MICROORGANISMS ASSOCIATED WITH SPOILAGE OF TOMATOES

BY

FABIYI KEHINDE ESTHER

2211920061

SUBMITTED TO
DEPARTMENT OF MICROBIOLOGY, FACULTY OF PURE AND APPLIED
SCIENCES, OSUN STATE COLLEGE OF TECHNOLOGY
ESA-OKE, OSUN STATE, NIGERIA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD

OF HIGHER NATIONAL DIPLOMA (H.N.D)

AUGUST, 2024
 CERTIFICATION

This is to certify that FABIYI KEHINDE ESTHER with MATRIC NO 2211920061

carried out this research work and have been read and approved as meeting part of the

requirement of the faculty of pure and applied science, Osun State College of Technology

Esa Oke for the award of Higher National Diploma (HND) degree in Microbiology.

________________ ________
MISS OMOLE O.Fr Date
Supervisor

________________ _________
AGBESANYA Date
Head of Department

________________
Date

ii
 DEDICATION

This project is dedicated to Almighty God, the beginning and the end, the author and

finisher of our faith.

iii
ACKNOWLEDGEMENT

TABLE OF CONTENTS

iv
Content Page

Title page i

Certification ii

Dedication iii

Acknowledgement iv

Table of Content vi

Abstract xi

CHAPTER ONE

1.0 INTRODUCTION 1

1.1. Background to the Study 1

1.2 Statement of the Problem 4

1.3 Justification for the study 5

1.4 Objective of the study 5

1.5 Significance of the Study 6

1.6 Scope of the Study 6

1.7 Limitation of the study 6

1.8 Operational Definition of Terms 7

CHAPTER TWO

2.0. LITERATURE REVIEW 8

2.1. A brief description of the tomato 8

2.1.1. Scientific classification of tomato 9

v
2.1.3. The economic, health and nutritional values of tomatoes 10

2.2. Tomato production 11

2.3. Fungal diseases of tomatoes 13

2.4. Current methods to control post-harvest losses 14

2.4.1. Physical control 15

2.4.2. Chemical control 16

CHAPTER THREE

3.0 Materials and Methods 19

3.1 Materials and Reagents 19

3.2 Collection of samples 19

3.3 Media preparation 19

3.4 Isolation of micro-organisms from the tomatoes samples 19

3.5 Purification and Maintenance of Isolates 20

3.6 Biochemical characterization and Identification of Isolates 21

3.6.1 Identification of Isolates 21

3.6.2 Biochemical Characterization 21

3.6.2.1 Gram Staining 21

3.6.2.2 Catalase test 22

3.6.2.3 Coagulase test 22

3.6.2.4 Citrate test 23

3.6.2.5 Indole test 23

3.6.2.6 Methyl red (MR) test 23

3.6.2.7 Voges-proskaeur (VP) test 24

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3.6.2.8 Hydrogen sulphide test 24

3.7 Sugar fermentation 24

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION 25

4.1 Results 25

4.2 Discussion 28

CHAPTER FIVE

5.0 DISCUSSION AND RECOMMENDATION 30

5.1 Discussion 30

5.2 Recommendation 31

REFERENCES 33

TABLE OF CONTENTS

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Content Page

4.1: Total viable counts from samples of tomatoes obtained from

Esa Oke, Osun State 25

4.2: Gram Reaction of Bacterial isolated from Tomatoes purchased

from Esa Oke, Osun State 26

4.3: Biochemical characteristics and Sugar fermentation of

bacterial isolated from Tomatoes obtained from Esa Oke, Osun State 27

ABSTRACT
viii
Tomato is very important mainly for its dietary needs, it can be consumed in diverse ways;

it can be cooked as vegetable, as an ingredient in many dishes and sauces, in the making

of stew, fruit juices and can be eaten raw in salads. This study is design to examine

microorganisms associated with spoilage of tomatoes in Esa oke, Osun State. The

microbial deterioration on tomato fruits causes reduction in its market values and

nutritional qualities. A total of five (5) samples of tomatoes were collected from Esa Oke

in Osun State. All the samples were collected aseptically in sterile universal containers

and immediately transported to the laboratory for analysis. About 10g of each of the

tomatoes samples were weighed and homogenized in 90ml of sterile distilled water and

then blended in a sterile blender after which 1 mL of the homogenate was then constituted

in 9 mL of sterile peptone water. Gram staining and other biochemical tests were carried

out for the identification of the bacteria isolates. The biochemical tests that were used to

further characterize the bacteria. The result of the total viable counts in colony forming unit

per ml (CFU/mL).The results shows that the sample T 1 has highest total viable plate count of 12.0

× 10-4, followed by sample T4, 5.7 × 10-4, while sample T3 has 3.6 × 10-4 , sample T5 with 2.5 × 10-

4
and sample T2 with the least counts of 2.4 × 10-4. All the bacterial isolates with sample

code T1, T3 and T4 are rod-shaped and gram negative except for T 2a, T2b and T5 are Gram

positive cocci. The probable identity of the microorganisms include; Erwinia sp,

Lactobacillus sp, Micrococcus sp, Proteus sp, Pseudomonas sp and Staphlococcus sp. The

presence of enteric bacteria on the tomato fruits may be due to the exposure of the tomato

fruits to fecal contaminated irrigation water and also the organic manure used for the

bioremediation process. This study revealed that several opportunistic pathogens

associated with tomato fruits and this is of high potential hazard to consumers especially

ix
the illiterate majority who are not aware of such risks and can consume tomato fruits even

without any proper washing.

x
 CHAPTER ONE

1.0 INTRODUCTION

1.1. Background to the Study

Tomato (Solanum lycopersicum) is an important crop that belongs to the

Solanaceae family, together with potato (Solanum tuberosum), hot pepper (Capsicum

frutescens), pepper (Capsicum annum) and eggplant (Solanum melongena) (Shah, et al.,

2013). It ranks first among plants widely grown in the world, and accounts for over 14%

of the world’s fruit production (FAO, 2011). According to the Food and Agriculture

Organisation Corporate Statistical Database (FAOSTAT), China is the leading tomato

producer in the world, followed by India, United States, Turkey, Egypt, Iran and Italy with

these countries accounting for more than 80% of global tomato production (FAO, 2011).

Egypt is the only African country amongst the top ten world’s tomato producers (DAFF,

2015).

In South Africa, tomatoes are the second most important and popular crop after

potato from the Solanaceae family and also one of the main plants sold on both local and

export markets (DAFF, 2015). Furthermore, the Department of Agriculture Forestry and

Fisheries (DAFF) reported that 75% of the tomatoes in South Africa are produced in the

northern areas of Limpopo province while the remaining 25% are produced between

Onderberg area of Mpumalanga province and the border area of the Eastern Cape province

(DAFF, 2015).

Food spoilage refers to various changes to food in which the food becomes less

palatable or even toxic to consumers these changes may be accompanied by alterations in

1
smell taste appearance or texture (Akinmusire, 2011). Tomatoes are a widely consumed

fruit eaten in both raw and processed forms. It has the botanical name Lycopersicum

esculentum and belongs to the plant family solanaceae. It is rich in vitamins such as

vitamin B, C, and E. Carbohydrates such as fructose and glucose; and trace elements like

iron, copper, zinc, and dietary fiber, which are all vital nutrients in man. The high water

content of tomatoes makes it more susceptible to spoilage by the action of microorganisms

(Obunkwu et al, 2018).

Tomato is very important mainly for its dietary needs, it can be consumed in

diverse ways; it can be cooked as vegetable, as an ingredient in many dishes and sauces, in

the making of stew, fruit juices and can be eaten raw in salads (Onuorah and Orji, 2015).

Tomatoes spoilage can be referred to as those adverse changes in the quality of

tomatoes caused by the action of predominantly biological and physical factors. These

changes may include changes in taste, smell, appearance or texture of the fruits. (Onuorah

and Orji, 2015). Estimates have shown that about one third of the produce is lost before

reaching the consumer (Mbajiuka and Emmanuel, 2014).This loss has been attributed to a

number of factors which include; physical (mechanical breakage, bruises), and also

damages caused by microbes such as fungi and bacteria (Onuorah and Orji, 2015). Tomato

spoilage usually occurs during storage, transportation and also while waiting to be

processed. The microbial deterioration on tomato fruits causes reduction in its market

values and nutritional qualities. The tomato fruits are rendered unsafe for consumption due

to contaminations with mycotoxins that produces aflatoxins in human, following

inhalation or ingestion and thus resulting to food poisoning (Bello et al., 2016).

Some studies have been carried out to identify both bacteria and fungi associated

with the spoilage of tomato Wogu and Ofuase (2014) isolated Bacillus subtilis, Klebsiella
2
aerogenes, Pseudomonas aeruginosa, Salmonella typhi, Proteus mirabilis, and

Staphylococcus aureus from spoilt tomatoes in Benin City. A similar study also revealed

high levels of Staphylococcus sp, Bacillus sp, and Escherichia coli in Lagos State, Nigeria

(Ogundipe et al., 2012). Akinmusire (2011) reported that Rhizopus sp were associated

with the spoilage of tomatoes.

Tomatoes are susceptible to fruit spoilage caused by numerous fungal pathogens

(Barkai-Golan and Paster, 2008; Samuel and Orji, 2015; Sanzani et al., 2016; Tournas and

Katsoudas, 2005). Examples of fungal induced tomato diseases include Alternaria rot

caused by Alternaria solani and Alternaria tenuis, Phytophthora rot caused by

Phytophthora infestans and Phytophthora nicotianae var. parasitica, Anthracnose ripe rot

caused by Colletotrichum phomoides, Phoma rot caused by Phoma destructiva and

Fusarium rot caused by Fusarium spp. (Wani, 2011).

Diseases caused by the above mentioned fungi may be due to large nutrient

composition or other factors such as high water content and low pH (Droby et al., 1992).

These factors may make the produce to become highly susceptible to pathogenic attack.

Diseases reduce the yield of tomatoes by up to 25% in industrialised countries and more

than 50% in developing countries (Nunes, 2012). However, there are numerous methods

for preventing diseases in fruits and one of them is the use of fungicides.

Wogu and Ofuase (2014) isolated Aspergillius sp, Penicillum sp, Fusarium sp and

Saccharomyces sp from spoilt tomato fruits. Mbajiuka and Emmanuel (2014) also isolated

Aspergillius spp, Penicillum sp and Saccharomyces cerevisiae from spoilt tomatoes.

Ghosh (2009) reported that fungi were responsible for more tomatoes spoilage than

bacteria.

3
 Spoilage refers to any change in the condition of food in which the food becomes

harmful for human consumption. The susceptibility of tomatoes to microbial spoilage is

largely attributed to its high moisture content. In northern Nigeria, freshly harvested

tomato fruits are stored, conveyed and marketed in wooden boxes and baskets. These

baskets are often used until they become infected with bacteria and or fungal spores.

Pathogenic inoculums on these wooden boxes and baskets can initiate spoilage upon

contact with healthy tomato fruits resulting in losses, which translate to a waste of the

farmers’ resources, a reduction in their income and ultimately their welfare. These

pathogenic inoculums could also originate from infected farm tools, or during

transportation.

Proper isolation and characterization of these organisms in tomatoes will greatly

reduce the spoilage of this perishable fruit and as such producers and consumers will be

able to protect their vegetables (tomato) and also identify spoiled tomatoes that have been

attacked by fungi and bacteria. Tomato is one of the most popular and widely grown

plants in the world as well as in Africa. It is the second most important vegetable

worldwide, in terms of the amount of vitamins and minerals it contributes to the diet

(Osemwegie, et al., 2010). The current research aimed at identifying the various

microorganisms associated with spoilage of tomatoes.

1.2 Statement of the Problem

Tomatoes are an attractive cash crop for small scale farmers and provide potential

source of employment to many rural and urban Kenyans. The tomato fruits have been

marketed freshly picked from the field and is the best selling fresh market vegetable crop

(AVRDC, 2006). Despite the human need of tomato, damage as a result of post-harvest
4
spoilage micro-organisms has been of serious concern. Microbial decay is one of the main

factors that determine losses and compromises the quality of the produce. The extent of

the losses especially through microbial decay has not been quantified in most areas and

where this has been quantified the results are short lived.

1.3 Justification for the study

Microbial spoilage is the main cause of postharvest losses of tomatoes (Deribe et

al., 2016; Osman, 2015; Suprapta, 2012). The quality of tomato fruit deteriorate after

harvesting, thus resulting in reduction of tomato yield. To prevent yield loss, pesticides are

used, but their residues on fresh fruits and vegetables have been and will continue to be

one of the main concerns of the regulatory agencies (Dukare et al., 2018).

1.4 Objective of the study

The broad objective of the study is to examine microorganisms associated with spoilage of

tomatoes while the specific objectives are to:

i. Isolate microorganisms from spoilage tomatoes purchased from market in Esa

oke, Osun State.

ii. Identify microorganisms isolated from spoilage tomatoes through

morphological characterization.

iii. Characterize microorganisms isolated from spoilage tomatoes through

biochemical test.

iv. Identify microorganisms isolated from spoilage tomatoes through Sugar tests.

5
1.5 Significance of the Study

The outputs of this study will be important in reducing tomato post-harvest losses

by improving the efficiency of post-harvest handling due to better post-harvest

management strategies

The study would be of great benefit to policy makers, the management of various

agricultural organizations, investors, stakeholders, shareholders and academic researchers.

The study would enable governments and farmers to have an in-depth understanding of

microorganisms associated with the spoilage of tomatoes. It would also benefit investors,

stakeholders, manufacturers, industries, shareholders and the public

Future researchers who will be researching on a similar or related topic will benefit

from the findings by borrowing a leaf in form of related literatures. The study will also

help build on the researcher’s knowledge and understanding of the variables and gain

more skills of conducting research.

1.6 Scope of the Study

This study focused on the microorganisms associated with spoilage of tomatoes. This

study was limited to microorganisms responsible for tomatoes spoilage only.

1.7 Limitation of the study

This study is subjected to the following limitations.

i. Time frame of the work

ii. Power supply to the laboratory is inadequate

iii. Accessibility to the laboratory during the weekends for reading of results is

tedious
6
1.8 Operational Definition of Terms

Spoilage refers to any change in the condition of food in which the food becomes harmful

for human consumption

Food spoilage refers to various changes to food in which the food becomes less palatable

or even toxic to consumers these changes may be accompanied by alterations in smell

taste appearance or texture

Tomatoes spoilage can be referred to as those adverse changes in the quality of tomatoes

caused by the action of predominantly biological and physical factors.

7
 CHAPTER TWO

2.0. LITERATURE REVIEW

2.1. A brief description of the tomato

Tomato (Lycopersicon esculentum) is one of the most important vegetables

worldwide. It is a self-pollinated fruit that belongs to the Solanaceae family (Arah et al.,

2015). The family also includes potato (Solanum tuberosum), eggplant (Solanum

melongena), pepper (Capsicum annum), tomatillo (Physalis philadelphica) andpepper

(Capsicum annuum, Capsicum frutescens, and Capsicum chinense). The family also

includes plant drugs such as Tobacco (Nicotiana tabacum), deadly nightshade (Atropa

belladonna), mandrake (Mandragora officinarum), jimson weed (Datura stramonium) and

petunia (Petunia hybrida) (Shah et al., 2013).

Tomato is widely cultivated in tropical, sub-tropical and temperate climates and is

ranked third in the world for vegetable production (FAO, 2011). Tomato is known by

different names worldwide, for example, tomate (German, France), tomati (West Africa),

tomaatti (Finish), tomat (Indonesia), pomidoro (Italy), kamalis (Malay), jitomate (Spain,

Mexico), pomidor (Russia), faan ke’e (China), tomatl (Nahuatl), nyanya (Swahili) and

tamatar (Hindi) (Naika et al., 2005).

Tomato originated in the South America Andes, in the mountains of Peru (Shnain

et al., 2017). It was taken to other parts of the world by the early travellers where it was

planted as an ornamental curiosity but not eaten (Arah et al., 2015). By 500 BC it had

been moved to Mexico for the purposes of domestication. Tomato was brought to Europe

in 1554 by the Spanish conquistadors. It was later cultured in the U.S. in 1710, and

introduced from Europe into southern and eastern Asia, Africa and the Middle East.

8
Thereafter, tomato became popular and was exported around the world by 1850 for

commercial production (Shnain et al., 2017).

2.1.1. Scientific classification of tomato

Kingdom: Plantae

Sub kingdom: Tracheobionia

Class: Magnoliopsoda

Sub class: Asterielae

Order: Sultanates

Family: Solanaceae

Genus: Lycopersicon

Species: esculentum

2.1.2. Different botanical varieties of tomato

There have been numerous changes to the botanical name for tomato. For several

years it was known as Solanum lycopersicum, which later changed to Lycopersicon

esculentum(Naika et al., 2005). Tomato is a true diploid with 2n = 24 (Stack and

Anderson, 1986). The plant is annual with a herbaceous prostrate stem having determinate

or indeterminate growth habit (Naika et al., 2005). Tomato has three vine types, namely,

indeterminate (sprawling, staggered ripening and tall type), semi-determinate

(intermediate response and semi-bush type) and determinate (compact, uniform ripening

and bush type) (Naika et al., 2005). Table 2.1 represents the names of tomato according to

diversities.

9
2.1.3. The economic, health and nutritional values of tomatoes

Tomato has become an important cash and industrial crop in many parts of the

world. This is not only because of its economic importance but also its nutritional value in

the human diet and subsequent importance for human health as a result of the essential

nutrients it provides (Ayandiji and Adeniyi, 2011; Yadav et al., 2017). It is also a versatile

crop that can be classified according to use into two categories as fresh market tomatoes

for direct consumption and processing tomatoes which are cultivated for industrial

canning and processed foods, respectively (Osman, 2015).

Tomato is rich in vitamins A, B, C and E; carbohydrates such as fructose and

glucose; minerals such as phosphorus, sodium, potassium, calcium and magnesium and

trace elements such as iron, copper, zinc and dietary fibers (Ayandiji and Adeniyi, 2011;

John et al., 2016; Yadav et al., 2017). It therefore serves as a source of essential nutrients

when consumed (Arah et al., 2015; Guil-Guerrero and Rebolloso-Fuentes, 2009).

An average size (70 - 150 g weight and 50 - 70 mm diameter) tomato fruit contains

energy (18 kcal), protein (0.95 g), fat (0.11 g), carbohydrate (4.01 g), total sugar (2.49 g),

niacin (0.731 mg), calcium (11.0 mg), iron (0.68 mg), magnesium (9.0 mg), phosphorus

(28.0 mg), potassium (218.0 mg), sodium (11.0 mg), zinc (0.14 mg), thiamin (0.036 mg),

riboflavin (0.022 mg), carotene (vitamin A) 320 IU, vitamin B (60.079 mg), vitamin C

(16.9 mg), and ascorbic acid (31 mg) per 100 g pulp of fruit (Arah et al., 2015; Yadav et

al., 2017). Tomatoes are ready-to-eat food, and are thus minimally processed (John et al.,

2016). They are consumed in various ways such as raw in salads and sandwiches, cooked

or processed in ketchup, sauces, soup, chutney, pickles, paste, puree, juices, dried powder

10
and whole canned fruits, while it also forms an important ingredient in the cocktail known

as a Bloody Mary (Ayandiji and Adeniyi, 2011; Chaudhary, 2014; Yadav et al., 2017).

The deep-red coloration of the ripened tomato is due to the high amount of

lycopene, a form of B-carotenoid pigment and a notable antioxidant that is beneficial in

reducing the incidence of certain chronic diseases such as prostate cancer, cardiovascular

disease and diabetes (Ram et al., 2014; Wu and Tanksley, 2010). Tomato juice promotes

gastric secretion, acts as a blood purifier and works as an intestinal antiseptic (Chaudhary,

2014). Tomatoes are good sources of vitamin C and vitamin A which are vital in warding

off muscular degeneration and improving eyesight. It is also believed to be a powerful

blood purifier and clear up urinary tract infections. Tomatoes are high in fibre which aids

easy digestion and may assist in weight loss (Arah et al., 2015). Tomatoes have numerous

advantages that make them economically important (Naika et al., 2005). These advantages

include the following: relatively short-duration vegetable crop, short production period,

growth as an uncovered field crop and in protected cultivation, easy fitting into different

cropping systems, high economic value, and high micronutrient content (Naika et al.,

2005).

2.2. Tomato production

On a global scale, the annual production of fresh tomatoes amounts to

approximately 159 million tons with more than a quarter of these 159 million tons grown

for the processing industry, thus making tomatoes the world’s leading vegetable for

processing (Noonari et al., 2015). Tomato is cultivated in both the tropics and subtropics

of the world and is also cultivated in kitchen gardens, commercial fields under greenhouse

and polyhouse conditions and soil-less culture or hydroponic systems (Chaudhary, 2014).
11
 Although the root structure of a tomato plant is able to penetrate various soil types

up to depths of two metres, the highest percentage of the roots will be found in the top 600

mm of the soil. Tomatoes are grown and produced optimally when the mean temperatures

are between 20 °C and 24 °C. When average daily temperature is above 32 °C and the

night temperature fails below 21 °C the fruit set is poor (Starke Ayres, 2014). It takes

tomato plants three to four months to bear fruits that are ready for harvesting. Tomato

planting involves different techniques and methods for determinant (generally grown

under open-field condition) and indeterminate (normally grown under poly-house

condition) varieties (Yadav et al., 2017). Moreover, tomato can grow well in soil, organic

substrates, soilless mixes, perlite, sand or hydroponics (Shamshiri et al., 2018).

The following countries are the highest producers, users and exporters of tomato in

the world namely China (largest producers and dominates production of processing

exports), US and Europe (major users of fresh tomatoes), Mexico (the largest exporter of

fresh tomatoes), and Turkey (a major exporter into Europe) (Kahan, 2010; Yadav et al.,

2017). As illustrated in Table 2.2, the top ten tomato producers are as follows; China,

USA, India, Turkey, Egypt, Italy, Iran, Spain, Brazil and Mexico in that order, and these

countries account for 80% of the total world tomato production (Chandio et al., 2016;

FAO, 2011). Egypt is the only African country amongst top ten world tomato producers

while South Africa (580 851 tons) ranks as the seventh highest tomato producing country

in Africa (FAOSTAT, 2018).

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2.3. Fungal diseases of tomatoes

In total, there are more than 200 species of fungi that may infect the tomato crop,

with diseases often being the limiting factor in tomato production (Agrios, 2004; Suprapta,

2012). The epidemics of a disease depend on complex interactions between host, pathogen

and environment as well as cultural practices such as fertilisation and irrigation (Osman,

2015; Aust and Hoyningen Huene, 1986). Plant pathogens use different strategies to

survive and spread to new hosts (Osman, 2015). Most pathogens have a life cycle that

includes both plants and soil, although they usually need to infect a specific host to

increase their population (Abdul-baki, 1996; Berlin, 2005). Fresh vegetable fruits are

fairly perishable because their high moisture content renders them vulnerable to microbial

diseases as well as to physiological deterioration (Deribe et al., 2016; Naika et al., 2005;

Osman, 2015; Peet and Welles, 2005).

A lack of adequate pre-harvest and post-harvest handling factors may lead to

diseases such as those caused by certain pests, namely, Aculops lycorpersici (causes rusty

brown and coarse surface cracking), and Thrips tabaci (causes blossom drop and scarring

of the fruit) as well as some virus species such as fruit necrosis caused by the Tomato

marchitez virus (ToMarV), fruit marbling caused Pepino mosaic virus (PepMV) (Hanssen,

2010). There are also some bacterial diseases such as bacterial speck caused by

Pseudomonas syringae, bacterial wilt (Rhizopus solanacearum), bacterial Spot

(Xanthomonas campestris) and bacterial canker (Clavibacter michiganensis) (Rashid et

al., 2016). Tomatoes are also affected by the physiological disorders such as blossom end

rot which is caused by a shortage in the availability of calcium, and growth cracks caused

by the fruit expansion which stretches the epidermis (skin) beyond its capacity, as well as

13
diseases caused by viruses such as the tomato mosaic virus which have been reported on

tomato (Arli-Sokmen and Sevik, 2006; Kennelly, 2009).

In addition, many of the smallholder farmers in South Africa encounter attacks of

pathogenic fungi because they possess inadequate technical information, in particular

relating to crop diseases (Mandiriza-Mukwirimba et al., 2016). It has been reported that

the highest percentage cause of the PHLs of tomato fruit are associated with different

species of soil-borne phytopathogenic fungi (Etebu et al., 2013; Fatima et al., 2009).

These species cause diseases such as early blight (Alternaria solani), anthracnose

(Colletotrichum spp.), Sclerotium wilt (Sclerotium rolfsii), damping off (R. solani), tomato

wilt (Fusarium oxysporum), Phoma rot (Phoma destructive), Fusarium wilt (Fusarium

oxysporum), late blight wilting (Phytophthora capsici), Septoria leaf spot (Septoria

lycopersici) and Rhizopus rot (Rhizopus stolonifer) (Fatima et al., 2009; Ignjatov et al.,

2012; Kleemann et al., 2008; Kumar et al., 2008; Osman, 2015).

These pathogens are severe wound pathogens that may infect the fruit in the

packing house, and throughout subsequent handling or storage, thereby limiting

production and reducing both crop yield and crop quality (Palou et al., 2008). Pathogenic

microorganisms in tomato are recognised as a source of potential health hazard to both

man and animals following ingestion as a result of their production of mycotoxins, which

are capable of causing diseases such as respiratory infection, meningitis, gastroenteritis

and diarrhoea in man (Beuchat, 2006).

2.4. Current methods to control post-harvest losses

The response of tomatoes during storage and the post-harvest qualities depend to a

certain extent on pre-harvest factors such as cultural practices, the use of natural plant
14
extracts, fertilizers, manure, and genetic and environmental conditions (Meaza et al.,

2007; Pretorius et al., 2003). The losses of untreated fruit from fungal decay have been

estimated to be as high as 90% during post-harvest handling and marketing (Albrigo,

1978). Nevertheless, decay in tomato fruits can be controlled by various methods that are

explained below.

2.4.1. Physical control

Controlling the storage temperature is the most well-known physical treatment.

Such treatment may be applied in the form of a hot water dip, hot water rinsing and

brushing, vapour, hot air and curing (Conway et al., 2004; Fallik, 2004). The temperature

is calculated using an adaptive management framework and the TOMGRO model (Jones

et al., 1992; Shamshiri et al., 2018). During the entire tomato growing season, optimal air

temperatures from 18 to 32.2 °C are considered with 50 to 70% humidity (Peet and

Welles, 2005; Shamshiri et al., 2018).

In the green house, the cultivation of tomato temperature is maintained at 17 to

28°C in coastal areas and 17 to 22 °C in inland areas with 85 to 95% humidity (Puyaubert

& Baudouin, 2014). During storage, temperature greatly encourages the rate of respiration

of fruits and vegetables, and is certainly one of the most important factors in maintaining

the post-harvest quality of tomato fruits (Žnidarčič et al., 2010). The chilling injury and

ripening rate is minimal at 10 to 15 °C temperature and 85 to 95% relative humidity which

may extend the postharvest life of fruits (Žnidarčič et al., 2010) . Ultraviolet light (UV-C,

254 nm) hormesis has been identified as one of the physical methods which may be used

15
to stimulate positive responses in order to induce resistance to storage diseases and extend

the shelf-life of fruits and vegetables (Liu et al., 1993).

Tomatoes are treated with UV-C doses from 1.3 to 40 KJ/m2 in order to induce

resistance to the various fungal pathogens that lead to spoilage (Buzby and Hyman, 2012;

Tang et al., 2015; Vaklounakis, 1991). However, it must be noted that the use of

temperature and UV-C lights during storage changes the aroma profile and the taste of

fruits after six days of storage (Baloch & Bibi, 2012; de León-Sánchez et al., 2009; Ponce-

Valadez et al., 2016).

2.4.2. Chemical control

Strategies such as synthetic fungicides and pesticides applications, resistant-variety

cultivation and crop rotation are used to control fungal diseases in crops with pesticide

application remaining as the most common control strategy (Gao et al., 2017). These

strategies are fairly inexpensive, easy to apply and demonstrate both curative and

preventive actions against various infections. The azoxystrobin, fludioxonil, and

pyrimethanil fungicides were introduced for the post-harvest management of citrus mould

(Kanetis et al., 2007). These are also chemicals such as sporekill, vinclozolin, copper

oxychloride, benomyl and kitazin that are being used against various fungal pathogens that

cause spoilage in fruits and vegetables (Amini and Sidovich, 2010; Lee et al., 2012;

Leroux, 2007; Nel et al., 2007; Sahu et al., 2013; Stansly et al., 2004).

However, the intensive use of synthetic pesticides and fungicides may cause

pathogen resistance and pesticide residues and release fungicides in the environment (Ma

et al., 2015; Yang et al., 2015). Their use is becoming more restricted because of the

concerns of the consumers and the administration about human health (De Curtis et al.,

16
2010; Usall et al., 2016). Moreover, effective chemical treatments cannot inhibit the

growth of some plant diseases and consumers are increasingly demanding pesticide-free

food Wang et al., 2009).

2.4.3. Biological control

The non-biodegradable nature and the environmental pollution caused by chemical

control applications have led to the alternative production of naturally derived substances

(Migliori et al., 2017). Among these alternatives, biological control using microorganisms

with a strong fungal activity such as growth and ecological fitness has been identified (Pal

and Gardener, 2006; Shafiq, 2015; Zong et al., 2010).

There are mechanisms that have been suggested as being liable to the antagonistic

activities of biocontrol agents, including competition for nutrients and space,

mycoparasitism of the pathogen, emission of antifungal compounds, antibiotics, volatile

metabolites, induction of host resistance, biofilm development and the participation of the

reactive oxygen species (ROS) in the defence response (Dukare et al., 2018; Liu et al.,

2013). These biocontrol agents are safe for the environment, they improve crop production

and they limit pesticide resistance (Khonglah & Kayang, 2018; Shafiq, 2015).

The successful application of these agents, by spraying, dipping or drenching,

occurs during the postharvest period (Di Francesco et al., 2016; Liu et al., 2013). The

antagonists used to manage postharvest diseases include bacteria and yeast and it is only

recently that fungi have been reviewed as well (Liu et al., 2013; Lledó et al., 2016; Nunes,

2012). Antagonism is a phenomenon whereby a microorganism inhibits the growth or

interferes with the development of another microorganism (Liu, et al., 2013; Rodrigo et

17
al., 2017). Fungal antagonists such as Debaryomyces hanseniis, Candida guilliermondii,

Byssochlamys spectabilis, Trichoderma harzianum, Trochoderma viride, Phythium

debaryanum, Gliocladium roseum, Aureobasidium pullulans, Phythophthora cryptogea

and Cryptococcus laurentii are among the effective antagonists that have been identified

as the best alternatives to monitor postharvest diseases on citrus fruits (Agrios, 2004;

Castoria et al., 2001; De Curtis et al., 2010; Gomathi and Ambikapathy, 2011; Naglot et

al., 2015; Zong et al., 2010).

18
 CHAPTER THREE

3.0 Materials and Methods

3.1 Materials and Reagents

The materials and reagents used during the course of this research include:

weighing balance, beakers, conical flasks, autoclave, petri-dishes, 70% ethanol, non-

absorbent cotton wool, aluminium foil, test tubes, wire loops, incubators, microscope,

blender, nutrient agar, potato dextrose agar, mannitol salt agar, salmonella-shigella agar,

MacConkey agar, peptone water and distilled water.

3.2 Collection of samples

A total of five (5) samples of tomatoes were collected from Esa Oke in Osun State.

All the samples were collected aseptically in sterile universal containers and immediately

transported to the laboratory for analysis.

3.3 Media preparation

The different media which included nutrient agar, mannitol salt agar, eosin methylene blue

agar, MacConkey agar, salmonella-shigella agar; and peptone water were prepared

according to the manufacturer’s instruction.

3.4 Isolation of micro-organisms from the tomatoes samples

About 10g of each of the tomatoes samples were weighed and homogenized in 90ml of

sterile distilled water and then blended in a sterile blender after which 1 mL of the

homogenate was then constituted in 9 mL of sterile peptone water. From there, 10-fold

19
serial dilution was performed and 0.1 mL of last two dilutions (10 -4 and 10-6) was

inoculated in triplicate on appropriate prepared media using pour plate technique. The

plates were then incubated at 37oC for 24-hours.

After incubation, the plates were examined for the presence of discrete colonies. Colony

were counted using the colony counter and expressed as colony forming per unit gram

(CFU/g) of sample homogenate. Specifically, a total aerobic count was performed on

nutrient agar, while Escherichia coli were enumerated on Eosin methylene blue agar.

Mnnitol salt agar and MacConkey agar were used to enumerate Staphlococcus aureus and

non E.coli coliforms respectively, while Salmonella Shigella agar was used for Salmonella

counts after 24h pre enrichment of sample homogenate in Selenite-Flouch according to

Oranusi and Olorunfemi (2011). Characteristics discrete colonies on the different media

were isolated and purified by repeated sub culturing on the same media. Pure colonies

were stored on agar slants at 4oC for further characterization.

3.5 Purification and Maintenance of Isolates

Each discrete colony on a petri dish was transferred using sterile inoculating loop into

plates containing freshly prepared nutrient agar and were incubated at 37 oC for 24-48

hours. After incubation, the colonial morphologies (Cultural characteristics) of the isolates

were recorded and compared with descriptive features contained in Holt et al., (1995). The

isolates were then preserved on nutrient agar slants and stored in the refrigerator at 4oC.

20
3.6 Biochemical characterization and Identification of Isolates

The method of Oranusi et al. (2004) was employed for the identification of the bacteria

isolates. The biochemical tests that were used to further characterize the bacteria are:

catalase, mrthyl red, oxidase, citrate utilization, and coagulase and indole tests. Oxidase

test was also carried out on the gram negative isolates to know if they are oxidase positive

or negative. The identities of coliforms and bacteria was then confirmed using the

identification and outlined in the Bergy’s Manual for Determinative Bacteriology (Holt et

al., 1994)

3.6.1 Identification of Isolates

Gram staining and other biochemical tests were carried out based on the method of

Cheesbrough (2006). The colonial appearance of each bacterial isolate on the plate was

observed and classified based on the following characteristics; colour, shape, edge,

elevation, surface and opacity (Olutiola et al., 200).The biochemical tests performed here

included catalase test, oxidase test, indole test and coagulase test.

3.6.2 Biochemical Characterization

3.6.2.1 Gram Staining

Gram staining was used in this study to differentiate the bacterial isolates into

Gram positive and gram negative bacterial groups based on their ability to retain the

primary stain (Gram positive bacterial) or the secondary stain (gram negative bacterial). A

smear of the test isolate was emulsified in a drop of sterile distilled water on a clean glass

slides forming smooth suspension, air dried, heat fixed by slightly passing the inoculated

21
glass slide through the Bunsen burner flame for about 2-3 times and then stained with the

primary stain, crystal violet for 60 seconds and was further stained with Gram`s iodine for

60 seconds and rinsed off in a slow running tap water. It was then decolourized with 70%

ethanol for about 5 seconds followed with a thick rinsing with water to avoid

decolourization.

Finally, the smear was counter stained with safranin for 60 seconds and was rinsed

with water, allowed to air dry before a drop of immersion oil was put on the smear and

was observed under oil immersion objectives lens of microscope. Cells of the isolates that

retained the purple colour of the primary stain, crystal violet were labelled and noted as

gram-positive bacteria while those that were unable to retain the colour of the primary

stain but were stained with pink colour of the counter stain were labeled and recorded as

gram-negative bacteria (Fawole and Osa, 2001).

3.6.2.2 Catalase test

2-3 ml of hydrogen peroxide was purred in a test tube. A colony of test organism was

taken with sterile wooden or glass rod and immersed it into hydrogen peroxide solution.

Generation of bubbles indicated oxygen production. If bubbles were produced, the

organism was catalase positive, if bubbles were not produced, the organism was catalase

negative.

3.6.2.3 Coagulase test

The test was performed as described by Barrow and Feltham (1993). 0.1ml of 18-24 h old

broth culture of the tested organism was added to 0.5 ml of 1:10 dilution of human plasma

22
in saline, then incubated at 37 ° C and examined after 2-24 hrs for coagulation. Definite

clot formation indicated a positive result.

3.6.2.4 Citrate test

The test is used to detect the ability of an organism to use citrate as a sole source of carbon

and energy. About 2.4 g of citrate agar was dissolve in 100 mL of distilled water. 10 mL

of citrate medium was dispensed into test tubes and covered, then sterilized and allowed to

cool in a slanted position. The tubes were inoculated by streaking with the 24 h old culture

organisms once across the surface. A change from green to blue indicates utilization of the

citrate (Cheesbrough, 2006).

3.6.2.5 Indole test

Test bacterial colony were inoculated in tryptophan broth and incubated at 37°C for 24-28

h. Then 0.5 ml of Kovac’s reagent was added. Positive test Pink colored rink was

observed after addition of reagent. Negative test: no color change after reagent addition.

3.6.2.6 Methyl red (MR) test

5 mm of glucose phosphate broth (1 g glucose, 0.5% KH2PO4, 0.5% peptone and 100 mL

distilled water) were dispensed into clean test tubes and sterilized. The tubes were then

inoculated with the isolated test organisms and incubated at 37°C for 48 h after which few

drops of methyl red solution were added to each test and colour change was observed. A

red colour indicates a positive reaction (Olutiola et al., 2000).

23
3.6.2.7 Voges-proskaeur (VP) test

5 mm of glucose phosphate broth (1 g glucose, 0.5% KH2PO4, 0.5% peptone and 100 mL

distilled water) were dispensed in clean test tubes and sterilized. The tubes were then

inoculated with the test organisms and incubated at 37 °C for 48 h. After incubation, 6%

α-naphhtol and 6% sodium hydroxide were added to about 1 mL of the broth culture of the

isolated organisms. A strong red colouration formed within 30 min indicates positive

reaction (Olutiola et al., 2000).

3.6.2.8 Hydrogen sulphide test

A small quantity of 24 h old culture was inoculated into already prepared tubes of triple

sugar iron agar and inoculated at 37oC for 48 hours. After the incubation period the tube

were observed for hydrogen sulphide (black coloration) gas production (Oyeleke and

Manga, 2008).

3.7 Sugar fermentation

Sugar fermentation test was carried out to determine the ability of organisms to ferment

sugars (Lactose, Glucose and sucrose) with production of acid and gas. Sugar indicator

broth was prepared using peptone water medium containing 1% fermentable sugar and

0.01% phenol red. About ten milliliters of sugar broth was dispensed into each of the test

tubes, Durham tube which would trap the gas if produced was inverted carefully. The test

tubes were autoclaved and inoculated with a loopful of 24 h old culture of the test

organisms after then incubated for 2-7 days at 36±1 °C and observed daily for acid and

gas production. Yellow coloration indicates acid production while gas production was

indicated by displacement of the medium in the Durham tube (Fawole and Oso, 2004).

24
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 Results

Table 4.1 shows the result of the total viable counts in colony forming unit per g

(CFU/ml). It shows that the sample T 1 has highest total viable plate count of 12.0 × 10- 4,

followed by sample T4, 5.7 × 10-4, while sample T3 has 3.6 × 10-4 , sample T5 with 2.5 ×

10-4 and sample T2 with the least counts of 2.4 × 10-4 Table 4.1.

Table 4.1: Total viable counts from samples of tomatoes obtained from Esa Oke, Osun

State

S/N Sample Code Total Viable Count

1 T1 12.0 × 10-4

2 T2 2.4 × 10-4

3 T3 3.6 × 10-4

4 T4 5.7 × 10-4

5 T5 2.5 × 10-4

25
Table 4.2: Gram Reaction of Bacterial isolated from Tomatoes purchased from Esa Oke,

Osun State

S/N Sample Code Positive/Negative (+/-) Shape

1 T1 - Rod

2 T2a + Cocci

3 T2b + Cocci

4 T3 - Rod

5 T4 - Rod

6 T5 + Cocci

All the bacterial isolates with sample code T 1, T3 and T4 are rod-shaped and gram

negative except for T2a, T2b and T5 are Gram positive cocci (Table 4.2).

26
Table 4.3: Biochemical characteristics and Sugar fermentation of bacterial isolated from

Tomatoes obtained from Esa Oke, Osun State

Isolate Cat Coag Ci I M V H G Lac Gl Suc SP A Probable

code t n R P S H u F
Identity

T1 - + + - + - + + + + - - Erwinia sp

T2a - - - - - - - + + + + - - Lactobacillus
sp

T2b + + + - - + - - + + + - - Micrococcus sp

T3 + - + - - + + + - + + - - Proteus sp

T4 + - - + + - + + + + + - - Pseudomonas
sp

T5 + + - - + + - - A A A - - Staphlococcus
sp

Keys: Cat= catalase, Coag=Coagulase, Cit=Citrate, In=Indole, MR=Methyl Red,

VP=Voges Prosekeaur, HS= Hydrogen sulphide, GH= Gas production, Lac=Lactose, Glu-

Glucose, Suc-Sucrose, SP=Spore formation, AF=Acid fast, - = negative, + = positive

From Table 4.3 above, Probable identity of bacterial isolates were determined after

biochemical and sugar fermentation test were conducted on isolates. The results of

biochemical and sugar tests were compared with Bergy’s manual for bacterial

identification (Table 4.3). The six isolates were probably identify as Erwinia sp,

Lactobacillus sp, Micrococcus sp, Proteus sp, Pseudomonas sp and Staphlococcus sp

27
4.2 Discussion

The total viable counts recorded in this study are within acceptable standard but the

value is significant. Factors such as storage, transportation, packaging and distribution

(loading and off-loading) at various channels and selling outlets may be responsible for

the significant microbial growth recorded for the tomato fruits obtained from the market.

This study is in accordance with the work of Baiyewu et al. (2007) who reported that

tomato fruits in the market are often displayed in baskets and on benches thereby exposing

them to opportunistic microorganisms and further justified by the work of Beuchat (2002)

who concluded that the natural protective cover (epidermal layer) of tomato fruits are

hindered and contaminated during field cultivation, harvesting, post-harvest handling,

storage and distribution. The probable identity of the microorganisms include; Erwinia sp,

Lactobacillus sp, Micrococcus sp, Proteus sp, Pseudomonas sp and Staphlococcus sp

which is in agreement with the findings of previous researchers who had earlier isolated

these microorganisms from tomato fruits (Chinedu and Enya, 2014; Oyemaechi et al.,

2014; Bello et al., 2016).

The bacteria isolated from the tomatoes sample were Erwinia sp. and

Staphylococcus aureus. The bacteria isolated in this study is similar to that of Ogundipe et

al.( 2012) and Wogu and Ofuase (2014) who also isolated these bacteria as organisms

associated with tomato spoilage. The occurrence of bacteria species could be as a result of

feacal contamination due to poor hygienic practices by the farmers and /or the sellers. The

implications of microbial contamination and growth on tomatoes, causes spoilage,

decreased sensory appeal and also decreased shelf life, leading to loss and wastage of

28
products which have significant economic consequences as reported by Obunkwu et al.

(2018).

The presence of enteric bacteria on the tomato fruits may be due to the exposure of

the tomato fruits to fecal contaminated irrigation water and also the organic manure used

for the bioremediation process (Amoah et al, 2009). These enteric bacteria may be

transferred from the manure to the soil and then to the tomato fruits. The presence of

Staphylococcus aureus may also be through nasal passages of healthy food handlers or

through contact with an infected skin (Adams et al., 2009). Staphylococcus sp. was

isolated from all the samples of tomato examined; it is a known micro flora of nostrils,

skin and hands of man. It might have originated from the traders bodies or the

contaminated air.

29
 CHAPTER FIVE

5.0 DISCUSSION AND RECOMMENDATION

5.1 Discussion

The source of some of the common contaminating microorganisms which were

isolated from tomato fruits could be from the farm during harvesting while others may be

acquired as post-harvest contaminants probably during storage or in transit. Also the

careless handling of these fruits by traders and consumers account for the major cause of

tomato spoilage, this involves the use of direct river or stagnant water in washing and

wetting of these fruits by markets traders. This can create a condition which predisposes

the fruits to easy contamination.

The use of dirty tables and rags used in covering tables increase the incidence of

contamination. The use of dirty case and baskets for loading tomatoes and the pouring of

these fruits on the floor as a way of preservation by the vendors lead to easy

contamination of these tomato fruits.

Various market condition and varying environmental situation also increases rate

of contamination of these fruits.

This study revealed that several opportunistic pathogenic microorganisms are

associated with tomato fruits and this is of high potential hazard to consumers especially

the illiterate majority who are not aware of such risks and can consume tomato fruits even

without any proper washing.

30
5.2 Recommendation

It is recommended that;

1. The thorough washing of harvested tomatoes with clean or chlorinated water, proper

cleaning and sanitation of ware houses and disinfection of packaging containers, proper

handling of the vegetable during harvest should be done to prevent bruises and scars or

other mechanical injuries.

2. The inhibition of bacterial and fungal growth by lowering storage temperature through

storage under refrigeration of less than 100 oC but not freezing and the use of appropriate

antimicrobial agents when stored by drying are encouraged

3. The presence of microorganisms on the surface of tomato fruits has an adverse effect on

their shelf life. It is therefore advisable to surface sterilize tomato frui t s af ter harves t or

before consumption particularly if the fruits are to be eaten raw.

4. We also found in this study that wounds on the surface of tomato fruits can serve as

portal of entry for these microbial pathogens. Better harvest practices to minimize cuts

could help in reducing health hazards that might be associated with the consumption of

raw tomatoes.

5. Tomatoes can be contaminated by certain decaying pathogens from contaminated air,

insects and animals. It is important that packaging and storage areas be kept free of

rodents, birds and insects which can disperse various undesirable microorganisms or even

be the source of such organisms.

6. Thorough sorting of injured fruits is essential to minimize later development of post-

harvest decays.

31
7. Baskets used for loading tomatoes should be inspected for cleanliness and where

necessary should be cleaned and sanitized prior to loading.

8. Finally, good sanitary measures should be upheld from harvest through handling and

distribution to consumers.

32
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