STUDIES ON FUNGI ASSOCIATED WITH SPOILT TOMATO FRUITS AT KASUWAN DAJI MARKET SOKOTO
Monday, July 20, 2020
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A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY, FACULTY OF SCIENCE, USMANU DANFODIYO UNIVERSITY, SOKOTO IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF BSc. DEGREE (HONS) IN MICROBIOLOGY.
OCTOBER, 2018.
CERTIFICATION
This research project entitled “Studies on Fungi Associated with spoilt tomato fruits at kasuwan daji market Sokoto state Nigeria” by Abbas Muhammad Auwal (Adm. Number: 1410307043) has been read and approved by the underdesigned as meeting part of the requirement for the award of Bachelor of Science (BSc. Hons) Degree in Microbiology, Usmanu Danfodiyo University, Sokoto.
Mal. A. A Usman Date
(Project Supervisor)
Dr. A. B. Rabah Date
(Head of Department)
Date
(External Examiner)
DEDICATION
This research work is dedicated to my beloved parents, Alhaji Abbas Abubakar and Hajiya Mariya Haruna for their parental guidance, supports, encouragements and prayers against all evils of darkness.
ACKNOWLEDGEMENTS
All praises, greetings and adorations are to almighty Allah (Subhanahu Wata’ala) for seeing me through from the beginning of my program in this prestigious institution. Greetings, salutations and benedictions to His noble prophet (Sallallahu Alaihi Wa sallam) for being a guide to me and all of mankind. Oh! Allah, we continue to seek for your help and blessings in all our endeavours.
I wish to express my profound gratitude and deep appreciation to my supervisor Mal A.A Usman for his immense contribution in terms of suggestions, corrections, constructive criticisms and his being ever ready to give me a helping hand throughout the course of this research work. May Allah (Subhanahu Wata’ala) crown all his effort with success.
My incalculable debt of profound gratitude goes to my parents Alhaji Abbas Abubakar and Hajiya Mariya Haruna, who if not for their moral, financial and material support and prayers against all evil of darkness, I would not have been where I am today. May almighty Allah (Subhanahu Wata’ala) grant them the very best here and hereafter.
I am greatly indebted to my beloved brothers and sisters Muhammad, Umar, Rukayyah, Musa, Rahma, Saratu, Haruna, Izzatu, Idris, Fatima, Abbas, Rabiu, Ibrahim, Abdullahi and AbdulRahman, for their encouragement, prayers and supports. May Allah (Subhanahu Wata’ala) reward them all abundantly.
My sincere appreciation also goes to Aunty Hadiza, Aunty Bilkisu and Aunty Fatima, the following friends Usman Ahmad, Salim Sabo, my course mates especially Abdullahi Ibrahim, Abdullahi Sani and my beloved one in person of Aisha Yusuf Zurmi, my lecturers and others too numerous to mention, for their support in one way or the other. May Allah (Subhanahu Wata’ala) reward them all abundantly, Ameen.
TABLE OF CONTENTS
Title Page
Title page I
Certification II
Dedication III
Acknowledgements IV
Table of contents V
List of Tables VIII
Abstracts IX
CHAPTER ONE
1.0 Introduction 1
1.1 Background of the study 1
1.2 Aim and Objectives of the research 2
1.3 Statement of the research problem 3
1.4 Justification for the research 3
CHAPTER TWO
2.0 Literature Review 4
2.1 Tomato Fruits 4
2.1.1 Soil and Climate that support the Growth of Tomato Fruits 5
2.2 General Properties of Fungi 6
2.2.1 Classification of Fungi 6
2.2.1.1 Based on Sexual Reproduction 7
2.2.1.2 Based on Morphology 9
2.3 Spoilage 12
2.3.1 Spoilage of Fruits 12
2.3.2 Spoilage of Tomato Fruits 14
2.3.3 Mode of Activity of Microbes on Causing Spoilage 15
2.4 Factors Affecting the Growth of Microorganisms in Fruits 17
2.4.1 Intrinsic Factors 17
2.4.2 Extrinsic Factors 19
2.5 Some Tomato Fruits Diseases 20
2.5.1 Fungal Diseases 20
2.5.1.1 Sour Rot 21
2.5.1.2 Rhizopus Rot 22
2.5.1.3 Buckeye Rot 22
2.5.1.4 Black Mold 22
CHAPTER THREE
3.0 Materials and Methods 24
3.1 Study Area 24
3.2 Sample Collection 24
3.3 Preparation of Media 24
3.4 Sample Preparation 25
3.5 Fungi Count 25
3.6 Identification of isolated fungi 25
CHAPTER FOUR
4.0 Results 26
CHAPTER FIVE
5.0 Discussion, Conclusion, and Recommendations 31
5.1 Discussion 31
5.2 Conclusion 33
5.3 Recommendations 34
REFERENCES 35
LIST OF TABLES
Tables Page
Fungal load in the spoilt tomato fruits 27
Percentage occurrence of fungal isolate in the spoilt tomato 28
Fungal isolates relevant to different sellers 29
Images of fungal isolates 30
ABSTRACT
This research was aimed at studying fungi associated with spoilt tomato fruits. Samples of spoilt tomato fruits were collected from ten different sellers at kasuwar daji market in Sokoto North local government area. Standard procedure was employed. The highest fungal load (4×103) is found to be at seller 3 and the lowest fungal load (1×103) is found to be at seller 7 and seller 10 (Table 4.1). Sacchromyces cerevesiae was found to have the highest occurrence (43.5%) and Fusarium oxysporum have the lowest occurrence (8.7%). From the samples of tomatoes collected in kasuwan daji, it can be deduced that most of the spoilt tomatoes are contaminated with microorganisms, especially fungi. Some of these fungi were Sacchromyces cerevesiea, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Fusarium oxysporum. As such, more hygienic practices should be taken on to ensure healthy and microbe free tomato. These will go a long way in preventing the consumption of contaminated tomato fruits thereby reducing the health hazards posed by the mycotoxins produced by these fungi isolated in this study. Good quality control measures must therefore be employed by the farmers, marketers and consumers during the harvesting, transportation, handling and processing of the fruits. Frequent inspection of the fruits for sale by food inspectors is also recommended.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Tomato is a widely consumed fruit eaten in both raw and processed forms (Moneruzzaman et al., 2008). It has the botanical name Lycopersicum esculentum and belongs to the plant family solanaceae. It is rich in vitamins including vitamin A and vitamin C, carbohydrates such as glucose and fructose, proteins, fats, and fibres (Talvas et al., 2009). Minerals which include Phosphorous, Sodium, Potassium, Calcium, Magnesium and trace elements like Iron, Copper Zinc and Dietary fibers (Oyemaechi et al., 2014). It is rich in lycopene which has many beneficial health effects. It contains large amount of water which makes it more susceptible to spoilage by the action of microorganisms (Bai and Lindhout, 2006). Tomato has much lower sugar content than other fruits and is therefore not sweet.
Tomatoes may be pear-shaped, elongated, flattened and heart shaped. They are edible, fleshy and reddish when ripe and vary in their acid composition, with white and yellow ones being less acidic. Tomatoes can be used as savoury or flavouring in soups and cooked foods or can be eaten as fruits. It is used in many dishes, salads, sauces and drinks and can also be dried and ground into pancakes (Effiuwevwere, 2000).
The consumption of tomatoes throughout the world is believed to benefit the heart and other organs. The richest source of lycopene is tomato and tomato-based products (Evangelia et al., 2005). Lycopene has been found to prevent prostate cancer, improve the skin’s ability to protect itself against the harmful ultra violet rays, decrease the risk of breast, lung, stomach, bladder, uterine, head and neck cancers, protect against neurodegenerative diseases, lower urinary tract infections and reduce the cardiovascular risk associated with type 2 diabetes (Freedman et al., 2008.; Borguini and Torres., 2009.; Zhang et al., 2009.; Shidfar et al., 2010.; Zdenka et al., 2010). Tomatoes have serious challenges to their existence. These include changes in climate conditions, pests, inadequate rainfall and microorganisms particularly fungi. One of the limiting factors that influence tomato economical value is its relatively short shelf life.
Tomato contains large amount of water which makes it more susceptible to spoilage by fungi. Spoilage of tomatoes are those adverse changes in the quality of tomatoes that are brought about by the action of predominantly biological and physical factors. These may be changes in taste, smell, appearance or texture of the fruits. (Ghosh, 2009) reported that fungi were the source of spoilage of most of the tomato samples accessed than bacteria. Fungi affecting tomatoes include Aspergillus phoenicis, Absidia species, Trichoderma species, Alternaria alternata, Fusarium oxysporum, Fusarium moniliformis, Aspergilliu sniger, Mucor species, Rhizopus stolonifer, Penicillium species, Geotrichum species and Phytophthora species (AL-Hindi et al., 2011.; Akintobi et al., 2011.;Etebu et al., 2013).
Fungal spoilage of tomatoes has been recognized as a source of potential health hazard to humans and animals due to the fact that they produce mycotoxins which are capable of causing mycotoxicoses in man following ingestion or inhalation. The mycotoxins are not limited to their areas of infections. Since tomatoes contain large amount of fluid, these mycotoxins diffuse rapidly throughout them, contaminating all parts and making the fruits unfit for consumption (Baker, 2006).
1.2 Aim and Objectives of Research Work
The aim of this research is to study fungi associated with spoilt tomato fruits.
The objectives of this research are;
1. To isolate the fungi associated with spoilt tomato fruits.
2. To identify the fungi associated with spoilt tomato fruits.
3. To determine the percentage of occurrence of isolated fungi from spoilt tomato fruits.
4. To determine the fungal load on spoilt tomato fruits.
1.3. Statement of the Research Problem
Tomatoes are referred to as ready-to-eat fruit since they are minimally processed and many people take tomatoes raw directly or via meals of salad usually served cold. Microbial spoilage and contaminating pathogens on this product poses a serious problem in food safety. The centre of disease control and prevention (CDC) estimates that there are 76 million cases of food borne illness every year. Outbreaks with identified etiology are predominantly of microbial origins, it is therefore necessary to study microbes associated with tomatoes spoilage.
1.4. Justification for the Study
Tomato is the most perishable fruit during handling, transportation and storage. This is because tomato contains large amount of water which make it susceptible to spoilage by microorganism such as fungi, bacteria and protozoa. Since microorganisms have been identified with the major cause of spoilage in tomato fruits due to its high water content, proper isolation and identification of fungi in tomatoes will greatly reduce the spoilage of these perishable fruit and as such producers and consumers will be able to protect their fruit (Tomato) and also identify spoiled tomatoes that have been attacked by fungi.
CHAPTER TWO
2.0 LITRETURE RREVIEW
2.1 Tomato Fruits
The Lycopersicon esculentum (tomato) is an important vegetable crop across the world, originated in West South America (Kimura and Sinha, 2008). The fruits of tomato are popular throughout the world and are used in all kind of stews, soups and also eaten raw in salads. Ripe tomato fruits have high nutritive values, being a good source of vitamin A, B, C and minerals (Elsayed and Edrees, 2014). Because of the importance of tomato as food, it has been bred to improve productivity, fruit quality, and resistance to biotic and a biotic stresses. Tomato has been widely used not only as food, but also as research material. Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in every country of the world-in outdoor fields, greenhouses and net houses (Bihn and Gravani, 2006).
Tomato plants are perennial, have a weak stem that often sprawls over the ground and vines over other plants (Sravanthi and Gangadhar, 2015). Fruit of tomato are diverse in size and shape, ranging from small and round to large and variable shapes (Brewer et al., 2006). Tomato fruits contain high amount of carbohydrates, fats, organic acids, water, minerals, vitamins and pigments. Tomato fruits are used in garnishing various cooked food in Nigerian dishes as well as dishes in many other parts of the world. It is estimated that ripe tomato fruits contain approximately 94 % of water, 4.3 % carbohydrates, 1 % protein, 0.1 % fat, 0.6 % fibre and vitamins. Antioxidant phytochemicals such as the carotene and lycopene are contained in tomatoes (Wogu and Ofuase, 2014). They are good sources of natural antioxidants which include carotenoids, vitamins, phenolic compounds, flavonoids, dietary glutathione, and endogenous metabolites and have been shown to eliminate free radicals, (Pitchaon et al., 2007).
2.1.1 Soil and Climate That Support the Growth of Tomato Fruits
Tomato grows well on most mineral soils that have proper water holding capacity and aeration, and are free of salt. It prefers deep, well drained, sandy loam soils. The upper layer needs to be permeable. Soil depth of 15 to 20 cm is needed to grow a healthy crop. In heavy clay soils, deep ploughing allows better root penetration. Tomato is moderately tolerant to a wide range of pH (level of acidity), but grows well in soils with a pH of 5.5 – 6.8 with adequate nutrient supply and availability. Addition of organic matter is, in general, favorable for good growth. Soils with very high organic matter content, like peat soils, are less suitable due to their high water holding capacity and nutrient deficiencies (Shankara et al., 2005).
Tomato requires a relatively cool, dry climate for high yield and premium quality. However, it is adapted to a wide range of climatic conditions from temperate to hot and humid tropical. The optimum temperature for most varieties lies between 21 and 24°C. The plants can survive a range of temperatures, but the plant tissues are damaged below 10 °C and above 38 °C. Tomato plants react to temperature variation during the growth cycle, for seed germination, seedling growth, flower and fruit set and fruit quality. If cool or hot weather spells persist during flowering, pollen production will be low. This will influence fruit formation. Frost will kill the plants. To avoid frost damage, it is best to wait until the winter is definitely over before sowing. It is possible to sow indoors earlier (in pots or trays). Light intensity affects the color of the leaves, fruit set and fruit color (Shankara et al., 2005).
2.2 General Properties of Fungi
They are eukaryotic cells contain membrane bound cell organelles including nuclei, mitochondria, golgi apparatus, endoplasmic reticulum, lysosomes etc. They also exhibit mitosis. They possess ergosterolsin their membranes and possess 80S ribosomes, having rigid cell wall and are therefore non-motile, a feature that separates them from animals. All fungi possess cell wall made of chitin. Fungi are chemoheterotrophs (require organic compounds for both carbon and energy sources) and fungi lack chlorophyll and are therefore not autotrophic. Fungi are osmiotrophic; they obtain their nutrients by absorption. They obtain nutrients as saprophytes (live off of decaying matter) or as parasites (live off of living matter). All fungi require water and oxygen and there are non obligate anaerobes. Typically reproduce asexually and/or sexually by producing spores. They grow either reproductively by budding or non-reproductively by hyphal tip elongation. Food storage is generally in the form of lipids and glycogen (Sridhar, 2006).
2.2.1 Classification of Fungi
Fungi were initially classified with plants and were a subject of interest for botanists; hence the influence of botany can be seen on their classification. In 1969 R.H Whittaker classified all living organisms into five kingdoms namely Monera, Protista, Fungi, Plantae and Animalia. Traditionally the classification proceeds in this fashion: Kingdom - Subkingdom- Phyla/phylum - Subphyla - Class - Order - Family - Genus- Species This classification is too complicated to be dealt here. There are alternate and more practical approaches, one based on sexual reproduction and the other based on morphology of the thallus (vegetative structure)( Sridhar, 2006).
2.2.1.1 Based on Sexual reproduction
Zygomycetes
Commonly known as bread moulds, these are fast growing, terrestrial, largely saprophytic fungi. Hyphae are coenocytic and mostly aseptate. Asexual spores include chlamydoconidia, conidia and sporangiospores. Sporangiophores may be simple or branched. Sexual reproduction involves producing a thick-walled sexual resting spore called a zygospore.
Medically important orders and genera include:
1. Entomophthorales: Conidiobolus and Basidiobolus are involved in subcutaneous zygomycosis
2. Mucorales: Rhizopus, Mucor, Rhizomucor, Absidia and Cunninghamella are involved in subcutaneous and systemic zygomycosis (formerly called Mucormycosis).
Ascomycetes
They exist as saprophytes and parasites of plants. Hyphae are septate with simple septal pores. Asexual reproduction is by conidia. Sexual reproduction is by the formation of endogenous ascospores, typically eight, in anascus.
Medically important genera include the:
1. Teleomorphs of known pathogenic fungi e.g. Arthroderma (of Trichophyton and Microsporum), Ajellomyces dermatitidis (of Blastomyces dermatitidis), Pseudallescheria boydii(of Scedosporium apiospermum)
2. Agents of mycetoma, like Leptosphaeria
3. Agents of black piedra, like Piedraiahortae.
Basidiomycetes
They exist as saprobes and parasites of plants. Hyphae are dikaryotic and can often be distinguished by the presence of clamp connections over the septa. Sexual reproduction is by the formation of exogenous basidiospores, typically four, on a basidium. Occasional species produce conidia but most are sterile.
Genera of medical importance include:
1. Teleomorph of Cryptococcus neoformans, which is Filobasidiella neoformans
2. Agents of basidiomycosis such as Coprinus and Schizophyllium
3. Mushroom poisoning by Aminita, Lepiota, Coprinus and Psilocybe etc.
Deuteromycetes
Deuteromycetes are also known as Fungi Imperfecti because of absence of sexually reproducing forms (teleomorphor perfect stage). As their teleomorph continue to be discovered, they would be classified among the previous categories, until then this remains an artificial and heterogeneous group.
There are three classes of Fungi Imperfecti.
1. Blastomycetes: These include asexual budding forms of Cryptococcus, Candida, Torulopsisand Rhodotorula.
Depending on the presence of melanin in their cell walls, they may be non-dematiaceous or dematiaceous.
2. Hyphomycetes: A class of mycelial moulds which reproduce asexually by conidia on hyphae. Hyphae are septate. This class contains the majority of medically important fungi. Dematiaceous hyphomycetes are those conidial fungi that produce dark brown, green-black, or black colonies and are the causative agents of phaeohyphomycosis. Hyaline hyphomycetes include those conidial fungi, which are not darkly pigmented; colonies may be colourless or brightly coloured. These include the agents of hyalohyphomycosis, aspergillosis,dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum.
3.Coelomycetes: These produce acervuli, which are tightly bound mats of hyphae on which conidia are produced (Sridhar, 2006).
2.2.1.2 Based on Morphology
Moulds (Molds): Filamentous fungi eg: Aspergillus species,Trichophyton rubrum
Yeasts: Single celled cells that buds eg: Cryptococcus neoformans, Saccharomyces cerviciae
Yeast like: Similar to yeasts but producepseudohyphaeeg: Candida albicans
The large and diverse group of microscopic foodborne yeasts and molds (fungi) includes several hundred species. The ability of these organisms to attack many foods is due in large part to their relatively versatile environmental requirements. Yeasts tend to grow within food and drink matrices in planktonic form and they tend to ferment sugars, growing well under anaerobic conditions. Molds, on the other hand, tend to grow on the surface of objects in the shape of a visible ‘mycelium’ made up of many cells (Sridhar, 2006).
Both yeasts and molds cause various degrees of deterioration and decomposition of foods. They can invade and grow on virtually any type of food at any time. They invade crops such as grains, nuts, beans, and fruits in fields before harvesting and during storage. They also grow on processed foods and food mixtures (Valerie et al., 1960; Barnett, 1960; Lodder,1970 and DOA U.S, 2012).
Several foodborne molds, and possibly yeasts, may also be hazardous to human or animal health because of their ability to produce toxic metabolites known as mycotoxins. Even though the generating organisms may not survive food preparation, the preformed toxin may still be present. Certain foodborne molds and yeasts may also elicit allergic reactions or may cause infections. Although most foodborne fungi are not infectious, some species can cause infection, especially in immunocompromised populations, such as the aged and debilitated, HIV-infected individuals, and persons receiving chemotherapy or antibiotic treatment. This is particularly problematic in plants producing high sugar, low water activity, lowpH products. Factories producing fruit products, baked goods, confectionary, and fermented dairy products can be at real risk from yeast and mold contamination (Valerie et al., 1960; Barnett, 1960; Lodder,1970 and DOA U.S, 2012).
Yeast had long been considered the organism of choice for the production of alcoholic beverages, bread, and a large variety of industrial products. All of these products are currently making a huge impact in the agriculture and food industry. Traditionally, yeasts have been very important in the food industry and nowadays it would be almost impossible to imagine a world devoid of fermented products such as wine, beer or cheese. Nevertheless, given their ability to grow at low pH levels, low water activity and even in the presence of some chemical preservatives, they have become a classic food contaminant causing huge losses to the food industry as well as illnesses to consumers. Yeasts are slow growing organisms when compared to bacteria. If yeasts and bacteria were placed in the same optimum environment and both could grow, it is most likely that the faster growing bacteria would quickly outgrow and outcompete the slower growing yeast, becoming the dominant flora. However, if we move outside the ‘optimum’ growth conditions of most bacteria, into environments that are acidic, or of low water activity (high in sugar), then the yeasts have advantage and would rapidly overtake the growth of bacteria. It is in these specialist food niches that the yeast spoilage has become a problem (Valerie et al., 1960; Barnett, 1960; Lodder,1970 and DOA U.S, 2012).
Molds have both positive and negative effects on the food industry the same way that yeasts do. Some molds are perfectly safe to eat and, in some cases, even desirable (the classic example would be cheese made with mold, such as blue, Brie, Camembert, and Gorgonzola). Other molds can be quite toxic and may produce allergic reactions and respiratory problems, or produce poisonous substances called mycotoxins. Aspergillus mold, for instance, which is most often found on meat and poultry (as well as environmentally), can cause an infection called Aspergillosis, which is actually a group of illnesses ranging from mild to severe lung infections, or even whole-body infections. One of the greatest concerns regarding mold in food is the mycotoxins that some varieties produce. One of the most researched mycotoxins is aflatoxin, a cancer-causing poison (Valerie et al., 1960; Barnett, 1960; Lodder,1970 and DOA U.S, 2012).
Dimorphic: Fungi existing in two different morphological forms at two different environmental conditions. They exist as yeasts in tissue and in vitro at 370C and as moulds in their natural habitat and in vitro at room temperature. Eg: Histoplasma capsulatum, Blastomyces dermatidis, Paracoccidiodes brasiliensis, Coccidioides immitis Some 200 "human pathogens" have been recognized from among an estimated 1.5 million species of fungi (Sridhar, 2006).
2.3 Spoilage
Spoilage is the process in which food deteriorates to the point in which it is not edible to humans or its quality of edibility becomes reduced. Various external forces are responsible for the spoilage of fruits. Food that is capable of spoiling is referred to as perishable food. Harvested foods decompose from the moment they are harvested due to attacks from enzymes, oxidation and microorganisms. These include bacteria, mold, yeast, moisture, temperature and chemical reaction (Anita, 1997).
Bacteria can be responsible for the spoilage of food. When bacteria breaks down the food, acids and other waste products are created in the process (Anita, 1997). While the bacteria itself may or may not be harmful, the waste products may be unpleasant to taste or may even be harmful to one's health (Jill, 2001). Spoilage bacteria do not normally cause "foodpoisoning "; typically, the microorganisms that cause foodborne illnesses are odorless and flavourless, and otherwise undetectable outside the lab. Eating deteriorated food could notbe considered safe since for example mold produces mycotoxins (Michelle, 2015).
2.3.1 Spoilage of fruits
The main sources of microorganisms in fruits are soil, water, air, and other environmental sources, and can include some plant pathogens. Fresh fruits 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 fruits. The presence of air, high humidity, and higher temperature during storage increases the chances of spoilage. The common spoilage defects are caused by molds belonging to genera Penicillium, Phytophthora, Alternaria, Botrytis and Aspergillus. Among the bacterial genera, species from Pseudomonas, Erwinia, Bacillus, and Clostridium are important. Microbial fruits 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 (Hozbor et al.,2006).
Fruits are another tempting source of nutrients for spoilage organisms because of their near neutral pH and high water activity. Although tomato fruits are exposed to a multitude of soil microbes, not all of these can attack tomato and some spoilage microbes are not common in soil, for example, lactic acid bacteria. Most spoilage losses are not due to microorganisms that cause plant diseases but rather to bacteria and molds that take advantage of mechanical damage to tomato surfaces (Tournas, 2005).
Bacterial spoilage first causes softening of tissues as pectins are degraded. Starches and sugars are metabolized next and unpleasant odors and flavors develop along with lactic acid and ethanol. Some examples of important spoilage bacteria are lactic acid bacteria and Pseudomonas species (Cocolin et al., 2004).
Molds belonging to several genera, including Rhizopus, Alternaria and Botrytis, cause a number of fruits rots described by their color, texture, or acidic products. The higher moisture content of tomato fruits as compared to others allows different fungi to proliferate examples are Aspergillus niger, Aspergillus flavus and Fusarium oxysporum (Thomas et al.,2004). Infact, healthy fruits have many microbes on their surfaces but can usually inhibit their growth until after harvest. Ripening weakens cell walls and decreases the amounts of antifungal chemicals in fruits, and physical damage during harvesting causes breaks in outer protective layers of fruits that spoilage organisms can exploit. Molds are tolerant of acidic conditions and low water activity and are involved in spoilage of citrus fruits, apples, pears, and other fruits. Penicillium, Botrytis and Rhizopusare frequently isolated from spoiled fruits (Calvo et al., 2007).
Lactic acid bacteria can spoil orange and tomato juices, and some Pseudomonas and Enterobacteriaceae also spoil juices. These bacteria are not as heat tolerant but may be post-pasteurization contaminants colonization by many, but not all, microbes and are the most important first step in delaying the spoilage process. Microbes require certain conditions for growth, and therefore management of the environment of fruits can change these factors and delay spoilage. Many, but not all, microbes grow slowly or not at all at low temperatures, and refrigeration can prolong the lag phase and decrease growth rate of microbes. Many microbes require a high water activity and therefore keeping fruits such as tomatoes and onions. Some microbes require oxygen, others are killed by oxygen, and still others are facultative(Walker and Phillips, 2007).
2.3.2 Tomato Spoilage by Fungi
Tomatoes have serious challenges to their existence. These include changes in climate conditions, pests, inadequate rainfall and microorganisms particularly fungi. One of the limiting factors that influence tomato economical value is its relatively short shelf life caused by pathogen attack. Spoilage of tomatoes are those adverse changes in the quality of tomatoes that are brought about by the action of predominantly biological and physical factors. These may be changes in taste, smell, appearance or texture of the fruits. (Ghosh, 2009) reported that fungi were the source of spoilage of most of the tomato samples accessed than bacteria. Fungi affecting tomatoes include Aspergillus phoenicis, Absidia species, Trichoderma species, Alternaria alternata, Fusarium oxysporum, Fusarium moniliformis, Aspergillius niger, Mucor species, Rhizopus stolonifer, Penicillium species, Geotrichum species and Phytophthora species (Etebu et al., 2013).
Fungal spoilage of tomatoes has been recognized as a source of potential health hazard to humans and animals due to the fact that they produce mycotoxins which are capable of causing mycotoxicoses in man following ingestion or inhalation. The mycotoxins are not limited to their areas of infections. Since tomatoes contain large amount of fluid, these mycotoxins diffuse rapidly throughout them, contaminating all parts and making the fruits unfit for consumption (Baker, 2006).
2.3.3 Mode of Activity of Microbes on Causing Spoilage.
Fruit (tomato) present nearly ideal conditions for the survival and growth of many types of microorganisms. The internal tissues are nutrient rich and have a pH near neutrality. Their structure is comprised mainly of the polysaccharides cellulose, hemicellulose, and pectin. The principal storage polymer is starch. Spoilage microorganisms exploit the host using extracellular lytic enzymes that degrade these polymers to release water and the fruit other intracellular constituents for use as nutrients for their growth. Fungi in particular produce an abundance of extracellular pectinases and hemicellulases that are important factors for fungal spoilage (Miedes and Lorences, 2004). Some spoilage microbes are capable of colonizing and creating lesions on healthy, undamaged plant tissue (Tournas, 2005).
Spoilage microorganisms also can enter plant tissues during fruit development, either through the calyx (flower end) or along the stem, or through various specialized water and gas exchange structures of leafy matter. Successful establishment, however, requires the spoilage microbe to overcome multiple natural protective barriers. Fruit (tomato) possess an outer protective epidermis, typically covered by a natural waxy cuticle layer containing the polymer cutin (Leque et al., 2003). A diverse community of epiphytic microorganisms that present a further competitive barrier to the spoilage organism also typically colonizes the outermost fruit surface. Overcoming these barriers requires an exquisite set of biochemical tools that allow the spoilage microorganism to
(1) Identify and recognize the plant surface.
(2) Employ one or more strategies to achieve irreversible attachment to the plant surface.
(3) Initiate steps leading to internalization of the tissue (Mandrell et al., 2006).
The natural acidity of most fruits also serves as a barrier to many spoilage microbes, especially bacteria. By contrast, spoilage fungi that typically produce more diverse and greater amounts of extracellular depolymerases successfully attack and spoil both fruits. Colonization and lesion development more typically and more rapidly occurs within damaged or otherwise compromised plant tissue. External damage such as bruising, cracks, and punctures creates sites for establishment and outgrowth of the spoilage microbes. Lesion development can be relatively rapid, occurring within days or weeks. This presents the risk that rapidly reproducing spoilage microorganisms will arrive within open wound sites at the packing facility, and thereby, through shedding from the asymptomatic wound, present the potential for crosscontamination within the facility during handling, culling, washing, sorting and packing before storage. Such cross-contamination to some degree is inevitable and, if not carefully managed with a robust facility sanitation program, could lead to the establishment of a population of spoilage microbes endemic to the facility that may be difficult to eradicate. A further and potentially more serious complication is the introduction into the cold storage facility of spoilage microorganisms already established in wound sites on product, whether the product is in bins or boxed and palletized (Mandrell et al., 2006).
2.3.4 Factors Affecting the Growth of Microorganism in fruits
Several factors related to the environment and the conditions in which fruit is stored influence the growth of micro-organisms in fruit. These factors canbe divided into intrinsic and extrinsic elements. Growth is possible over a wider range of temperatures than that of toxin production. For example, Aspergillus flavus can grow from 10 - 12 °C to 43 - 48 °C, where as aflatoxins are produced from 13 - 15 °C up to 37 °C. The minimum water activity (aw) for Aspergillus flavus is 0.78 - 0.80, but aflatoxins are produced above aw 0.82 - 0.83. Some organisms have the ability to produce spores when exposed to conditions outside their typical growth range. These organisms pose difficulties for the fruits as the spores are more resistant to the intrinsic and extrinsic factors that are lethal to vegetative cells. Unless a factor or treatment is targeted at destruction of the spores, they can survive in the fruits, and when the environmental conditions return to suitable levels, the spores are able to germinate and grow. The issue is compounded by the fact that spores sometimes germinate earlier than would be expected as a result of heat shock if they are exposed to temperatures outside their growth range but less than their lethal limit, and can make the spores more resistant to other factors than normal. It is for this reason that fruits which are subject to contamination with spore-forming organisms are often subjected to high temperature processing (Wareing et al., 2011).
2.4.3.1 Intrinsic Factors
The inherent physical, chemical and biological properties of the fruits, such as pH, redox potential, water activity and the presence of antimicrobial substances have the capacity to either stimulate or retard the growth of micro-organisms. Some intrinsic factors are interlinked with some extrinsic factors. For example, water activity rises with increasing temperature; there is an increase in water activity of 0.03 with a 10 °C rise in temperature (Wareing et al., 2011).
1. pH
The intracellular pH of any organism must be maintained above the pH limit that is critical for that organism. The control of intracellular pH is required in order to prevent the denaturation of intracellular proteins. Each organism has a specific requirement and pH tolerance range; some are capable of growth in more acid conditions than others. Most micro-organisms grow best at neutral pH (7.0). Yeasts and moulds are typically tolerant of more acidic conditions than bacteria but several species of bacteria will grow down to pH 3.0. These species are typically those that produce acid during their metabolism such as the acetic or lactic acid bacteria. Bacterial pathogens are usually unable to grow below pH 4.0. The type of microbial growth typically seen in a particular food is partly related to the pH of that product. Fruits like tomato are naturally acidic, which inhibits the growth of many bacteria, therefore spoilage of these products is usually with yeasts and moulds (Wareing et al., 2011).
Different fruits tend to spoil in different ways. For example, carbohydrate-rich fruits often undergo acid hydrolysis when they spoil; this usually reduces the pH, and tends to reduce the risk of pathogen growth. Protein-rich fruits tend to increase in pH when they spoil, making them possibly less safe, as the pH rise to the zone where more pathogens can grow (Wareing et al., 2011).
2. Redox Potential
Also known as the oxidation-reduction potential or Eh, the redox potential of a fruit has an impact on microbial growth. Aerobic organisms require a food to have a positive redox potential (an oxidised state) whereas anaerobes require a negative potential (a reduced state) for growth. It should be noted that the presence of oxygen is not an absolute requirement for oxidation reduction reactions as other compounds can accept electrons. Different foods have distinct redox potentials and these influence the type of microbial growth typically seen in that fruits. Fruits of typically have a redox potential of +300 to 400 mV thereby favouring the growth of aerobic bacteria and moulds (Wareing et al., 2011).
3. Water Activity
Water activity (aw) is a measure of the amount of freely available water within a fruit. The aw of a fruit can be expressed as the ratio of the water vapour pressure of the fruit to the water vapour pressure of pure water at the same temperature. Equilibrium relative humidity values can be converted to aw by dividing by 100. Water is required for microbial growth; therefore fruits with low water activities cannot support the growth of microorganisms. Pathogenic and spoilage bacteria do not grow in fruits with a water activity of less than 0.85. Many yeasts and moulds however are capable of growth at much lower water activities than this; some can even grow at aw 0.60 (Wareing et al., 2011).
2.4.3.2 Extrinsic Factors
The characteristics of the environment in which the fruits are maintained, such as the temperature, atmosphere and relative humidity can affect the properties of the fruits as well as the potential for the growth of microorganisms (Wareing et al., 2011).
1. Temperature
As temperature influences enzymatic reactions it has an important role in promoting or preventing microbial growth. Micro-organisms can be categorised into one of four groups depending on their optimum growth temperature and the temperature range at which they will grow.
Thermophiles have optimum growth of 55 °C and a growth range of30 - 75 °C
Mesophiles have optimum growth of 35 °C and a growth range of10 - 45 °C
Psychrotrophs have optimum growth of 20 - 30 °C and a growth rangeof 0 - 40 °C
Psychrophiles have optimum growth of 15 °C and a growth range of-5 - 20 °C
At temperatures higher than an organisms optimum growth range, cells die rapidly. Lower temperatures still result in cell death but at a slower rate. Temperature can therefore be used to eliminate or control the growth of microorganisms. Refrigeration of a fruits can prevent spoilage by controlling the growth of thermophilic or mesophilic organisms. Most pathogens are capable of growth at refrigeration temperatures and therefore cannot be controlled via refrigeration alone (Wareing et al., 2011).
2. Atmosphere
As all micro-organisms have specific requirements for oxygen and carbondioxide, by altering the atmosphere within a fruit package the growth of micro-organisms can be controlled. Vacuum packing fruit removes available oxygen and thereby prevents the growth of aerobic organisms; it does however still allow the growth of anaerobes such as C. botulinum (Wareing et al., 2011).
3. Relative Humidity
The relative humidity in which a fruit is stored can have an influence on the water activity of that product and an influence on the growth of microorganisms on the surface of a product. If the growth of micro-organisms in a fruit is controlled by the water activity of a product then it is very important that the fruit be stored under relative humidity conditions which will not allow the uptake of moisture from the air, and therefore an increase in water activity. Packaging can be used to limit the migration of moisture into the product (Wareing et al., 2011).
2.4 Some Tomato Fruits Diseases
Many microorganisms can cause a variety of tomato diseases. Tomato fruits diseases can be caused by bacteria, fungi, protozoan etc. Below are some of tomato fruit diseases caused by fungi.
2.4.1 Fungal diseases
Fungi are mostly filamentous microorganisms commonly known as molds. In nature, they often appear threadlike, cottony, or as yeast-like scum. Many fungal species can cause fruit decay in tomatoes. Fungi are generally more difficult to eradicate than bacteria, because fungal cells are much larger and produce spores that are highly resistant to drying and other environment stresses. The major postharvest diseases caused by fungi are Sour rot, Rhizopus rot, Buckeye rot and Black mold rot. Descriptions of these diseases have been well articulated by various workers (Bartz et al., 2004).
2.4.1.1 Sour rot
This is caused by the yeast Geotrichum candidum. The disease is characterized by lesions whose growth resembles a thick, gelatinous mass similar in appearance to cottage cheese. The lesions are usually watery in the early stages of the disease and later become coated with pathogen growth and remain relatively firm. Lesions give off an odor similar to that produce by lactic acid bacteria hence, the name, sour-rot (Bartz et al., 2004).
2.5.1.2 Rhizopus rot
This disease is caused by Rhizopus stolonifer. Disease symptoms first appear as water-soaked lesions which exudes a clear liquid with time. Resulting lesion surfaces are covered with thin, cotton-like structure. Infection is usually through natural openings or wounds created by mechanical damage. (Bartz et al., 2004)
2.5.1.3 Buckeye rot
Another postharvest disease of tomato is Buckeye rot caused by Phytophthora parasitica. The pathogen attacks both ripe and unripe tomato fruits. Symptoms first appear as water-soaked circular spots. As the disease progresses, the center of the spots become darkened and overgrown with sparse white mycelia of the fungus. The disease derived its name from the manner the fungal mycelia spreads from diseased fruit to adjacent healthy fruit (Bartz et al., 2004).
2.5.1.4 Black mold
Unlike the fungal diseases described so far, Black mold rot is caused by any of several different fungal pathogens which include Alternaria arborescens, Stemphyllinm botryosum and S. consortiale. The disease symptoms are characterized by rots observed on the shoulder, stem scar or on the blossom end of the tomato fruit. Lesions are initially sunken and later quickly covered with a dark brown to black mold. Lesions also develop internally if the stylar pore or a vascular strand that is connected to the stem scar is infected. Severe internal bruise greatly predisposes the fruits to infection that forms internal black spots (Mehrotra and Ashok, 2005).
Several other fungi have been described as potential postharvest pathogens. Such fungi are able to infect tomato fruit in the field and later lead to postharvest decays. They are usually not considered serious problems in themselves because like, black mold rot, the causal fungi hardly spread among fruits within a box. However it has rightly been pointed out that they could predispose postharvest tomato fruits to more destructive disease pathogens (Bartz et al., 2004).
2.5.2 Physiological disorders of tomato
These are problems or disorders that are not caused by infectious microorganisms but rather by environmental stresses on the plant. In a study involving three varieties of tomato in Southwestern Nigeria, physiological damage was responsible as much as 36 and 44% of damage respectively among two of the varieties studied (Adeoye et al., 2009). Although, detail reports of works on the different types of physiological disorders in Nigeria are scarce, postharvest tomato fruits are known to suffer a good number of defects not primarily attributed to animate pathogens. Some physiological disorders common to tomato are Blossom end rot, fruit cracking, Cat faced fruit, Sun scald and Blotchy ripening (Peet, 2009).
CHAPTER THREE
3.0 MATERIALS AND METHOD
3.1 Study area
The study will be carried out in sokoto north of sokoto state. The area is located within latitude 130 30 0” N, and longitude 50 140 0” E of the equator. Sokoto north is geographical located in the north east part of the state. The estimated population of the L G A is 232,846 people in 2006. It has an area of 51km2.
3.2 Sample collection
Samples of spoilt tomato fruits was purchased from ten different sellers at kasuwan daji market in Sokoto north local government area. The samples been collected were placed in separate sterile polyethylene bags respectively and were brought to Usmanu Dan Fodio University, Sokoto mycology laboratory of biological science department for further studies.
3.3 Preparation of media
The media that was used for the research work is Potato Dextrose Agar (P D A). The media was prepared using manufacturer instruction. Appropriate gram of the agar was dissolved in appropriate ml of distilled water. The mixture was autoclaved at 1210C for 15 minutes. The autoclaved media was allowed to cool to a temperature of about 450C before dispensing into petri dishes. Antibiotic (streptomycine) was added also before dispensing.
3.4 Sample preparation
The spoilt tomato fruits were blended using sterile distilled water by blender in order to have all microorganism involved in the spoilage. 1ml from blended sample undergo serial dilution of to factor 10-3.
3.5 Fungal Count
Total fungal count was determined using standard pour plate technique method. Potato Dextrose Agar was used for isolating fungi. The spoilt tomato fruits were blended using sterile distilled water by blender in order to have all microorganism involved in the spoilage. 1ml from blended sample undergo serial dilution of to factor 10-3.1ml from the 10-2 and 10-3 for each were inoculated in a sterile Potato Dextrose Agar plate respectively and undergo incubation at 280C for 3 to 5 days. Plates were examined for growth after 72 hours. After which it was counted and subcultured.
3.6 Identification of isolated fungi
The fungal isolate were identified using cultural and morphological features such as colony growth pattern, conidial morphology and pigmentation. The technique of Oyeleke and Manga (2008) was adapted for the identification of the isolated fungi using cotton blue in lactophenol stain.
CHAPTER FOUR
4.0 RESULT
4.1 Fungal load in the spoilt tomato fruits are shown in table 1.Where saller 3 have the highest fungal load (4×103) and saller 7 and 10 have the lowest fungal load (1×103).
4.2 The percentage % occurrence of isolated fungi from spoilt tomato fruits are shown in table 2.Where Sacchromyces cerevesiae have the highest % occurrence (43.5%) and Fusarium oxysporum have the lowest % occurrence (8.7%).
4.3 The fungal isolates from the spoilt tomato fruits are shown in Table 2.They were Sacchromyces cerevesiae, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus and Fusarium oxysporum.
4.4 Images of the fungal isolate from the spoilt tomato fruits are shown
Table4.1Fungal load in the spoilt tomato fruits.
Samples Fungal load (cfu/ml)
S1 2×103
S2 3×103
S3 4×103
S4 2×103
S5 2×103
S6 3×103
S7 1×103
S8 2×103
S9 3×103
S10 1×103
Table 4.2 Percentage occurrence of fungal isolate in the spoilt tomato
Fungi No. of isolates % Occurrence
Saccharomyces cerevesiae 10 43.5
Aspergillus niger 4 17.4
Aspergillus fumigates 4 17.4
Fusarium oxysporum 2 8.7
Aspergillus flavus 3 13.0
Total 23 100
4.3 Fungal isolates relevant to the different sellers
Sallers Fungal Isolates
S1 S. cerevesiae and A. fumigatus
S2 S. cerevesiae, A. niger and A. fumigatus
S3 S. cerevesiae, A. niger, A. flavus and F. oxysporum
S4 S. cerevesiae and A. niger
S5 S. cerevesiae and A. flavus
S6 S. cerevesiae, A. niger and A. fumigatus
S7 S. cerevesiae.
S8 S. cerevesiae and A. flavus
S9 S. cerevesiae, A. fumigatus and F. oxysporum
S10 S. cerevesiae.
4.4 Images of fungal isolate
Image 1 shows S. cerevesiae Image 4 shows A. flavus
Image 2 shows A. niger Image 4 shows A. fumigatus
CHAPTER FIVE
5.1 DISCUSSION
The fungi associated with the spoilt tomato fruits sold in kasuwan daji markets in Sokoto, Nigeria were studied and the result revealed the presence of a teeming population of fungi. The highest fungal load (4×103) is found to be at saller 3 and the lowest fungal load (1×103) is found to be at saller 7 and 10 (Table 4.1). The fungal isolates from the fruits were Aspergillus niger, Fusarium oxysporum, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus fumigatus. Ibrahim et al., 2011 isolated Aspergillus niger as one of the major fungi responsible for the production of volatile compounds in spoilt tomatoes. Baker, 2006 also isolated Aspergillus niger from rotten tomato fruits and reported that they are pathogenic on tomato fruits. Akinmusire, 2011 reported that Rhizopus species were associated with the spoilage of tomatoes which is not in this research. Wogu and Ofuase 2014 isolated Aspegillius species, Penicillum species, Fusarium species and Saccharomyces species from spoilt tomato fruits. Mbajiuka and Enya 2014 also isolated Aspergillius species, Penicillum species and Saccharomyces cerevisiae from spoilt tomatoes while Fatih et al., 2005 reported the presence of Alternaria alternata and Fusarium oxysporum in the spoilt tomato fruits they studied. Ghosh, 2009 also isolated Fusarium oxysporum, Aspergillius niger and Rhizopus stolonifer from the spoilt tomato fruits studied.
The percentage occurrence of the fungi in relation to the marker’s showed that the fungi (Sacchromyces cerevisiae) had the highest percentage occurrence of 43.5% while fungi (Fusarium oxysporum) had percentage occurrence of 8.7% in the fruits (Table 4.2). Fungus may be the major organism responsible for the spoilage of tomato fruits. Fungal spoilage of tomatoes is attributable to the high water content, environmental conditions, state of handling, state of storage facilities, the fungal load of the handlers and the quality of the tomatoes. These fungi isolated in this study are sources of potent mycotoxins which are detrimental to health. Aspergillus niger is a source of Ochratoxin which is considered to be a potent Carcinogen, therefore spoilt tomatoes must not be consumed but disposed off, since such consumption could be detrimental to health. Farmers and marketers of the produce are also advised to take appropriate precautions during the harvesting, transportation, storage and sale of tomatoes to reduce the risk of these toxins and other metabolites that are deleterious to health (Onuorah and Orji 2015).
5.2 CONCLUSION
Spoilt tomato fruits houses pathogenic fungi which includes Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Fusarium oxysporum, and these oraganisms can cause mycotic diseases when ingested.
5.3 RECOMMENDATIONS
Good quality control measures should be employed by the farmers, marketers and consumers during the harvesting, transportation, handling and processing of the fruits.
Frequent inspection of the fruits for sale by food inspectors is also recommended.
It is also recommended that spoilt tomato should not be taken because the pathogenic fungi been isolated are mycotoxins producers and these mycotoxins are heat resistance.
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