Isolation, identification and characterization of fungi from stored vegetables elufer akram

ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF
FUNGI FROM STORED VEGETABLES
A Dissertation submitted in partial fulfilment of the requirements for the
Award of the degree of Master of Science in Microbiology
Assam downtown University
Session 2017-19
Submitted by-
ELUFER AKRAM
Examination Roll No:170545
Enrolment No: ADTU/2017-19/MSc(M)/045
Department of Microbiology
Assam downtown University
Panikhaiti,Guwahati-781026
Assam, India

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ACKNOWLEDGMENT
Success is the sweet & succulent fruit of vision, inspiration, guidance & execution. Words are
very poor comforters to express the deep gratitude which one feels in one’s corner in heart
when one is helped to achieve the ultimate goal, in this boundless & endless field of research
work. An acknowledgement sheet with a few words inscribe as it may not be enough to describe
the kind of help we have received in the form of guidance & inspiration from several person,
during the tenure of our project work. We wish to take this opportunity to thank everyone who
has directly or indirectly contributed their ideas and energies in successful completion of my
project. Teacher is a Guide, Philosopher and friend, which we experienced in my respected
Guide .We consider our self the luckiest person to work under his excellence guidance. As we
commence this project, we would like to humbly acknowledge and my profound gratitude to
my guide Mr. KIRAN TELENGA. If this work has any credit it’s just beyond my capabilities
to express my heartfelt thanks & regards to him or the great intent he has shown, with kind
guidance, able supervision, constant encouragement as well as omnipresence in every aspects
of my project work. We gratefully wish to express our immense gratitude, thanks, deep sense
of respect and to for their support, constructive and meticulous guidance which exonerated us
to consummate the small work assigned to us. We deeply convey our regards to our entire
teaching staff For their inspiration, support and valuable criticism throughout the course of my
work and beyond that. We also thankful to all the Lab technician, Lab assistant and other
supporting staff or their support during work. We express our deep gratitude to our faculty of
Allied Health, Adtu & Principal, Assam down town University Prof. Dr. P.C. Sharma,
Chancellor Dr. N. N. Dutta. And the other trustee member of down town charity trust for
providing the facilities so that we can complete our research work in due course time. Our
acknowledgement would be incomplete if we don’t offer sincere gratitude to the almighty God
& our beloved Parents Mr. INTAZ ALI, Mrs. IRANI BEGUM, & others family members
for sharing my dream for encouragement & moral support throughout my life. The completion
of this thesis is not only fulfilment of our dream but also the dreams of our Parents who have
taken lots of pain for us in completion of our higher studies.
With thanks to one and all......
Date:
Place: Signature of candidate

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CONTENTS PAGE NO.
 ABSTRACT
 CHAPTER 1 :INTRODUCTION----------------------------------- 2-5
 CHAPTER 2 :REVIEW OF LITERATURE---------------------7-13
 CHAPTER 3 :MATERIALS AND METHODS-----------------14-18
 CHAPTER 4 :RESULT-----------------------------------------------19-22
 CHAPTER 5 :DISCUSSION-----------------------------------------27-27
 CHAPTER 6 :CONCLUSION---------------------------------------- 28
APPENDIX
 REFERENCES----------------------------------------------------------34-36

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ABSTRACT
Experiment was carried out to determine the fungi involved in the spoilage of vegetables that
are stored in the local market such as tomato, carrot, capsicum etc. Vegetables are exposed to
contamination by microorganisms through direct contact with soil, dust, water and by handling
at harvest or during postharvest processing. This makes them to harbour a wide range of
microorganisms including plant and human pathogens. Spoiling microbes are considered
toxigenic or pathogenic as a lot of toxigenic fungi have been isolated from spoilt vegetables.
Even during refrigeration some microbes such as moulds and other fungi produce mycotoxins
of various types that are harmful to the consumers. These mycotoxins have low molecular
weight and toxic secondary metabolites from some species of fungi. They are dangerous even
in minute quantities and present extreme toxicity due to their ability to withstand heat In this
investigation different fungi and their morphology are observed macroscopically and
microscopically. Fungal isolation was done by using Sabouraud Dextrose Agar and
Aspergillus, Penicillium, Fusarium were isolated.

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CHAPTER-1
 Introduction
Vegetables are fertilized ovaries that served as vital sources of nutrient to human. They give
the body the necessary vitamins, fats, minerals and oil in the right proportion for human growth
and development [1]
. Also carotenoids like beta-carotene from carrots, lycopene from tomatoes,
zeaxanthin from green veggies, and lutein from spinach and collard greens. Salad greens are
packed with important nutrients and phytochemicals that support the maximum benefit to our
day to day vegetable requirement. Eating a diet rich in vegetables may reduce risk for stroke,
cancer, heart diseases. So vegetables need to be in proper edible condition as they are obligatory
part of our daily diet.
However, despite such tremendous benefits of vegetables, their existence and life span is
threatened by several factors among which are Climate change, pests, inadequate rainfall and
fungal attack. Vegetables contain high levels of sugars and nutrient elements and their low pH
values make them particularly desirable to microbial decay. These vegetables are usually
displayed on benches and in baskets for prospective customers in the open markets until sold,
thereby exposing them to further microbial infection beside those associated with fruit surface
and those from adjacent infected vegetables [2]
. The main sources of microorganisms in
vegetables are soil, water, air, and other environmental sources, and can include some plant
pathogens. Fresh vegetables are fairly rich in carbohydrates (5% or more), low in proteins
(about 1 to 2%), and, except for tomatoes, have high pH. Microorganisms grow more rapidly
in damaged or cut vegetables. The presence of air, high humidity, and higher temperature
during storage increases the chances of spoilage. Microbial vegetable spoilage is generally
described by the common term rot, along with the changes in the appearance, such as black rot,
gray rot, pink rot, soft rot, stem-end rot etc.
Fungi are increasingly implicated as the agents of spoilage of economically important
vegetables such as Alternaria, Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus and
Trichoderma species. Some microbes are found in only a few types of vegetables while others
are widespread. Molds belonging to several genera, including Rhizopus, Alternaria and
Botrytis, cause a number of vegetable rots described by their colour, texture, or acidic products.

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The higher moisture content of vegetables as compared to grains allows different fungi to
proliferate, but some species of Aspergillus attack onions.
Consumption of vegetable products has dramatically increased in India by more than 40%
during the past few decades. It is also estimated that about 30% of all vegetables produced are
lost each year due to spoilage. The contamination of vegetables by fungi could also be as a
result of poor handling practices in food supply chain, storage conditions, distribution,
marketing practices and transportation .The prevalence of fungi as the spoilage organism of
some edible vegetables abound in different locations. Vegetables are exposed to contamination
by microorganisms through direct contact with soil, dust, water and by handling at harvest or
during postharvest processing. This makes them to harbour a wide range of microorganisms
including plant and human pathogens [2]. Microorganisms responsible for spoilage of
vegetables exploit the host using extracellular lytic enzymes that degrade these polymers to
release water and the plant’s other intracellular constituents for use as nutrients for their growth.
Fungi in particular produce an abundance of extracellular pectinases and hemicelluloses that
are important factors for fungal spoilage [3]. Spoilage of vegetables refers to various changes
in which they becomes less palatable or taste, smell, appearance or texture. Numerous
microbial defects of agricultural crops are characterized by the types of micro-organisms
responsible for their deterioration.
Generally, spoiling microbes are considered toxigenic or pathogenic as a lot of toxigenic
fungi have been isolated from spoilt vegetables. Even during refrigeration some microbes such
as moulds and other fungi produce mycotoxins of various types that are harmful to the
consumers. These mycotoxins have low molecular weight and toxic secondary metabolites
from some species of fungi. They are dangerous even in minute quantities and present extreme
toxicity due to their ability to withstand heat. The most significant mycotoxins are: aflatoxin,
zearalenone, ochratoxin, trichotecen, fumonisin and palutin. One of their features refers to
thermo-stability and resistance in certain medium. They are causal agents of mycotoxicoses of
acute or chronic course, affecting the central nervous system, lungs, liver, digestive or
cardiovascular systems. Disorders are manifested as poisoning, with special accent on their
cumulative, carcinogenic, teratogenic and mycogenic, mutagenic, immunosuppressive effects
or represent a cause of endemic nephropathy. All mycotoxins do not have the same features;

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their effects on animals and humans depend on certain factors (dose, affinity towards organs,
gender and age), as well as on their possibilities of metabolic changes in organism. One of their
features is stability, longevity and resistance in certain medium. The most significant genera of
fungi, the species of which can excrete mycotoxins, include: Aspergillus, Penicillium,
Fusarium, Rhizopus, Alternaria, etc
Genus Aspergillus involves over 180 species of fungi; a small number of them are related
to human infections and diseases. A. flavus, A. fumigatus, A. niger and A. parasiticum are the
most significant for human medicine, being able to excrete certain types of aflatoxins and
ochratoxins.
Genus Penicillium involves a number of species are potentially significant causal agents of
diseases and they are found in several fruits and vegetables. Their structure includes septate,
branched hyphae. Conidiophores can be pigmented, smooth or rough; they carry a bottle-
shaped group of phialides. Genus Fusarium involves ubiquitous, saprophytic fungi that are
pathogenic for herbs and able to infect or intoxicate animals and humans. They can cause
keratitis, onichomicosis, infections of skin and wounds or other localizations of the infection
in humans. Their structure includes hyphae and they possess two types of conidias
(macroconidias and microconidias). They can excrete toxins, causing mycotoxicoses.
Some spoilage microbes are capable of colonizing and creating lesions on healthy, undamaged
plant tissue [4] .Improper pre-harvest fungicide application, poor washing, and/or inadequate
culling of fruits and vegetables usually lead to expanding infestation of spoilage
microorganisms which can destroy a substantial portion of a stored lot of fruits [3]. Despite the
high water activity of most vegetables, the low pH gives fungi a competitive advantage over
the majority of bacteria and mould spoilage is not uncommon. However, the plant and fungal
kingdoms have evolved together and many plants have endophytic and mycorrhizal fungi
associated with them. In a healthy plant these relationships may be beneficial to both partners
and, in addition, plants have evolved many mechanisms to prevent overt fungal attack of the
living tissue. These may involve physical barriers, chemical barriers or, at a more sophisticated

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level, the production of antifungal metabolites, known as phytoalexins, in response to fungal
attack.
Many food products and vegetables are perishable by nature and require protection from
spoilage during their preparation, storage and distribution to give them desired shelf-life.
Because food and vegetables are now often sold in areas of the world far distant from their
production sites, the need for extended safe shelf-life for these products has also expanded. The
development of food preservation processes has been driven by the need to extend the shelf-
life of foods. Food preservation is a continuous fight against microorganisms spoiling the food
or making it unsafe. Several food preservation systems such as heating, refrigeration use of
food additives and addition of antimicrobial compounds can be used to reduce the risk of
outbreaks of food poisoning; however, these techniques frequently have associated adverse
changes in organoleptic characteristics and loss of nutrients.

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 Aim and Objectives
Aim &Objectives:
This study was undertaken to isolate and identify fungi that are associated with spoilt
vegetables commonly sold in our local Market and recommend appropriate control measure.
 To isolate and identify various type of fungus causing spoilage to the selected
vegetables.
 To determine specific type of fungus for a particular type of vegetables.

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CHAPTER-2
 Review of Literature:
 Mairami Fatima Mukhtar et al 2019 carried out an investigation to identify the types
of fungal flora associated with the spoilage of fruits at Utako market, Abuja, Nigeria.
They concluded that the presence of 7 different fungal species: Aspergillus niger, A.
flavus, A. fumigatus, Aspergillus sp., Rhizopus stolonifer, Mucor mucedo and
Alternaria sp. However, Aspergillus niger was the most predominant species while
Alternaria sp. was the least common. However, the pathenogenicity of Rhizopus
stolonifer was higher than that of the remaining fungal species isolated, while that of
Alternaria sp. was the least. All the fungal species were found to be responsible for the
deterioration of fruits in Utako market. Some of these isolated fungal species produce
toxins that could cause severe food poisoning and other potential health hazards.
 Samaila Samaila Yaradua et.al 2018 done the Isolationand identification of fungi
from some selected vegetables in Kankara local government area, Katsina state.
Samples of vegetables (infected and healthy) were investigated in this study to identify
and isolate fungal species growing present. The vegetables are Tomato
(Lycopersicumesculentum), Pepper (Capsicum annum), Cucumber (Cucumissativus)
and Onion (Allium cepa) which were collected from local farms. Potato dextrose agar
(PDA) was used for isolation of the fungi and morphological features and fungal
identification. All the vegetables collected showed the presence of fungi. The fungi
isolated are Aspergillusflavus, Rhizopusstolonifer, Aspergillusniger,
Aspergillusparasiticus, Penicilliumdigitatum.,Penicilliumcitrinumand Mucor spp.
among all, Penicilliumcitrinum, Aspergillusniger., and Aspergillus. parasiticus had the
highest rate of occurrence followed by Penicilliumdigitatumand Aspergillusflavuswhile
Mucor spp. and Rhizopusstolonifer were the least.

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 Suleiman et. Al , 2017 studied about Fungi and Aflatoxin Occurrence in Fresh and
Dried Vegetables Marketed in Minna, Niger State, Nigeria. In this study Mycoflora and
aflatoxin contamination was determined in 200 samples of fresh and dried vegetable,
which are widely used because of their nutritional and medicinal principles. Fifty (50)
fresh pumpkin (Curcurbitaspp), fresh spinach (Spinanciaoleracea), bitter leaf
(Vernoniaamygdalina) comprising of (25 dried and 25 fresh) and 50 samples of tomato
(Solanumlycopersicum) (25 dried and 25 fresh) were collected from local vegetable
vendors in Minna metropolis, Nigeria for investigation. Identification of isolates was
carried out based on their morphological and microscopic characteristics.
 AMINU. F et.al, 2017 studied about Microorganisms associated with spoilage of
Cabbage (Brassica oleracea) using standard microbiological methods (Gram staining,
bacteriological analysis and Biochemical tests) for bacteria and (macroscopic and
microscopic examination) for fungi. The analysis was done on fifty (50) samples each
of cabbage with soft rot symptoms purchased from different vendors within Sabon –
Gari Market, Kano.
 Samuel Mailafia et al 2017 perform a study and found A. niger causes black mold in
certain fruits and vegetables. Some strains of A. niger have been reported to produce
potent mycotoxins called ochratoxins that can be harmful to human beings and animals.
The pathogenic fungi species associated with fruits spoilage are of economical and
public health significance. And also investigate that Aspergillus niger had the highest
occurrence in pineapple, watermelon, oranges, pawpaw, and tomatoes with a frequency
of 38%. Fusarium avenaceum followed with the frequency of occurrence of 31% in
fruits such as pineapple, watermelon, oranges, pawpaw, and tomatoes while Penicillium
digitatum and Rhizopus stolonifer had the least frequency of 4% each in tomato; and
orange and tomato, respectively. Other fungal species were identified as yeast
(Saccharomyces species) (10%), Fusarium solani (8%), and Aspergillus flavus (5%).
The highest prevalence rate was 70% of A. niger from orange followed by F.
avenaceum of which 65% isolates were recovered from pawpaw. Other fungal

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organisms such as yeast (Saccharomyces species), P. digitatum and R. stolonifer were
isolated with varying prevalence (40%, 20%, and 5%) from watermelon, tomato, and
orange, respectively. However, there was no significant difference in the fungal load of
the various fruits studied.
 Digambar et. al, 2016 studied about isolation and identification of pathogenic fungi
.The fungal isolation were Colletotrichumcapsici, Phytopthorainfestans,
Fusariumoxysporum, Fusariummoniliforme, Taphrinamaculans, Alternariaalternata
and Alternariasolani which were isolated from vegetable and were identified on the
basis of colony morphology, microscopic examination by using selective and
differential media. The morphological characteristics of these fungal elements showed
various kinds of spores have been identified up to genus/species level.
 D.S.R. Rajender Singh 2016 studied on Fungi Associated with Tuber Vegetables
during Storage in Markets of Telangana State, India. They attempt to isolate and
identify storage rot fungi associated with tuber vegetables collected from local markets.
Fungi were isolated and identified from rotten tuber vegetables namely carrot, beet root,
sweet potato and corm .Fusarium species was found to be the most dominant fungi.
Among the Fusarium sp., F. solani was the common fungus isolated from all four root
vegetables. Among the four tuber vegetables, carrot had the highest number of fungi ,
while Sweet potato had the lowest number of fungi.
 Khatoon Akhtari et. al. 2016 studied on Fungi Associated with Storage Rot of Carrot
(Daucuscarota L.) and Radish (Raphanussativas L.) in Odisha, India. An extensive
survey on fungi associated with post-harvest deterioration of carrot and radish storage
roots was conducted during 2014-2015 in different market places of Odisha, India.
Rotten samples were collected from five different localities such as Bhubaneswar,
Cuttack, Jajpur, Puri, Balasore and Bhadrak. Three fungal species such as
Aspergillusniger, Geotrichumcandidumand Rhizopusoryzaewere isolated from the

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rotten samples. Of these, Geotrichumcandidumhas highest percentage frequency of
occurrence in both carrot and radish. In carrot next to Geotrichumcandidum, Rhizopus
oryzaeshowed more frequency of occurrence than Aspergillus niger but in case of
radish the case is just opposite, here the percentage frequency of occurrence of
Aspergillus niger was found to be more than Rhizopusoryzae. Pathogenicity tests
revealed that all the isolated fungi were pathogenic to their respective host storage roots
 Dimphana et. Al 2016 carried out a study on isolation and identification of fungi
associacted with post harvest decay of Lycopersicumescukntum M. Sold in Abakaliki,
Nigeria. This work identified preponderant of Aspergillus ,Penicillium and Fusarium
in rotten tomatoes sold in five different markets in Abakaliki.
 Muhammad HL et al 2016 investigated on determination of mycotoxicity. Mycoflora
and aflatoxin contamination was determined in 200 samples of fresh and dried
vegetable, which are widely used because of their nutritional and medicinal principles.
Fifty (50) fresh pumpkin (Curcurbita spp), fresh spinach (Spinancia oleracea), bitter
leaf (Vernonia amygdalina) comprising of (25 dried and 25 fresh) and 50 samples of
tomato (Solanum lycopersicum) (25 dried and 25 fresh) were collected from local
vegetable vendors in Minna metropolis, Nigeria for investigation. Identification of
isolates was carried out based on their morphological and microscopic characteristics.
A total of 165 isolates made up of 4 genera (Aspergillus, Penicillum, Fusarium and
Mucor) were identified. Aflatoxins (AF) were determined using High Performance
Liquid Chromatography technique with UV detection. The report calls for a need for
enlightenment on proper agricultural, storage and hygienic practices alongside the
hazards related with aflatoxin-contaminated foods.
 Onuorah Samuel et al 2016 studied on the microorganisms associated with spoilt
carrots obtained from Ose Market, Onitsha, Nigeria were carried out using standard
cultural techniques. Nutrient agar, sabouraud dextrose agar and Eosin methylene blue

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agar were the growth media for the isolation of the heterotrophic bacteria, fungi and
coliforms. The bacteria were identified as Serratia marcescens, Escherichia coli and
Corynebacterium bovis while the fungi was identified on the basis of their colonial and
microscopic characteristics as Penicillium digitatum, Rhizopus stolonifer, Aspergillus
niger and Alternaria alternata. Escherichia coli was predominantly isolated among the
bacterial isolates (50%) while Aspergillus niger occurred most frequently than the other
fungal species (40%).
 Zahra Ibrahim El-Gali et. al 2016 carried out an investigation about Isolation and
Identification of Fungi Associated with Fruits Sold in Local Markets. A total of 12
species of fungi representing eight genera were found to be associated with the rotten
fruits collected from the markets of El-Beida city. The fungi isolated from the fruits
were Alternariaalternata, A.niger, Colletotrichumgloeosporioides
Colletotrichumacutatum, FusariumoxysporumGeotrichumc and idum,
Penicilliumdigitatum.,Penicilliumitalicum, and Rhizopusnigricans. Species of G.
candidum, B. cinerea, and R. nigricans., were found to be associated as the
predominantfungus with the rotten fruits. The higher diversity 4 species of fungi was
found in guava, and the lowest one species in banana, pear and rock melon fruits.
 Akinro, E.B et. al. 2015 studied Isolation and Identification of Fungal Species
Associated With the Spoilage of Some Selected Edible Fruits in IreeTown OfBoripe
Local Government, Osun State, Nigeria. Four Spoilage fruits were investigated in this
study. They were pawpaw (Carica papaya), Pineapple (Ananascomosus), Orange
(Citrus sinensis) and Tomato (Lyeoptersieonesculentum), which were collected from a
market at Iree town. All the fruits collected showed signs of spoilage by fungal species.
The fungal species isolated were Aspergillusflavus, Rhizopusstolonifer,
Aspergillusniger, Candida tropicalis, Phytophthora sp., Fusariumoxysporum and
Mucus sp. Among all, Aspergillus species had the highest rate of occurrence followed
by Rhizopus and Candida species while Phytophthora sp., Mucus sp. and Fusarium sp.
were the least. Finally, the pathogenicity tests showed that all the fungi isolated were
pathogenic to all the fruits

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 G.D. SHARMA et.al 2013 examined the Isolation and Identification of Fungi
Associated with Local Fruits of Barak Valley, Assam. They studied the fungal diseases
of eight selected local fruits in Cachar district and twenty three fungal pathogens were
isolated which caused spoilage of fruits. Samples were plated out on potato dextrose
agar (PDA) medium and incubated at 28°C±2°C. Resulting growth microscopically
screened for fungal species. Aspergillus was commonest fungus found in all fruits
during storage of fruits.Other genera like; Acremonium, Alternaria, Aspergillus,
Chalaropsis, Cladosporium, Curvularia, Fusariumm, Mucor, Penicillium, Rhizopus,
and Trichodermawere common in fruits stored stored in warm and humid condition.
 Rashad R. Al-Hindi et al 2011 made an investigation on the current spoilage fruit fungi
and their plant cell wall degrading enzymes of various fresh postharvest vegetables sold
in Jeddah city and share in establishment of a fungal profile of fruits. Ten fruit spoilage
fungi were isolated and identified as follows Fusarium oxysporum (banana and grape),
Aspergillus japonicus (pokhara and apricot), Aspergillus oryzae (orange), Aspergillus
awamori (lemon), Aspergillus phoenicis (tomato), Aspergillus tubingensis (peach),
Aspergillus niger (apple), Aspergillus flavus (mango), Aspergillus foetidus (kiwi) and
Rhizopus stolonifer (date). The plant cell wall degrading enzymes xylanase,
polygalacturonase, cellulase and α-amylase were screened in the cell-free broth of all
tested fungi cultured on their fruit peels and potato dextrose broth (PDB) as media.
Xylanase and polygalacturonase had the highest level contents as compared to the
cellulase and α-amylase. In conclusion, Aspergillus spp. are widespread and the fungal
polygalacturonases and xylanses are the main enzymes responsible for the spoilage of
fruits.
 A.Adebanjo et al 1993 studied on the identification of the sources of mycoflora of
Abelmoschus esculentus, Corchorus olitorius, Solanum macrocarpon and Capsicum
annum. The major fungal flora of the stored vegetables over a period of 8 weeks were
Aspergillus flavus, A. niger, A. fumigatus, Rhizopus oryaze, Penicillium oxalicum,
Rhizomucor pusillus and Fusarium equiseti however, the mycoflora isolated from the
fresh vegetables and after sundrying differed. Some micro-organisms were isolated
immediately after sundrying and throughout storage. There was also evidence of field

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acquisition of some of these species which were carried into storage. Also, post-harvest
contaminants, handling, air spora during sundrying and storage fungi were other
sources of contamination of these vegetables.

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CHAPTER-2
 Materials and Methods
 Materials :
GLASSWARES OTHERS REAGENTS EQUIPMENTS
Conical flask Spatula Agar agar Hot air oven
Beakers Aluminium foil PDA Microscope
Petri plates Spirit lamp SDA Laminar air flow
Micropipette Cotton Antibiotic Incubator
Glass rod Test tube stand Distill water Autoclave
Measuring cylinder Ethyl alcohol
Pipette
Test tube
Slide
Spreader
 Methods:
 Media Preparation-
(I) 4.7 gm of Sabouraud Dextrose Agar (SDA) was measured and properly mixed with
100 ml of distilled water in a conical flask.
(II) Then the solution was heated with frequent agitation and boil for one minute to
completely dissolve the medium.
(III) Then autoclaved at 121° C for 15 minutes.
(IV) After autoclaving the solution was cool to 45 to 50°C.
(V) Antibiotics are added to the solution to inhibit any bacterial growth.

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 Sample Collection-
Fungal isolates were obtained from randomly selected symptomatic vegetables (decaying or
rotting plant substrate) such as L. esculentus (tomatoes), D. carota (carrot), Capsicum annuum
(capcicum) from five different markets and farm lands in Guwahati, Assam. The samples were
collected in zip-lock pouches and transferred to the laboratory.
SAMPLE-1 SAMPLE-2
Capcicum (Capsicum annum) Carrot (Daucus carota)
Tomato (Solanum lycopersicum)

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 Fungal Isolation-
(I) Culture procedure of Isolation:
Fungal isolation was done with the help of a sterile cotton swab. The swab was rubbed
all over the skin surface of the vegetable sample and then the swab is spreaded over the agar
medium in the plate. Then the plate was kept in the room temperature for 5-7 days.
 SAMPLE-1
Capcicum (Capsicum annuum)
CONTROL(S1) Isolate obtained from sample-1 (S1)

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 SAMPLE-2
Carrot (Daucus carota)
CONTROL(S2) Isolate obtained from sample-2 (S2)

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 SAMPLE-3
Tomato (Solanum lycopersicum)
CONTROL(S3) Isolate obtained from sample-3
(S3)

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CHAPTER-4
 RESULTS:
 Morphological Characterization:
SAMPLE-1
Specimen Form Elevation Margin Pigmentation Texture
COLONY-1
COLONY-2
COLONY-3
COLONY-4
Filamentous
Irregular
Irregular
Filamentous
Raised
Raised
Raised
Flat
Filiform
Entire
Undulate
Filliform
White
Brown
Dark brownish
Bottle green
Soft
Rough
Rough
Soft
SAMPLE-2
COLONY-1
COLONY-2
COLONY-3
COLONY-4
Filamentous
Irregular
Irregular
Filamentous
Raised
Raised
Raised
Flat
Filiform
Entire
Undulate
Filliform
White
Brown
Dark Brownish
Bottle green
Soft
Rough
Rough
Soft

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SAMPLE-3
COLONY-1
COLONY-2
COLONY-3
Filamentous
Filamentous
Irregular
Raised
Flat
Corvex
Filiform
Filliform
Undulate
White
Bottle green
Brown
Soft
Soft
Rough

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 Microscopic Observation:
SL
No.
LPCB MOUNT
KOH MOUNT CHERECTERS IDENTIFIED FUNGI
1
Septate, hyaline, acute angle
branching, tree or fern like branching,
conidial head biseriate, conidia was in
chain and detached.
Aspergillus spp.
2
Bluish green with white border,
hyphae septate, hyaline. Conidio
phores, branched phialides group in
brush like clusters at the end of the
conidio phores, conidia unicellular,
round hyaline arranged in chain.
Penicillin spp.
3
Macroconidia arehyaline, two to
several- celled, fusiform to sickle-
shaped, mostly with an elongated
apical cell and pedicellate basal cell.
Microconidia are one or two celled
hyaline, smaller than macroconidia.
Fusarium spp.
4
non septate hyphae having branch
sporangio phores with sporangium at
terminal ends Mucor Spp.

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5
Dark mycelia, one-celled conidia,
vegetative hyphae are present.
Conidiophores and conidia are equally
pigmented. Shield-shaped conidia, a
distinct hilum , conidia chains are
found.
Cladosporium spp.
6
Plant body is eucrapic and consists of
white cottony, much branched
mycelium. Body differentiated into
nodes and internodes. Stolon is
present. Hyphal wall is microfibrillar.
Rhizopus spp.
7
Spores can be formed singly, but most
often in chains. Club shaped, highly
septate. Divisions are found in both
vertical and horizontal. Spore surfaces
are smooth.
Alternaria spp.

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 Aspergillus sp.
Aspergillus is a genus of fungi that consists of about 300 identified species of mold (mould).
Aspergillus can be found in a variety of environments throughout the world given that their
growth is largely determined by the availability of water.
They grow is largely determined by the temperature range in the environment they grow in.
Regardless, studies have shown Aspergillus to be able to tolerate extreme conditions only if
all of the other conditions are ideal. Most of them reproduce asexually, they are often
described as conidial fungi. A majority have been shown to be terricolous, which means that
they are largely found in soil/land. Aspergillus are saprophytes that obtain their nutrition from
dead and decaying matter, they can also be pathogenic to human beings and animals with
some also affecting and damaging plants. Because they lack chlorophyll, and are therefore
unable to produce their own food, they are dependent on other material in their surroundings
for nutrition. They release different types of enzymes such as amylase capable of breaking
down these materials into simpler compounds that can be absorbed through the vegetative
hyphae.
 Fusarium sp.
Fusarium spp. grow rapidly on many media such as SDA or PDA. The colonies are fast
growing and bright in colour. Fusarium typically produce both macro- and microconidia
from slender phialides
 Penicillium sp.
Colonies of Penicillum spp. are fast growing. It has green colour shades. It also contain
conidospores. Chains of single-celled conidia are produced in basipetal succession from a
specialised conidiogenous cell called a phialide. The youngest conidium is at the basal or
proximal end of the chain. Phialides may be produced singly, in groups or from branched
metulae, giving a brush-like appearance.

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 STUDY OF MYCOTOXINS:
Mycotoxins are the secondary metabolic products of fungi. Introduced in organism they can
cumulate and cause certain disorders, disease or death. They expresss their effect towards
various animals species, including humans. They can express carcinogenic(liver, uterus and
esophagus), mutagenic, teratogenic and immunosuppressive effects or represent a cause of
endemic nephropathy.
All mycotoxins do not have same features; their effects on animals and human depend on
certain factors(dose, affinity towards organs, gender and age) aas well as on their possibilities
of metabolic changes in organism.
One of their features is stability, longevity and resistance in certain medium. Mycotoxins are
introduced in organism through digestive system or inhalation, as well as absorption through
the skin. The influence of mycotoxins in organism can be manifested in the form of acute or
more frequently chronic toxic effects, affecting the central nervous system, lungs, liver,
digestive or cardiovascular systems. Besides their significance as possible cause of disorders
of human and animal health, they are also significant from both economic and trade aspect.
Mass poisonings with mycotoxins are extremely rare, but we are exposed to their long term
accumulation in organism in certain situation.
The story of mycotoxins is very new comparing with the old known story of fungi. It began
with the detection of ergot, trichothecines, aflatoxins and recently fumonisins. Nowadays, more
than one thousand different chemically identified mycotoxins are isolated. They are of low
molecular weights. Some of them acts with each other synergistically as fumonisin-B1 and
aflatoxin-B1, ochratoxin-A and aflatoxin, T2 toxin and aflatoxin. Mycotoxins cause a wide
variety of adverse clinical signs depending on the nature and concentration of mycotoxins
present, duration ofexposure, the animal species, its age and nutritional and health status at the
time of exposure to contaminated feed.
 STRUCTURE
They are peptide derivatives (Cyclochoritme, Ergot, Gliotoxin, Sporidismine),
quinone derivatives (Lotuskirin, Rogulosin), peron derivatives (Aflatoxin, Citrinin,
Kojic acid, Sterigmatocystin), terpine derivatives (Fusarinone, Satratoxin,
Trichothecines, Vomitoxin), nonadrid (Rubratoxin), alkaloid (Lesergic acid, Slaframin),
xanthine (Sterigmatocystin), lacton (Patulin, Penicillic acid, Rubratoxin, Zearalenone),

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botnolid (Patulin), phynol (Zearalenone), glucose (Kojic acid), qumarin (Aflatoxin,
Ochratoxin, Sterigmatocystin) as citd by Abdelhamid (2000-b).
Fig : structure of mycotoxin
 MYCOTOXIN DETECTION:
The method of mycotoxin analysis depends mainly on the mycotoxin it self (or its
metabolites) and the contaminated tissue or substance will be tested. Therefore, there are
many detection methods for each mycotoxin, and there are screening methods for detecting
more than one mycotoxin simultaneously in the same sample. However, each method has
specific accuracy, sensitivity, recovery and reproducibility within a specific range of the
mycotoxin levels (Abdelhamid, 1981, 1995a and 1996). The principles of analysis consist of
precise sampling, sample preparation and toxin extraction, purification, derivation, elution,
concentration, qualitative detection, confirmation, and quantitative detection. Methods of
mycotoxins examination includebiological methods (e.g. cells, tissues, eggs, shrimp, fish,
chicks…etc), physical methods (e.g. UV-light), physico-chemical methods {e.g.
spectrophotometer and chromatography (Paper, Column, TLC, HPTLC, LC, HPLC, GLC –
MS)} and immuno-enzyme methods, e.g. ELISA (Schweighardt et al., 1980-a & b and
Abdelhamid, 1985, 1996 & 2000-b).

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FACTORS AFFECTING MYCOTOXINS PRODUCTION
 The substrate (on which the fungus will grow) and its nutritious content.
 Water content {water activity (aw)} of the substrate and ambient relative humidity.
 Ambient temperature (dry growing season).
 Ambient oxygen content (is required for fungal growth).
 Ambient carbon dioxide (not required for fungal growth).
 Mechanical damage (enable fungal invasion and mycotoxin production).
 Insect’s invasion (enable fungal invasion and mycotoxin production).
 Increased count of fungal spores accumulates the produced mycotoxin.
 The growth of non-toxic fungal strains inhibits the production from the toxigenic
fungi.
 Presence of specific biota inhibit growth of fungi and mycotoxin production.
 Time

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CHAPTER-5
 Discussion:
In this study we found that certain vegetables such as Tomato (Solanum lycopersicum),
capcicum (Capsicum annum) Cucumber (Cucumissativus) and Onion (Allium cepa) which
were collected from local farms are effected by several fungi. They are Aspergillus flavus,
Aspergillus niger, Aspergillus parasiticus, Penicillium digitatum. Penicillium citrinumand
Mucor spp. Among all, Penicillium citrinum, Aspergillus niger. and Aspergillus. parasiticus
had the highest rate of occurrence followed by Penicillium digitatumand, Aspergillus
flavuswhile Mucor spp.
The most significant mycotoxins that are produced by fungi such as: aflatoxin, zearalenone,
ochratoxin, trichotecen, fumonisin and palutin. One of their features refers to thermo-stability
and resistance in certain medium. They are causal agents of mycotoxicoses of acute or
chronic course, affecting the central nervous system, lungs, liver, digestive or cardiovascular
systems. Disorders are manifested as poisoning, with special accent on their cumulative,
carcinogenic, teratogenic and mycogenic, mutagenic, immunosuppressive effects. Genus
Penicillium involves a number of species are potentially significant causal agents of diseases
and they are found in several fruits and vegetables. Genus Fusarium involves ubiquitous,
saprophytic fungi that are pathogenic for herbs and able to infect or intoxicate animals and
humans. They can cause keratitis, onichomicosis, infections of skin and wounds or other
localizations of the infection in humans
Most vegetables get infected at the point of harvest, during handling, processing, and storage
or at point of sale. The findings of this study showed that several fungi at different frequency
of occurrences were found to be associated with contamination of tomato, capsicum, carrot
etc commonly sold at local market. Aspergillus spp. Mucor spp. Penicillium spp. The
deterioration could be probably due to mechanical injuries such as cuts that occur during
harvesting, post harvesting or storage periods which could provide infection sites for spoilage
fungi, improper handling and lack of good storage facilities, ability of the fungi to produce
spores and their ubiquitous nature, environmental conditions such as temperature and relative
humidity etc.

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CHAPTER-6
 Conclusion :
The project work entitled “Isolation, Identification and Characterisation of Fungi from Stored
Vegetables” was conducted to investigate the diversity of various fungi from spoiled
Capcicum (Capcicum annuum), Carrot (Daucus carota), and Tomato (Solanum
lycopersicum).
In the study several types of fungi species were isolated, such as Aspergillus, Penicillium,
Fusarium, Mucor and Molds. These species were isolated based on morphological and
microscopic examination.
The most significant genera of fungi, the species of which can excrete mycotoxins, include:
Aspergillus, Penicillium, and Fusarium. These mycotoxins causes serious health disease like
affecting the central nervous system, lungs, liver, digestive or cardiovascular systems etc. So
eating spoiled vegetables is very dangerous. And prevention of spoilage of vegetables is very
important.

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APPENDIX.
 Sabouraud Dextrose Agar (SDA).
Principle:
 Sabouraud Dextrose Agar (SDA) is used for the isolation, cultivation, and
maintenance of non-pathogenic and pathogenic species of fungi and yeasts. SDA was
formulated by Sabouraud in 1892 for culturing dermatophytes. The pH is adjusted to
approximately 5.6 in order to enhance the growth of fungi, especially dermatophytes,
and to slightly inhibit bacterial growth in clinical specimens.
 Peptone (Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue)
provide the nitrogen and vitamin source required for organism growth in
SDA. Dextrose is added as the energy and carbon source. Agar is the solidifying
agent.
 Chloramphenicol and/or tetracycline may be added as broad spectrum
antimicrobials to inhibit the growth of a wide range of gram-positive and gram-
negative bacteria. Gentamicin is added to further inhibit the growth of gram-negative
bacteria.
 The neutral pH of the Emmons modification seems to enhance the growth of some
pathogenic fungi, such as dermatophytes.

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Composition of SDA:
Ingredients In gm/L
Dextrose (Glucose) 40 gm
Peptone 10 gm
Agar 15 gm
Distilled Water 1000 ml
Utility:
 SDA is primarily used for the selective cultivation of yeasts, molds and aciduric
bacteria.
 The medium is often used with antibiotics for the isolation of pathogenic fungi from
material containing large numbers of other fungi.
 This medium is also employed to determine microbial contamination in food,
cosmetics, and clinical specimens.
Procedure:
Weight out of all the ingredients in a conical flask or any other suitable container that can
stand heating. Solution is made by dissolving, heating, and constant stirring. Autoclaved at
121° C for 15 minutes. After autoclaving the solution was cool to 45 to 50°C. Then
antibiotics are added to the solution to inhibit any bacterial growth. And for isolation the
media is poured into sterile petri dishes or test tubes.

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 Lactophenol Cotton Blue (LPCB) mounts:
Principle
Lactophenol Cotton Blue Solution is a mounting medium and staining agent used in
the preparation of slides for microscopic examination of fungi. In this staining
technique fungal elements are stained intensely blue.
The lactophenol cotton blue (LPCB) wet mount preparation is the most widely
used method of staining and observing fungi and is simple to prepare.
It has three preparation components-
Phenol: Kills any live organisms;
Lactic acid: It preserves fungal structures, and
Cotton blue: It stains the chitin in the fungal cell walls.
Preparation of lactophenol cotton blue (LPCB) slide mounts
 A drop of seventy percent alcohol placed on a clean microscope slide.
 Material from cultures of filamentous fungi was removed using a stiff inoculating
wire not the loop used for manipulations with bacteria or yeasts.
 Flamed the wire by holding it upright in the hottest part of the Bunsen flame, just
above the blue cone, until the whole length of the wire glows red hot.
 The inoculating wire cooled before placing it in a fungal culture.
 A small amount of the culture was removed. For fungal cultures, it is often useful to
take a little of the agar medium together with the fungus. In any case, the material
should be disturbed as little as possible when being transferred to the slide.
 Immersed the fungal material in the drop of seventy percent alcohol. This drives out
the air trapped between the hyphae.
 Teased out the material very gently with mounted needles.

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 The inoculating wire and the needles was steriled after use by heating to red heat in a
Bunsen flame
 Fungal structures readily visualised after staining with a lactophenol cotton blue dye
preparation.
 Before the alcohol dried out one or at most two drops of the stain added. A common
fault is to add too much to the preparation. Holding the coverslip between your index
finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
 The coverslip was gently lowered onto the slide, trying to avoid air bubbles.
 The specimen was observed under 40X magnification.
 KOH Mount
Principle
Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal
elements in clinical specimen, as it clears the specimen making fungal elements more
visible during direct microscopic examination.
This staining method softens, digests and clears the tissues surroundings of the
fungi so that the hyphae and conidia (spores) of fungi can be seen clearly under
microscope.
Utility:
KOH mount is one of the main methods of investigating fungal infections. It is used
as a primary screening tool, it detects fungal elements present but may not necessarily
identify the species of the fungi.
Procedure:
 A drop of KOH solution was added on a slide.
 The specimen was transferred to the drop of KOH, and covered with glass.
 The slide then placed in a petri dish, or other container with a lid
 As soon as the specimen has cleared, examination was done microscopically
using the 10X and 40X objectives.

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 References:
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