ECOLOGICAL ROLES OF CLPs PRODUCED BY THE

RHIZOSPHERE-DERIVED ENTEROBACTER SPECIES LPb1-1 IN THE

CONTROL OF FUSARIUM SP. GP5 WHICH CAUSE WILTING OF

SCOTCH BONNET PEPPER

BY

RAJI, RIDWAN OLANREWAJU

MCB/2016/331

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY, FACULTY OF SCIENCE, OBAFEMI AWOLOWO

UNIVERSITY, ILE-IFE, OSUN STATE, NIGERIA. IN PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

BACHELOR OF SCIENCE (B. Sc.) HONS. DEGREE IN

MICROBIOLOGY.

JANUARY, 2022.

i
 CERTIFICATION

This is to certify that this work was carried out by Raji, Ridwan Olanrewaju with the

matriculation number MCB/2016/331, in partial fulfilment of the requirements for the award

of Bachelor of Science (B. Sc.) Hons. degree in Microbiology, Department of Microbiology,

Faculty of Science, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, under the

supervision of Dr. Ir. Olumide O. Omoboye.

______________________________ _________________________
Dr. Ir. O. O. Omoboye Date
Project Supervisor

______________________________ _________________________
Prof. B. O. Omafuvbe Date
Head of Department

ii
 DEDICATION

This project is dedicated to the Almighty Allah who has been my source of strength, grace

and wisdom throughout the hurdles of my academic pursuit.

iii
 ACKNOWLEDGEMENTS

All praises to the Almighty Allah and His blessing for all the opportunities, trials and strength

that have been showered on me from the start of this academic pursuit to its successful

completion.

My sincere appreciation goes to my project supervisor, Dr. Ir. Olumide O. Omoboye for his

guidance, understanding, patience and most importantly, he has provided positive

encouragement and a warm spirit to finish this project. It has been a great pleasure and

honour to have him as my supervisor.

I also offer my special thanks to the Head of Department, Prof. B. O. Omafuvbe, all lecturers

and all the technical staffs of the Department of Microbiology, Obafemi Awolowo

University, Ile-Ife, Osun State.

I would also like to appreciate Dr. T. O. Fadare, Mr. Michael Aghughu, Mr. Damilola

Orasakin, Miss Hannah Onipede and Mr. Taofiq Sulayman, for their words of encouragement

and support during the course of this project work.

I would sincerely like to thank all my beloved friends, Yusuf Adam, Babarinde Favour,

Adesola Salmah, Atanda Sofiat, Akinola Suliyat, Osunyomi Deborah and Ademola Adam,

who were with me and support me through thick and thin. I pray that the Almighty God

continue to bless you.

I will not forget to appreciate my project colleagues, Aduloju James, Eze Godswill, Oginni

Omolara, Balogun Clement, Olowolaju Tolulope, Ojedele Oluwatobi, Ogunba Fehintola,

Oyewole Yetunde and my other project colleagues, for their help and support in the

successful completion of this project.

Special thanks to myself for a job well done and my parents, Mr. and Mrs. Rafiu Raji, and my

siblings, Ahmad, Semiat and Muhammed Raji, for their moral, spiritual and financial support

since the beginning of my studies and towards the success of this project.

iv
 TABLE OF CONTENTS

CERTIFICATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

TABLE OF CONTENTS v

LIST OF TABLES v

LIST OF FIGURES ix

ABSTRACT x

CHAPTER ONE 1

INTRODUCTION AND LITERATURE REVIEW 1

1.1. INTRODUCTION 1

1.2. LITERATURE REVIEW 5

1.2.1. Pepper as a plant 5

1.2.2. Taxonomy of Pepper 6

1.2.3. Types of Pepper 8

1.2.4. Uses and Benefits of consuming Pepper 13

1.2.5. Fungal Pathogens of Pepper 13

1.2.6. Control of plant pathogens 17

1.2.7. Beneficial bacteria associated with plants 24

1.2.8. Cyclic lipopeptides (CLPs) 31

CHAPTER TWO 39

MATERIALS AND METHODS 39

2.1 MATERIALS 39

2.1.1 Glassware 39

2.1.2 Equipment, Instruments and other materials 39

2.1.3 Reagents 39

2.1.4 Culture Media used 39

v
 2.2 METHODS 40

2.2.1 Sterilization of glassware and other materials used 40

2.2.2 Media preparation 40

2.3 SAMPLING 41

2.3.1 Sample Collection 41

2.3.2 Method of Isolation 41

2.4 Microbiological tests 44

2.4.1 Antagonism assay 44

2.5 Microscopic identification of fungal isolates 44

2.5.1 Principle 44

2.5.2 Procedure 45

2.6 Molecular characterization of bacterial isolate LPb1-1 by 16S rRNA sequencing 45

2.6.1 DNA Extraction 45

2.6.2 Polymerase Chain Reaction (PCR) 46

2.7 Morphological characterization of bacteria for CLP production 47

2.7.1 Drop Collapse Assay 47

2.7.2 Swarming Motility test 47

2.8 Extraction of cyclic lipopeptides (CLPs) 48

2.9 Antagonistic assay of CLPs against fungi on microscopic slide 50

2.9.1 Microscopic examination using Photomicrograph (Olympus BH-2

Microscope) 50

CHAPTER THREE 52

RESULTS 52

3.1. Isolation 52

3.2. In vitro antagonistic activity of bacterial isolates against fungal isolate 52

3.3. Morphological and microscopic characteristics of the fungi GP5 implicated in the
wilting of scotch bonnet pepper 52

3.4. Molecular characterization of bacteria (16S rRNA sequencing) 57

vi
 3.5. Drop collapse assay 57

3.6. Swarming Motility Test 57

3.7. Antagonistic activity of CLP produced by Enterobacter sp. LPb1-1 on Fusarium sp.
GP5 57

3.8. Morphological damage exerted by CLP produced by Enterobacter sp. LPb1-1 on the
Fusarium sp. GP5 63

CHAPTER FOUR 66

DISCUSSION, CONCLUSION AND RECOMMENDATION 66

4.1. DISCUSSION 66

4.2. CONCLUSION 68

4.3. RECOMMENDATION 68

REFERENCES 69

APPENDIX 108

vii
 LIST OF TABLES

Table Title Page

1.1 Classification of Capsicum annuum 8

2.1 Forward and Reverse primers used 46

3.1 Colonial Morphology of Fusarium sp. GP5 55

3.2 In vitro antagonistic activity of CLP produced by Enterobacter sp. 64

LPb1-1 against Fusarium sp. GP5.

viii
 LIST OF FIGURES

Figure Title Page

1.1 Cayenne pepper – Shombo 10
1.2 Scotch bonnet pepper – Ata rodo 12
1.3 Symptoms and damages caused by fungal disease in leaves, plants 16
and fruits
1.4 Different structure of various cyclic lipopeptides 33
2.1 Representative pictures showing diseased (a) and healthy (b) 43
scotch bonnet pepper plants (Capsicum chinense); and healthy (c)
cayenne pepper plant (Capsicum annuum)
2.2 Flow chart diagram of cyclic lipopeptide (CLP) extraction 49
3.1 In vitro antagonistic effect of Enterobacter sp. LPb1-1 on 53
Fusarium sp. GP5.
3.2 In vitro antagonistic effect of Enterobacter sp. LPb1-1 on 54
Fusarium sp. GP5. Representative pictures showing the inhibition
of Fusarium sp. GP5 by Enterobacter sp. LPb1-1.
3.3 Colonial morphology of Fusarium sp. GP5 56
3.4 Representative picture of agarose gel electrophoresis of the 58
bacteria isolates
3.5 The result of drop collapse test of Enterobacter sp. LPb1-1 using 59
water as control.
3.6a Diameter of swarming motility of the bacterial isolates 60
3.6b Representative pictures showing swarming motility of the 61
bacterial isolates
3.7 Diameter of the zones of inhibition of CLP produced by 62
Enterobacter sp. LPb1-1 against Fusarium sp. GP5 on microscopic
slide.
3.8 Representative picture showing the various microscopic effect of 65
CLP produced by Enterobacter sp. LPb1-1 on Fusarium sp. GP5 at
different concentrations

ix
 ABSTRACT

Pepper is a domesticated species belonging to the Capsicum genus. It is the most widely used

spice for flavour, taste enhancement and food colouring owing to its high vitamins and

antioxidant composition. The production of pepper is frequently hampered by fungal

pathogens such as Phytophthora capsici, Rhizoctonia solani, Verticillium dahliae, Leveillula

taurica, Sclerotium rolfsii, Botrytis cinerea, Sclerotinia sclerotium, Fusarium oxysporum, and

so on; which could result in high losses to the farmers. Cultural and chemical methods have

been used to control these diseases, with some drawbacks. This study is therefore set up to

investigate the biological control agents of fungal diseases of pepper. Antagonistic

rhizosphere-derived Enterobacter sp. LPb1-1 isolated from healthy cayenne pepper plants,

was investigated against Fusarium sp. GP5 implicated in the wilting of scotch bonnet pepper,

using direct antagonism on Potato Dextrose agar plate. The antagonistic bacteria was

identified via 16S rRNA sequencing, and the fungi morphologically. The antagonistic activity

of cyclic lipopeptide (CLP) produced by Enterobacter sp. LPb1-1 was determined on

microscopic slide, and observed under Olympus BH-2 Microscope. The result of the study

showed that Enterobacter sp. LPb1-1 had direct antagonistic activity against Fusarium sp.

GP5 on PDA plates, with the diameter of zones of inhibition of 3 and 11 mm, after day 3 and

day 5, respectively. The direct antagonistic potential of CLPs produced by Enterobacter sp.

LPb1-1 against Fusarium sp. GP5 was confirmed on microscopic slides with diameter of

zones of inhibition of 15, 18, 20 and 26 mm, at CLP concentrations of 5, 10, 25 and 50

µg/ml, respectively. In addition, the CLP at the different concentrations exerted

morphological damages on the fungal hyphae; 5, 10, 25 and 50 µg/mL of the CLP caused

extensive hyphal branching; 5, 10 and 50 µg/mL displayed blackening of hyphal edge; 10 and

25 µg/mL caused vacuolization; 5 µg/mL showed curling; while 25 µg/mL caused hyphal

digestion of the fungal hyphae. It could therefore be concluded that rhizosphere-derived

bacteria Enterobacter sp. LPb1-1 could serve as a potential, viable, suitable and eco-friendly

x
alternative to chemical pesticides in the strengthening of scotch bonnet pepper against

pathogens such as Fusarium sp. GP5 implicated in the wilting of scotch bonnet pepper.

xi
 CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1. INTRODUCTION

Pepper is a domesticated species of the Capsicum genus, which belongs to the Solanaceae

family (Greenleaf, 1986). Pepper is the second most important vegetable in the world, after

tomatoes, and is the most widely used spice for flavour, taste enhancement and food coloring

while supplying vitamins and minerals (Rehima, 2006; UzunalicAmra et al., 2004). Peppers

contains some of the most important carotenoids (Jozsef et al., 1996), which are widely

employed in the culinary industry as food colorants (Matsufuji et al., 1998), spices and

flavouring compounds (Famurewa et al., 2006). Most countries’ cuisines benefit from their

spiciness and fragrance (Paik et al., 2003).

Pepper is a major commercial commodity grown for vegetable, spice, and value-added

processed foods (Kumar and Rai, 2005). The fruits include a variety of antioxidants,

including carotenoids, ascorbic acid, flavonoids, and polyphenols, in addition to vitamins A

and C

(Nadeem et al., 2011). As a result, it is a vital component of many meals, providing flavor,

color, and spiciness, as well as being a key source of nourishment for humans (Norman,

1992). Peppers can be used whole, sliced, or in a variety of processed forms, including fresh,

dried, and made into powder (with or without seeds), or as an extract (Norman, 1992). Fresh

fruits can be processed into paste and bottled for sale in supermarkets in most developed

countries. Pepper is also used for medical purposes, such as the treatment of fevers and colds

(Norman, 1992). Bell peppers are high in vitamins A, C, B6, folic acid, and beta-carotene,

making them a good source of nutrition for humans (Nadeem et al., 2011). Sweet bell peppers

have antioxidative potential due to antioxidant chemicals found in the various colors (green,

1
yellow, orange, and red) that help protect the body from oxidative damage caused by free

radicals when consumed (Simonne et al., 1997). It lowers the chances of heart disease,

asthma, sore throat, headaches, and diabetes. On the other hand, red pepper includes

lycopene, which is thought to have anti-cancer qualities. It's also utilized by security

organizations to make tear gas for crowd control (Simonne et al., 1997).

Despite this unique importance of pepper, its production is restricted by diseases caused by

biotic and abiotic factors. Several abiotic factors such as sunlight, nutrient deficiency and

high temperature impair its production. Biotic agents of pepper diseases are fungi, bacteria,

nematodes, and viruses (Roberts et al., 2004). Fungi is the most devastating pathogen

affecting pepper worldwide (Dewitt and Bosland, 1993). Pepper is infected by a wide variety

of fungal pathogens which includes Phytophthora capsici, Rhizoctonia solani, Verticillium

dahliae, Leveillula taurica, Sclerotium rolfsii, Botrytis cinerea, Sclerotinia sclerotium,

Fusarium oxysporum, and so on (Sarath et al., 2011; Pitrat, 2012; Djian-Caporalino et al.,

2014).

As a result of these pathogens, cultural, chemical, and biological controls are used to achieve

disease and pest management. Cultural control describes the activities of humans aimed at

controlling disease through the cultural manipulation of pepper plants. Cultural methods such

as organic amendments, soil solarization, and cover cropping have been observed to control

Pythium-related diseases in Pepper. However, it may be difficult to assess their effectiveness

because the farming operations are often performed well in advance of expected pathogen

attacks (Ogle and Dale, 1997). Pesticides have become a viable alternative due to the

persistence of some diseases. Soil-borne disease has been controlled with chemical pesticides

such as fungicides. Fungicides are utilized as seed treatments, soil drenching, and fumigation

(Arora et al., 2021). However, the increasing use of pesticides in agriculture has sparked

public concern and scrutiny due to potential environmental impact, unfavorable effects on

2
non-target organisms, and the possibility that some compounds are carcinogenic (Agrios,

1988; Cook, 1993; Heydari, 2007 and Heydari et al., 2007). As plant pathogenic agents are

quickly becoming resistant to chemical pesticides and considering the high price of pesticides

as well as their accumulation in plant or soil which has harmful effects on human, there is an

obvious need to discover non-chemical alternatives to control plant diseases such as plant-

associated pathogen antagonists in a phenomenon known as biological control.

Biological control of plant diseases has already been seen as a potential alternative to control

plant diseases (Cook, 1993). Biological control is the use of another organism to prevent one

organism from growing, infecting, or reproducing (Cook, 1993; Baker, 1987). Biocontrol is

both ecologically friendly and a suitable way to protect plants from infections (Cook, 1993).

Natural enemies of pests or pathogens are used in biological control to destroy or manage

their populations. This may entail the introduction of foreign species or the use of whatever

biological control mechanisms present naturally in the environment. The use of beneficial

microbes to induce plant resistance or antagonize plant pathogens is also a form of biological

control (Cook, 1993; Schouten et al., 2004). Biological control agents (BCAs) are able to

inhibit diseases in an ecologically friendly and more sustainable manner (Yuliar et al., 2015).

Pathogens are directly antagonized by biological control agents through hyper-parasitism,

predation, and the production of antibiotics and lytic enzymes; and indirectly by biological

control agents through competition for space and nutrients, inducing systemic resistance, and

promoting plant growth (Cook, 1993; Latha et al., 2009; Brimner and Boland, 2003; Pal and

Gardener, 2006). Control of disease using biological control agents have been reported to

result from a combination of mechanisms including the production of enzymes as well as

antibiotics that play important roles in inhibiting growth of the plant pathogens (Kumar et al.,

2019). Furthermore, bacterial BCAs can also play significant role in disease management by

producing extracellular proteins that induce plant defense genes and resistance against P.

3
capsici (Kim et al., 2010; Bacon et al., 2015; Kumar et al., 2019). Some BCAs such as

Bacillus and Pseudomonas produce secondary metabolites such as linear/cyclic lipopeptides

(L/CLPs) which contributes to their bioactivity. Bhusal and Mmbaga (2020) revealed that

endophytic Bacillus species exhibited biocontrol potential against Phytophthora capsici and

promote seedling shoot length, weight and chlorophyll content of bell pepper. However, the

mode of action of the biological control potential of lipopeptide-producing bacteria against

fungi implicated in the wilting of pepper plants have not been studied. This study is therefore

designed to investigate the biological control potential of rhizosphere-derived bacteria against

the fungi implicated in wilted pepper.

Objectives

The objectives of the study are to:

(a) isolate fungi from wilted pepper root and stem; and bacteria from the root of healthy

pepper plants,

(b) identify the bacteria by 16S rRNA sequencing; and the fungi morphologically,

(c) extract cyclic lipopeptides (CLPs) from the bacteria isolates, and

(d) investigate the direct antagonistic potential of the isolated bacteria and CLPs against

the fungal pathogens.

4
1.2. LITERATURE REVIEW

1.2.1. Pepper as a plant

Pepper is the second most important vegetable in the world, after tomatoes, and is the most

widely used spice for flavour, taste enhancement and food coloring while supplying vitamins

and minerals (Rehima, 2006; UzunalicAmra et al., 2004). Peppers include the oldest and

most important carotenoids (Jozsef et al., 1996), which are widely employed in the culinary

industry as food colorants (Matsufuji et al., 1998), spices and flavouring compounds

(Famurewa et al., 2006). Most countries’ cuisines benefit from their spiciness and fragrance

(Paik et al., 2003).

Pepper is a domesticated species of the Capsicum genus, which belongs to the Solanaceae

family (Greenleaf, 1986). The berry-like fruit of the hot pepper (Capsicum annum L.) can be

green, yellow or red when fully ripe. Some woody forms of this species have been named C.

frutescens in the past, however the characteristics that distinguished those forms may be

found in many populations of C. annuum and there is no reliably identifiable C. frutescens

species (Zhang et al., 2002). Capsicum annuum is hard to differentiate from cultivated C.

chinense (the hottest pepper) and C. frutescens (tabasco pepper) and their morphological

characteristic sometimes overlap. These three species share the same ancestral gene pool,

therefore the terms pepper, chilli, chile, chili, aji, paprika, and Capsicum are sometimes used

interchangeably to describe the plant (DeWitt and Bosland, 2009; Mcmullan and Livsey,

2013).

The most significant vegetable on the planet is the hot pepper (Capsicum annum L.). It is

used as a vegetable, spice, or condiment in fresh, dried or processed form. This species is

known by more than 200 different names. Chili pepper, paprika (sweet variety); bell pepper,

5
cayenne, jalapenos, chiltepin (hot variety); Christmas peppers (ornamental) are some of the

most often used common names (Zhang et al., 2002; Latham, 2009).

Peppers are thought to be the first spice used by humans, with archaeological evidence of

pepper and other fossil foods dating back as far as 6000 years ago (Hill et al., 2013). There

are five domesticated species in the genus Capsicum (C. annuum, C. frutescens, C. chinense,

C. pubescens and C. baccatum) of which C. annuum is the most extensively grown species on

the planet (Andrews, 1984).

In the sixteenth century, Columbus and other early new explorers introduced Pepper to

Europe, and cultivation increased throughout the world (Greenleaf, 1986). It is a small

perennial shrub with a white or greenish-white corolla, one or more pedicels at a node, and

fruit of varied sizes and shapes. Pungency, which varies by cultivar but is often higher in

smaller fruit types than bigger thick-fleshed varieties, is another distinguishing feature of the

crop. With a maturation period of 3-4 months, pepper grows relatively quickly (Norman,

1992).

1.2.2. Taxonomy of Pepper

The potato (Solanum tuberosum), tomato (Lycopersicon esculentum or Solanum

lycopersicum), tree tomato (Cyphomandra betacea or Solanum betaceum), eggplant

(Solanum melongena), African eggplants (Solanum macrocarpon, S. aethiopicum), husk or

strawberry tomato (Physalis pruinosa) and Cape gooseberry (P. peruviana) are all members of

the Solanaceae family, which also includes tobacco (Nicotiana tabacum), medicinal plants

such as deadly nightshade (Atropa belladonna) and Datura stramonium, ornamentals such as

tree daturas (Brugmansia) (which are also hallucinogenic) and Petunia, and weeds such as

black nightshade (Solanum nigrum) (George, 1985; Hunziker, 2001; Knapp, 2002).

6
Capsicum belongs to the subfamily Solanoideae and tribe Capsiceae (Olmstead et al., 1999;

Hunziker, 2001; Knapp, 2002; Knapp et al., 2004). There are approximately 25 wild and 5

domesticated species in the genus Capsicum (IBPGR, 1983; Eshbaugh, 1993; Bosland and

Votava, 2000). Capsicum annuum, C. frutescens, C. chinense, C. baccatum and C. pubescens

are the five domesticated species (Heiser and Smith, 1953; Smith and Heiser, 1957; Heiser,

1985).

Capsicum annuum, C. frutescens and C. chinense belong to a taxonomic complex containing

three, two or one species (Pickersgill, 1988), with the three domesticated plant clusters

appearing to be more divergent than their wild progenitors (Heiser, 1985; Eshbaugh, 1993;

Prince et al., 1995; Idu and Ogbe, 1997; Park et al., 1999; Bosland and Votava, 2000; Walsh

and Hoot, 2001; Jarret and Dang, 2004; Ryzhova and Kochieva, 2004; Baral and Bosland,

2004).

Other taxonomic complexes of the genus include the remaining two domesticated species –

C. baccatum and C. pubescens (Eshbaugh, 1993; Tong and Bosland, 1999; Walsh and Hoot,

2001). Both are rarely used outside of Latin America, although C. baccatum var. pendulum

(Willd.) Eshbaugh, a widely domesticated cultivar, is widely utilized. The ability of

members of the genus Capsicum to effectively interbreed can also be used to categorize them.

These include Annuum, which comprises of the species C. annuum (varieties glabriusculum

and annuum), C. frutescens, C. chinense, C. chacoense and C. galapagoensis; the baccatum

group which consists of the species C. baccatum (varieties baccatum, pendulum and

praetermissum) and finally C. tovari, and the pubescens group which also consist of the

species C. cardenasii, C. eximium and C. pubescens (Pickersgill, 1997).

7
 Table 1.1. Classification of Capsicum annuum

Taxonomic placement Scientific name

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Solanales

Family Solanaceae

Genus Capsicum

Species annuum

var. glabriusculum

Botanical varieties (synonym var. aviculare)

var. annuum

(Karpate, 2009; Pawar, 2011)

1.2.3. Types of Pepper

Some varieties of pepper commonly grown include:

1. Cayenne pepper or red pepper—Shombo (Capsicum annuum)

2. Scotch bonnet pepper - Atarodo (Capsicum chinense)

3. Tatase (Capsicum annuum)

4. Bell pepper (Capsicum annuum)

8
5. Bird peppers—atawere (Capsicum frutescens)

6. Habanero pepper (Capsicum chinense) etc.

Some of these varieties are discussed below:

1.2.3.1. Cayenne Pepper (Shombo)

Capsicum annuum is the botanical name for cayenne pepper, which belongs to the capsicum

family. Cayenne pepper is cultivated all over the world. Pungent, small-fruited types are a

staple of exotic Asian and African cuisines, whether fresh, dried, or crushed (Csilléry, 2006).

It's the red chili pepper, which is used to spice meals and has a lot of therapeutic properties.

Vitamin E, vitamin C, vitamin K, carotenoids, and the entire B complex vitamins are all

found in cayenne pepper. It also contains organic calcium, potassium, manganese, and dietary

fiber (Zsombok, 2013; Nilius and Szallasi, 2014).

9
 Figure 1.1. Cayenne pepper – Shombo

(Image from https://www.supermart.ng/product/shombo-pepper-x10)

10
1.2.3.2. Scotch Bonnet pepper (Ata Rodo)

Scotch Bonnet pepper is a Capsicum chinense Jacquin cultivar from the Solanaceae family.

C. chinense is one of five domesticated Capsicum species (Andrews, 1984), and it is most

closely related to C. annuum and C. frutescens (Pickersgill et al., 1979). C. chinense has been

shown to hybridize with C. annuum and C. frutescens (Schweid, 1989; DeWitt and Bosland,

1996). C. chinense is mostly grown in the Caribbean and South America (Purseglove et al.,

1981, Andrews, 1984), and is assumed to have originated in the Andes (Purseglove et al.,

1981) or Amazon Basin (DeWitt and Bosland, 1996). The term Scotch Bonnet originates

from the shape of the fruit. It's also noted for its distinct flavor and spiciness, putting it in the

high echelon of hot pepper heat levels (Andrews, 1984; DeWitt and Bosland, 1996).

11
 Figure 1.2. Scotch bonnet pepper – Ata rodo

(Image from https://www.123rf.com/photo_88990173_scotch-bonnet-peppers.html)

12
1.2.4. Uses and Benefits of consuming Pepper

Pepper is a major commercial commodity grown for vegetable, spice, and value-added

processed foods (Kumar and Rai, 2005). The fruits include a variety of antioxidants,

including carotenoids, ascorbic acid, flavonoids, and polyphenols, in addition to vitamins A

and C

(Nadeem et al., 2011). As a result, it is a vital component of many meals, providing flavor,

color, and pungency, as well as being a key source of nourishment for humans. Peppers can

be used whole, sliced, or in a variety of processed forms, including fresh, dried, and made

into powder (with or without seeds), or as an extract. Fresh fruits can be processed into paste

and bottled for sale in supermarkets in most developed countries. Pepper is also used for

medical purposes, such as the treatment of fevers and colds (Norman, 1992).

Bell peppers are high in vitamins A, C, B6, folic acid, and beta-carotene, making them a good

source of nutrition for humans (Nadeem et al., 2011). Sweet bell peppers have antioxidative

potential due to antioxidant chemicals found in the various colors (green, yellow, orange, and

red) that help protect the body from oxidative damage caused by free radicals when

consumed. (Simonne et al., 1997). It lowers the chances of heart disease, asthma, sore throat,

headaches, and diabetes. On the other hand, red pepper includes lycopene, which is thought

to have anti-cancer qualities. It's also utilized by security organizations to make tear gas for

crowd control (Simonne et al., 1997).

1.2.5. Fungal Pathogens of Pepper

Despite their benefits to man, pepper is infected by a wide variety of fungal pathogens which

includes Phytophthora capsici, Rhizoctonia solani, Verticillium dahliae, Collectotrichum

gloeosporioides, C. capsici, C. coccodes, C. scovillei and truncatum, Leveillula taurica,

Sclerotium rolfsii, Botrytis cinerea, Sclerotinia sclerotium, Fusarium oxysporum, and so on.

13
To ensure a healthy and lucrative pepper crop, fungicides and cultural treatments are used.

Given the growing demand for sustainable agriculture, the deployment of resistant plants is

the most effective way to protect pepper cultivation from biotic pressures (Sarath et al., 2011;

Pitrat, 2012; Djian-Caporalino et al., 2014).

1.2.5.1. Fungal Diseases

The diseases caused by these pathogens are as follows:

A. The powdery mildew

Pepper powdery mildew is found all throughout the world, but it is more prevalent in warm,

dry, or humid areas, where it produces significant output losses. Grayish white patches on the

undersides of leaves and light green-yellow lesions on the top leaf surface are symptoms of

Leveillula taurica (asexual stage: Oidiopsis taurica) (Figure 1.3a) (Anand et al., 1987; De

Souza and Café-Filho, 2003; Lee et al., 2001).

B. Phytophthora Root Rot and Foliar Blight

Phytophthora capsici is one of the most damaging diseases of pepper, especially if the soil is

damp and temperatures are low (15–23 oC) (Quirin et al., 2005). Stem and fruit rot, wilting,

stunting, dumping-off, plant mortality, and stem and leaf blight are all symptoms caused by

the oomycete (Figure 1.3b).

C. Anthracnose or Ripe Rot of Pepper

Anthracnose causes significant fruit losses throughout the pre-harvest and post-harvest

periods (Mishra et al., 2018 and Ridzuan et al., 2018). It can also cause damage to the stem

and foliage. Circular water-soaked patches with concentric rings of black acervuli growing

beneath the skin are the characteristic fruit symptoms (Figure 1.3c). Fruits soften and perish

as a result of the spots, which are often many and coalesce (Montri et al., 2009).

Colletotrichum species can cause anthracnose in a variety of ways. C. scovillei (formerly

14
classified as C. acutatum), C. truncatum (syn. C. capsici), and C. siamense (previously

identified as C. gloeosporioides) are the most prevalent pathogenic species that infect pepper.

The latter is not as deadly as the former (Ridzuan et al., 2018; Mongkolporn and Taylor,

2018).

D. Vascular Diseases

Vascular wilt is caused by four genera of fungi, Fusarium, Verticillium, Ceratocystis and

Ophiostoma. Each genus causes diverse and fatal diseases of crop, forest and ornamental

plants. Ceratocystis and Ophiostoma mostly cause vascular wilts of trees including oak wilt

(Ceratocystis fagacearum) and Dutch elm disease (Ophiostoma ulmi) (Brown and Ogle,

1997).

Verticillium wilt is a major danger to the production of peppers all over the world

(Goicoechea, 2006). The soilborne fungus Verticillium dahliae is the primary cause of the

disease, with V. alboatrum playing a minor role. Both pathogens infect plants directly or by

wounds, then spread acropetally through the xylem, causing vascular tissue browning,

stunting, foliar epinasty, chlorosis and necrosis, wilting, and plant death (Figure 1.3d).

Fusarium is the other vascular disease that causes agricultural output losses ranging from

10% to 80% (Loganathan et al., 2013). Pepper wilt has been associated with a number of

isolates from the Fusarium species complex. The most common species include F.

oxysporum (Lomas-cano et al., 2014), F. solani (Maruti et al., 2014), F. oxysporum f. sp.

vasinfectum (Miller et al., 1996), F. redolens (formerly classified as F. oxysporum var.

redolens) (Rahin et al., 1985), and F. oxysporum f. sp. capsici (Rivelli, 1989). In some parts

of India, F. verticillioides (syn. F. moniliforme) and F. pallidoroseum induce pepper

withering (Naik et al., 2008).

E. Rhizoctonia Solani

15
Rhizoctonia solani (teleomorph Thanatephorus cucumeris) is a soil-borne pathogen that

causes seedling damping-off, root rot, stem rot, and canker, among other symptoms (Figure

1.3e) (Mannai et al., 2018).

Figure 1.3. Symptoms and damages caused by fungal disease in leaves, plants and fruits:
(a) powdery mildew on leaf; (b) shriveled plants attacked by Phytophthora root rot;
(c) anthracnose of fruit; (d) Verticillium wilt with discolored vascular tissue of infected stem;
(e) Root and stem rot caused by Rhizoctonia solani (Parisi et al., 2020).

16
1.2.6. Control of plant pathogens

The production of pepper is affected by diseases caused by biotic and abiotic factors. Several

abiotic factors such as sunlight, nutrient deficiency and high temperature impair its

production. Biotic agents of pepper diseases are fungi, bacteria, nematodes, and viruses

(Roberts et al., 2004). Fungi is the most devastating pathogen affecting pepper worldwide

(Dewitt and Bosland, 1993). As a result of these pathogens, cultural, chemical, and biological

controls are used to achieve disease and pest management.

1.2.6.1. Cultural control

Cultural practices (CPs) can be used for the management of foliar and soilborne diseases by

establishing an environment that is beneficial to the crop while being detrimental to the

pathogen. Certain cultural practices, such as floods and sanitation, are primarily utilized for

pest control, whilst others, such as irrigation, can be used for crop management as well as

pest control. Some cultural methods, such as deep plowing, crop rotation, flaming sanitation,

soil solarization, and biofumigation, are employed as pre-planting measures, while others,

such as water and mineral nutrition management, tillage, and soil temperature alteration, are

used both pre- and post-planting (Katan, 2010).

Pythium-related illnesses in C. annuum crops have been controlled using cultural approaches

such as organic amendments, soil solarization, and cover cropping. Solarization of soil has

long been used in greenhouses and nursery beds to control numerous soil-borne diseases. As

observed by Akhtar et al. (2012), soil solarization reduces colony-forming units (CFUs) or

propagules of various soil-inhabiting pathogens as well as other microbes, with CFUs of

Pythium spp. reduced to 1.67 x 104 after 8 weeks of solarization compared to 5.00 x 104 in

presolarized soils at a depth of 15 cm. The use of isothiocyanates (ITCs) producing

Brassicaceae cover crops for soil biofumigation as an alternative to restricted chemical

fumigants like methyl bromide for soil-borne disease control is increasingly becoming

17
popular (Baysal-Gurel et al., 2020). Isolates of Pythium spp. were efficiently inhibited by

ITCs in in-vitro conditions (Smith and Kirkegaard, 2002), however field experiments

utilizing Brassica cover for biofumigation were ineffective in reducing Pythium populations

in pepper crops (Hansen and Keinath, 2013). However, after the pulverization and

incorporation of Brassica cover crops in soils, high ITC levels were reported in field soils

(Hansen and Keinath, 2013). When the ITC-producing intact seed meals of Brassica napus

and Brassica juncea were incorporated into P. ultimum-infested soils, Handiseni et al. (2012)

reported a 40%–60% improvement in pepper seedling emergence. The variation in sensitivity

to ITC observed by Hansen and Keinath (2013) and Handiseni et al. (2012) can be related to

the sensitivity of Pythium species to ITC.

Cultural practices frequently provide the potential to alter the environment, the host's

condition, and/or the behavior of the causal agent in order to achieve economic management

of disease. On this basis disease control by cultural practices is mainly preventive. Inoculum

density and activity are reduced as a result of these activities. To provide alternatives for

controlling economically important plant diseases, it may be essential to combine cultural

techniques with host resistance, fungicides, and bio-control agents (Baker, 1983).

1.2.6.2. Chemical control (Pesticides)

Chemical pesticides are still used because of their effectiveness, despite their well-known

negative impacts such as environmental toxicity and accumulation at various trophic levels

(Chowdhary et al., 2018). Fungicides have been used to manage soil-borne disease through

soil drenching, fumigation, and seed treatment. In a four-year study, Saha et al. (2011)

investigated the effect of treating chili seeds with the fungicidal formulations Thiram 75 WS

and Captan 50 WP on incidences of damping-off caused by P. aphanidermatum. Treatment of

seeds using Thiram 75 WS at the dose of 2.5 kg/g of seeds reduced the preemergence

damping-off losses from 29.06% in untreated control to 6.52% in treatment as well as post-

18
emergence damping-off losses from 59.12% in the untreated control to 16.15% in treatment

conditions. Metalaxyl was even used with the biocontrol agent Trichoderma harzianum as a

seed treatment, which yielded positive results, with the proportion of seed rot dropping from

93.75 percent in untreated seeds to 52.08 percent in treated seeds (Zagade et al., 2012).

However, as observed in metalaxyl-resistant Pythium spp., using a single active component

has resulted in the emergence of resistant species (White et al., 2019; Wang et al., 2020).

A combination of several fungicides, creating a cocktail of alternative compounds, has

provided the solution to overcome the resistance developed by Pythium spp. (White et al.,

2019; Wang et al., 2020). In most circumstances, the two or more active compounds in this

pesticide cocktail work additively, however synergism has been observed in some cases

(Rizzati et al., 2016). Previcur 840 SL, a combination of active substances combining

propamocarb (47.3%) and fosetyl (27.7%), is recommended for the efficient treatment of

damping-off disease caused by Pythium spp. in chili peppers (DOA, 2021). Dubey et al.

(2020) investigated the efficacy of combined fungicides on isolates of Pythium isolated from

diseased parts of chili plants in vitro and observed Vitavax (Carboxin 37.5% + Thiram

37.5%) to be extremely effective, inhibiting mycelia growth by 93.3% at a concentration of

100 ppm. Because high moisture content in the soil encourages the growth of Pythium spp.,

severe disease outbreaks can result from extreme rainfall events. After Hurricane Frances,

Saha et al. (2005) observed severe mortality in the Capsicum crop, which was exacerbated by

the biological vacuum created by pre-plant metalaxyl fumigation. The fumigation caused a

significant drop in beneficial soil bacteria, which eventually facilitated the outbreaks

by Pythium spp. Pythium outbreaks were also seen in methyl bromide-treated plots of pepper

plant in later years, which were attributed to the biological vacuum generated by methyl

bromide fumigation (Kokalis-Burelle et al., 2017). Pythium had no competition since there

were fewer soil microorganisms in the soil. As a result, disease forecasting based on criteria

19
including the amount of pathogen propagules, the susceptible host, and the conducive

environment is crucial in determining when and how to administer fungicides (Jørgensen,

2017).

1.2.6.3. Biological control of fungal pathogens

Biological control, in its most primitive sense, is the use of a living organism to combat a

specific plant pathogen or pest through parasitism, antibiosis, or competition for resources or

space (Eilenberg et al., 2001). Plant diseases and pests, however, are triggered and regulated

by complex processes at various levels, including the invader (the plant pathogen or pest), the

environment, and the plant itself. They can only thrive in ideal conditions on all three levels

(Michael and Pelczar, 2020). In order to achieve biological control's actual potential in

disease and pest management, a broader definition of biological control is required, one that

encompasses all levels. This broader term refers to the use of living organisms and their

derivatives to control plant diseases and pests, not only through direct antagonistic impact

against pathogens and pests, but also indirectly through induced resistance (Köhl et al.,

2019). As a result of the differences with the restricted definition of biocontrol, derivatives of

living organisms and inducers of resistance, which activate plant defense systems, are also

classified as biocontrol agents (BCAs) (Raymaekers et al., 2020).

The presence of a phytopathogenic complex formed by the genus Fusarium and others, such

as Phytophthora spp., Pythium spp., and Rhizoctonia spp., which are agents that cause

seedlings to fall, is one of the main concerns that negatively affects Capsicum production.

Together, these can result in output losses ranging from 60% to 100% (Villa-Martinez et al.,

2015). Despite this, conventional control measures have proven ineffective and difficult to

implement, due to issues such as soil pollution, phytotoxicity, and the development of

resistance in the target pathogen (Rajkumar et al., 2005). As a result of these problems, the

use of beneficial microorganisms is proposed as an alternative to chemical pesticides for the

20
control of these pathogens, not only for soil-borne diseases but also for pathogens that can

proliferate in the aerial part of the plant, and can also compensate for the negative

environmental effects of chemical pesticides (Das et al., 2019). Studies on the biological

control of diseases through the use of microorganisms have increased recently and the

function of beneficial microorganisms in interactions with the plant and/or pathogen is

attracting increasing attention, not only because of their antagonistic activity but also because

of their potential to boost plant growth, contributing to more sustainable output over time

(Gautam and Shubhi, 2019).

Fungi of the genera Trichoderma, Clonostachys, and Penicillium, as well as bacteria

Pseudomonas and Bacillus, are among the most widely used microorganisms for the control

of pathogens. In respect to their antagonistic activity, these exhibit a variety of modes of

action that can work independently or jointly. The probability of the pathogen developing

resistance is lessened if the antagonist may express many mechanisms of action. Competition

for resources and space, antibiosis, parasitism (considered an antagonistic symbiosis between

organisms), and activation of systemic resistance are among the mechanisms of action

described. The use of these control agents is considered to be a more effective control

strategy; nevertheless, various factors should be investigated in order for them to reach their

full potential, including the method of application, the combined use of antagonists,

formulation, and condition during application. Moreover, the likelihood that beneficial

microbes may remain in the soil could be an important advantage over the usage of

pesticides. An additional advantage is that if they remain in the rhizosphere, they are in the

first line of defence against attack by soil pathogens (Abeysinghe, 2009).

Biocontrol of plant pathogens using CLPs may involve direct antagonism and/or indirect

activity via induced systemic resistance (ISR).

21
1.2.6.3.1. Induced Systemic Resistance (ISR)

Induced resistance is a physiological state in which a plant's defensive capacity is increased in

response to biological or chemical inducers, protecting plant tissues that were not exposed to

the initial attack from future pathogen and herbivorous insect attacks (Van Loon et al., 1998).

Plants can develop induced resistance in response to pathogen infection, insect herbivory, or

root colonization by specific rhizosphere mutualistic microorganisms. Two of the most

studied forms of induced resistance are SAR (Systemic Acquired Resistance) which is

activated by plant pathogens, and ISR (Induced Systemic Resistance) which is caused by

root-colonizing mutualistic microbes, like Pseudomonas simiae (syn. P. 241 fluorescens),

Paenibacillus polimyxa or Trichoderma spp. (Zhang et al., 2009; Pieterse et al., 2012, 2014;

Alizadeh et al., 2013; Zamioudis et al., 2014, 2015; Zhao et al., 2014; Martínez-Medina et

al., 2017; Verbon et al., 2017).

The redundancy of the microbial elicitors that cause ISR is a general characteristic. Because

of this redundancy, microbial mutants that are deficient in one elicitor can still trigger ISR via

other elicitors (Meziane et al., 2005; Pieterse et al., 2014; Zamioudis et al., 2015). For

example, the siderophore pseudobactin was just as effective as live bacteria in causing ISR,

but a mutant defective in pseudobactin production was just as effective (Meziane et al.,

2005). Beneficial ISR-eliciting microorganisms do not directly activate defense responses;

instead, they sensitize the entire plant (a process known as priming) so that defense responses

are activated sooner and more effectively when pathogens invade (Choudhary et al., 2007;

Berendsen et al., 2012; Jung et al., 2012; Pieterse et al., 2014 and Martínez-Medina et al.,

2016). To enable the establishment of a mutually beneficial association with the host root, the

ISR-eliciting microorganisms repress a large majority of the genes produced by the elicitors,

such as flagellin, which are largely linked with defense responses (Stringlis et al., 2018). New

22
findings have shown that Plant hormone signaling pathways are being hijacked by beneficial

soil-borne bacteria to decrease host defenses (Pieterse et al., 2012).

Similar to microbe (or pathogen)-associated molecular patterns (MAMPs or PAMPs), ISR

can occur in plants as a result of the sensing of elicitors released by microbes (or pathogens)

(Newman et al., 2013). Plant innate immunity is elicited when MAMPs or PAMPs are

recognized by pattern recognition receptors attached to the cell membrane (Boutrot and

Zipfel, 2017). ISR can be triggered by a variety of elicitors secreted by plant beneficial

bacteria (De Vleesschauwer and Höfte, 2009; Mariutto and Ongena, 2015), including CLPs

generated by Pseudomonas spp. (massetolide A, orfamide, and sessilin) (Tran et al., 2007;

Ma et al., 2016, 2017), as well as Bacillus spp. (surfactin, fengycin and iturin) (Ongena et al.,

2007; Jourdan et al., 2009; Henry et al., 2011). However, there is no evidence that plants

detect CLPs through specialized receptors. Perception is based on a lipid-driven mechanism

at the plasma membrane level, according to results with Bacillus CLP surfactin (Henry et al.,

2011).

1.2.6.3.2. Direct antagonism

Pathogens are antagonized by the presence and activities of other organisms they come into

contact with. By the mechanism(s) expressed by the Biological Control Agents (BCAs),

direct antagonism is caused by physical contact and/or a high degree of selectivity for the

pathogen. Hyperparasitism by obligate parasites of plant pathogens would be considered the

most direct type of antagonism in such a scheme because no other organism's activities would

be necessary to provide a suppressive effect (Iavicoli et al., 2003).

The most effective biocontrol active microorganisms researched appear to use a variety of

mechanisms to antagonize plant pathogens (Cook, 1993). Pseudomonads that produce the

antibiotic 2, 4-diacetylphloroglucinol (DAPG) may, for example, trigger host defenses

(Kloepper et al., 1980; Lafontaine and Benhamou, 1996; Leeman et al., 1995; Maurhofer et

23
al., 1994; Silva et al., 2004). Furthermore, DAPG-producing bacteria can colonize roots

aggressively, a characteristic that may contribute to their ability to limit pathogen activity in

the rhizosphere of plants through competition for organic nutrients.

Hyperparasitism occurs when a specialized biocontrol agent (BCA) attacks the pathogen

directly, killing it or its propagules. Generally, there are four major classes of hyperparasites:

obligate bacterial pathogens, hypoviruses, facultative parasites, and predators (Tjamos et al.,

2010). There are different fungal parasites of plant pathogens, including those that attack

sclerotia (i.e. Coniothyrium minitans) while others attack living hyphae (i.e. Pythium

oligandrum) and, a single fungal pathogen can be destroyed by multiple hyperparasites. For

example, Acremonium alternatum, Acrodontium crateriforme, Ampelomyces quisqualis,

Cladosporium oxysporum, and Gliocladium virens are just a few of the fungi that are capable

of parasitizing powdery mildew pathogens (Heydari and Pessarakli, 2010).

Unlike hyperparasitism, microbial predation is more general and pathogen agnostic, resulting

in less predictable levels of disease control. Under nutrient-limited environments, some

BCAs engage in predatory behavior. However, such activity is rarely seen under normal

growth conditions. Some Trichoderma species, for example, release a range of enzymes that

attack fungi's cell walls (Sharma and Bhat, 2011).

1.2.7. Beneficial bacteria associated with plants

The rhizosphere is a thin layer of soil that surrounds plant roots and serves as a highly

essential and active area for root activity and metabolism. The rhizosphere was first defined

by Hiltner (1904), a German researcher, as the soil area surrounding the root that is directly

or indirectly influenced by the root and has a high microbial activity. The original notion has

since been expanded to encompass the soil surrounding a root, which has been altered by root

24
growth and activity in terms of physical, chemical, and biological aspects (McCully, 2005;

Sivasakthivelan and Saranraj, 2013).

The rhizosphere is home to a diverse range of microorganisms, including bacteria, fungus,

protozoa, and algae. Bacteria are the most numerous of these microorganisms. Plants choose

the bacteria that are most beneficial to their health by releasing organic chemicals through

exudates, resulting in a highly selective ecosystem with little variety (Garcia et al., 2011).

Because bacteria are the most prevalent microbes in the rhizosphere, it's likely that they have

a stronger impact on plant physiology, especially given their competition in root colonization

(Barriuso et al., 2008).

The rhizosphere, which refers to the thin layer of soil around plant roots as well as the soil

inhabited by the roots, is home to enormous colonies of bacteria known as plant growth

promoting rhizobacteria (PGPR). Rhizobacteria that promote plant growth are known to

infiltrate the rhizosphere quickly and suppress soil-borne pathogens at the root surface

(Rangarajan et al., 2003). These bacteria can also aid in the growth of the plant by stimulating

it (Bloemberg and Lugtenberg, 2001). Fluorescent Pseudomonas are the most promising

category of plant growth-promoting rhizobacteria for biocontrol of plant diseases among

these organisms. Pseudomonas spp. is frequently employed as a model bacteria for root

colonization (Lugtenberg et al., 2001).

A variety of bacteria from the genera Azospirillum, Alcaligenes, Arthrobacter,

Acinetobacter, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Pseudomonas,

Rhizobium, and Serratia are present in the plant rhizosphere and have the ability to promote

plant growth (Tilak et al., 2005).

1.2.7.1. Bacillus spp.

Bacillus species are excellent candidates for biological control. To begin, they produce anti-

fungal and anti-bacterial antibiotics. Their second distinguishing characteristic is their ability

25
to produce spores; as a result of this property, they are simple to formulate because they

survive longer than tube cells. It is worth noting that rhizospheric bacteria have a huge

potential for synthesizing and releasing a variety of chemicals that control plant growth as

well as the physical and chemical texture of the soil. As a result, Bacillus species' charcoal-

based formulations can be used to promote plant growth and activity of a wide range of crops

(Pahari et al., 2017). Bacillus species have been found to inhibit phytopathogens by

producing antibiotic compounds that protect plants against pathogens via antibiosis (Cawoy

et al., 2011; Bacon et al., 2015). Another distinguishing aspect is their permanent existence in

the soil (due to their role in the soil's general microflora) (Balouiri et al., 2015). Among the

activities of antibiotics produced by this bacterium, one can name their direct effect on fungi,

competition with microorganisms which attack root systems. Bacillus bacteria, because of

their propensity to colonize the plant's internal organs, appear to be acceptable alternatives for

controlling vascular pathogens. Strains of Bacillus subtilis produce a broad spectrum of

antimicrobial compounds (Mannanov and Sattarova, 2001). As a result of this activity, they

are potential biological control agents for a variety of plant pathogens (McSpadden-Gardener

and Driks, 2004).

Because of the potential of a varied collection of Bacillus species to treat plant diseases,

various disease control products based on these strains have been registered and

commercialized. It has been proven that incorporating these products into integrated pest

management systems is an effective disease control technique (Jacobsen et al., 2004). The

synthesis of secondary metabolites, particularly antibacterial cyclic lipopeptides, has been

linked to the biological activity of these strains. Bacillus species is one of the most common

genus in the rhizosphere, and the PGPR activity of some strains has been long known,

resulting in a thorough understanding of the mechanisms involved . These strains emit a

number of metabolites (Charest et al., 2005), which have a significant impact on the

26
environment by enhancing plant nutrition availability. Bacillus species are normally found in

close proximity to plant roots. Bacillus subtilis can sustain persistent contact with higher

plants and encourage them to grow. From the standpoint of the establishment of the soil

microbiota rhizosphere, bacterial inoculation at the start of the acclimation phase can be

detected in a micro-propagated plant system. When Bacillus licheniformis is inoculated on

tomato and pepper plants, it colonizes rapidly and can be used as a biofertilizer without

affecting conventional greenhouse management (Garcia et al., 2004).

Bacillus species used as biofertilizers are thought to have direct effects on plant development

by producing plant growth hormones (Amer and Utkhede, 2007). Phosphate-solubilizing

Bacillus spp. increases plant growth by boosting the uptake of N, P, potassium (K), and iron

(Fe). By enhancing the effectiveness of biological nitrogen fixation and the availability of

iron (Fe) and zinc (Zn) through the creation of plant growth promoting chemicals, phosphate

biofertilizers could help increase the availability of phosphates accumulated in the soil and

enhance plant growth. Pseudomonas and Bacillus use a variety of direct and indirect

mechanisms to promote growth and control disease, including metabolite synthesis (auxin,

cytokinin, and gibberellins), induction of 1-amino cyclopropane-1-carbocylate (ACC)

deaminase, siderophore production, antibiotics, hydrogen cyanide (HCN), and volatile

compounds. Mineral solubilization (e.g. phosphorus), competition, and induced systemic

resistance are also some of the mechanisms involved (Hamid and Ahmad, 2010).

1.2.7.2. Pseudomonas species

Pseudomonas spp., which belong to the genus Proteobacteria, are abundant in the rhizosphere

and soil microorganisms (Omoboye et al., 2019; Philippot et al., 2013). The biocontrol

abilities of Pseudomonas species associated with the rhizosphere have been studied

extensively (Haas and Défago, 2005; Weller, 2007; D’aes et al., 2010; Höfte and Altier,

2010; Olorunleke et al., 2015b; Stringlis et al., 2018 and Omoboye et al., 2019).

27
Pseudomonas species have a diverse metabolic profile, producing a variety of secondary

metabolites such as antibiotics and cyclic lipopeptides (CLPs) (Omoboye et al., 2019; Gross

and Loper, 2009).

Many characteristics of Pseudomonas spp. make them biocontrol agents, including

colonization and proliferation within the plant, competition with other microorganisms,

adaptation to environmental stresses, and the production of a wide range of active

biometabolites, including antibiotics, siderophores, volatile substances, and growth stimulant

compounds (Stockwell and Stack, 2007). As a result, plant pathogens are suppressed by such

bacteria. Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas aureofaciens,

Pseudomonas putida, and Pseudomonas syringae are among the most frequent Pseudomonas

species that play a role in biological control; P. syringae being non-pathogenic.

Fluorescent Pseudomonas spp. has proven to be the most effective Pseudomonas strains.

Fluorescent Pseudomonas contribute to soil health and have the largest metabolic and

functional diversity (Lata et al., 2002). Pseudomonas spp. are essential plant growth

promoting rhizobacteria (PGPR) which are used as biofertilzers and can increase crop output

through both direct and indirect processes (Walsh et al., 2001). Several studies have proved

that fluorescent Pseudomonas is common in the rhizosphere of various crops. (Kumar et al.,

2004). They effectively produce a wide range of biologically active chemicals, with growth-

promoting molecules being of particular relevance (Rodriguez, 2006).

Pseudomonas strains can solubilize phosphorus in soil, making it more available to plants.

Some strains of Pseudomonas produce siderophores, which are chelating agents with a high

affinity for iron absorption. By increasing iron solubility in the plant rhizosphere, microbial

siderophores can promote plant development. Pathogens' negative impacts on plant growth

can also be mitigated with these products (Sundara et al., 2002).

28
1.2.7.3. Enterobacter species

The genera within the family Enterobacteriaceae that contain members known as plant

growth promoting bacteria (PGPB) are Citrobacter, Enterobacter, Erwinia, Klebsiella,

Kluyvera, Pantoea and Serratia, although some of these genera also contain species reported

to be plant pathogens, such as Erwinia carotovora (Rodrı´guez-Dı´az et al., 2008).

Hormaeche and Edwards were the first to describe the Enterobacter genus in 1960

(Hormaeche and Edwards, 1960).

Several strains of Enterobacter spp. have been described as effective biological control agents

antagonistic to a variety of fungal phytopathogens. Several Enterobacter cloacae isolates have

been identified as biocontrol agents for different rots and pre-emergence damping-off of pea,

beet, cotton, and cucumber plants incited by Pythium spp., as well as of Fusarium wilt of

cucumber and some other plant diseases caused by fungal pathogens (Hadar et al., 1983;

Nelson, 1988; Sneh et al., 1984).

Enterobacter aerogenes B8 significantly decreased infections of apple crown and root rot

caused by Phytophtora cactorum (Uthkede, 1986). Some Enterobacter agglomerans strains

have been demonstrated to be effective in controlling plant diseases caused by several

bacterial and fungal pathogens (Hebbar et al., 1992; Nelson, 1988; Park and Kim, 1989). The

effect of these strains as biological control agents is thought to be due to a variety of features

exhibited synchronously or in a regulated sequence (Nelson and Maloney, 1992). E.

aerogenes B8 was discovered to have the ability to produce an antibiotic-like compound

(Utkhede and Gaunce, 1983).

Attachment to fungal hyphae was thought to be an essential mechanism in the biocontrol

activity of E. cloacae strains against Pythium ultimum (Nelson et al., 1986), and chitinolytic

enzymes from Trichoderma harzianum were shown to stimulate this binding (Lorito et al.,

1993).

29
Ahmad and Khan (2010) studied Enterobacter for fungicide tolerance and production of PGP

traits in the presence and absence of fungicides. Deepa et al. (2010) studied PGP potential of

strains NII0907 (E. aerogenes), NII-0929 (E. aerogenes), NII-0931(E. cloacea) and NII-0934

(E. asburiae) members of the genus Enterobacter. The four Enterobacter species are very

good phosphate solubilizers (60.1–79.5 mg/ml/day after tenth day of incubation); indole

acetic acid (IAA) producers (23.8–104.8 mg/ml/day after 48 h of incubation); HCN producers

and siderophore producers. Studies have been carried out to investigate their impact on

cowpea and recorded significant increase in higher root and shoot lengths when compared

with uninoculated control. Further testing of the isolates exhibiting multiple PGP traits on

soil–plant system is needed to determine their efficacy as effective PGPR.

E. cloacae and E. agglomerans strains have been shown to produce hydroxamate siderophores

(Berner et al., 1988; Loper et al., 1993) as well as a variety of volatile and non-volatile

antifungal metabolites (Howell et al., 1988; Truitman and Nelson, 1992). In E. cloacae

(Wisniewski et al., 1989) and E. agglomerans (Hebbar et al., 1992), competition for nutrients

and rhizosphere colonization abilities were proposed as probable mechanisms of antifungal

activity.

E. cloacae is a bacterial species that has been found in a wide range of environments,

including plants, soil, and humans. Plant pathogenic strains of E. cloacae have been known to

cause Enterobacter bulb decay in onion plants and bacterial wilt in mulberry (Zhu et al.,

2011; Schroeder et al., 2010); endophytic E. cloacae strains have been shown to colonize and

benefit plant growth in a variety of crops, including soybean, cucumber, corn, rice and ginger

(Liu et al., 2007; Hinton and Bacon, 1995). Previous biological studies of a variety of plant-

origin isolates have revealed that E. cloacae is antagonistic to the oomycete pathogen

Pythium ultimum (Nelson, 1988), the fungal pathogens Fusarium moniliforme and Fusarium

oxysporum (Hinton and Bacon, 1995; Suárez-Estrella et al., 2007), and the bacterial pathogen

30
Ralstonia solanaceae (Xue et al., 2009). Several strains of E. cloacae have also been

identified as plant growth-promoting rhizobacteria (PGPR).

Enterobacter have the potential to aid in the development of sustainable agricultural systems.

In general, Enterobacter function in three different ways: synthesizing particular compounds

for the plants, aiding the uptake of specific nutrients from the soil, and lessening or

preventing the plants from diseases (Jha et al., 2011).

1.2.8. Cyclic lipopeptides (CLPs)

CLPs are a structurally diversified family of natural products (Roongsawang et al., 2011) that

are mostly biosynthesized by bacteria from the genera Streptomyces (Kügler et al., 2015),

Bacillus (Ongena and Jacques, 2008), and Pseudomonas (Gross and Loper, 2009). The

amphiphilic nature of CLPs, which makes them great biosurfactants, is responsible for much

of their biological activity. As a result, a large number of CLPs are essential for bacterial

swarming motility and biofilm formation (Banat et al., 2010).

CLPs are multifunctional molecules that have antibacterial, cytotoxic, and surfactant

characteristics. Several plant-associated Pseudomonas spp., including pathogenic

Pseudomonas syringae, P. tolaasii, P. fuscovaginae, P. corrugata, and P. fluorescens (Bender

et al., 1999), as well as antagonistic Pseudomonas fluorescens and P. putida, produce

CLPs (Nielsen et al., 2002; Nybroe and Sørensen, 2004).

1.2.8.1. Lipopeptide producing bacteria

Despite the fact that a vast variety of strains were capable of producing surfactants,

lipopeptide-producers were less diversified, according to research statistics. About 70 strains

of the species Bacillus (Kim et al., 1997; Lv et al., 2005), Pseudomonas (Kuiper et al., 2004;

Raaijmakers et al., 2006), Streptomyces (Hojati et al., 2002), Serratia (Anyanwu et al.,

2011), etc. Bacillus and Pseudomonas lipopeptide has been extensively studied.

31
Enterobacter studies have always focused on properties related to human pathogenicity and

antibiotic sensitivity, but less on biotechnology, such as enzyme production and metabolites

(Barraquioa and Watanabea, 1981; Sone et al., 1987; Wang et al., 1989; Dahiya et al., 2005).

Mandal et al. (2013) discovered a group of lipopeptide-producing bacteria belonging to the

Enterobacteriaceae family. These strains were able to co-produce iturins, fengycins,

kurstakins, and surfactin, among other lipopeptides. However, they did not provide much

information regarding surfactin; instead, they believed that a component produced by

Citrobacter was surfactin based on its molecular mass.

1.2.8.2. Structure of Cyclic lipopeptides

CLPs are amphiphilic compounds with a fatty acid tail and a cyclic oligopeptide lactone ring

(Raaijmakers et al., 2006, 2010). CLPs are made up of an oligopeptide with a peptidically

connected fatty acid at the N-terminus. The length (usually C 6-C18) and degree of oxidation of

the linear or branched lipid tail might vary (Cochrane and Vederas, 2016). The oligopeptide's

C-terminus (up to 25 amino acids) forms a lactone or lactam with a hydroxyl, phenol, or

amino functional group present in the peptide's side chains or portion of the lipid moiety,

resulting in macrocycles of various sizes (typically 4-16 amino acids) (Schneider et al.,

2014). Nonproteinogenic (e.g., D-configured or -amino acids) and modified amino acids

(e.g., 4-chlorothreonine) amino acids can be found in CLPs since they are biosynthesized by

nonribosomal peptide synthetases (NRPS) (Marahiel, 2016).

32
Figure 1.4. Different structure of various cyclic lipopeptides (Lee and Kim, 2015).

33
1.2.8.3. Mode of action of Cyclic lipopeptides

CLPs have received a lot of attention for their antimicrobial, cytotoxic, antitumour,

immunosuppressant and surfactant properties (Cameotra and Makkar, 2004; Donadio et al.,

2007; Gross and Loper, 2009; Pirri et al., 2009). CLPs are thought to work by causing pore

formation in membranes, which leads to an imbalance in transmembrane ion fluxes and cell

death (Bender et al., 1999; Baltz, 2009). As a result, there is increased interest in the

discovery, combinatorial synthesis, and application of ‘new' and ‘old' CLPs for a variety of

environmental and medicinal purposes (Pirri et al., 2009). Pseudomonas and Bacillus have

attracted the most attention among the bacterial CLP producers. Both genera are found in a

variety of natural habitats, harbor pathogenic and beneficial species, and have a wide range of

lifestyles. There is a wealth of knowledge on the structural variety of LPs, their production,

and broad-spectrum antibacterial activity in Pseudomonas and Bacillus species (Bender et al.,

1999; Nybroe and Sørensen, 2004; Raaijmakers et al., 2006; Ongena and Jacques, 2008;

Gross and Loper, 2009).

Antimicrobial activity of cyclic lipopeptides is mediated by either cell wall production or cell

membrane integrity. Friulimicin B, produced by Actinoplanes friuliensis, and CLP malacidin,

recently found in soil microbiomes, both hinder cell wall biosynthesis (Schneider et al., 2009;

Hover et al., 2018). Streptomyces roseosporus produces daptomycin, and Bacillus and

Paenibacillus species generate tridecaptin A1, which has antibacterial potential by altering

membrane integrity (Straus and Hancock, 2006; Cochrane et al., 2016).

Antibiosis activity is also mediated by Pseudomonas CLPs like as syringopeptins, cormycin,

and massetolides, which alter membrane integrity. The majority of CLPs' molecular targets

are unknown (Masschelein et al., 2017). Many CLPs produced by Pseudomonas spp. are

involved in a variety of biological processes, including motility, biofilm formation, and

pathogenicity, in addition to antibacterial action. As biosurfactants, most CLPs generated by

34
Pseudomonas spp. have effect on bacterial motility and biofilm formation (Raaijmakers et

al., 2010).

Due to the structural diversity of CLPs produced by Pseudomonas spp. and other bacterial

genera, Ron and Rosenberg (2001) postulated that CLPs and, more broadly, microbial

surface-active compounds (biosurfactants) have various natural roles, some of which may be

unique to the physiology and ecology of the microorganism producing them. Several natural

roles of CLPs and other biosurfactants were proposed (Ron and Rosenberg, 2001), including

their function in

(i) Pathogenicity,

(ii) Antimicrobial activity,

(iii) Regulation of attachment and detachment to and from surfaces, and

(iv) Motility

Another natural function of CLPs, which may be limited to spore-forming bacteria, is as

signal molecules for coordinated development and differentiation (Marahiel et al., 1997).

1.2.8.4. Roles of CLPs in direct antagonism

Against Gram-positive bacteria, cyclic lipopeptides have a pronounced antagonistic effect. At

similar concentrations, syringopeptin 22A, syringopeptin 25A, corpeptin-(SP), tolaasin, and

WLIP-(V) all suppress Bacillus megaterium growth (Emanuele et al., 1998; Grgurina et al.,

2002; Bassarello et al., 2004; Rokni-Zadeh et al., 2012) while syringomycin E and

syringotoxin-(SM) have contradictory actions (Lavermicocca et al., 1997; Scaloni et al.,

2004). Early reports of the biological activity of syringomycin Ps-CLPs, however, may have

been conducted using impure preparations containing syringopeptins. In general,

syringopeptins are more effective against Rhodococcus and Micrococcus species than

syringomycins and syringotoxins-(SM) (Lavermicocca et al., 1997).

35
CLPs from the viscosin and syringopeptin families have antagonistic activity against a variety

of Staphylococcus aureus subspecies, including methicillin-susceptible and methicillin-

resistant strains. Only WLIP-(V) (D-aIle4, D-Leu5) and massetolide A-(V) (D-Val4, L-Leu5)

were ineffective against all S. aureus strains tested (Gerard et al., 1997; Grgurina et al., 2005;

Andolfi et al., 2008; Reybroeck et al., 2014; Geudens et al., 2017).

The vast majority of Ps-CLPs have been found to have no antimicrobial activity against

Gram-negative bacteria (Lavermicocca et al., 1997; Grgurina et al., 2005; Lo Cantore et al.,

2006; Andolfi et al., 2008; Sinnaeve et al., 2009a, b; Reybroeck et al., 2014). Pseudomonas

CLPs are therefore thought to be ineffective against Gram-negative bacteria. The existence of

the outer membrane or peptidoglycan layer, which prevents access to the plasma membrane,

is commonly attributed to this (Nybroe and Sørensen, 2004; Raaijmakers et al., 2006).

However, the situation is less apparent as CLPs of the tolaasin group (Lo Cantore et al.,

2006), and the recently identified xantholysin group (Li et al., 2013; Molina-Santiago et al.,

2015) can inhibit Gram-negative bacteria. Furthermore, WLIP-(V) had some activity against

Erwinia carotovora subsp. carotovora, a Gram-negative bacterium (Lo Cantore et al., 2006).

To add to the seeming uncertainty, contradictory activity against several Xanthomonas

species have been observed, whereby WLIP-(V) did (Rokni-Zadeh et al., 2012) or did not

(Lo Cantore et al., 2006; Andolfi et al., 2008) have any activity against this Gram-negative

bacterium. Despite this, it is obvious that Ps-CLP activity is not confined to Gram-positive

bacteria.

Antifungal activity has been investigated for a number of CLPs as well as plant- and human-

pathogenic fungi and yeasts, including Rhizoctonia solani, Phoma lingam, Alternaria

brassicae, Sclerotinia sclerotiorum, Geotrichum candidum, Botrytis cinerea , Ophiostoma

ulmi, Aspergillus and Fusarium spp., Penicillium digitatum, Cryptococcus neoformans,

Candida albicans, and C. glabrans (Nybroe and Sørensen, 2004). The studies with

36
viscosinamide produced by antagonistic Pseudomonas sp. strain DR54, in particular, give

numerous lines of evidence that CLPs are significant components in the biological control of

plant-pathogenic fungi. In vitro studies revealed that viscosinamide damaged R. solani and

Pythium ultimum mycelium, causing decreased growth and intracellular activity, hyphal

swellings, increased branching, and rosette formation (Hansen et al., 2000 and Thrane et al.,

1999, 2000a).

In situ, strain DR54 produces viscosinamide (Nielsen and Sorensen, 2003), and several of the

negative effects on R. solani and P. ultimum observed in vitro, such as reduced mycelium

density and intracellular activity in P. ultimum and reduced sclerotia formation in R. solani,

were also observed in situ (Thrane et al., 1999, 2000b, 2001). A comparison of the biocontrol

activity of a wild type strain and CLP-deficient mutants was not included in these and earlier

experiments with antagonistic CLP-producing Pseudomonas strains. The development of

CLP-deficient mutants as well as mutants overproducing CLPs has arisen from advances in

our understanding of CLP biosynthesis. For example, a Bacillus subtilis 6051 mutant

deficient in surfactin synthesis was significantly less effective than the wild-type strain in

preventing Pseudomonas syringae root infection in Arabidopsis (Bais et al., 2004).

Furthermore, on tomato seedlings, a B. subtilis BBG100 variant that overproduces

mycosubtilin demonstrated increased activity against Pythium spp. (Leclère et al., 2005).

The dual function of CLPs in the interactions between antagonistic Pseudomonas strains and

plant pathogens is a complicating factor in studying the role of CLPs. In addition to their

direct effects on pathogen membranes, their surface activity may increase or perhaps be

required for the delivery of and exposure of target pathogens to other antagonistic features.

CLPs produced by Pseudomonas syringae acted synergistically in antagonism against many

plant-pathogenic fungi when combined with CWDE from Trichoderma atroviride (Fogliano

et al., 2002).

37
In this study, we will present the use of direct antagonism and ISR as a mechanism for the

biological control of Pepper plant pathogens as it is applied to the suppression of plant

diseases. Also, morphological identification of fungi; and molecular characterization of

bacteria by 16S rRNA sequencing will be carried out. Extraction of CLPs from bacterial

isolates as well as the direct antagonistic potential of the isolated bacteria and CLPs against

the fungal pathogens will be investigated.

38
 CHAPTER TWO

MATERIALS AND METHODS

2.1. MATERIALS

2.1.1. Glassware

Glass wares used include measuring cylinder, conical flask, beakers, test tubes, McCartney

bottles, measuring pipettes and microscopic glass slides.

2.1.2. Equipment, Instruments and other materials

The equipment and instrument used in performing the research include weighing balance,

spatula, cotton wool, micropipettes, inoculating loop, forceps, test-tube rack, gas cylinder,

Bunsen burner, pressure pot, incubator, hot air oven, autoclave, refrigerator, cryovials,

eppendorf tubes, centrifuge tubes, mini-centrifuge tubes, PCR tubes, centrifuge, sterile petri

dishes, photomicrograph, glass spreader, toothpick, foil paper, paper tape, lighter, permanent

markers, disinfectant (ethanol), votex, extraction chamber, heating block, thermocycler and

microscope.

2.1.3. Reagents

These include NEB OneTaq 2X MasterMix with Standard Buffer, Genomic DNA, Tris-

borate-EDTA (TBE) buffer, forward and reverse primer, nuclease free water, DNA template,

hypochlorite solution, normal saline, methanol, paraffin, lactophenol cotton blue solution,

hydrochloric acid and distilled water.

2.1.4. Culture Media used

Culture Media used include Potato Dextrose Agar (PDA) for the cultivation and sub-culturing

of fungi; Nutrient broth, Nutrient Agar, King’s B broth, King’s B Agar, Luria Bertani (LB)

39
broth and Luria Bertani (LB) agar for the cultivation and sub-culturing of bacteria and also

for obtaining pure colonies.

2.2. METHODS

2.2.1. Sterilization of glassware and other materials used

Sterilization of glassware and other materials were performed via:

1. Autoclaving: All test tubes, conical flasks, beakers and other glassware used for this

project work were properly washed with detergent, then dried and sterilized in hot air oven at

160 oC for 1h. All media prepared, according to the manufacturers’ specification, were

sterilized using the autoclave at 121 oC for 15 minutes and 15N/m2. Distilled water and

normal saline prepared were also sterilized in the autoclave.

2. Disinfecting: Heat-sensitive materials such as plastic containers were thoroughly

washed with detergent, rinsed in clean water, air-dried and sterilized using 70% ethanol.

Work surfaces were disinfected by scrubbing with cotton wool soaked in 70% ethanol before

and after use to avoid contamination.

3. Flaming: During the process of pouring media from the conical flasks into sterile

petri dishes, they were flamed before and after use and any form of contamination such as

talking and eating was avoided. The inoculating loops and the cork borer were sterilized to

red-hot before and after use.

Aseptic technique was ensured at the workbenches throughout the research period so as to

avoid contamination in any form in a bit to achieving reliable and standard results.

2.2.2. Media preparation

All culture media used were prepared following the manufacturer’s instruction. With the aid

of a weighing balance, accurate measurements were taken and poured into conical flasks.

40
Distilled water was measured and poured into the conical flask and homogenized in a water

bath. Liquid media such as nutrient broth, LB broth, KB broth and normal saline were

dissolved in distilled water and dispensed into test tubes or conical flasks before autoclaving.

The media were sterilized in an autoclave at 121 oC and 15 N/m2 for 15 minutes after which

they were allowed to cool down. The media were poured into Petri-dishes aseptically,

allowed to solidify and were kept inverted in an oven overnight to check for contaminant and

to dry.

2.3. SAMPLING

2.3.1. Sample Collection

Stem and root of wilted scotch bonnet pepper plants (Capsicum chinense) affected by fungi,

and the root of a healthy cayenne pepper plant (Capsicum annuum) were collected from

Moremi Estate, Ile-Ife, Osun State. The residential setting of the site was characterized with

good decomposed activities in the soil that allows for good and efficient growth of plants.

The samples were collected in a clean container then taken to the laboratory at the

Department of Microbiology, Obafemi Awolowo University.

2.3.2. Method of Isolation

2.3.2.1. Isolation of fungi from wilted scotch bonnet pepper plant root and stem

The roots and stems of the wilted scotch bonnet pepper plant (C. chinense) were cut into

smaller pieces and carefully rinsed with sterile distilled water to retain the microorganisms,

after which it was washed with 2% hypochlorite solution for 2 minutes to remove surface

contaminant. The roots and stems were cut open longitudinally and placed aseptically on the

PDA plate and incubated at room temperature for 3-7 days. 0.4% of Antibiotics

(Streptomycin) was added prior to pouring of the PDA to suppress the growth of bacteria.

The PDA plates were checked for growth and different colonies were sub-cultured on PDA

plates and incubated at room temperature for 3-7 days to obtain pure culture.

41
For preservation of the fungal isolates, a standard volume of Potato Dextrose agar was

prepared inside a conical flask and homogenized after which 15 ml each were dispensed into

the McCartney bottles. The culture media were autoclaved at 121 oC for 15 minutes. After

sterilization, the agar was allowed to cool and solidify in a slanting position. Each surface of

the slanted agar was inoculated with 5-day old pure fungal isolates and labeled appropriately.

The cultures were incubated at 30 oC for 5-7 days. The slants were stored in the refrigerator

and the preserved cultures were used for the subsequent tests that were carried out on the

isolates.

2.3.2.2. Isolation of bacteria from the root of a healthy cayenne pepper plant

The roots of the healthy cayenne pepper plant (C. annuum) were carefully washed with sterile

distilled water and damped to remove moisture after previously cutting into smaller pieces.

All the root was weighed and macerated using mortar and pestle with 10 ml sterile normal

saline, with the addition of little amount of sterile sand until it is very smooth. This was

transferred into test tubes containing normal saline and a 10-fold serial dilution was

performed. Exactly 100 µL of the serially diluted suspension (10 -3 and 10-4) were inoculated

into King’s B agar plate using the glass spreader and incubated at 30 oC for 24 h. The KB

plates were checked for growth and different colonies were sub-cultured on KB plates by

streaking and were incubated again at 30 oC for 24 h to obtain pure culture.

Luria Bertani (LB) broth was prepared and poured into test tubes after which they were

autoclaved at 121 oC for 15 minutes. The broth was allowed to cool and few colonies of the

bacteria from King’s B agar plates were inoculated aseptically into the test tubes and labeled

according to the isolate codes. The test tubes were incubated overnight at 28 oC. Exactly 1 ml

of overnight broth culture of the bacteria and another 1 ml of 40% glycerol were added to

sterile cryovial tubes. The cryovial tubes were stored in the freezer for subsequent tests that

will be carried out on the isolates.

42
43
Figure 2.1. Representative pictures showing diseased (a) and healthy (b) scotch bonnet
pepper plants (Capsicum chinense); and healthy (c) cayenne pepper plant (Capsicum
annuum).

44
2.4. Microbiological tests

2.4.1. Antagonism assay

The antagonism test of the bacterial isolates against the fungi was carried out as described by

Omoboye et al. (2019). Potato Dextrose agar was prepared, homogenized and then sterilized

in an autoclave after which it was allowed to cool. The agar was poured into sterile petri

dishes and allowed to solidify. Fungal plugs, labelled as GP5, were obtained from 5-7 day old

fungi using 5mm cork borer and the plug was transferred onto the prepared PDA at the centre

of the plate. Exactly 3 µL of overnight broth culture of bacterial isolates LPb1-1 were then

transferred to each of the sides of the mycelium at a distance of 2 cm from it. The plates were

incubated at 30 oC for 3-5 days after which the diameter of the zones of inhibition was

measured.

2.5. Microscopic identification of fungal isolates

2.5.1. Principle

The staining reagent used for the identification of fungi is Lactophenol Cotton blue (LPCB).

This is a simple histological staining method used for the microscopic examination and

identification of fungi. Lactophenol Cotton Blue (LPCB) staining method works on the

principle of aiding the identification of the fungal cell walls.

Fungi are eukaryotic organisms with both macroscopic and microscopic characteristics. The

fungal spore cell wall is made up of chitin, which is the components which lactophenol cotton

blue solution stains for identification. The lactophenol cotton blue solution acts as a mounting

solution as well as a staining agent. The solution is clear and blue in colour and it is made up

of a combination of three main reagents: phenol, which acts as a disinfectant by killing any

living organisms; lactic acid, to preserve the fungal structures; and cotton blue, to stain or

give color to the chitin on the fungal cell wall and other fungal structures.

45
The stain will give the fungi a blue-colored appearance of the fungal spores and structures,

such as hyphae.

2.5.2. Procedure

A 4-5 days old pure fungal culture was first prepared due to the fact that a fungus starts

sporulation at day 4 or 5. Then, drops of 70% ethanol was used to clean the microscopic glass

slides. A sterile mounter (i.e. an inoculating loop), previously dipped in 70% ethanol, was

used to pick the fungal specimen unto the sterile slide. The fungal sample was teased using a

needle mounter, to ensure the sample mixed well. About one or two drops of Lactophenol

Cotton Blue solution (prepared stain) was added using a dropper before the ethanol dries off.

Then, a smear was prepared from the cultured fungi and the lactophenol stain. The stain was

covered with a clean sterile cover slip so as to prevent air bubbles to the stain.

A compound microscope was used for viewing and the fungal spores and other fungal

structures were observed at 150X objective lens. The different structure of the mycelium such

as the conidia, philadia, philalides, sporangia, sporangiospores and conidiophores were

closely observed and recorded as shown in Chapter three of this project. The viewed fungal

structure was compared with Benette and Hunter’s manual for fungi identification.

2.6. Molecular characterization of bacterial isolate LPb1-1 by 16S rRNA sequencing

The molecular characterization of the bacteria was carried out at Inqaba Biotech, Ibadan,

Nigeria.

2.6.1. DNA Extraction

DNA extraction was carried out according to the protocol described in Zymo research DNA

purification kit (Quick-DNA 96 Plus kit). For total DNA extraction from bacteria cells, the

culture media were removed before processing by pelleting cells (at approximately 500 x g

for 2 minutes depending on volume and cell type). The supernatants were then removed.

46
2.6.2. Polymerase Chain Reaction (PCR)

PCR was performed by mixing 10 μl of NEB OneTaq 2X MasterMix with Standard, 1 μl of

Genomic DNA (10-30 ng/μl), 1 μl of both Forward primer (10 μM) and Reverse primer (10

μM) and 7 μl of Nuclease free water). The PCR tubes were arranged into the thermocycler.

The thermocycling temperature is set depending on the primers used. The forward and

reverse primers used are referred to in the table below:

Table 2.1. Forward and Reverse primers used

Name Sequence Reference

27F (16S rRNA F) 5`-AGAGTTTGATCCTGGCTCAG-3` Heuer et al., 1997
1492R (16S rRNA R) 5`-TACGGYTACCTTGTTACGACTT-3` Heuer et al., 1997

The initial denaturation temperature is 94 oC for 5 minutes,

The denaturation temperature is 94 oC for 30 seconds,

The annealation temperature is 50 oC for 30 seconds and

The elongation temperature is 68 oC for 1 minutes.

The cycle was run 35 times. The final elongation temperature is 68 oC for 10 minutes and the

hold temperature is 4 oC until removed for further studies.

Agarose gel electrophoresis

The integrity of the PCR amplicons was visualized on a 1% agarose gel (CSL-AG500,

Cleaver Scientific Ltd) stained with EZ-vision® Bluelight DNA Dye.

PCR products were cleaned using ExoSAP Protocol by the preparation of the Exo/SAP

master mix by adding 50 µl of Exonuclease I and 200 µl of Shrimp Alkaline Phosphatase.

Then, a reaction mixture of 10 µl of Amplified PCR Product and 2.5 µl of ExoSAP Mix were

prepared. The reaction mixtures were mixed and incubated at 37 oC for 15 minutes. Finally,

the reaction was stopped by heating the mixtures at 80 oC for 15 minutes.

47
Sequencing of the purified PCR product

Fragments were sequenced using the Nimagen, BrilliantDye Terminator Cycle Sequencing

Kit, according to manufacturer’s instructions. The labelled products are then cleaned with the

ZR-96 DNA Sequencing Clean-up Kit (Catalogue No. D4053). The cleaned products were

injected on the Applied Biosystems ABI 3500XL Genetic Analyser with a 50 cm array, using

POP7. Then, Sequence chromatogram analysis is performed using FinchTV analysis

software.

Extraction of DNA sequencing and identification of the bacteria

The nucleotides obtained after sequencing were processed using BioEdit software after which

it was blasted on the NCBI website: http://www.blast.ncbi.nlm.nih.gov/Blast.cgi. The identity

of the sequenced bacteria was revealed with the percentage identity.

2.7. Morphological characterization of bacteria for CLP production

2.7.1. Drop Collapse Assay

Jain et al. (1991) developed the drop-collapse assay. The assay was carried out on a glossy

surface. Luria Bertani (LB) broth culture of bacterial isolates LPb1-1 was prepared and

incubated in a shaker incubator for 24 h. Exactly 100 µL of the bacteria and 100 µL of water

were dropped on a glossy surface to check for the drop collapse. The bacteria collapsed easily

because the interfacial tension between the liquid drop and the hydrophobic surface was

reduced due to the presence of CLP.

2.7.2. Swarming Motility test

Swarming motility is a rapid and coordinated translocation of a bacterial population across

solid or semi-solid surfaces. One particular feature is the formation of dendritic fractal-like

patterns formed by migrating swarms moving away from an initial location. Exactly 2 mL of

the overnight broth culture of the bacteria were dispensed into eppendorf tubes and

48
centrifuged at 13,400 rpm for 2 min. The supernatant was discarded and 1 mL of normal

saline was added so as to re-suspend the bacteria cells and also centrifuged at 13,400 rpm for

2 min. This process was repeated again after which the supernatant was discarded and 1 mL

of normal saline was added to the pellets to re-suspend it.

Swarming motility tests were conducted on 0.6% Luria Bertani agar for strains used during

this study, using a similar method as previously described (Oni et al., 2019). Representative

strains producing CLP was tested for swarming motility capacity. The bacteria strain was

LPb1-1. CHAO and B2W1-7 are used as control. The swarming motility capacity was

evaluated after incubation at 28 oC for 12-17 h. Measurement of the diameter of the swarming

was taken from the plates.

2.8. Extraction of cyclic lipopeptides (CLPs)

Extraction of crude CLPs from bacteria strain LPb1-1 was carried out following an

established protocol (Oni et al., 2019). Specifically, LPb1-1 was grown on KB agar overnight

after which seed cultures were obtained by growing bacterial cells in 5 ml KB broth in test

tube. The seed culture was transferred in 50 mL KB broth and incubated at 150 rpm and 28
o
C for 24 h in a shaker incubator. Cell-free supernatants were obtained by centrifugation of

the culture at 4,000 rpm and 4 oC for 25 minutes followed by acidification to pH 2 using 6N

hydrochloric acid with constant stirring. Acidified culture supernatant was kept overnight at 4
o
C in order to precipitate the CLPs. Precipitated CLPs were collected by centrifugation at

4,000 rpm for 25 minutes after which crude CLPs were extracted using 100% methanol. The

solvent was evaporated at room temperature under fume hood to obtain crude CLP samples.

Crude CLP was collected.

Extraction and Purification process

Growing bacterial cells in KB Broth

49

Removal of cells by centrifugation at

4000 rpm for 25 minutes
 Supernatant acidified (pH 2.0 with
6N HCl)

Centrifuging again and precipitate

extracted with methanol

Extract concentrated by evaporation

in fume hood

Figure 2.2. Flow chart diagram of cyclic lipopeptide (CLP) extraction (Malfanova et al.,

2012; Sun et al., 2006; Zhang et al., 2012).

50
2.9. Antagonistic assay of CLPs against fungi on microscopic slide

Exactly 10 mg of the concentrated CLP was added to a previously weighed eppendorf tube

after which 1 mL of 100% Dimethyl sulfoxide (DMSO) was added into the eppendorf tube

and thoroughly mixed on a votex. Another 1mL of the DMSO was added to prepare the stock

solution (5 mg/mL). Also, 10 mL of 0.1% DMSO was prepared in an eppendorf tube by

adding 10 µL of 100% DMSO to 9990 µL of sterile water. Dilution of 50 µg/mL was

prepared by transferring a calculated amount of the stock solution to a labeled eppendorf tube

and a calculated amount of 0.1% DMSO was added to make it up to 2 mL. The process was

repeated for dilutions of 25, 10 and 5 µg/mL consecutively.

The sterile Petri dishes were labeled accordingly in batch numbers of previously prepared

solutions and filter paper were aseptically transferred into each of the Petri dishes. Two

toothpicks were put on each end of the Petri dish and 2 ml each of sterile water was added to

the Petri dish. The glass slides were placed aseptically into the Petri dish with the aid of a

sterile forceps. About 350 µL of water agar was dispensed on the glass slides and spread

throughout for proper growth of the organisms to be inoculated. The 5 mm plug of the fungi

(GP5) from the PDA plates were inoculated at the centre of the slides and 15 µL each of the

CLPs (previously prepared with different dilutions) were dispensed on both sides of the plug

at 2 cm distance. Also, 0.1% DMSO solution was used as control for the experiment. The

plates were incubated at room temperature for 3 days after which the zones of inhibition was

measured and a microscopic examination was performed using a Photomicrograph.

2.9.1. Microscopic examination using Photomicrograph (Olympus BH-2 Microscope)

A micrograph or photomicrograph is a photograph or digital image taken through a

microscope or similar device to show a magnified image of an object. 

51
The morphology of the fungi hyphae was observed after 3 days of incubation using the

Olympus BH-2 Microscope, Japan. Images were taken and the kind of branching was

observed for any distinct morphology. Also, the morphology of the fungi (antagonism with

bacterial CLPs) was observed for any change that occurs in its morphology when compared

to its control. The changes may be extensive branching, coiling, vacuolization and so on. The

images taken are shown in Chapter 3 of this project.

52
 CHAPTER THREE

RESULTS

3.1. Isolation

Bacterial isolate LPb1-1 and fungal isolate GP5 were isolated from the roots of a healthy

cayenne pepper plant (Capsicum annuum) and wilted scotch bonnet pepper plants (Capsicum

chinense), respectively.

3.2. In vitro antagonistic activity of bacterial isolates against fungal isolate

Figure 3.1. shows the result of the antagonistic assay for the inhibitory effects of bacteria on

fungi isolated from the roots of a healthy cayenne pepper and diseased scotch bonnet pepper

plant performed on Potato Dextrose Agar (PDA) plate. The zone of inhibition of the

Enterobacter sp. LPb1-1 against Fusarium sp. GP5, after day 3 and day 5, was measured to

be 3 and 11 mm, respectively. GP5 cultured on PDA plate, without the bacteria antagonist, is

used as a control. The picture of the antagonistic activity of LPb1-1 at day 3 and 5 are

presented in Figure 3.2.

3.3. Morphological and microscopic characteristics of the fungi GP5 implicated in the

wilting of scotch bonnet pepper

The morphological and microscopic characteristics of the fungal isolate GP5, which was

inhibited by the antagonistic bacteria LPb1-1, were observed under the microscope and

recorded as shown in Table 3.1. The morphological characteristics was observed to have a

mycelium white in culture, chlamydospores with rounded conidia, and presence of non-

septate hyphae. The fungal isolate GP5 was suspected to be Fusarium sp. Representative

picture of the colonial morphology of GP5 was shown in Figure 3.3.

53
 12

10
Antagonistic effect of Enterobacter sp. LPb1-1 (mm)

0
Control LPb1-1 x GP5 Control LPb1-1 x GP5
Day 3 Day 5

Treatment

Figure 3.1. In vitro antagonistic effect of Enterobacter sp. LPb1-1 on Fusarium sp. GP5.

54
55
 Figure 3.2. In vitro antagonistic effect of Enterobacter sp. LPb1-1 on Fusarium sp. GP5.
Representative pictures showing the inhibition of Fusarium sp. GP5 by Enterobacter sp.
LPb1-1.
Table 3.1. Colonial Morphology of Fusarium sp. GP5

Morphological characteristics Fungal isolate GP5

Colour White with powdery colonies

Chlamydospores Rounded conidia

Hyphae Non-septate

56
Figure 3.3. Colonial morphology of Fusarium sp. GP5.

57
3.4. Molecular characterization of bacteria (16S rRNA sequencing)

The representative picture of agarose gel electrophoresis of the PCR performed on the

bacteria isolates is presented in Figure 3.4. The sequenced bacterial isolate LPb1-1 was

blasted on the NCBI website: https://www.ncbi.nlm. nih.gov/Blast.cgi and the identity of the

bacteria was revealed to be Enterobacter sp. with a percentage identity of 99.73% in

comparison to Enterobacter cloacae strain CIFRI S-32 with the accession number

OL519118.1.

3.5. Drop collapse assay

The result of the drop collapse assay of Enterobacter sp. LPb1-1 in comparison to water

dropped on a glossy surface, revealed that it produces CLP as shown in Figure 3.5. Water is

used as control.

3.6. Swarming Motility Test

As a result of the ability of the bacteria to produce lipopeptides or cyclic lipopeptides (CLPs),

they tend to promote swarming. Two bacterial isolates CHAO and B2W1-7 were used as

control because they are slow swarmers. After the evaluation of the surface swarming

motility of Enterobacter sp. LPb1-1, the diameter of the measurement of spread or swarm as

well as the representative pictures were measured and recorded as shown in Figure 3.6a and

Figure 3.6b respectively. The results obtained confirmed that Enterobacter sp. LPb1-1 are

CLP producer.

3.7. Antagonistic activity of CLP produced by Enterobacter sp. LPb1-1 on Fusarium sp.

GP5

The antagonistic activity of CLP produced by Enterobacter sp. LPb1-1 on Fusarium sp. GP5

was carried out on microscopic slide with concentration 5, 10, 25 and 50 µg/ml, while 0.1%

DMSO was used as a control. The result of the study showed that the CLP inhibited the fungi

58
at all the concentrations tested. A representative picture showing the zone of inhibition of

Enterobacter sp. LPb1-1 on Fusarium sp. GP5 is presented in Figure 3.7.

Figure 3.4. Representative picture of agarose gel electrophoresis of the bacteria isolates.

59
Figure 3.5. The result of drop collapse test of Enterobacter sp. LPb1-1 using water as control.

60
 100.00

90.00

80.00

70.00
Diameter of measurement of swarm (mm)

60.00

50.00

40.00

30.00

20.00

10.00

0.00
LPb1-1 CHAO (Control) B2W1-7 (Control)

Bacterial isolates
Figure 3.6a. Diameter of swarming motility of the bacterial isolates.

61
Figure 3.6b. Representative pictures showing swarming motility of the bacterial isolates.

62
 30

25

20
Diameter of zones of inhibition (mm)

15

10

0
50 25 10 5 Control

Concentration of LPb1-1 CLP (µg/ml)

Figure 3.7. Diameter of the zones of inhibition of CLP produced by Enterobacter sp. LPb1-1
against Fusarium sp. GP5 on microscopic slide.

63
3.8. Morphological damage exerted by CLP produced by Enterobacter sp. LPb1-1 on the

Fusarium sp. GP5

Structural damages on the hyphae were observed on Olympus BH-2 Microscope

photomicrograph and recorded for the various concentrations as shown in Table 3.2.

Representative pictures of the structural damages caused by the interaction of the CLP with

the fungi hyphae were presented in Figure 3.8. CLP at concentration 5 µg/mL showed

extensive hyphal branching, curling and blackening of the hyphal edge; 10 µg/mL of the CLP

caused extensive hyphal branching, vacuolization and blackening of the hyphal edge; 25

µg/mL of the CLP lead to extensive hyphal branching, vacuolization and hyphal digestion of

the fungi; while concentration 50 µg/mL showed extensive hyphal branching and blackening

of the hyphal edge. However, 0.1% DMSO used as control did not show structural damage on

the hyphae.

64
Table 3.2. In vitro antagonistic activity of CLP produced by Enterobacter sp. LPb1-1 against

Fusarium sp. GP5.

LPb1-1 CLP (μg/ml)

Structural damage on the hyphae
5 10 25 50 Control

Extensive hyphal branching + + + + -

Curling + - - - -

Vacuolization - + + - -

Blackening of the hyphal edge + + - + -

Hyphal digestion - - + - -

65
Figure 3.8. Representative picture showing the various microscopic effect of CLP produced
by Enterobacter sp. LPb1-1 on Fusarium sp. GP5 at different concentrations.

66
 CHAPTER FOUR

DISCUSSION, CONCLUSION AND RECOMMENDATION

4.1. DISCUSSION

In this study, we investigated the antagonistic activity of CLP-producing Enterobacter sp.

LPb1-1 isolated from the rhizosphere of healthy cayenne pepper plants (Capsicum annuum)

against Fusarium sp. GP5 implicated in the wilting of scotch bonnet pepper (Capsicum

chinense). We identified the fungi by cultural and microscopic characteristics, while the

bacteria was identified molecularly using 16S rRNA sequencing. The in vitro bioassay of

Enterobacter sp. LPb1-1 against Fusarium sp. revealed the interesting antagonistic potential

of the bacteria against the test fungi, with observable inhibition of the fungi after three (3)

and five (5) days, respectively, as presented in Figure 3.2. It has been reported that bacteria

antagonists produce secondary metabolites including antibiotics and CLPs, which could

contribute to their direct antagonistic potential. It could therefore be inferred from the result

of this study that Enterobacter sp. LPb1-1 might be a potential secondary metabolite such as

antibiotic or CLP, producer.

The drop collapse assay of Enterobacter sp. LPb1-1 revealed that they produce CLP. Since

Enterobacter sp. LPb1-1 contains surfactants, the drops spread or even collapse because the

force or interfacial tension between the bacteria drops and the hydrophobic surface is

reduced. However, water used as a control does not contain surfactants, the polar water

molecules are repelled from the hydrophobic surface and the drops remain stable. Hence, we

were able to deduce that the stability of drops is dependent on surfactant concentration and

correlates with surface and interfacial tension. Therefore, Enterobacter sp. LPb1-1 is a

surfactant such as CLP-producing strain. CLP production is known to play an important role

in surface motility and biofilm formation (De Bruijn et al., 2007; Raaijmakers et al., 2006).

67
Representative strains producing CLP was tested for swarming motility capacity. It was

revealed that Enterobacter sp. LPb1-1 showed a very high diameter of swarm on 0.6% LB

agar plate; while bacteria strains CHAO and B2W1-7, which are known CLP-producers were

used as control. As a result, we were able to deduce that Enterobacter sp. LPb1-1 produces a

CLP which contributes to its characteristic swarming motility. Interestingly, previous report

by Mandal et al. (2013), showed that Enterobacter sp. strain S-11 produce fengycin-,

kurstakins- and iturin-like lipopeptide. These lipopeptides have been reported to possess

antimicrobial potentials.

In order to decipher the metabolite responsible for the antagonistic activity of Enterobacter

sp. LPb1-1 against the fungi GP5 on PDA plate observed in Figure 3.2, we extracted the CLP

produced by Enterobacter sp. LPb1-1 and investigate its direct antagonistic effect on the

fungi GP5 on a microscopic slide using different CLP concentrations of 5, 10, 25 and 50

µg/mL, while 0.1% DMSO serves as the control. Interestingly, the CLP exerted

morphological damage on the mycelia of the fungi with characteristic extensive branching,

vacuolization, curling, blackening of the hyphal edge and hyphal digestion. The literature is

replete with information about the direct antagonistic effect of CLPs against fungal and

oomycetes pathogens of plants with the characteristic features observed under the microscope

in this study. An example is the report of Omoboye et al. (2019), who demonstrated the

hyphal damage caused by CLP on the oomycetes Pythium myriotylum CMR1, and

Pyricularia oryzae VT5M1 and GUY11.

So far, previous studies have not investigated the role of CLP-producing bacteria in the

biological control of fungi implicated in the wilting of pepper plants. However, a number of

studies have been carried out on the biological control of the diseases of pepper, which

include the use of endophytes in promoting plant growth and controlling diseases of pepper,

with potential application as biofertilizers and biopesticides. Our results confirmed previous

68
studies in which bacterial endophytes suppressed P. capsici in green pepper (Irabor and

Mmbaga, 2017). Also, in a previous study carried out by Bhusal and Mmbaga (2020), it was

revealed that endophytic Bacillus species exhibited biocontrol potential against Phytophthora

capsici and promote seedling shoot length, weight and chlorophyll content of bell pepper.

These results clearly indicate that our antagonistic bacteria Enterobacter sp. LPb1-1 could

serve as a viable, suitable and eco-friendly alternative to chemical pesticides in the

strengthening of scotch bonnet pepper against pathogens such as Fusarium sp., and

guaranteeing disease management, and possibly promoting pepper growth.

4.2. CONCLUSION

We could conclude from the result of our study that healthy cayenne pepper (Capsicum

annuum)-derived Enterobacter sp. LPb1-1 exhibited direct antagonistic activity against fungi

GP5 implicated in the wilting of scotch bonnet pepper plant. The antagonistic activity of

Enterobacter sp. LPb1-1 against Fusarium sp. GP5 on the PDA plates, was due to the

capacity of Enterobacter sp. LPb1-1 to produce CLP(s) which played a key role in the

antagonism, as confirmed microscopically. In a broader context, our results add support to an

emerging picture that certain biocontrol microorganisms and mechanisms contribute to some

level of natural suppression of soil-borne diseases.

4.3. RECOMMENDATION

1. Plant-growth promotion potential of Enterobacter sp. LPb1-1 should be investigated

on scotch bonnet pepper in green house experiment and on the field.

2. The identity and structure of the CLP produced by Enterobacter sp. LPb1-1 should be

characterized using appropriate chemical method.

3. The antagonistic potential of Enterobacter sp. LPb1-1 against fungal pathogen

implicated in the wilting of scotch bonnet pepper should be determined in green house

experiment and on the field.

69
4. Molecular characterization of the fungi should be carried out in order to guarantee the

identity of the fungi.

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 APPENDIX

Preparation of media and reagents

1. King’s B (KB) agar

Composition g/l

Proteose peptone 20

MgSO4.7H2O 1.5

K2HPO4 1.5

Glycerol 10 ml

Agar 15

pH 7.2 ± 0.2

Preparation

The above composition of King’s B (KB) agar was added to 1000 ml of distilled water in a

conical flask, homogenized using pressure pot, and was sterilized by autoclaving at a

temperature of 121 °C for 15 minutes. After cooling, the medium was aseptically dispensed

into sterile Petri dishes.

2. King’s B (KB) broth

Composition g/l

Proteose peptone 20

MgSO4.7H2O 1.5

K2HPO4 1.5

Glycerol 10ml
110
pH 7.2 ± 0.2

Preparation

The above composition of Luria Bertani (LB) agar was added to 1000 ml of distilled water in

a conical flask and transferred into test tubes. The broth medium was sterilized by

autoclaving at a temperature of 121 °C for 15 minutes.

3. Luria Bertani (LB) agar

Composition g/l

Tryptone 10

Yeast extract 5

Sodium chloride 10

Agar 15

pH 7.5 ± 0.2

Preparation

The above composition of Luria Bertani (LB) agar was added to 1000 ml of distilled water in

a conical flask, homogenized using pressure pot, and was sterilized by autoclaving at a

temperature of 121 °C for 15 minutes. After cooling, the medium was aseptically dispensed

into sterile Petri dishes.

4. Luria Bertani (LB) broth

Composition g/l

Tryptone 10

Yeast extract 5

111
Sodium chloride 10

pH 7.5 ± 0.2

Preparation

The above composition of Luria Bertani (LB) broth was added to 1000 ml of distilled water

in a conical flask and transferred into test tubes. The broth medium was sterilized by

autoclaving at a temperature of 121 °C for 15 minutes.

5. Potato Dextrose agar (PDA)

Composition g/l

Potatoes, infusion from 200

Dextrose 20

Agar 15

pH 3.5

Preparation

Exactly 39.0 g of the medium was suspended in 1000 ml distilled water, according to the

manufacturer’s instruction, in a conical flask, homogenized using pressure pot, after which it

was sterilized by autoclaving at a temperature of 121 °C for 15 minutes. After cooling, the

medium was aseptically dispensed into sterile Petri dishes.

6. Nutrient agar (NA)

Composition g/l

Beef extract 3.0

Peptone 5.0

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NaCl 5.0

Agar 15.0

pH 7.3

Preparation

Exactly 28.0 g of nutrient agar was dissolved in 1000 ml distilled water, according to the

manufacturer’s instruction, in a conical flask and the dissolved agar was homogenized using

pressure pot, after which it was sterilized by autoclaving at a temperature of 121 °C for 15

minutes. The medium was aseptically dispensed into sterile Petri-dishes after cooling.

6. Nutrient broth

Composition g/l

Beef extract 3.0

Peptone 5.0

NaCl 5.0

pH 7.3

Preparation

A quantity of 25.0 g of nutrient broth was dissolved in 1000 ml distilled water, according to

the manufacturer’s guide, in a conical flask and transferred into test tubes. The broth was

sterilized by autoclaving at a temperature of 121 °C for 15 minutes.

7. Normal saline

Composition g/l

NaCl 8.5

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Preparation

8.5 g of NaCl was dissolved in 1000ml of distilled water, and was sterilized by autoclaving at

a temperature of 121 oC for 15 minutes, after which it was allowed to cool to room

temperature.

114