFB1

(µg/kg)

A. flavus

% Incidence

T1- Control (inoculated with A.flavus) 15.00 a 29.85 a 9.84 a 87.27 a* 50.17 c*

T2- Control (uninoculated ) 18.75 a 32.16 a 11.42 a ND b 0.73 a

T3-S. postorianus 109

+Moderate stress (45% AWC)

in solarized soil 20.35 a 43.60 a 13.01 a ND b 1.13 a

T4- S. postorianus 109

+ Moderate stress (45% AWC)

in non-solarized soil 14.65 a 29.84 a 9.44 a 64.64 a 29.87 b

*Means in column followed by the same letters are not significantly different according to DMRT

108

Table 4.21 Integrated management of aflatoxin production and A. flavus growth in

red chilli by best treatments selected from laboratory trial with the most

effective bacterial cultures

Treatments Aflatoxins

(µg/kg)

T1-Control

64.64 a*

T2- F. auranitiacum (culture filtrate)

25.25 b

T3- L. rhamnosus (culture filtrate)

8.08 c

*Means in column followed by the same letters are not significantly different

according to DMRT

109

Figure 4.8. Reduction of aflatoxin B1 in red chilli by post-harvest treatments

87.5

60.94

0

10

20

30

40

50

60

70

80

90

100

L. rhamnosus F. aurantiacum

Per

cen

tag

e

Bacteria

110

Figure 4.9. Combined effect of pre and post-harvest treatments on seed germination

of red chilli

0

2

4

6

8

10

12

14

16

18

20

Negative Control L. rhamnosus F. aurantiacum Positive Control

No

of

seed

s

Treatments

111

SUMMARY

Red chilli is a food commodity of high susceptibility to fungal infection and

mycotoxin production. Aspergillus flavus produces four types of aflatoxins: AFB1,

AFB2, AFG1 and AFG2. The presence of AFB1 in red chilli is the most important

issue, since it is acutely toxic and highly carcinogenic. In Pakistan, Kunri is a hub

of red chilli farming, producing 85% of red chilli in the country. However, the

problem of aflatoxin contamination has disrupted the Pakistani red chill production

and now Pakistan is unable to compete in the international market. Keeping this

rationale in mind, the work described here was designed to develop an integrated

management strategy against mycotoxin contamination in red chilli in Pakistan. For

this purpose, red chilli pods were collected from the fields of Kunri, Pakistan. A

total of sixty nine samples of red chilli pods from six cultivars (Nagina, Kunri,

Maxi, Drooping Type, Tall Round and Tall pointed) were obtained. The samples

were dried in the laboratory and subjected to mycoflora and mycotoxin analysis. A.

flavus was the most common species from all samples, however aflatoxins were

detected in 46 samples. The occurrence of certain fungi does not necessarily

involve in the production of toxins. After analysis, the most susceptible cultivar

was found to be Nagina while, two cultivars, Kunri and Drooping Type, were

resistant, less contaminated with A. flavus and aflatoxins as compared to other

cultivars. The Nagina cv and an isolate A. flavus were subjected to laboratory and

greenhouse trials to determine the best treatments to reduce A.flavus growth and

mycotoxin contamination. In laboratory trials radial growth of A. flavus was

inhibited by diffusates of L. camara, E. hirta, A. viridis and T. portulacastrum.

AFB1 content was significantly (P < 0.05) reduced by the E. hirta treatment. Hence

111

112

this plant was selected for the integrated management trial. Out of seven biological

agents, L. rhamnosus showed highest inhibition of the growth of A. flavus,

followed by F. aurantiacum, B. subtilis and P. flourescens. Production of AFB1

was highly inhibited by L. rhamnosus and F. aurantiacum. Bacillus subtilis was not

active against AFB1, even though it inhibited growth of A. flavus. Among three

yeast species Saccharomyces cerevesiae and S. bayanus inhibited AFB1 by 47%.

All red chilli samples treated with plant diffusates and bio agents were analysed to

evaluate nutritional and quality profiles. L. rhamnosus and F. aurantiacum had

little effect on red chilli quality and were selected to develop the integrated

management strategy. In greenhouse management trials aflatoxins were reduced

by maintaining water level at 45 % AWC. The literature supports foliar sprays of

yeast in different crops to increase resistance against different diseases, and

especially for the healthy growth of plants. The present study found the best results

in the plants treated with S. cerevisae and S. bayanus, as they completely inhibited

aflatoxin production and reduced the occurrence of A. flavus. However, the plants

treated with S. postorianus were highly contaminated, except at the highest dose

(109) applied, when the contamination level was low. Finally the most effective

treatments selected from these experiments were combined in the last trial of

integrated management of mycotoxins in chillies. In this trial, pre-harvest

management was carried out by maintaining an irrigation level of 45 % AWC and

spraying with an appropriate dose of S. postorianus. The experiment was

conducted on both solarized and non-solarized soils. In solarized soil the treatments

completely eliminated AFB1 contents and reduced the occurrence of A. flavus down

to 1.13 %. At the time of harvest the fruits of non-solarized treatment were

113

subjected to the optimal post-harvest treatments to determine the combined effect

of pre and post-harvest treatments. The experiments confirmed that L. rhamnosus

significantly reduced AFB1 contents to 87.5% of contents followed by F.

aurantiacum (60.9%). These treatments had no significant (P > 0.05) effect on

seed germination as compared to controls. Therefore, it can be concluded that an

integrated management strategy from pre to post-harvest stage provide a possible

solution to the issue of aflatoxin contamination in red chilli. Farmers can be trained

in the use of resistant cultivars, like Kunri and Drooping type, for healthy

production of red chilli. Fungal attack should be prevented by maintaining

appropriate irrigation levels and applying yeast in the fields. The final produce

should also be treated with L. rhamnosus to control the proliferation of A. flavus

and its toxins during storage. This approach is not only safe but inexpensive.

114

CONCLUSIONS AND RECOMMENDATIONS

The use of resistant or tolerant varieties (Kunri and Drooping Type) is an

inexpensive, easy and eco friendly mean of controlling fungal infection in

crops where such varieties are available.

At the pre-harvest stage, formulations of Saccharomyces cerevisae and S.

bayanus should be sprayed in fields during flowering.

At the post-harvest stage, formulations of Lactobacillus rhamnosus should

be applied to pods to avoid contamination in storage and transport.

Farmers should solarize the soil as a routine.

The integration of all treatments from pre- to post-harvest stages is crucial

for the management of mycotoxin contamination in red chilli.

114

115

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