OCTOBER, 2018.
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)

(External Examiner)

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.

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.

Title          Page
Title page                                                                                                                      I
Certification                                                                                                                          II
Dedication                                                                                                                          III
Acknowledgements                                                                                                              IV
Table of contents                                                                                                                     V
List of Tables                                                                                                                          VIII
Abstracts                                                                                                                          IX
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
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 Based on Sexual Reproduction                                                                                     7 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 Sour Rot                                                                                                                        21 Rhizopus Rot                                                                                                                22 Buckeye Rot                                                                                                                 22 Black Mold                                                                                                                   22
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                                                                                                       

4.0 Results                                                                                                                            26
5.0 Discussion, Conclusion, and Recommendations                                                                31
5.1 Discussion                                                                                                                31
5.2 Conclusion                                                                                                                33
5.3 Recommendations                                                                                                                34
REFERENCES                                                                                                                35

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 

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.

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.

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). Based on Sexual reproduction
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).
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.

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 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). 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). 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). 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). 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). 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) 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). 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).
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.

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

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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