ISOLATION AND IDENTIFICATION OF FUNGI ASSOCIATED WITH SPOILAGE OF

FRUITS.
(ORANGE AND TOMATO)

BY

MICHEAL GRACE AMARACHI. 20/DSLT/6193

ADEYELU IREOLUWATOMIDE 0. 20/DSLT/6377
BADRU DAMILOLA LATEEF. 20/DSLT/6184
OLAWALE OLALEKAN BASHIR. 20/DSLT/6190

A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF SCIENCE

LABORATORY TECHNOLOGY, FACULTY OF SCIENCE, OSUN STATE
POLYTECHNIC, IREE

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

NATIONAL DIPLOMA (ND) IN SICIENCE LABORATORY TECHNOLOGY,
FACULTY OF SCIENCE, OSUN STATE POLYTECHNIC, IREE, OSUN STATE
NIGERIA

NOVEMBER, 2022

CERTIFICATION
This is to certify that this research project was written by MICHEAL GRACE AMARACHI,
20/DSLT/6193, ADEYELU IREOLUWATOMIDE O. 20/DSLT/6377, BADRU DAMILOLA

1
LATEEF 20/DSLT/6184, and OLAWALE OLALEKAN BASHIR 20/DSLT/6190. The project
has been red and approved has meeting the requirement for the award of National Diploma (ND)
in Science Laboratory Technology, Faculty of Science, Osun State Polytechnic, Iree.

…………………….. ……………………..
MR BUSARI T. DATE
SUPERVISOR

…………………….. ……………………..
DR OLAKUNLE T. P. DATE
COORDINATOR

2
 DEDICATION
This project work is dedicated to Almighty “ALLAH” and His prophet MUHAMMED (SAW).

3
 ACKNOWLEDGEMENT
My profound gratitude is due to almighty ALLAH for sparing my life throughout the course of
my HND program one in my academic carrier.
Special thank goes to ALLAH for his presence in my presence in my life, directing and
protecting me in all my endeavors.
First , I really appreciate the effort of sweet mother and father, for their love and care
shown by them for their moral, financial and spiritual support which are unquestionable, I thank
them for bringing me to life and making me what I am today may almighty ALLAH who never
sleep or slumber bless their life now and forever.
My sincere appreciation also goes to my supervisor MR. T. BUSARI for his fatherly love
and guidance, encouragement and necessary correction he made during this project work. May
you never known lack of the rest of your life .
I will also appreciate and express my love to my sweetheart ISIAKA OWOLABI A

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 TABLE OF CONTENTS
Title......................................................................................................................................i
Declaration..........................................................................................................................ii
Certification.......................................................................................................................iii
Dedication..........................................................................................................................iv
Acknowledgement..............................................................................................................v
Table of Contents...............................................................................................................v
List of Tables.....................................................................................................................vi
List of Figures....................................................................................................................vi
Abstract.............................................................................................................................vii

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1

1.0 Introduction and Literature review 1

CHAPTER TWO: METHODOLOGY 7

2.1 Materials 7
2.2 Method 7
2.3 Sterilization of materials 7
2.4 Preparation of Media 9
2.5 Method of Isolation 10
2.6 Method of identification 10
2.7 Pathogenicity test 12

CHAPTER THREE: RESEARCH AAND DISCUSSION 24

3.1 Result for pathogeniority test 24

CHAPTER FOUR: CONCLUSION AND RECOMMENDATION 27

4.1 Conclusion and Recommendation 27

REFERENCES 35

5
 LIST OF TABLES

Table 1: colonial, morphological and cellular characteristics of fungi associated with the spoilage
of orange fruit.

Table 2 colonial, morphology and cellular characteristics of fungi associated with the spoilage of
tomato fruits

6
 LIST OF FIGURES

Figure1: cellular structure of Aspergillums niger

Figure2: cellular structure of Rhizopus, stolonifer

Figure3: cellular structure of Penicilliumdigitatum

Figure4: Cellular structures of fusariumSolani

Figures: cellular structure of Yeast

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ABSTRACT

Heathy orange and tomato fruits were subjected to spoilage on laboratory bench. The
spoilagemicro organisms were isolated by using potato dextrose agar (PDA) for fungi isolated
from orange and tomato fruit. Pure cultures of the isolate were obtained. The fungi found
associated with the spoilage of these fruits (orange and tomato ) were identified to be fungi.
Aspergillusniger, Penicillimdigitatumand Rhizopusstolonifer, Yeast, fusariumsolani were
associated with the spoilage of fruits.

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

A fruits is the edible part of mature ovary of a flowering plants usually eaten raw (ZITTER,
1985). Fruits are the succulent or fleshy covering of nuts which are pulpy often juicy in
character. Since they developed from flower of plants, they consist of ripped seed or seeds with
some tissue attached (NAGY AND SHAW 1980).

Some fruits like apple, tangerine, sweets orange and banana are sweets while some that
are not sweets such as tomatoes cucumber and peppers are commonly called vegetables. Each
fruits has it own specific flavour due to esters compounds in them.

Studies by N- COHEN and BRUHN (2002) had shown that fungi can survive and /or
grow on fresh nutrient content (carbohydrate, protein and fats) of fresh produces support
pathogens.

Generally, fruits contain about 85% waters while fats and protein occurs in very small
and varying amounts. A fairs proportion of carbohydrates are presents as cellulose while starch
occurs in small quantity. However, sugar presents are chiefly glucose, lesser amounts of fructose
and sucrose, which producer sweetness in the fruits (NAGY and SHAW, 1980) fruits also

8
contain some organic acid like malic, citric and ascorbic some mineral such as calcium,
potassium and iron are presents (PUBOLS, 1960).

According to NAGY and SHAW (1980), fruits are also rich in vitamin. For instance,
orange had been found to contain 86% waters, 0.6% protein, 0.1% fat while micronutrients per
100g are calcium, 24mg are iron, 0.3mg. Other nutrients in the edibles portion of orange are
vitamin A, 1201U, thiamine 0.6 mg, riboflavin 0.02mg and ascorbic acid, 36mg.

The composition of fresh tomatoes composition 93% waters 1.1% protein and 0.3% fat.
Micronutrients are calcium 11mg and iron 0.6mg. There are also vitamin A 700IU, thiamine,
0.06mg, riboflavin, 0.04mg, niacin, 0.5mg and ascorbic acid, 23mg (PUBOLS, 1960).

1.1 IMPORTANCE OF FRUITS TO MAN

Orange is more enriched with vitamin C and minerals. Also the cellulose in orange fruits
is in a form, which although not digested, serves as useful roughage in the intestine of man, this
promoting normal elimination of faeces. This is however aided by high water content of the
fruits (NAGY and SHAW, 1980). Orange is also eaten raw or used for making fresh
unpasteurised or processed juice (NGUYENTHE AND CARLIN, 1994). Deep coloured fruits
and vegetable are considered by nutrionist to be the best sources of antioxidants. Antioxidants
are group of naturally occurring compound know to neutralize notoriously destructive cellular by
products known as free radicals. Antioxidants are believed to counteract the negatives effects of
free radicals by binding to them transforming them into non---damaging compounds or repairing
the damage they have caused (IHESIE, 2005).

Researchers in the united states and Canada have discovered a compound in that citrus
fruits (orange and tangerine) that has the potential to lower cholesterol more effectively than
prescription drug without side effect. Although a variety of citrus fruits contain
polymethoxylated flavones are tangerines and nobiletin, which are found in the peels of
tangerines and oranges (OSARENTIN, 2006).

In case of tomato, it is rich in potassium and vitamin A and C the fruit often is
sometimes eaten raw or cooked and often processed to make tomato paste sauces, pure cat soup
or juice (THE NEW ENCYCLOPEPIA AMERICANA 1988). Fruits spoilages is generally

9
caused by opportunities pathogens which cannot directly infect fruits tissues unless their tissues
are stressed. These pathogen are ubiquitous in the natural environments.

Mechanical injuries like bruises, cuts and punctures that occurs during harvest and
handling are believe to be predominant causes for decay and spoilage because they provide
infection courts for decay pathogen (ZITTER, 1985). Fruits and juices are mainly spoiled by
yeast and moulds. Furthermore microorganisms that had Penicillium digitatuim fusarium Solani,
Rhizopus been associated with spoilage are Stolonifer, Aspergillus niger (PELCZAR AND
REID, 1965).

1.2 ORANGE.

The orange (sweets orange), citrus sinesis belongs to the family Rutaceae and sub 150 species
(Samson, 1986) familyAurantioideae with 130 genera and arouse The sweet orange probably is
natives to northern eastern India and southern china but has spread to other tropical and
subtropical regions of the world. The introduction of this delicious fruits into Mediterranean
regions in the 15 century aroused much interest in citrus The orange was first introduced into the
western hemisphere by Columbus and has become a major crops in Florida, Califonia and Brazil.
The united states is the largest producer of orange, followed by Spain, Italy, and other countries
(Science and Technology) Encyclopedia, 1982

1.3 DESCRIPTION OF ORANGE

SAMSON( 1986) described citrus as evergreen trees, which bears spines on the stem. They attain
a height of 6m or more After 5-6 citrus beings to bears fruits citrus fruits matures on the tree for
six to twelve months. The sweets orange is mostly grown on the citrus tree and the fruits are
either eaten raw or made into juices KEFFURD AND CHANDLE (1970) reported that citrus are
important source of vitamin C with the juices containing 86% waters, 0.6% protein, fat 0.1% and
44 59mg anaerobic acid per 100g the edible portion of the orange is referred to as endocarp and

10
consist of segment composed of carpels (6-20) united to form locules that contain the thin wall
juices vesicle surrounding the endocarp is the peels that comprise 20-50% weight of the fruits
and consist of the flavedo and aldedo. The flavedo or outer peel is a layer of tissue underlying
the epidermis and contains the chemolast and oil sacs The albedo ar Inner peel is a layer of
spongy, white tissue that is connected to the core and supplies waters and nutrient for fruits
growth and development (Unekoronye and Ngoddy. 1985) JANICK AND MOORE (1975)
Grouped sweets orange into four namely, the common orange The common oranges are orange
coloured and acidless navel and pigmented or have varying numbers of seeds. The acidless
oranges are prized in some countries because of their very low acidity but generally are not
majors cultivators. The pigmented or blood orange developed varying amount of pink or Red in
the flesh and peel and haves distinctive aroma and flavour Most originated in the Mediterranean
area and are popular there, but are littlesgrown in the united states Navel orange are so named
because they are usually seedless, some have become important cultivars for fresh fruits market.

1.4 TOMATO

The tomato belongs to the members of the night shade family Solanaceae, genus
Tycopercicon and is closely related to potatoes and egg plants (Solanum) Some scientist actually
have named tomato SolanumEsculentum (Hawkes 1997) Tomato originated in the high land of
south American near the equator and was introduced to Europe in the mid 1500's.

Tomatoes were eaten in Italy as early as 1554 and were grown as gold apple, probably
because most varieties were yellow The tomato plants had also been carried north wards inte
America but the fruits was not eaten. In the united states tomatoes were not cultivated until the
today, tomatoes are cultivated late 1700's and were not widely caten until about 1900's
commercially in every states of united states at annual values of about 8500 million (The new
Encylopedia Britannica 2005)

1.5 DESCRIPTION OF TOMATO

The tomato plants may be bushy or vine-like and range in height from 3-6 feet (09- 18meters).
Although it is a perennial plants it is grown as an annual. Its leaves are composed of many small
and large leaf lets and the small yellow nodding flower are borne in cluster form Once fruits

11
development starts, it takes 45-50 days for fruits to matures the size, shapes and colour of the
fruits vary according to the variety. Some tomatoes are to the small and round, some what
cherries. Others are shapes like pears or plums and some are nearly spherical. Still others are
very large and flattered weighted up to 2 pounds (0.9kg) The flesh may be white, yellow,
oranges, pink or red and the skin may be yellow or colorless (The new Encyclopedia Americana
1988) AND NYODDY( 1988), tomato juice, tomato puree,

According to IHEKORONYE tomato paste and ketch. The carbohydrates presents in tomato
fruits are in form of reducing sugars and the predominant acids are citric followed by malic.
Glutamic acid is the man az,,,,,,,;;mino acid that is rarely founds in other fruits. The pigments,
which develops in ripening are the yellow pigments which develops in ripening are the yellow
pigments and at the later to red pigments

1.6 TAXONOMY OF ORANGE AND TOMATO

Cronquist (1998) classified sweet orange and tomato as follows

ORANGE

Kingdom Plantae
Phylum Magnoliophyta
Class Angiospermae
Sub-class Rosidac
Order Sapindales
Family Rutacease
Acaus Citrus
Species Citrus sinensis

TOMATO Plantae
Kingdom Magnoliophyta
Phylum Magnolidae

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Class Asteridae
Subclass Solanales
Order Solanaceae
Family Lycopersion
Citrus Lycopercicon peruvianum
Species Lycopersicon esculentum
Lycoperscion parviflorum
Lycopersicion chilense

KING ALL AND PENSTER (1964) observer that a cut in the fleshy part of the fruits always
serves as an entrances for microbes into the fruits leading to their spoilage Research by FOX and
CAMERON (1989) had revealed that fruits like orange and tomato contain the essential for the
that are excellent medium for the growth of micro-organisms such as Yeast and Moulds One of
the reasons why moulds usually spoil fruits or Yeast is because they grow best over range PH 3.0
6.0 and PH level of fruits is low (high acidity) Some moulds and yeasts can grows extremely in
the presence of sugars However, most of the fruits contain a lot of sugar and waters and micro-
organisms that spoiled them were those that can use sugar as source of carbon, metabolizing all
the sugar through process of fermentation and the end product change the fruits taste, colour and
smell ( Krauses and micric, 1982) PARRY AND POWSERY 1973) discovered that most of the
yeast and moulds are widely distributed especially on the skin of fruits and vegetables are
responsible for most decay. Free waters on the wound surface of fruits had been found to
absolutely promote infection. Also high fruits temperature above 30 C to 35° C has been
associated with rapid developments of fruits soft rct may he less than 18 hours (Zintter, 1985).
are one of the fungi associated OPEKE (1987) reported that Aspergillus species with spoilage of
fruits This mould may be yellow, green or black on fruits producer septate mycelia STUMBO
that produce an extensive mycellium that is (1965) Also noted that Fusarium species cottony in
structures with ling of pink, purple or white is important in the spoilages of fruits Partner (1973)
reported that Geotrichum species is a yeast like fungus, which is always white and feathery in
appearances, are capable of infecting ripe fruits PITT (1979) observes that penicilliumsspieces is
a plants parasite that occurs on decaying fruits, which could be either green moulds or blue

13
mould Green mould has a pasty wrinkle Mycelium on fruits which shows much in advance of the
conidia and the spores are green

1.7. PURPOES OF STUDY

The main purpose of this work is to isolate and identity the fungi that are associated with the
spoilage orange and tomato fruits. Orange and tomato fruits like other fruits are perishables
because they are highly susceptible to attack by fungi, which easily cause their spoilage resulting
in their limited

shelf life after harvesting However, if enough is known about these spoilage organisms, some
preservatives methods could be formulated that can be employed to improved the shell life of
these fruits during post-harvesting storages. This may makes them to be available throughout the
years

CHAPTER TWO

METHODOLOGY

2.1 MATERIALS

Equipment1 and apparatus

Light microscope, incubator, inoculating needle, autoclave, beam balance, oven, spirit lamp,
lamina flow chamber and 4mm cork borer GLASS WARE Petri dishes, microscope slides, cover
slips, 1000ml measuring cylinder, 1000ml conical flask 1000ml breakers and glass stirrer

REAGENTS AND CHEMICALS

Distilled waters, s 95% ethanol, 75% ethanol, methane blue stain.

MEDIA

Potato Dextrose Agra (PDA)

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FRUITS

Orange and Tomato

OTHER MATERIAL

Aluminum foil, cotton wool

2.2 METHOD

Collection of Samples

Healthy orange and Tomato fruits were bought from the market and brought into the laboratory
in sterile ploythene bags

2.3 STERILIZATION OF MATERIALS

All the glassware were washed with detergent them rinsed with tap water they were

drained by arranging them on a track after which they sterilized for two hours in an oven at a

temperatures of 180°C inoculating needles was sterilized by dipping it in 95% ethanol and then

flamed before and after each use. The fruits were rinsed with tap waters followed by distilled

water and there after surface sterilized with 75% ethanol.

2.4 PREPARATION OF MEDIA

Potato Dextrose Agra (PDA)

According to the manufacturer's instruction, 39g of potato dextrose agar dissolved in 1 little of
distilled water in a 1000ml conical flask. The mixture was them, stirred using a stirrer while s
heating on a hot plate in order to obtain a homogenous solution. After then, the month of the
conical flask was plugged with cotton wool and the whole flask wrapped with foil. It was then
sterilized in the autoclave at 15 pounds per squares inch for aluminium 15minutes at 121°C.
After cooling the medium to 60° C, medium was there after poured aseptically into sterile Petri
dishes. In the lamina flow chamber.

2.5 METHODS OF ISOLATION

15
 The healthy orange and tomato fruits were washed with tap waters and there after rinsed
with distilled water after which they were surface s sterilized with 75% ethanol. They were then
kept. In separate petri dishes and placed on a laboratory bench.

The fruits were than allowed stay for 7 days to rot. after 7 days, the spoilt fruits were taken to
inoculating chamber. Fungal colonial on the fruits were scraped with inoculating needle and
inoculums introduced into the plates of potato dextrose Agar

The plates were labeled as follow: orange I as O₁. plate2 as O₂ and plates 30, while tomatoplatel
T₁, plates2 as Tz, and plate 3 as T₁, all the plates were incubated at 30°C for 48hours. After
48hours, different types of colonies were obtained and later subculture on to another freshly
prepared plates of PDA for fungal colonies from orange and tomato plates. The plates were
labeled from O₁, O2, O, for orange and T₁, T2. Ty for tomato plates. The were incubated for
48hours at 30°C, after Incubation period, pure culture of the colonies was obtained

2.6 METHOD OF IDENTIFCATION

The colonial and cellular characteristics of individual colony of pure culture were
examined. The colonials morphology of the pure culture of each fungus was determined.
Microscopic examination of each pure fungus isolates was also made. The colonial
characteristics determined includes, colour of the colony on the surface of the agar, and the
colour changes of the reveres of the colony and t growth rate on the media. The variousfungal
isolates were identified by references to (Francisco, 1973), LUNN, 1977, Raper and Fennel,
1965, Booth, 1971, Raper and Thom, 1949). After then, temporary slide of cach fungal colony
were prepared to be able to examine the morphological structures of each colony under
microscopes.

TEMPORARY SLIDE PREPARATION

A drop of methylene blue stain was place at the center of the slide. A sterile
innoculcating needle was them used to pick small portion of colony from 0₁, 0₂, 03 T₁ T2 and T₁
plates. Each specimen was then spreads in the drop of methjlene blue stain and there after
covered with cover slip. These slide were labeled O, to O. The same procedure was applied to
remaining slide Ti to Ty observation was made on each slide under the light microscope.

16
2.7 PATHOGENICITY TEST

Each of the fungi isolated was tested on healthy fruits its ability includes spoilage. The
healthy fruits (orange and Tomato) were washed with tap waters and rinsed with distilled water
after which they were surface sterilized with 75% ethanol. A sterile 4mm cork broer was used to
punch each of the fruits.

A colony of fungi isolates (from pure culture) was used to inoculates the fruits and the
core of the fruits were replaced. The point of inoculation was sealed with petroleum jelly to
prevent contamination. The fruits (orange and Tomato) were places on a laboratory bench for
seven days after which the colonies from the spoilt fruits were subjected to identification
procedures.

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 RESULTS

After passing through all the procedure necessary for the isolation and identification of

fungi associated with the spoilage of orange and tomato fruits, different colonies were observed

growing on the spoilt fruit.

The fungi colonics spoiled the orange and tomato fruits, causing their deterioration. A

mixed colony was obtained when the fungi were first isolated on potato dextrose agar after this,

each colony of the fungi was then sub-cultured on a freshly prepared medium which resulted to

pure culture of each of the spoilage fungi

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3.2 OBSERVED SYMPTOMS ON THE SPOILED FRUIT

Physical observation of the diseased fruit showed brownish necrotic patches on the skin
of orange and tomato fruits. The patches were a bit sunken and turn black by 3days. A mass of
mycelia was observed growing on the skin of orange and tomato fruits while it took 7days for
orange to get spoiled.

The main type of spoilage observed in these infected fruits was the soft rot (zitter 1985)
The table below shows both the colonial, morphological and cellular characteristics of the
spoilage fungi isolated (lunn, 1977, Raper and the thom, 1949, Raper and funnel, 1965, both
1971)

Table 1: colonial, morphological and characteristics of fungi associated with the spoilage
of orange fruits.
ISOLATE COLONIAL MOPHOLOGICAL ORGANISM
D CHARACTERISTICS STRUCTURE AND CELLULAR OBSERVED
STRUCTURE
O1 Colonies velvety Colnidiopjores smooth, relatively short, Penicillium
Yellow green penicillin arranged very irregularly and digitatum
asymmetrical with branches of various
length. Sparse and irregular metulae
with philides on them conida smooth
O2 Colonies light grey A bundle of sporangiophore smooth Rhizopus
growing extrimly walled aspate light brown. Simple stolonifer
rapidly and filling the arising in group of 3-5 from stolons
petridish with dense opposite rhizoids. Sporangia globes or
cottony mycelium flattened base contained many spores
producing mass of white at first then black
sporangia
O3 Colonies with loose Vesicles light yellow-brown. Philalide Aspergillus
white to mycelium growing radially along the whole Niger
rapidly becoming dark periphery of phialides primary phialides
brown toblack on the and secondary wesicle both are brown.

18
 development of conidia

Key; orange plate=>O1,O2 and O3

Table 2; Colonial, morphological and cellular characteristics of fungi associated with

spoilage of tomato fruits.
ISOLATED COLONIAL MOPHOLOGICAL ORGANISM
CHARACTERISTICS STRUCTURE AND CELLULAR OBSERVED
STRUCTURE
T1 Shiny creamy white The structure found where single Yeast
colonies called
T2 Colonies light grey A bundle of sorangiophore was Rhizopus
growing extreimely formed. Sopangiophore smooth stolonifer
rapidly and filling the walled asptate, light brown. Simple
petridish with dense arising in group of 3-5 from stolons
cottony mycelium opposite rhizoids. Soprangio globes
producing mass of sub-globose with some flattened
spotangia base white at first then black and
many spores.
T3 Growth moderately rapid Micro conidia oval micro conidial Fusarium
covering agar plate within produced on richly branched Solani
4 days with sperse,grayish conidiophores cylindrical to falcate.
with mycelium. A bluish
brown decploration
developed in the agar
Key: tomato plate T1, T2, T3.

3.3 RESULT FOR PATHOGENICITY TEST

PATHOGENICITY TEST

19
 On the seventh day, the healthy fruits (Orange and Tomato) got spoilt. When the moulds
in the infected part were subjected to the identification procedures by examining their colonial,
morphological and cellular characteristics the moulds seen were the same as those of isolated
fungi of fresh fruits which were subjected to spoilage.

3.4 DISCUSSION

The results of this work shows that Penicilium digitatum, Rhizopus stolonifer and Aspergillus
niger, had been implicated in the deterioration of orange fruits also the Rhizopus stolonifer, yeast
and Fusarium Solani had been implicated. These agree partly with the findings of JAMES
(1997), NITRA (1997) who discovered that the species of fungi associated with spoilage of
orange and tomato fruits include Aspergillus fusarium, Penicillium Rhizopus and Yeast.

The result of pathogenicity test also established that fungi caused deterioration of fresh orange &
tomato fruits where then gained entrance to them through mechanical injuries like bruised and
wound asd noted by Zitter (1985) in his research work.

20
21
22
23
24
 CHAPTER FOUR

CONCLUSION AND RECOMMENDATION

Mechanical injuries like bruises or cuts that occur during harvesting or handling could provide
infection site for spoilage pathogens.

Fruit spoilage can be controlled by putting into practice the following techniques:

(1) washing of the harvested fruits with clean or pure water.

(2) Proper cleaning and sanitation of warehouse and dis-infection of packaging and transit
containers.
(3) Proper handling of the fruit during harvest to prevent bruises and scars or other
mechanical injury.
(4) Inhibition of fungi growth by lowering storage temperature through storage under
refrigeration.
25
 (5) The use of fungicides such as Benlate, Tecto and Botran (Maharaj, 1990).

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