(ADM. NO: 1410307077)


OCTOBER, 2018.

This research project entitled “Determination of fungi associated with the spoilage of Solanum tuberosum (Irish potato)” by Abdulkadir Salisu Ango (Adm. Number 1410307077) has been read and approved by the undersigned as meeting part of the requirements for the award of Bachelor of Science (Bsc. Hons) Degree in Microbiology, Usmanu Danfodiyo University, Sokoto.

________________ ________________
Dr. A.B. Rabah              Date
(Project supervisor)

_________________ ________________
Dr. A.B. Rabah             Date
(Head of Department)

Prof. D.W. Taura _______________
(External Examiner)              Date

This work is dedicated to Almighty Allah and my beloved parents (Alhaji Salisu Ango and Hajiya Khadija Abubakar), my able and willing brothers (Tukur Salisu Ango and Aminu Salisu Ango) for their supportive in all my academic pursuit. May the Almighty Allah SWT reward them in abundance (AMEEN).

I am most grateful to Allah Almighty, the sole provider of knowledge, wisdom, love, mercy and grace for his protections throughout the period of the programme. I sincerely appreciate my supervisor, Dr. A.B. Rabah who offered timely criticism and corrections that led me through the various stages of this research project.I also wish to acknowledge the contributions of my lecturers Prof. S. B. Manga, Dr. A. A. Faruq, Dr. U-K Muhammad, Dr. A. S. Baki, Dr. (Mrs.) M. L. Riskuwa, Dr. A. I. Dabai, Mal. A. A. Usman, Mal. A. Y. Fardami, Mal. U.B. Ibrahim, Mal. A. Muhammad, Mal. M. K. Nata’ala, Mal. A. M. Jodi and all laboratory technicians for their support.  I appreciate my parents Alhaji Salisu Ango and Hajiya Khadija Abubakar, my able and lovely brothers (Tukur Salisu Ango and Aminu Salisu Ango), my siblings and friends for their unquantifiable love and financial assistance during this period. May Allah S.W.T bless you all  Ameen thumma ameen.
My profound gratitude goes to my siblings Tukur , Aminu , Abubakar , Ibrahim, Kabir ,Bilyaminu ,Buhari , Sani ,  Suleiman, Ismaʼil, Imrana, Maryam, Jamila, Bilkisu, Hadiza, Hauwaʼu, Rabiʼa, Maryam, Hassana , Hussaina, Fatima, Saadat, Hassana, and Hussaina Salisu Ango for their encouragement, prayers and support. May Allah (SWT) reward them abundantly. My sincere appreciation also goes to my friends Mustapha Abubakar Halliru, Mustapha Danfuloti, Ibrahim Abubakar, Ibrahim Lawal, Nura Bala, Yusuf Kabir, Kawu, Isma’il Rabi’u Danzaria, my course mates, and other family members for their support in one way or the other. May Allah reward them abundantly, Ameen.
 Lastly I wish to express my gratitude to Mal. A.U Alkammawa,  Baba Sanusi Yusuf and all those who their names could not appear here, you will always be remembered and I thank you all.
Title Page
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of contents v
List of Tables viii
Abstracts ix
Introduction 1
1.1 Background of the Study 1
1.2 Statement of the research problem 3
1.3 Justification for the Research 4
1.4 Aim and Objectives 4
2.0 Literature Review 5
2.1 Solanum tuberosum (Irish potato ) 5
2.1.1 Habitat of Solanum tuberosum 8
2.1.2 Related Species of Solanum tuberosum 8
2.2 Food Spoilage 9
2.2.1 Food Spoilage Microorganisms 10 Yeast 11 Molds 12
2.2.2 Bacteria 14
2.3 The Fungi 17
2.3.1 Nutrition in Fungi 17
2.3.2 Mycotoxigenic Fungi and Mycotoxins 18
2.3.3 Food-borne Mycotoxins 18 Aflatoxins 19 Mycotoxigenic 20
2.4 Some examples of Fungi associated with spoilt Irish potato 21
2.4.1 Aspergillus niger 21
2.4.2 Fusarium solani 22
3.0 Materials and Methods 23
3.1 Sample Collection 23
3.2 Mycological Studies 23
3.3 Preparation and Sterilization of Culture Media 23
3.4 Isolation of Fungi from Spoilt Irish potato 24
3.5 Identification of the Isolates 24
3.5.1 Microscopic Morphology 24
3.6 Tests for Pathogenicity of Fungal Isolates 24
                        CHAPTER FOUR 26
              4.0 Results 26
  4.1Fungi Isolated from Rotting SolanumTuberosum 26
4.2 Pathogenicity of fungi on SalanumTuberosum 26
5.1 Discussion 30
5.2 Conclusion 31
5.3 Recommendations 31

Table Page
4.1    Fungi Isolated from Rotting SolanumTuberosum    27
4.2 Pathogenicity of fungi on Solanumtuberosum     28
4.3 Percentage Frequency of occurrence of Fungi     29    

A total of ten (10) samples of spoiled Solanum tuberosum (Irish potato) tubers were obtained from ten different vendors and mould isolation was carried out. Mould isolation was done to determine the fungi associated with the spoilage of Solanum tuberosum (Irish potato) and pathogenicity test was performed. Six fungal species were isolated and identified as the spoilage fungi. These Organisms are Aspergillus niger(39.13%), Aspergillus flavus(4.35%), Fusarium solani(26.09%), Penicillium digitatum(17.39%), Rhizopus oryzae(4.35%), and mucor(8.69%). Aspergillus niger employed for the pathogenicity test seen to be most pathogenic, followed by Fusarium solani, then Penicillium digitatum and mucor. Pathogenicity test revealed that all the isolated fungi were pathogenic to the different fruit. It showed that each infected fruit gave initial organisms that cause spoilage of the fruit. The rot symptoms obtained were similar to those observed previously on the fruits when subjected to identification procedures of all the isolated fungi. Aspergillus spp. was highly pathogenic leading to rapid disintegration. As the decay proceeds water soak lesion occurred. Good sanitary, storage and handling measures should be practiced to limit the extent fungal infestation.

Background of the  Study
Irish Potato (Solanum tuberosum L.)  tuber  is  a nutritious  but  highly  perishable  crop  that  is  subject  to high  fungal  spoilage  and  wastage  due  to  non availability  of  appropriate  storage  techniques  (FAO, 2008). Despite  its  nutritional  importance,  the  tubers have  short  storage  life,  generally,  less  than  four  weeks in  the  tropics.  Their  skin  is  easily  damaged  during harvest  and  post-harvest  handling  leaving  the  crops highly  perishable in microbial  spoilage  (FAO; 2008).
Ensuring food safety is becoming an important part of consumers’ daily lives. The search for healthy eating has favoured the increase consumption of fresh and ready-to-eat vegetables worldwide. Since ready-to-eat vegetables are often consumed raw or minimally processed, and not subjected to any cooking processes that could considerably kill the foodborne pathogens, they may act as potential vehicles for the transmission of pathogens (Elexson et al., 2017; New et al., 2017).
Irish potato (Solanum tuberosum L.)  is an annual herbaceous, dicotyledonous plant belonging  to the family Solanaceae.  It  is  a  perennial  that  grow  about  60cm  high  depending  on  variety  (Kudi et al., 2008).  Irish  potato  originated  from  the Andes  highlands  of  south  America,  Bolivia  and  Peru.  It later spreads to Europe in 16th century.  It was brought to Africa in 19th century (Mih et al., 2011).  Irish  potato  was  introduced  into  Nigeria  in  the  later  part  of  19th  century  and  early  20th century  by  Europeans  notably  the  tin  miners  in  Jos  Plateau.  Most  important  potato  producing  areas  in  Nigeria  are  Jos Plateau,  Kano,  Zaria,  Mambila,  Biu  and  Obudu  highlands  (Cip, 2010).  About 95% of the total production comes from Jos Plateau. The current irish potato production in Nigeria is 800,000 tonnes per annum (NRCRI, 2011). Irish  potato  is  ranked  fourth  in  importance  after  rice,  wheat  and  maize.  Potatoes are eaten boiled, fried and in stews (Badiru et al., 1985).  It  is  also  grown  for  livestock  feed  and  industrial  purposes  (Odebunmi et al., 2007). It  has  high  nutritive  value  and  the  tubers  contain carbohydrate  14.2%,  protein  4.7%,  ash  5%,  fat  0.7%,  fibre  0.8%,  vitamin  69%,  calcium  1%,  iron  14%,  magnesium 6%, phosphorus 8%, potassium 9%, and sodium 6% (Okonkwo et al., 1995).

In the quest for food and the struggle for human survival, Irish Potato has historically played important role and is suitable in addressing the problems previously highlighted. This is due to their yield per unit area (hectare) per unit time and their nutritional value (i.e. ratio of protein to carbohydrate) (Tewe, et al., 2003). African farmers are faced with several constraints in the production of food and cash crops. Some of these constraints are poor soils, poor farm practices, use of local varieties, land tenure and damages by diseases and pests (FAO, 1997).
The greatest of these constraints is that of post-harvest spoilage of farm produce, out of the several  tons harvested,  just a fraction is utilized while  most of the amount is lost to post harvest diseases  especially  rot diseases.  (Alexandratos, 1995). These pathogens cause great losses and reductions in the value of these crops but with a good system of control this can be eradicated. Pathogenic fungi associated with post-harvest Irish potato tuber rot are Alternaria solani (early blight).  Rhizogospora  subtranea  (Powdery  Scab),  Fusarium  roseum  and Fusarium solani (fusarium  rot disease),  Helminthosporium solani  (skin blemishes disease),  Colletotrichum atramentarium (black rot disease), Aspergillus niger,  Pythium ultimum (Pythium tuber rot disease), Phytophthora erythroseptica (Pink rot disease), and Phytophthora infestans (late  blight disease) (Clark  and Moyer,  1988).
The mechanisms by which diseases are produced vary considerably with the causal agents and sometimes with the plant. Parasitic diseases commonly arise as a result of an interchange of metabolites between a parasite and the invaded host plant (Agrios, 1998). This is because enzymes are the first substances secreted by the pathogen on establishing contact with the surface area  of their hosts. Many plant pathogenic and non-pathogenic organisms are capable of producing an array of cell-wall-degrading enzymes (Agrios, 1998). 
         Statement of the research problem
Fusarium dry rot of potatoes is a worldwide economic problem.  There  are  many  species  of  Fusarium reported  to  cause  dry  rot  of  potato  Worldwide (Nielson, 1981).  The  disease  may  cause  greater  losses of  potatoes  than  any  other-post  harvest  disease.  Crop losses attribute to dry rot have been estimated to an average of 6 to 25% (Powelson et al., 1993).
 Potato blight is one of worst diseases problem for the Irish Potatoes grower. It can wipe out  the plants almost  overnight  and,  worst  still,  it  can  infect;  causing them  to  rot  in  storage.  In  a  sack  it  will  travel  from potato  to  potato  ruining the  lot. Fusarium  dry  rot  is  one  of  the  most  important  diseases of  potato,  affecting  tubers  in  storage  and  seed  potato pieces  after  planting. Fusarium dry rot of feed tuber can reduce crop  establishment  by  killing  developing potato  sprouts,  and  crop  losses  can  be  up  to  25%, while more than  60% of  tuber can  be infected in storage (Hanson  et  al., 1996).

Justification for the Research
The present study is of public health significance in that it evaluate the risk of intoxication posed to the consumers of fungi contaminated of the skin of freshly bought Irish potato, so as to suggest appropriate control, measures and rapid detection to the relevant regulatory authority.
             Aim and Objectives
The aim of the research is:
To identify fungi associated with spoilage of Solanum tuberosum (Irish potato).

The objectives of this work are:
  To isolate the fungi responsible for the spoilage of Solanum tuberosum (Irish potato).
To determine the pathogenicity of the isolated fungi.

2.0                                            LITERATURE REVIEW
2.1 Solanum tuberosum (Irish Potato)
Potato is a starch tuberous crop from the perennial Solanum tuberosum of the solanaceae family (also known as the nightshades). The word may refer to the plant itself as well as the edible tuber. In the region of the Andes, there are some other closely related cultivated potato species. Potatoes were introduced outside the Andes region four centuries ago, and have become an integral part of much of the world’s cuisine. It is the world’s fourth-largest food crops, following rice, wheat and maize (Hawkes, 2000). Long term storage of potato requires specialized care in cold warehouse (Lang, 2001). Wild potato species occur throughout the Americas, from the United States to Southern Chile (Langar, 2005).
The potato was originally believed to have been domesticated independently in multiple location (McNeill, 2009), but later genetic testing of the wide variety of cultivars and wild mild species proved a single origin for potatoes in the area of present-day southern and extreme north western Bolivia (from a species in the Solanum berevicaule complex) where they were domesticated 7,000-10,000 years ago (McNeill, 2008). Following centuries of selective breeding, there are now over a thousand different types of potatoes (Afrios, 2008), of these sub-species, a variety that at one point grew in the Chile Archipelago (the potato’s south central Chilean sub-center of origin) left its garn plasum on over 99% of the activated potatoes world-wide (Salaman, 2009). The animal diet of an average global citizen in the first decades of the 21st century included about 33kg (731b) of potato (Hawkes, 2000).
In Nigeria, However, the local importance of potato is extremely variable and rapidly changing. It remains an essential crop in Europe (especially eastern and central Europe), where per capital production is still the highest in the world, but the most rapid expansion over the past few decades has occurred in Southern and Eastern Asia. China is now the world’s largest potato producing country, and meanly a third of the world’s potatoes are harvested in China and India (Stevenso et al., 2001). Many people in poor countries, who cannot afford high-calorie diet such as milk products, meat and pulses use potatoes as their prime source of calories but microorganisms such as Bacillus spp., Erwinia spp., etc reduce the nutritional contents of Irish potato and this necessitated the curiosity of this study. Therefore, the aim of this research was to isolate and characterize bacteria that are associated with the spoilage of Irish potato (Stevenso et al., 2001).
Potatoes are responsible for more than half the total carbohydrate requirements of the populace in localities where potato is cultivated and consumed as a staple food. In comparison with other roots and tubers, the protein content of potato is very high. In many developing countries especially the urban areas, rising levels of income are driving a “nutrition transition” towards more energy dense foods, as part of that transition; demand for potato is increasing (FAO, 2008). 
Potato tubers suffer from post harvest losses as a result of physical, physiological or pathological factors or a combination of all three factors (Booth, 1974). The principal factors responsible for losses during storage of  potatoes have been reported to include the natural processes of  the dormant but living tubers which result in the conversion of  starch in the tubers into carbon dioxide and water, evaporation of  water from the tuber, and sprouting and infection by microorganisms resulting in tuber decay (Amadioha and Adisa, 1993).   Fungi and bacteria causing rots in potato have been reported to produce a wide range of hydrolytic enzymes such as cellulases, pectinases, xylanases, and proteases (Olivieri et al., 2004). These enzymes are responsible for tissue maceration and cell death, following which the microorganisms have access to the nutritional resources of the dead plant tissues (Aveskemp  et al., 2008).
Bacterial soft rot is one of the most common potato diseases in the tropics and induces quick and heavy spoilage losses. Its causal bacterium, Erwinia carotovora, has been extensively studied (Harrison and Nielson, 1990). Among the fungi reported to be associated with dry rot of potato, Fusarium solani has been reported as the most virulent (Mayea et al., 1980; Adisa, 1986). Understandably, preventing spoilage of potato during storage carries a great economic significance. One way to prevent this spoilage is to protect healthy tubers from mechanical and biological injuries. As potato , the world`s largest non-cereal crop, is frequently damaged by  E. carotovora leading to huge economic losses, a cost effective strategy for spoilage control is therefore critical to potato farmers , retailers, and processors. In line with this, some efforts to inhibit E. carotovora development on postharvest potatoes using salts, antimicrobial agents and irradiation and /or disinfectants have been previously described (Afek  et al.,  1999; Tsai  et al., 2001; Cladera   Olivera  et al., 2006).
Irish potato, Solanum tuberosum L. is a basic food crop in the temperate zones of the world, having originated from South America around 200BC. (Macdonald and Low, 1984). It is an annual herbaceous (30-100cm tall) but highly perishable crop in terms of deterioration in storage. The tubers vary in size; shape and colour depending on the cultivar, while the flesh can be white, yellowish-white or dark yellow (Burton, 1966).
In Nigeria today, this crop is being cultivated in commercial quantities in two states namely; Plateau (Barakin-Ladi, Bokkos, Jos South, Mangu, Bassa and Pankshin L. G. A. s) and Taraba (Sardauna L. G. A. Mambilla Plateau). Other states where Irish potato (IP) is grown in “pockets include Jigawa, Kaduna, Kano, Yobe, Nassarawa, Adamawa and Zamfara States. It is possible to grow the crop as a rain – fed crop (April – August) as well as under irrigation i.e.  Fadama (Okunade, 2004).  Wills et al., (1998) reported that IP are stored in piles in ventilated stores.
2.1.1 Habitat of Solanum tuberosum (Irish potato)
Solanum tuberosum rarely exists as a wild plant other than as a volunteer (Burton 1989; Simon et al., 2010). S. (Solanum) tuberosum is cultivated around the world, although in the tropics it is grown in the cool highlands, typically at elevations over 1000 m (metres), and in the subtropics it is grown during the cooler winter, autumn, and spring seasons or at midelevations (Hijmans, 2001). S. (Solanum) tuberosum grows best in cool climates, with higher temperatures favoring foliar development over tuberization (Haverkort 1990). S. (Solanum) tuberosum is not frost tolerant and will be killed at temperatures of 3°C (celsius) or lower (Li 1977). It can grow in a range of soil types, but is sensitive to drought stress and therefore can only be cultivated where there is adequate rainfall or the ability to irrigate (Bohl and Johnson 2010; Haverkort 1990). Differences in tolerance to frost and drought occur within the species. Thus, cultivars have been selected with greater adaptation to these stresses (Burton, 1989).
2.1.2 Related species of Solanum tuberosum
There are more than 1000 species in the genus Solanum (Fernald 1970; Spooner and Knapp, 2012). The Solanum genus is divided into several sections, and S. tuberosum belongs to the section Petota, which contains approximately 200 wild species that can be found from southwestern United States to Argentina and Chile (Spooner and Hijmans, 2001). Wild relatives of S. tuberosum grow in a range of habitats, including semi-desert, subtropical and temperate 800m and up to 4000m (Spooner et al., 2004). Most of these are weedy and are commonly found in habitats that have recently been disturbed (Spooner et al., 2004).
The three species in the section Etuberosum (S. etuberosum Lindl., S. fernandeziaum phil. And S. palustre poepp. Ex Schltdl) are closely related to S. tuberosum and the section Petota (Anderson and de Vicente 2010). They grow in Argentina and Chile, both in the mainland and on the màs a Tierra Island, in moist deciduous forests to upland dry scrub forests beside streams or near to waterfalls at elevations from 40 m to 2500 m (CFIA, 2015).
No wild relatives from the section Petota grow in Canada, nor do relatives from the section Etuberosum. The solanum species that can be found in Canada include: from the section Solanum, S. ptychantum Dunal ex DC, S. nigrum L., S. interius Rydb, S. americanum P. Miller, and S. sarrachoides auct. non Sendtner ex Martius; from the section  Dulcamara, S. dulcamara L.; from the section  parasolanum, S. triflorum Nutt.; from the section Lathyrocarpum, S. carolinense L.; from the section Androceras, S. rostratum Dunal; from the section Cryptocarpum, S. sisymbriifolium Lam.; and from the section Lycopersicon, tomato, S. lycopersicum (Brouillet et al., 2013; GOC, 2013).
2.2 Food Spoilage
Spoilage is the process in which original nutritional value, texture, flavor of food are damaged, such food become harmful to people an unstable to eat. Most foods deteriorate in quality following harvest, slaughter or manufacture, in a manner that is dependent on food type, its composition and storage conditions. Various external forces are responsible for the spoilage of food (Archer et al., 2004).

2.2.1 Food spoilage microorganisms
Chemical reactions that cause offensive sensory changes in foods are mediated by a variety of microbes that use food as a carbon and energy source. These organisms include prokaryotes (bacteria), single-celled organisms lacking defined nuclei and other organelles, and eukaryotes, single-celled (yeasts) and multicellular (molds) organisms with nuclei and other organelles. Some microbes are commonly found in many types of spoiled foods while others are more selective in the foods they consume; multiple species are often identified in a single spoiled food item but there may be one species (a specific spoilage organism, SSO) primarily responsible for production of the compounds causing off odors and flavors. Within a spoiling food, there is often a succession of different populations that rise and fall as different nutrients become available or are exhausted. Some microbes, such as lactic acid bacteria and molds, secrete compounds that inhibit competitors (Gram et al., 2002).
Spoilage microbes are often common inhabitants of soil, water, or the intestinal tracts of animals and may be dispersed through the air and water and by the activities of small animals, particularly insects. It should be noted that with the development of new molecular typing methods, the scientific names of some spoilage organisms, particularly the bacteria, have changed in recent years and some older names are no longer in use. Many insects and small mammals also cause deterioration of food but these will not be considered here (Gram et al., 2002). Yeasts
Yeasts are a subset of a large group of organisms called fungi that also includes molds and mushrooms. They are generally single-celled organisms that are adapted for life in specialized, usually liquid, environments and, unlike some molds and mushrooms, do not produce toxic secondary metabolites. Yeasts can grow with or without oxygen (facultative) and are well known for their beneficial fermentations that produce bread and alcoholic drinks. They often colonize foods with a high sugar or salt content and contribute to spoilage of maple syrup, pickles, and sauerkraut. Fruits and juices with a low pH are another target, and there are some yeasts that grow on the surfaces of meat and cheese (Gram et al., 2002).
There are four main groups of spoilage yeasts: Zygosaccharomyces and related genera tolerate high sugar and high salt concentrations and are the usual spoilage organisms in foods such as honey, dried fruit, jams and soy sauce. They usually grow slowly, producing off-odors and flavors and carbon dioxide that may cause food containers to swell and burst. Debaryomyces hansenii can grow at salt concentrations as high as 24%, accounting for its frequent isolation from salt brines used for cured meats, cheeses, and olives. This group also includes the most important spoilage organisms in salad dressings (Mandrell et al., 2006).
Saccharomyces spp. are best known for their role in production of bread and wine but some strains also spoil wines and other alcoholic beverages by producing gassiness, turbidity and off flavors associated with hydrogen sulfide and acetic acid. Some species grow on fruits, including yogurt containing fruit, and some are resistant to heat processing (Martinezz et al., 2004).
Candida and related genera are a heterogeneous group of yeasts, some of which also cause human infections. They are involved in spoilage of fruits, some vegetables and dairy products (Casey and Dobson, 2003). Dekkera/Brettanomyces are principally involved in spoilage of fermented foods, including alcoholic beverages and some dairy products. They can produce volatile phenolic compounds responsible for off-flavors (Couto et al., 2005). Molds
Molds are filamentous fungi that do not produce large fruiting bodies like mushrooms. Molds are very important for recycling dead plant and animal remains in nature but also attack a wide variety of foods and other materials useful to humans. They are well adapted for growth on and through solid substrates, generally produce airborne spores, and require oxygen for their metabolic processes. Most molds grow at a pH range of 3 to 8 and some can grow at very low water activity levels (0.7–0.8) on dried foods. Spores can tolerate harsh environmental conditions but most are sensitive to heat treatment. An exception is Byssochlammys, whose spores have a D value of 1–12 minutes at 90ºC. Different mold species have different optimal growth temperatures, with some able to grow in refrigerators. They have a diverse secondary metabolism producing a number of toxic and carcinogenic mycotoxins. Some spoilage molds are toxigenic while others are not (Pitt and Hocking, 2009).
Spoilage molds can be categorized into four main groups: Zygomycetes are considered relatively primitive fungi but are widespread in nature, growing rapidly on simple carbon sources in soil and plant debris, and their spores are commonly present in indoor air. Generally they require high water activities for growth and are notorious for causing rots in a variety of stored fruits and vegetables, including strawberries and sweet potatoes. Some common bread molds also are zygomycetes. Some zygomycetes are also utilized for production of fermented soy products, enzymes, and organic chemicals. The most common spoilage species are Mucor and Rhizopus. Zygomycetes are not known for producing mycotoxins but there are some reports of toxic compounds produced by a few species (Pitt and Hocking, 2009).
Penicillium and related genera are present in soils and plant debris from both tropical and Antarctic conditions but tend to dominate spoilage in temperate regions. They are distinguished by their reproductive structures that produce chains of conidia. Although they can be useful to humans in producing antibiotics and blue cheese, many species are important spoilage organisms, and some produce potent mycotoxins (patulin, ochratoxin, citreoviridin, penitrem). Penicillium spp. cause visible rots on citrus, pear, and apple fruits and cause enormous losses in these crops. They also spoil other fruits and vegetables, including cereals. Some species can attack refrigerated and processed foods such as jams and margarine. A related genus, Byssochlamys, is the most important organism causing spoilage of pasteurized juices because of the high heat resistance of its spores (Restuccia et al., 2006).
Aspergillus and related molds generally grow faster and are more resistant to high temperatures and low water activity than Penicillium spp. and tend to dominate spoilage in warmer climates. Many aspergilla produce mycotoxins: aflatoxins, ochratoxin, territrems, cyclopiazonic acid. Aspergilli spoil a wide variety of food and nonfood items (paper, leather, etc.) but are probably best known for spoilage of grains, dried beans, peanuts, tree nuts, and some spices.  Other molds, belonging to several genera, have been isolated from spoiled food. These generally are not major causes of spoilage but can be a problem for some foods. Fusarium spp. cause plant diseases and produce several important mycotoxins but are not important spoilage organisms. However, their mycotoxins may be present in harvested grains and pose a health risk (Restuccia et al., 2006).

2.2.2 Bacteria
Spore-forming bacteria are usually associated with spoilage of heat-treated foods because their spores can survive high processing temperatures. These Gram-positive bacteria may be strict anaerobes or facultative (capable of growth with or without oxygen). Some spore-formers are thermophilic, preferring growth at high temperatures (as high as 55ºC). Some anaerobic thermophiles produce hydrogen sulphide (Desulfotomaculum) and others produce hydrogen and carbon dioxide (Thermoanaerobacterium) during growth on canned/ hermetically sealed foods kept at high temperatures, for example, soups sold in vending machine (Boor and Fromm, 2006).
Other thermophiles (Bacillus and Geobacillus spp.) cause a flat sour spoilage of high or low pH canned foods with little or no gas production, and one species causes ropiness in bread held at high ambient temperatures (Boor and Fromm, 2006).
 Mesophilic anaerobes, growing at ambient temperatures, cause several types of spoilage of vegetables (Bacillus spp.); putrefaction of canned products, early blowing of cheeses, and butyric acid production in canned vegetables and fruits (Clostridium spp.); and "medicinal" flavors in canned low-acid foods (Alicyclobacillus) (Chang & Kang, 2003). Psychrotolerant sporeformers produce gas and sickly odors in chilled meats and brine cured hams (Clostridium spp.) while others produce off-odors and gas in vacuum-packed, chilled foods and milk (Bacillus spp.) (Chang and Kang, 2003).
Lactic acid bacteria (LAB) are a group of Gram-positive bacteria, including species of Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, some of which are useful in producing fermented foods such as yogurt and pickles. However, under low oxygen, low temperature, and acidic conditions, these bacteria become the predominant spoilage organisms on a variety of foods. Undesirable changes caused by LAB include greening of meat and gas formation in cheeses (blowing), pickles (bloater damage), and canned or packaged meat and vegetables. Off-flavors described as mousy, cheesy, malty, acidic, buttery or liver-like may be detected in wine, meats, milk, or juices spoiled by these bacteria. LAB may also produce large amounts of an exopolysaccharide that causes slime on meats and ropy spoilage in some beverages (Hozbor et al., 2006).
Pseudomonas and related genera are aerobic, gram-negative soil bacteria, some of which can degrade a wide variety of unusual compounds. They generally require a high water activity for growth (0.95 or higher) and are inhibited by pH values less than 5.4. Some species grow at refrigeration temperatures (psychrophilic) while other are adapted for growth at warmer, ambient temperatures. Four species of Pseudomonas (P. fluorescens, P. fragi, P. lundensis, and P. viridiflava), Shewanella putrefaciens, and Xanthomonas campestris are the main food spoilage organisms in this group. Soft rots of plant-derived foods occur when pectins that hold adjacent plant cells together are degraded by pectic lyase enzymes secreted by X. campestris, P. fluorescens and P. viridiflava. These two species of Pseudomonas comprise up to 40% of the naturally occurring bacteria on the surface of fruits and vegetables and cause nearly half of post-harvest rot of fresh produce stored at cold temperatures. P. fluorescens, P. fragi, P. lundensis, and S. putrefaciens cause spoilage of animal-derived foods (meat, fish, milk) by secreting lipases and proteases that cause formation of sulfides and trimethylamine (off-odors) and by forming biofilms (slime) on surfaces (55;73). Some strains are adapted for growth at cold temperatures and spoil these foods in the refrigerator (Bruhn et al., 2004).
Enterobacteriaceae are gram-negative, facultatively anaerobic bacteria that include a number of human pathogens (Salmonella, E. coli, Shigella, Yersinia) and also a large number of spoilage organisms. These bacteria are widespread in nature in soil, on plant surfaces and in digestive tracts of animals and are therefore present in many foods. Erwinia carotovora is one of the most important bacteria causing soft rot of vegetables in the field or stored at ambient temperatures. Biogenic amines are produced in meat and fish by several members of this group while others produce off-odors or colors in beer (Obesumbacterium), bacon and other cured meats (Proteus, Serratia), cheeses (several genera), cole slaw (Klebsiella), and shell eggs (Proteus, Enterobacter, Serratia). Temperature, salt concentration, and pH are the most important factors determining which, if any, of these microbes spoil foods. Many Gram-negative bacteria, including pseudomonads and enterobacteriaceae, secrete acyl homoserine lactones (AHLs) to regulate the expression of certain genes, such as virulence factors, as a function of cell density. These AHL quorum-sensing signals may regulate proteolytic enzyme production and iron chelation during spoilage of some foods (Rasch et al., 2005).
Other bacteria are associated with spoilage of chilled, high protein foods such as meat, fish, and dairy products. They may not be the predominant spoilage organisms but contribute to the breakdown of food components and may produce off-odors.Most species are aerobic although some grow at lowoxygen levels and may survive vacuum packaging,and one (Brochothrix) is a facultative anaerobe. Some examples include: Acinetobacter and Psychrobacter, which are predominant bacteria on poultry carcasses on the processing line and have been isolated from a variety of spoiled meat and fish. Acinetobacter grows at a pH as low as 3.3 and has been detected in spoiled soft drinks. These two genera do not produce extracellular lipases, hydrogen sulfide, or trimethylamine (fishy odor) and so are considered to have a low spoilage potential (Rasch et al., 2005).

2.3 The Fungi
Fungi are eukaryotic, heterotrophic organisms, including both single-celled yeasts and multicellular filamentous fungi. They primarily function as recyclers of organic material. Many fungal species can survive in oligotrophic environments, through scavenging nutrients from the substrate which they colonized, or the air or water in which they live. To maximize nutrient uptake, filamentous fungi form mats of fine hyphae. Dispersion is via spores. Fungi also produce secondary metabolites, some of which are toxins. Some of the fungal species and the metabolites they produce are human pathogens or allergens (Paterson and Lima, 2005). The presence of fungi in water distribution systems may cause other indirect challenges for water companies. For instance, the secondary metabolites produced by some species can alter the taste and smell of water, generating complaints from end users. Organic acids produced by fungal metabolic processes can increase the rate of corrosion of water pipes, especially when it is difficult to maintain sufficient concentrations of water disinfectants, such as chlorine, throughout the distribution system (Grabinska-Loniewska et al., 2007).
2.3.1 Nutrition in fungi
Every living organism requires energy for its metabolic activities (growth, movement, reproduction). Fungi, being non-photosynthetic organisms, obtain their energy by feeding saprophytically on dead organic matters which contain high energy compounds (carbohydrates) or parasitically on living organisms which contain spores entry compounds (sugars, proteins, fats) in their tissues. Their parasitic activities cause a lot of damage to their hosts and these actions often give rise to different types of diseases of plants and animals including man (Grabinska Loniewska et al., 2007).
2.3.2 Mycotoxigenic Fungi and Mycotoxins
The most commonly known mycotoxigenic fungi are Aspergillus flavus, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus niger, Fusarium sp. and Penicillium sp. They are known for mycotoxin production in foods and feeds (Wilson et al., 2002). Mycotoxins are poisonous chemical compounds which are secondary metabolites produced by micro fungi that are capable of causing diseases in humans and other animals (Abraca et al., 2005). There are many such compounds, but only a few of them are regularly found in food and animal feedstuffs such as grains and seeds. Nevertheless, those that do occur in food have great implications to the health of humans and livestock. Since they are produced by fungi, mycotoxins are associated with diseased or moldy foods, although the visible mould contamination can be superficial (Cano, 2002). The effects of some food-borne mycotoxins are acute, where symptoms of severe illness appear very quickly. Other mycotoxins occurring in food have long term chronic or cumulative effects on health, including the induction of cancers and immunedeficiency. Information about food-borne mycotoxins is far from complete, but enough is known to identify them as a serious problem in many parts of the world, causing significant economic losses (Neus et al., 2008).
2.3.3 Food-borne Mycotoxins
There are five mycotoxins, or groups of mycotoxins, that occur quite often in food: deoxynivalenol/nivalenol; zearalenone; ochratoxin; fumonisins; and aflatoxins (Neus et al., 2008). The food-borne mycotoxins likely to be of greatest significance for human health in tropical developing countries are the fumonisins and aflatoxins (Wilson et al., 2002). To date, there is sufficient evidence in experimental animals for the carcinogenicity of cultures of Fusarium moniliforme that contain significant amounts of fumonisins; and there is limited evidence in experimental animals for the carcinogenicity of fumonisin B1 (Cano, 2002). F. moniliforme growing in maize may produce fumonisin B1, a suspected human carcinogen. Also, fumonisin B1 is toxic to pigs and poultry, and is the cause of equine leucoencephalomalacia (ELEM), a fatal disease of horses (Isabel et al., 2007). Mycotoxins can be neurotoxic, nephrotoxic, hepatotoxic, teratogenic, immunotoxic or mutagenic (Bennett and Klich, 2003). Neurotoxins alter the normal activity of nervous system in such a way as to cause damage to the nerve tissues. This can eventually disrupt or kill neurons which are key cells that transmit and process signals in the brain and other parts of the nervous system (Berger and Guss, 2005). Nephrotoxins damage the cells of the kidney making it difficult for the kidney to perform its excretory and osmoregulatory functions. Hepatotoxins damage the liver interfering with detoxification functions of the liver. Mutagenic mycotoxins cause genetic mutations and sometimes malfunctioning of certain proteins in the body (Fox and Howlett, 2008.) Aflatoxins
Aspergillus flavus and Aspergillus parasiticus are the molds that produce aflatoxin. These fungi can produce their toxic compounds on almost any food that will support their growth. The metabolites produced by these fungi are named AFB1, AFB2, AFG1, and AFG2, which occur naturally. Of the four, AFB1 is found in highest concentrations followed by AFG1, AFB2 and AFG2. Aspergillus flavus only produces AFB1 and AFB2 and Aspergillus parasiticus produces these same metabolites along with G1 and G2. Aflatoxins are secondary metabolites that are highly mutagenic and toxic to human and animal health (Goldbatt, 2011). Humans are exposed to aflatoxins by consuming foods contaminated with products of fungal growth. Such exposure is difficult to avoid because fungal growth in foods is not easy to prevent. Even though heavily contaminated food supplies are not permitted in the market place in developed countries, concern still remains for the possible adverse effects resulting from long-term exposure to low levels of aflatoxins in the food supply (Felicia et al., 2011). Evidence of acute aflatoxicosis in humans has been reported from many parts of the world namely, the Third World Countries, like Taiwan, Ouganda, India and many others. The syndrome is characterized by vomiting, abdominal pain, pulmonary edema, convulsions, coma and death with cerebral edema and fatty involvement of the liver, kidneys and heart (Finley et al., 1992). Conditions increasing the likelihood of acute aflatoxicosis in humans include limited availability of food, environmental conditions that favor fungal development in crops and commoditie and lack of regulatory systems for aflatoxin monitoring and control (Bankole and Adebanjo, 2003). Mycotoxigenic
The main mycotoxigenic fungi involved in the human food chain belong to three genera, Aspergillus, Fusarium and Penicillium. However, toxins have been detected in many other fungi under certain growth conditions. The kind and amounts of toxin produced depend on the fungal strain, the growing conditions, as well as the presence or absence of other organisms (Scotts, 2004). When present in foods in sufficiently high levels, these fungal metabolites can have toxic effects that range from acute (for example, liver or kidney deterioration), to chronic (for example, liver cancer), mutagenic and teratogenic. Resultant symptoms range from skin irritation to immunosuppression, birth defects, neurotoxicity, and death (ICMSF, 1996). Aflatoxin B1 (AFB1), fumonisins and patulin are suspected human carcinogens. Deoxynivalenol and other trichothecenes as well as AFB1 are likely to exert immune suppressive effects, and fumonisin B1 (FB1) may contribute to neural tube defects. Renal dysfunction due to ochratoxin A exposure (suspected in Balkan endemic nephropathy) is also a potentially significant problem, especially as this could exacerbate impaired renal function in individuals with diabetes, a burgeoning worldwide epidemic that is highly likely to grow (Aish et al., 2004). There is also uncertainty related to the effects of chronic, low-level, long-term exposure to single and/or multiple mycotoxins, which may be the case even for those individuals consuming a diverse diet (Lopez-Garcia +t al., 1999).
2.4 Some examples of fungi associated with spoilt Irish potato
Aspergillus niger
Fusarium solani
2.4.1 Aspergillus niger
Aspergillus niger is a filamentous ascomycete fungus that is ubiquitous in the environment and has been implicated in opportunistic infections of humans (Perfect et al., 2001). A. niger is most widely known for its role as a citric acid producer (Magnuson and Lasure 2004). With production of citric acid at over one million metric tons annually, A. niger citric acid production serves as a model fungal fermentation process. As a common member of the microbial communities found in soils, A. niger plays a significant role in the global carbon cycle. This organism is a soil saprobe with a wide array of hydrolytic and oxidative enzymes involved in the breakdown of plant lignocellulose. A variety of these enzymes from A. niger are important in the biotechnology industry. A. niger is also an important model organism for several important research areas including the study of eukaryotic protein secretion in general, the effects of various environmental factors on suppressing or triggering the export of various biomass degrading enzymes, molecular mechanisms critical to fermentation process development, and mechanisms involved in the control of fungal morphology (Magnuson and Lasure 2004).

2.4.2 Fusarium solani
F. solani is a cosmopolitan species and is classified into the section Martiella (Booth, 1971). F. solani can be distinguished into 50 subspecific lineages and most of them have not been further described formally (O’Donnell, 2000). The species is among a well known plant pathogen, causing various types of diseases on a wide range of plants and there are at least 111 plant species from 87 genera that are commonly infected by F. solani (Kolattukudy and Gamble, 1995).
The identification of Fusarium species is mainly based on distinctive characters of the shapes and sizes of macro and micro conidia, presence and absence of chlamydospores as well as colony appearances, pigmentations and growth rates on agar media (Leslie and Summerell, 2006). Polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) of intergenic spacer (IGS) region is commonly used as the IGS region appears to be rapidly evolving spacer regions (Hseu et al., 1996). This technique is highly dependable for the differentiation of strains at the intraspecific level in Fusarium taxonomic studies (Hillis and Dixon, 1991; Mirete et al., 2003).

3.0                                         MATERIALS AND METHODS
3.1 Sample Collection
Ten samples of spoilt Irish potato tubers were collected from ten different vendors , using sterile polythene bags. The samples were immediately transported to the Microbiology laboratory of the Department of Microbiology, Usmanu Danfodiyo University, Sokoto Nigeria for Microbial analysis.
3.2 Mycological Studies
For isolation of fungal pathogens from diseased part of irish potato, first we need preliminary operation for isolation including, sterilisation of glassware’s, petriplates and other materials needed.  These  petriplates  and  conical  flasks  were and  slides  were  properly  washed  in  chromic  acid  solution and then sterilized in hot air oven at 160°C for one hour. Sterlizedpetriplates were used for PDA medium and were put in petriplates in appropriate concentration.
3.3 Preparation and Sterilization of Culture Media
With little modification of procedure by (Difco™ Potato Dextrose agar, 2010): According to the manufacturer 39g of Potato Dextrose Agar was dissolved in 1000ml (1litre) of distilled water. The conical flask containing the media was plugged with a non-absorbent cotton wool and capped with aluminium foil; medium was incorporated with 0.4g streptomycin before autoclaving. It was then sterilized at 121°c for 15 minutes in an autoclave. Cooled to 45 - 50°C and poured into sterile Petri dishes and allowed to solidify for a minimum of 30 minutes. 

3.4   Isolation of Fungi from spoilt Irish potato
Infected samples were first washed with running tap water. An appropriate size of spoilt solanum tuberosum (irish potato tuber) were carefully cut with the aid of sterile blade then sterilized with 70% ethanol and rinsed in sterile distilled water. Sliced portion were then plated on sterile PDA medium and 2% streptomycin was used to inhibit bacterial growth and then incubated at 25°C. Incubation was carried out in inverted positions of petriplates for 4-6 days.  The colonies thus developed are repeatedly sub cultured on PDA medium to obtain pure cultures.  Isolations were identified based on cultural (Macroscopic) and microscopic characteristics and compared with standard mycological texts. (Donsch et al., 1980; Burnett and Hunter 1992).
3.5 Identification of the Isolates
The isolates were identified by comparison of their cultural and morphological characteristics to those in Larone (2002).
3.5.1 Microscopic Morphology (Wet Mount)
A drop of lactophenol blue was placed on a clean slide. Part of the isolate was emulsified in the
lactophenol blue with the aid of a sterile wire loop. This was done for each isolates on different slides. Each isolate was identified with the aid of a binocular microscope, using x 10 and x 40 objectives.
3.6 Tests for Pathogenicity of Fungal Isolates
The fungi isolated from spoiled potatoes were re-infected in healthy potato tubers. Sterilized cork borer (2mm) was used to bore the potato. Each of the isolated fungi was thereafter inoculated into the fruits after which the cores of the fruits were replaced. The inoculated tubers were preserved for one week. The extent of rottening was measured in cm. The fungi were re-isolated from the fruits and compared with the original isolates and the control set had just PDA without fungus (Parvati and Sihan, 2013).

4.0                                                         RESULTS
4.1    Fungi Isolated from Rotting Solanum Tuberosum
Six (6) fungal species were isolated from the rotting tuber of Solanum tuberosum (Table 4.1). They are Aspergillus niger, Aspergillus flavus, Fusarium solani, Penicillium digitatum, Rhizopus oryzae, and Mucor.
4.2   Pathogenicity of fungi on Salanum Tuberosum
Aspergillus niger employed for the pathogenicity test was seen to be most pathogenic, followed by Fusarium, then Penicillium and Mucor (Table 4.2).
4.3   Percentage Frequency of occurrence of Fungi Isolated from Spoilt Irish potato
A total of 6 storage fungi were recovered from spoilt Irish potato tubers. The result indicated that
Aspergillus niger, Aspergillus flavus, Fusarium solani, Penicillium digitatum, Rhizopus oryzae, and Mucor. Were responsible for storage rot of irish potato tubers in Sokoto. Aspergillus niger has the highest frequency of occurrence as shown in (Table 4.3)

Table 4.1        Fungi Isolated from Rotting Solanum Tuberosum
Fungal species
Macroscopic characters
Microscopic characters

Aspergillus niger
Black pin-like growth
Non-Branched conidiophore with bulb end

Aspergillus flavus
Green pin-like growth
Non-Branched conidiophore with bulb end carries conidia

Fusarium solani
Colonies appear dark green with white cottony growth
Spindle like conidia

Penicillium digitatum
Green  or Green-greyish colour colonies
Brush-like conidiophore carries conidia

Rhizopus oryzae
Colonies whitish-like

Cotton like white growth spotted with black colour
Sporangia contain spores, do not have rhizoids

Table 4.2: Pathogenicity of fungi on Solanum tuberosum
Length of Storage                                         Extent of spoilage in the tubers (cm)

Aspergillus niger
Fusarium solani
Penicillium digitatum










Table 4.3 Percentage Frequency of occurrence of Fungi Isolated from Spoilt Irish potato
Frequency of Occurrence
% Occurrence of Isolates

Aspergillus niger

Aspergillus flavus

Fusarium solani

Fenicillium digitatum

Rhizopus oryzae



5.1          DISCUSSION
This study shows conclusively that the microorganism that caused appreciable spoilage in the potato tubers were; Aspergillus spp, Fusarium spp, Penicillium spp, Rhizopus spp., and Mucor spp. This result agrees with the findings of Abiodun and Olumide (2007) and Udoh et al., (2015) who repoeted the isolation of the aforesaid fungi as pathogenic. The investigation also shows that Aspergillus niger has higher pathogenicity compared to the other pathogenic fungi responsible for the spoilage.
The presence of these fungi on irish potato may cause low economic yield of potato and serve as potential food safety hazard when consumed. This is because these microorganisms may cause disease to susceptible host. Most especially in immune compromised individual. Some strains of these fungi may as well produce toxic fungal secondary metabolite (mycotoxins) which when ingested in considerable amount may cause debilitation, various human maladies and even death (Zain, 2011).
The infestation by these fungi may be attributed to some constraints such as poor soils, poor farm practices, and use of local varieties, land tenure and damage by diseases and pest (Abiodun and Olumide 2007). The spoilage of irish potato by these fungi can promote attacked by various pathogens which cause diseases in them. Such pathogens include other fungi, bacteria and viruses (Abiodun and Olumide 2007). These pathogens cause great losses and reductions the value of these crops but with a good system of control this can be eradicated and as well limiting infestation by other pathogenic fungi associated with post-harvest irish potato tuber rost such as Alternariasolani ( early blight) (Abiodun and Olumide 2007). 
Irish potato contaminated with pathogenic microorganisms may be harmful to man and may also lead to huge economic losses of potato yield (Udohet al., 2015). This could be prevented by practice of proper handling and storage procedure, good sanitary measures and the use of resistant varieties. And therefore, the need to ensure necessary major precaution to these fungal pathogens that affects the tuber.
The present work identified preponderant of Aspergillus, Fusarium, Penicillium and Mucor in spoilt Irish potato bought from ten different vendors. These organisms and others identified are mycotoxigenic fungi. Their abundant presence in rotten Irish potato could lead to severe food poisoning. These pathogens lead to enormous loss of Irish potato tubers despite its economic and nutritive value. The disease is of great economic importance to this country. Some of these fungi are capable of producing mycotoxins which are hazardous to the health of consumers. As such urgent attention is required to safe the menace and increases the economic yield of the produce. This will ensure substantial contribution of the Irish potato to food supply and national economy.
Having known the economic value, nutritional value of Irish potato and the cause of its spoilage, the followings are therefore recommended:
Consumption of spoilt Irish potato should be avoided because it contains pathogenic microorganisms which may be harmful to human health.
Farmers and other individuals should practice good sanitary measures.
Developing specialized machinery for the potato handling and storage
Public enlightment should be enforced on proper storing and handling of Irish potato.
Breeding of disease resistance strains should be considered.
There is need to devise good storage facilities to prolong the shelf life of irish potato after harvest.
Employ adequate control measures especially biological measures which are safe and environmentally friendly such as the use of organisms that are parasites or antagonistic to the pathogens.
 Physical damage to the tubers should be avoided since they serve as major points of entry of pathogens into the tubers.

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