CHAPTER ONE

1.0 INTRODUCTION

1.1. Background to the study

Banana over the years have occupied considerable positions the agricultural

production across the continent of Africa representing the world's second largest fruit

crop with an annual production of 130 million metric tons (FAO, 2018). The African

continent is among the prominent producers with Cameron, Kenya, Nigeria, Uganda

and cote d'ivoire leading (FAO, 2006). In recent years, the status change of banana

from foods to food/cash crops further enhance their importance, apart from their

contribution to food and nutritional security in rural and urban not only traded within

a region but also exported to other countries of the world (Oladejo et al.,2008).

In West Africa, Cote d'Ivoire ranks the highest in production of Bananas with about

320,000 metric tons followed by Guinea, Mali and Liberia with about 216,000 metric

tons, 181,000 metric tons and 132,000 metric tons respectively. Only about 1.3

million metric tons of banana were produced representing 1.5% of worldwide

production in 2013(AJFND, 2020). In Nigeria, banana exports rose to 201% in 2021

against the 2013-2020 figure creating a significant growth in export of bananas and

are likely to continue in growth in immediate term causing it to hit the global market.

(Indexbox, 2023).

As perishable foodstuff, bananas are complex in marketing and distribution, the

process involves large number of producers and a few wholesalers who distribute

bananas to consumers on a large scale, they are mainly marketed fresh. Numerous

players usually organised in informal networks on chains may be involved in moving

the commodity from the site of production to the ultimate consumer (Akinyemi et

1
al.,2010). These players are usually organised in informal networks or chains. In

Nigeria, the distribution of banana is complex, farmers whose lands are nearer to the

major road harvest the crop at the mature green stage and display at the roadside or

transport it to nearby markets allowing small scale wholesalers, retailers and

consumers to purchase directly. In other cases, trade collectors moves around farms,

collect the produce from farmers and transport it to cities where they hand them over

to wholesalers who in turn pass the produce on to retailers or vendors for sale to

consumers.(Akinyemi et al., 2010)

Banana as a crop has many important and nutritional benefits, its different

parts is beneficial to man, the environment and the ecosystem.

Here is a concise economic and nutritional benefit of the banana;

Banana fruit can be eaten/ cooked as vegetables, and the leaves are commonly used as

plates on festive occasions and are widely used to wrap food items when cooking, its

fruit can also be eaten raw, it is sweet and contains starch and vitamins, the sheath leaf

base contains sap which is considered to be an antidote for cobra bites thereby serving

medicinal purposes and uses, fibres from banana plant( Musa textils) is woven and

made into abacea cloth used for twine, bagging and wrapping paper, plants such as M.

coccinea, M. accuminata, M. velutina have ornamental scarlet flowers and can be

cultivated as ornamentals and grown as ornamentals plants.

1.2. Statement of Problem

Agriculture being a biological system faces a lot of constraints that have the potential

to affect the production system. Across the West African continent, several

constraints related to the production of bananas had been identified in literature,

diseases and pest, environmental factors, low rate of adoption of technology,

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availability of labour, and access to inputs were some of the popular factors which

constraints banana production in this region, though much studies have not been

carried out on fungal pathogens of banana fruit, Musa spp fruit production is

adversely affected by a large number of fungal, viral and bacterial diseases, most of

which are widespread but only a few of them are economically significant. The major

disease includes anthracnose, bacteria wilt, Panama wilt, mycosphaerella leaf spot,

and banana bract mosaic virus. The fungi isolated from banana fruit are identified to

be Aspergillus niger, Colletotrichum musae, Fusarium spp. The Food and

Agricultural Organizations (FAO) estimates that 25% of the world’s fruits are

affected by contaminants, of which the most notorious are those resulted from

Aspergillus spp and Fusarium spp. In Nigeria, the impact of these fungi and their

secondary metabolite as fruit and food contaminants are well established (Yousif et

al., 2010)

1.3. Significance of Study

This study will be of significance to stake holders such as policy makers, researchers,

farmers, students and food Production Companies. For policy makers, this will enable

them enact laws to ensure that production companies employ the hazard analysis

critical control points at all levels of food production. For farmers, it will enlightened

them about danger associated with contamination of their produce with fungi thereby

enabling them to employed good agricultural practices that will reduce fungi

contamination with the help of the control measures. For researchers, it will provide

baseline information on the existing modern techniques available for controlling seed

borne fungi thereby providing more advanced methods of control. For students, it

3
will provide them with literatures for further studies on seed borne fungi. For

consumers, it will educate them on the contaminated produce.

1.4. Aim and Objectives

To isolate and identify fungal causing post-harvest decay of banana fruit and their

control using moringa seed powder extract in-vitro.

The set objectives are to;

• Isolate and identify fungi causing post-harvest decay of banana fruit and test

their pathogenicity.

• Determine the Antifungal activity of moringa extract on the control of post-

harvest decay of banana fruits.

• Determine the phytochemicals present in the moriinga.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Overview of Banana

Banana is a monocotyledonous, perennial herb within the order Zingiberales, and the

family Musaceae, it is divided into two genera: Musa and Ensete. Musa consist of

about 40 species and is distributed through India, New Guinea, Australia and

Southeast Asia (Agrios, 2005) The Musa genus is grouped into four sections: Eumusa,

Callimusa, Rhodochlamys and Australimusa, Eumusa is the most widespread and

contains the greatest number of species and forms, for it includes all the edible

seedless bananas. Almost all cultivars of the edible banana are now classified under

two species M. acuminata (AA) and M. balbisiana (BB), both belonging to Eumusa

section. According to Ocran et al., (2011) most cultivated bananas were derived from

natural hybridization between two diploid species M. acuminata and M. balbisiana.

Musa acuminate surpasses Musa balbisiana in variability and in diversity of species,

and at least nine sub species have been described (ssp.malaccensis, ssp. macrocarpa,

ssp. burmannica, ssp. burmannicoide, ssp. siamea, ssp. banksii, ssp. errans, ssp.

zebrine and ssp. truncate (Ocran et al., 2011), whereas Musa balbisiana is less diverse

with no subspecies recognized. Most of the edible types that are derived from these

species are triploid, although diploid (AB) and Tetraploid (ABBB) cultivars are also

known. A review of the global records taken for pathogenic fungi affecting bananas

was reported by Jones, (2000). The report cited about 39 fungal diseases worldwide

affecting bananas (Ocran et al., 2011). Some fungal diseases such as Panama disease,

Sigatoka, Septoria leaf spot, Cordana leaf spot, Anthracnose, Cigar end rot, Tip end

5
rot, Diamond spot, Brown spot, Pitting disease and Root rot were reported for bananas

across the world (Amani 2004).

These diseases have had an increasing trend in banana-growing regions.

Sixteen species of fungi were reported from banana trees in Iran. The fungi species

isolated from infected trees were as follows: Fusarium oxysporum, Cercospora sp.,

Colletotrichum musae, Aspergillus carnenus, Acremonium sp., F. verticillioides, F.

semi tectum, F. subglutinans, F. sambucinum, F. moniliform and Musicillium

theobromine that caused diseased fruit; Cylidrocarpon sp. and Rhizoctonia solani that

caused corm and root disease and Alternaria alternata, Drechslera gigantean and F.

proliferatum that caused diseased leaf. There is little information on ecological

relations between pathogenic fungi on post-harvest disease incidence in a banana.

2.2 Origin and distribution of banana

Banana ( Musa spp. L.) is the fourth food crop after rice, wheat and maize in tropical

and subtropical regions (Bakry et al., 2001). A Banana is an elongated, edible fruit-

botanically a berry (Alvindia, 2013). produced by different kinds of large herbaceous

flowering plants in the genus Musa. In some countries bananas used for cooking may

be called ‘’plantains’, distinguishing them from dessert bananas. The fruit varies in

size, colour, and firmness, but is usually elongated and curved, with soft flesh rich in

starch covered with a rind, which may be green, yellow, red, purple, or brown when

ripe. The fruit grows upward in clusters near the top of the plant. The banana plant

referred to as a ‘tree, is the largest herbaceous flowering plant. All the parts of a

banana plant just above the ground grow from a fleshy rhizome usually called a

‘corm’ (Ocran et al., 2011). The ripe banana is soft and delicate with a post-harvest

shelf life of 5- 10 days (Amani 2004). In developed countries, 40 – 50% of the annual

6
agricultural produce is converted into value-added commodities. Banana is largely

produced in the southern states of Nigeria as well as Plateau, Benue and Kogi in the

north-central zones of Nigeria. (Jones, 2000). Relevant statistics showed that Nigeria

was the fourth largest producer in sub-Sahara Africa, behind Rwanda and Ghana.

Nigeria is missing from the list of top-producing banana nations in the world. (Daniel

Workman, 2018). Nigeria produces 2.7 million tons of bananas per year, making it

one of the largest banana-producing nations in Africa. Western Nigeria accounts for

64% of production while Eastern and Central Nigeria accounts for 26% of production

(Alemu, 2014). lack of enough planting material is one of the reasons for low

production on a global scale. To increase production which will help alleviate

poverty, there is a need to use improved planting material. In Nigeria, banana shows a

high potential for increased productivity (Nelson, 2008).

2.2 Description of Banana Fruit

Banana fruit develops from the banana heart, in a large hanging cluster, made up of

tiers called ‘hands’ with up to 20 fruit in a tier. The hanging cluster is known as a

bunch comprising 3-20 tiers, or commercially as a banana stem and can weigh 30-

50kg. An individual banana or finger as it is known weighs 125 grams. Of which 75%

is water and 25 % is dry matter.

The fruit has been described as a ‘leathery berry’.(Sing, 2000) There is a protective

outer layer (a peel or a skin) with numerous long, thin strings which run lengthwise

between the skin and the edible inner portion. The inner part of the common yellow

dessert variety can be split lengthwise into three sections that correspond to the inner

portions of the three carpels by manually deforming the unopened fruit (Sing, 2000).

The seeds are diminished nearly to nonexistence; their remnants are tiny black specks

7
in the interior of the fruit. The end of the fruit opposite the stem contains a small tip

distinct in texture, and often darker in color. Often misunderstood to be some type of

seed excretory vein, it is just the remnants from whence the banana fruit was banana

flower (Sing, 2000).

2.2.1 Scientific Classification

Kingdom: Plantae

(Unranked): Angiosperms

(Unranked): Monocots

(Unranked): Commelinids

Order: Zingiberales

Family: Musaceae

Genus: Musa

Source: Sing, (2000)

2.3 Types of Bananas

"https://balconygardenweb.com/types-of-bananas-different-varieties/_blank"

2.3.1 Cavendish Bananas

These bananas are the most common variety, easily available in supermarkets in

Nigeria, they are available in young unripe green to fully ripe, smooth yellow to riper

dark yellow with brown spots. You can add them in smoothies, pancakes, or banana

bread.

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1

Fig1. Cavendish banana. (Source: google)

2.3.2 Gros Michel

This variety has a similar taste and size as Cavendish. It is not available as freely as

the other varieties. Gros Michel has a sweet taste, and a strong smell, with a creamier

texture, and can be used in the preparation of banana pies.

fig2. Gros Michel Banana (source= google image)

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2.3.3 Lady's finger

‘Lady Finger’ bananas are 4-5 inches long, cigar-shaped sweet fruit. They have a

bright yellow thin peel with dark flecks when fully ripen. The flesh is creamy and

more delicious than the other common banana varieties

Fig 3. Lady Finger Banana (Source: google)

2.3.4 Blue java

Blue Java bananas have vanilla ice-cream-like flavour, and are also famous as ‘Ice

cream bananas.’ They have silver-blue skin that turns pale yellow when ripe. The fruit

has aromatic soft and sweet flesh. Add them to smoothies or relish their distinctive

taste raw.

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 f

ig 3. Blue Java banana

2.3.5 Orinoco.

Orinoco banana is hardy that survives well in cold climates and winds. They require

support when bearing huge heads of fruit. The texture becomes silky and soft when

they mature, with a firm and fibrous peel. However, these bananas are comparatively

starchy but have a delicious sweet taste and smooth texture.

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Fig 4. Oronoico(google)

2.3.6 Apple Bananas:

These are the short, stubby bananas you sometimes see in speciality stores. They’re

very sweet and moist. (Parveen et al., 2016)

Fig6. Apple Banana

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2.4 Nutritional Value of Banana

Banana is a highly nutritious sweet fruit and staple starch for many tropical

populations. Depending upon cultivar and ripeness, the flesh can vary in taste from

starchy to sweet, and texture from firm to mushy. Both the skin and inner parts are

used for eating either cooked or uncooked. Banana provides exceptional nutrition and

supports muscles and nerves with potassium, helps lower bad cholesterol with soluble

fibre and it is a great source of vitamin C in addition to basic vitamins and

minerals(Sidhu and Zafar, 2018). Bananas contain prebiotics that supports the good

bacteria in our intestines. Despite their sweetness, they are rated low on the glycemic

index, so they have only a little to cause model rate impact on blood sugar. It is an

excellent source of vitamin B6, soluble fibre, and contains manganese and potassium.

Bananas are also an excellent food for people who want to lose weight. Although they

are rather high in calories (200 per banana) and carbs (51gm), they are a great source

of energy. A banana eaten before a workout can provide you with the necessary

energy to complete a longer workout. Bananas provide you with many health benefits,

but in addition to all of that, most people enjoy eating bananas as well. They can be

eaten alone or combined with a fruit salad, added to jello, or made into a smoothie or

a milkshake. They are one of the most affordable fruits in the marketplace and can be

found year-round nearly everywhere in the world (Parveen et al., 2016).

2.5 Economic Importance of Bananas

2.5.1 Industrial Uses

Antifungal properties of banana pulp and peel have been successfully used to treat

tomato fungus in an agricultural setting. In Western African countries like Nigeria and

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Ghana, Locals use banana leaves for everything from umbrellas to construction

materials. Bananas and plantain fibres are used throughout the world to weave ropes,

mats and other textiles. Tannins present in ripe banana peel act as tanning agents in

leather processing (Droby, 2006).

2.5.2 Medicinal properties of banana

In India, banana is considered a holy fruit and is distributed as consecrated food. It is

not only used as a favourite fruit but also used in treating a variety of health

conditions. They act as a fine tonic as well as an instant source of energy being a

favorite for athletes too. They are a boon for thin people who wish to gain weight.

Banana also contains a wide array of nutrients in good amounts. Banana has a mild

laxative property. Banana is a storehouse of minerals, vitamins and carbohydrates. It

contains potassium, calcium, magnesium, iron, zinc etc. Vitamins A, B, C, B-6 etc.

are all available in plenty. The presence of iron in bananas helps to boost the

production of haemoglobin. This helps persons who suffer from anaemia. Banana

contains plenty of potassium. This helps to balance sodium potassium levels and

reduce hypertension or high blood pressure. This fruit help to supply the required

vitamins and minerals to smokers who are trying to quit smoking. The craving for

nicotine is reduced by the consumption of this wonderful fruit (Suri, 2012). Banana

calms the nervous system and reduces stress and depression. According to a survey,

of people suffering from depression, many felt much better after eating a banana. This

happens because banana contains tryptophan, a type of protein that the body converts

into serotonin, known to make relaxed, improve mood and generally make them feel

happier. According to Japanese Scientific Research, a fully ripe banana with dark

patches of yellow skin produces a substance called TNF (Tumor Necrosis Factor)

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which can combat abnormal cells. The more dark patches it has, the higher will be its

immunity enhancement quality; hence, the riper the banana means the better the anti-

cancer quality of the banana. Yellow skin banana with dark spots on it is 8 times more

effective in enhancing the property of white blood cells than green skin version. As a

banana ripens and turns yellow, its level of antioxidants increases. These antioxidants

in ripe bananas protect our bodies against cancer and heart diseases (El-Tahtawi,

2005)

2.6. Requirements for Banana Cultivation

Bananas prefer rich, fertile soils and a sunny, sheltered location. These plants thrive

under uniformly warm or hot conditions. Plant growth slows if temperatures fall

below 60°F and irreversible freeze damage may occur below 32° Celsius. High winds

can cause leaf shredding and drying and may topple plants. Banana plants require

ample water and will suffer if the soil dries out; however, they are not flood-tolerant.

Each stem only produces flowers d fruits once, so the active stem must be cut away to

allow new suckers to emerge (Droby, 2006).

2.7 Fungal Diseases of Banana Fruit

The two primary post-harvest rots of banana (Musa spp.) fruits in Nigeria and around

the world are crown rot and anthracnose. The diseases usually appear on ripening

fruits either at points of sale (farmers’ markets, grocery stores) or later, after purchase.

Occurrence of these two diseases is closely linked to poor cultural and disease

management practices in the banana field, to unclean packinghouses, and to improper

post-harvest handling. The diseases can be serious problems for growers who fail to

15
manage them with a combination of integrated practices. Infected fruits are safe for

humans to consume; however, the infections reduce fruit quality, shelf life, and

marketability (El-Tahtawi, 2005)

2.7.1 The Pathogen

The fungus Colletotrichum musae can cause both crown rot and anthracnose; in

addition, crown rot diseases may also be caused by fungal pathogens in the genera

Fusarium, Acremonium, Verticillium, and Curvularia. These pathogens exist in

banana fields on dead banana leaf or inflorescence tissues. They disperse by wind and

water, r by some insects, birds, and rats (Prusky, and Yakoby, 2003)

Crown rot: One of the most serious post-harvest problems in bananas caused

by colletotrichum musae which appears as brown to black colour develops where the

hand was severed from the bunch. Frequently a layer of whitish mould forms on the

cut surface of the crown. The mould can penetrate deeply into the crown and necks of

fingers and cause a dry, black rot. Fingers may detach prematurely from severely

infected crowns. Disease may increase rapidly during banana fruit ripening (Prusky,

and Yakoby, 2003)

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fig7.crown rot disease of banana.(source:google)

Anthracnose: It is caused by the fungus Colletotrichum musae, which survives in dead

or decaying leaves and also on fruits. Its spores can be spread by wind or water, they

enter the banana fruit through wounds in the peels and later germinate and initiate the

expression of symptoms, the symptoms may develop on ripening fruits on tree

bunches or post-post-harvest during storage. It is the main disease affecting the

quality of banana fruit during transportation and storage. The fungus causes dark

brown to black sunken spots on the peel of the infected fruits. Initial symptoms are

visible on green fruits and are characterized by dark brown to black sunken lesions

with pale margin on the peel.

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 fig8

:Anthracnose disease of banana

2.8 Botany of Moringa oleifera

Moringa oleifera (MO) grows owing to its nutrientrich seeds, edible leaves and

flowers that can be used as food, medication, cosmetic oil or livestock feed. The

height varies between 5 to 10 meters. Different experiments have been demonstrated

positive effects on health (Abalaka et al., 2012). MO is also

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used in developing countries as a source of fruits, medicinal plants and edible oil. It is

an essential nutrient- rich vegetative plant and is commonly considered as a versatile

food that can be eaten in all sections (Abalaka et al., 2012). Most researchers consider

using MO as an alternative for preventive treatment or to relieve and avoid symptoms

of the disease. Nevertheless, Western (traditional) medicine has been very reluctant in

pursuing its dietary and medicinal potential, considering such research, observation,

recommendations and specific assertions. This tentative approach is surprising

because many “super foods” including garlic and green tea have been well known

(Ali et al., 2004). Moringa contain thirteen species from tropical and subtropical

ecosystems, varying from tiny to large trees in height. MO is a Moringa family vine,

the key crop in Asia and Africa, which is commonly grown in northwestern India. It is

an ancient inhabitant of Pakistan, India, Bangladesh and Afghanistan in the

Himalayan region and is popular here for various name of the field such as benzolive,

kroll, drumstick tree, horseradish tree, marango and malunggay. In tropical and

subtropical areas such as Asia, Eastern and Southern Asia, it has recently drawn

scientific and socioeconomic focus (Abdull Razis et al., 2014). Moringa claimed as a

nutrient-rich due to its antiulcer, anti-diabetic, hepatoprotective, diuretic and

cholesterol lowering capacity. It has also been used in skin and hair care products.

2.9. Benefits of Moringa oleifera

2.9.1 Nutritional Properties

Moringa oleifera is called a ‘Miracle Tree’ due to its multipurpose nutritional uses

and capacity to cure many diseases. Every part of plant has reserved nutrients. The

Leaves of Moringa oleifera are rich source of minerals like calcium, potassium, zinc,

magnesium, iron and copper. The Leaves has low calorific value so can be used in the

19
diet of obese. The leaves also contain all essential amino acids and are rich in protein

and minerals (Abdull et al., 2014).

Pods contains around 46.78% fiber, 20.66% protein and are highly valued for

curing digestive problem and colon cancer (Abdull et al., 2014). Vitamins like

vitamin A, Vitamin B, Beta-carotene, pyridoxine, nicotinic acid, vitamin C, Vitamin

D and E also present in abundant amount in Moringa oleifera.

Moringa oleifera also contains major phytochemicals such as: tannin, sterols,

terpenoids, flavonoids, saponins, anthraquinones, alkaloids and reducing sugar is also

present along with anti-cancerous agents like glucosinolates, isothio cyanates,

glycoside compounds and glycerol-1-9-octadecanoate. Oligosaccharides and oxalate

were reported as anti-nutrient factor in Moringa leaves (Adedapo et al., 2009).The

Dried leaves (M. oleifera) retained 87.5% and 50% of β‐carotene after 4 and 3 month

storage, respectively, and could therefore be processed for convenient use (Adedapo

et al., 2009).

The leaves of MO are considered to be a rich source of vitamins and minerals

and exhibits strong antioxidant activity, often attributed to the plants’ vitamins and

phenolic compounds such asquercetin and kaempferol. The leaves can be also taken

as vegetable and processed into tea, powder and other pharmaceuticals isolation.

Further-more, juice can be extracted from fresh leaves and act like growth harm one

and increase crop yield by 25-35% (Adedapo et al., 2009). According to Japanese

study in 2009, leaves act as a good sink for carbon dioxide absorption and utilisation.

The rate of assimilation of carbon dioxide by Moringa tree is 20 times higher than

general vegetation (Abdull Razis et al., 2014). Moringa is said to provide 7 times

more vitamin C than oranges, 10 times more vitamin A than carrots, 17 times more

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calcium than milk, 9 times more protein than yoghurt, 15 times more potassium than

bananas and 25 times more iron than spinach (Adedapo et al., 2009).

Moringa oleifera seed contains a range of phytochemicals, including

antioxidants such as vitamin C, β-carotene, α and γ-tocopherol, β-sitosterol, vitamin

A, the phenolic compounds quercetin and kaempferol, flavonoids, and anthocyanins,

along with a few rare classes of compounds, including alkaloids, glucosinolates, and

isothiocyanates. The mature seed of MO is rich in oil, containing between 22 and 40%

crude fat. Examination of the oil composition indicates that the oil has a high

proportion of monounsaturated fatty acids, particularly oleic acid. It has been

demonstrated that a higher dietary intake of monounsaturated fatty acid (mainly oleic

acid) is associated with decreased risk in coronary heart disease (Saucedo-Pompa et

al., 2018). The seed extract could be used to prevent some vectors such as for malaria

Anopheles mosquitoes. The phytochemicals derived from MO seeds extracts are

effective mosquito vector control agents and the plant extracts may be used for further

integrated pest management programs.

2.9.2 Medicinal properties

Moringa oleifera has several medicinal properties and has potentiality to cure many

diseases. It is used to treat diseases such as diabetes, heart disease, anaemia, arthritis,

respiratory problems, skin, liver problems, paralysis, sterility, rheumatism, digestive

disorders and many more. In India, it was named the ‘plant of the year’ in 2008 by the

National Institute of Health and Family Care. Several other countries like Africa, it is

also used for the treatment of ascites, pneumonia and venomous bites. According to

various research, the leaves are said to be anti-fungal, anti-viral, anti-abortifacient,

and act as flocculating agent and stimulants. Moringa powder can be used as a

21
substitute for iron tablet, hence a treatment for anaemia. The health benefits of this

wonderful tree appear to be boundless. Apart from all the goodness discussed above,

the regular intake of Moringa is said to help further (Adedapo et al., 2009).

2.9.3 Bioactive compounds of Moringa oliefera

Moringa plants have an extensive range of bioactive compounds that can be obtained

from different vegetative structures. These bioactive molecules include carbohydrates,

phenolic compounds, phenolic acids, flavonoids, isothiocyanates, tannins, and

saponins, oils and fatty acids, proteins and functional peptides and have great

potential to be used in several formulations of food products (Saucedo-Pompa et al.,

2018). Raw MO leaves are healthy source of Vitamin A which has a great importance

in the function of vision, fertility, growth and formation of the fetus, protection and

division of the cells. MO leaves also contain carotenoids with capacity for provitamin

A. The content of vitamin C in Moringa leaves is more as compare to oranges, act as

antioxidant which defends the body from the adverse effects of free radicals,

contaminants and toxins. It is also a great source of beta-carotene, vitamin C,

polyphenols and vitamin E, which are close in amount to those present in nuts, act as

an antioxidant; shows inhibit cell proliferation (Gopalakrishnan et al., 2016). Dried

moringa leaves are a major source of polyphenolic compounds including flavonoids

and phenolic acids. The synthesized flavonoids in planta are a reaction to microbial

infections and have a specific structure benzopyranone chain. Flavonoid intake has

been shown to reduce persistent oxidative stress- related diseases, including

respiratory disease and cancer. The main flavonoids found in moringa leaves are

Myrecytin, Quercetin and Kaempferol, and their concentrations are 5.8, 0.207 and

7.57mg/g, respectively. Quercetine is an effective antioxidant that has several

22
medicinal effects. For obese rats with metabolic syndrome Zucker it has

hypolipidemic, hypotensive and anti-diabetic results. Phenolic acids are a sub-class of

naturally occurring phenolic compounds in plants, originating from hydroxybenzoic

acid hydroxycinnamic acid. These substances have propensities to be antioxidant,

antiinflammatory, anti-mutagenic and anti-cancer. The concentration of gallic acid

1.034mg/g, chlorogenic acid 0.489mg/g and caffeic acid 0.409mg/g on a dry basis

(Hidayati et al., 2018). Chlorogenic acid (CGA) is a dihydrocinnamic acid ester and

the primary phenolic acid in moringa. CGA is an element which helps in the synthesis

of glucose, blocks the transportase of 6-phosphate glucose in the liver and helps to

increase liver gluconeogenesis and degradation of glycogen.

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 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1. Experimental Location

The study was carried out in the Botany Laboratory of Department of Biological

sciences, Benue State University, Makurdi. Makurdi is located in North central

Nigeria along the Benue-river, on latitude 07°43’N and longitude 08°35’E. It is

situated at elevation 104 meters above sea level within the Benue trough, at the lower

Benue valley and found in the Guinea Savannah region.

3.2. Sourcing and Collection of Materials

Ten decaying banana (Musa spp) fruits each showing signs of rot were purchased

from Wurukum, Wadata, Northbank, Highlevel Markets. Moringa seeds(Moringa

oleifera) were also purchased from Wurukum market all in Makurdi Local

Government Area of Benue State and taken to the Botany Laboratory of Department

of Biological Sciences, Benue State University Makurdi for fungal analysis.

3.3. Preparation of Saburoud Dextrose Agar (SDA) Medium

Saburoud Dextrose Agar (SDA) was the media for isolation of the fungi pathogens.

This was prepared according to the manufacturer’s recommended procedures by

dissolving 62g of powdered SDA in 1000 ml of sterile distilled water and stirred

vigorously to homogenize. The flask content was heated on a heating mantle until the

solution became clear. After heating, the flask was covered with foil paper and

autoclaved at 121°C for 15mins at 15 pounds per square inch (psi). The sterile

medium was allowed to cool to a temperature at which it could be held with hands

24
and 2-3 drops of chloramphenicol was added to inhibit bacterial growth. The medium

was poured aseptically into the Petri dishes by flame sterilizing the mouth of the flask

containing the medium at intervals before pouring into the petri dish. This was

allowed to solidify before being used for fungi culture.

3.4. Isolation of fungal Pathogens from the decaying banana (Musa spp) Fruits

Small sizes were cut from decaying banana fruits with sterile scalpel. They were first

surfaced sterilized by dipping them in a concentration of 5% sodium hypochloride

(NaOCl) solution for 1 minute. The pieces were then removed and rinsed in three

changes of sterile distilled water and placed on sterile filter paper to dry for 5 minutes.

They were then placed on solidified SDA medium. Three replicates were made for

each sample. The inoculated Petri plates were incubated at room temperature and

observations were made daily for possible microbial growth. After 5-7 days of

growth, subculturing was done to obtain pure cultures of the isolates as reported by

(Liamngee et al., 2015). To subculture the fungal isolates, a sterilized inoculation

needle was used to pick a little quantity of each pathogen and inoculated in another

Petri dish containing freshly prepared solidified SDA. The plates were sealed with

PVC tapes to avoid contamination. Plates were incubated for 5-7 days at room

temperature.

3.5. Percentage Occurrence of Fungi

Plates were observed for growth and the occurrence of fungi was determined by

counting the number of fungi per market divided by the total number of fungi and

expressed as a percentage using the formula adopted by Zakawa et al., (2008).

25
 Number of fungi per markets ×100
Total number of fungi

3.6. Percentage Occurrence of Individual fungi

Plates were observed for growth and the occurrence of fungi was determined by

counting the number of times each individual fungus occurred divided by the total

number of fungi and expressed as a percentage using the formula adopted by

Liamngee et al., 2016)

Number of time each fungi occurred ×100

3.7. Morphological Characterization and Identification of Fungal Isolates

The identification of fungi was done by observing the colour and the pattern of

growth of the fungi macroscopically in Petri plates. Microscopic identification was

done by staining a glass slide with a drop of Lactophenol in cotton blue and with the

aid of inoculation needle a small quantity of the fungal colony were placed on the

stained glass slide, covered with a cover slip and viewed under the ×40 objective lens

of the light microscope. The observed characteristics of the fungi were compared with

a standard chart for identification by Barnett and Hunter (1972) as reported by

Liamngee et al. (2016).

26
3.8. Pathogenicity Studies

Pathogenicity test was carried out on the fungi isolated from banana fruits to confirm

the pathogenic organisms. The pathogenicity of the isolated organisms was tested in

vivo on detached unripe healthy banana fruits using agar plug method of inoculation

(Ekhuemelo et al., 2016). Twelve healthy banana fruits were surface sterilized with

10% Sodium hypochlorite for one minute and washed in three changes of sterile

distilled water. A 3mm cork borer was used to punch into the healthy banana fruits

and the bored tissues were removed. A 1mm cork borer from pure cultures of the

fungi was used to inoculate the banana fruits (three banana fruits were inoculated per

replicate of each test fungus) and the tissue replaced. Lesion diameter was measured

based on the symptoms induced at six days after inoculation at ambient conditions of

light and temperature using a metre rule. The lesion depth and diameter were

measured 7 days after incubation and the area of rot was calculated using the formula

adopted by Ezeibekwe and Ibe (2010):

Area of rot = πdl (Where π = 22/7, d = diameter, l = depth)

3.8.1 Processing and Extraction of Moringa Extract

The outside covering (pod) of the moringa seed was removed (depodded) by peeling

with hands. The Seeds were weighed using an electric weighing scale to get 50g and

100g and thoroughly washed in distilled water to remove all forms of dirt. Moringa

seeds were surfaced and sterilized in 5% sodium hypochlorite for 1 minute, rinsed in

three changes of sterile distilled water and mashed using a wooden mortar and pestle.

27
To obtain extract concentrations in percentage weight per volume, 50g of the crushed

Moringa seeds were transferred into beakers and 100 ml of sterile distilled water was

added. This was allowed to stay for 30 mins and sieved using a sieve cloth to obtain a

filtrate. The same procedure was employed to get an extract concentration of 100%

w/v.

3.8.2. Antifungal Activity Test

Two (2) ml of each extract concentrations of 50 and 100% w/v were dispensed into

15-20 ml of molten Sabouraud Dextrose Agar to obtain an agar-extract mixture in

Petri plates as described by Liamngee et al. (2015). The Agar-extract mixture was

allowed to solidify and the isolated fungi were inoculated centrally on the medium in

9cm Petri dishes. Three replications were used for each fungi isolate in treatment.

Controls were Petri dishes containing Sabouraud Dextrose Agar with no plant extract.

All plates were properly labelled and arranged in a Completely Randomized Design

(CRD). After 5-7 days of incubation at room temperature, measurement of the growth

of the fungal colony was carried out using a metre rule at intervals of 48 hours.

3.8.3 Data Analysis

The data was analyzed using Analysis of Variance and treatment means were

seperated using Fisher's Least Significance Difference at 5% level of significance.

3.8.4 Phytochemical Analysis of Moringa oliefera Extract ( Qualitative Analysis)

28
The aqueous extraction of Moringa oleifera was subjected to phytochemical screening

to check for the presence of the following active ingredients. The phytochemical

screening was carried out using the method described by Kumar et al.,(2011)

3.8.5. Test for alkaloids (Dragendoff's Test)

Two (2) mls of aqueous moringa extract were mixed with 1% of HCL and about 6

drops of Mayor's reagents. A creamish or pale yellow precipitate indicated the

presence of respective alkaloids.

3.8.6 Test for Tannins (Ferric Chloride Test)

One (1)ml of the extract was treated with a few drops of 0.1 % ferric chloride and

observed for brownish green or a blue -black coloration.

3.8.7. Test for Anthraquinones (Borntrager's test)

one (1) ml of the samples was hydrolysed with diluted H2SO4 extracted with

benzene. 1ml of dilute ammonia was added to it. Rose pink coloration suggested the

positive response for anthraquinones.

3.8.8 Test for saponins (Froth Test)

One ml of the sample was weighed into a conical flask in which 10ml of sterile

distilled water was added to 10ml of sterile water was added and boiled for 5 min.

The mixture was filtered and 2.5ml of the filtrate was added to 10mls of sterile water

29
in a test tube. The test tube was stopped for about 30 seconds. It was then allowed to

stand for half an hour. Honeycomb froth indicated the presence of sapionins.

3.8.9. Test for Terpenoids (Salkowski Test)

Five (5) ml of aqueous Moringa oleifera extract was mixed in 2ml of chloroform, and

concentrated H2SO4 (3ml) was carefully added to form a layer. A reddish brown

coloration of the inner face was formed to show positive results for the presence of

terpenoids.

3.9.0 Test for Cardiac glycosides (Keller - Killani test)

Five (5) mls of aqueous extract of moringa was treated with 2ml of glacial acetic acid

containing one drop of ferric chloride solution. This was underlayed with 1 ml of

concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar

characteristics of cardenolides.

3.9.1 Test for Flavonoids (Lead acetate test)

Aqueous extract of Moringa was treated with few drops of soduim hydroxide

solution. Formation of intense yellow colour, which becomes colourless on further

addition of dilute acid indicated the presence of flavonoids.

3.9.2 Test for Phenolic compounds (Ferric Chloride Test)

30
The Moringa was treated with three to four drops of Ferric chloride solution.

Formation of bluish black colour indicated the presence of phenols.

3.9.3 Test for Steroids (Salwoski Test)

The 5 mls of aq extract was added to 2 ml of chlorroform and filtered. 5 drops of

concentrated H2SO4 acid was added to filtrate. Two layers were observed , a

colourless layer below and a brownish layer above. At interface, a reddish brown ring

colouration was observed.

31
 CHAPTER FOUR

4.0 RESULTS

4.0 RESULTS

A total of three organisms known to cause decay of Banana fruits were identified to

be; Aspergillus niger ,Rhizopus Stolonifer and Colletotrichum musae .The

macroscopic appearance of these organisms on SDA and under the microscope is

presented in plates. (Plate 1a) A.niger on SDA had a dark brown colony with black

and densely packed conidia. Conidiophores were hyaline to slightly brown (Plate 1b).

The colonies of Rhizopus Stolonifer on SDA grow from dark brown to black and

cottony after seven days of incubation (Plate 2a). The sporangiospores were branched

forming large terminal of globose sporangia. (Plate 2b). The isolates of C. musae

produced a hyaline cylindrical conidia showing in (plate 3b), C. musae has concentric

rings sporolating with masses of pinkish conidia after 3-5 days of culturing.(Plate 3a).

32
2

Plate1a.Macroscopic view of A. Niger Plate1b.Microscopic view of A.niger

Plate 2a.Macroscopic view of Plate 2 b. Microscopic view for

R. Stolonifer R.stolonifer

Plate 3a Plate 3b

Macroscopic view of C.musae Microscopic view of C.musae

33
The occurrence of fungi across the location is presented in Table 1. The result

revealed that Northbank market (3.67) , Wurnukum (3.67) and Railway market (3.67)

has the highest fungi occurrence but this was not statistically different from Wadata

market (3.00).

The percentage distribution of Rhizpus stolonifer across the location showed

that Northbank market (4.33) was higher than wurunkum market (2.00), wadata

market (3.33) and Railway market but no significant difference was observed between

the markets.

The percentage distribution of Colletotrichum spp accross the location shows

that Wadata market (5.00) was higher than Northbank market (0.33), Railway market

(0.67) and Wurukum market (0.33) respectively. a significant difference of (2.66) was

observed between the markets as shown in the Table 2

The percentage distribution of A.niger revealed that Northbank market (3.00)

and Wadata markets (2.33) was higher but no statistically difference were observed

when it was compared to Railway market (1.67) and wurukum market (1.33),

respectively as shown in Table 2.

The fungi isolates capable of causing disease (Pathogenicity) is shown in

Table 3. Aspergillus niger (4.27) cm2 Colletotrichum spp (3.08) cm2 and Rhizopus

Stolonifer (3.90) cm2 rot induced was statistically different when compared with

uninoculated healthy banana fruit serving as control. (1.50)cm2.

The effect of (Moringa oleifera) extract on radial growth of fungi is shown in

Table 4. Aspergillus niger was statistically higher in the control on all the days

3(3.23), 5(5.03) and 7(6.73) as compared with 50%w/v on days 3(1.70), 5(2.17) and

7(2.87) and 100%w/v on days 3(0.70), 5(1.17) and 7(1.63). A statistical difference

was observed in day 3(0.37), day 5(0.26) and day 7(0.35).

34
Similarly,radial growth in Colletotrichum spp is shown to have a statiscally higher in

the control on the number of days 3(2.43), day 5 (3.70) and day 7(5.13) and

significant difference was observed when compared to 50%w/v on day(s) 3(1.50),

5(2.07), and 7(2.80) and 100%w/v on day(s) 3(0.93), 5(1.23) and 7(1.70)

Furthermore, radial growth in Rhizopus Stolonifer were also higher in the control on

number of days 3(2.17), 5(3.20) and 7(4.80) and significant difference was observed

when compared to 50%w/v on days 3(1.57), 5(2.20) and 7(3.27) and 100%w/v on

days 3(0.57), 5(1.10) and 7(1.90) respectively.

Table 5, shows the phytochemical screening of Moringa oliefera extract.

Certain plants possesses some secondary metabolites which enable defence against

pathogens and these metabolites present are used for medicinal purposes. The

presence of alkaloids, saponins, tannins, flavonoids, sterols, phenols, glycosides and

terpenoids were all present in the Moringa oliefera extract after they were tested.

35
Table 1: Percentage Occurrence of Fungi in Banana Fruits obtained from

different markets in Makurdi.

LOCATION OCCURENCE (%)

Railway 3.67

Northbank 3.67

Wurukum 3.67

Wadata 3.00

FLSD(0.05) NS

Key; FLSD: Fisher’s Least Significance Difference

Table 2: Percentage Distribution of Individual Fungi from Banana Fruit across

the Different Markets in Makurdi.

LOCATION A. niger R.stolonifer C.musae

Railway 1.67 0.67 3.00

Northbank 3.00 0.33 4.33

Wurukum 1.33 0.33 2.00

Wadata 2.33 5.00 3.33

FLSD(0.05) NS 2.66 NS

Key; FLSD: Fisher’s Least Significance Difference

36
Table 3: Pathogenicity of Fungi Isolate on Healthy Banana Fruits in Makurdi,

Benue State.

FUNGI ISOLATE AREA OF ROT (Cm2)

A.niger 4.27

R.stonolifer 3.08

C.musae 3.90

Control 1.50

FLSD (0.05) 0.36

Key; FLSD: Fisher’s Least Significance Difference

37
Table 4: Effect of Aqueous Extract of Moringa oliefera on Radial Growth of

Fungi Isolated from Banana Fruits in Makurdi.

Organism

Concentration (w/v) 3 5 7

A.niger 0% 3.23 5.03 6.73

50% 1.50 2.07 2.80

100% 0.70 1.17 1.63

FSLD (0.05) 0.37 0.26 0.35

R.stolonifer 0% 2.43 3.70 5.13

50% 1.50 2.07 2.80

100% 0.93 1.23 1.70

FLSD (0.05) 0.15 0.36 0.31

C. musae 0% 2.17 3.20 4.80

50% 1.57 2.20 3.27

100% 0.57 1.10 1.90

FLSD (0.05) 0.26 0.31 0.31

Key; FLSD: Fisher’s Least Significance Difference.

38
Table 5:Phytochmical Analysis of Moringa showing the presence of secondary

metobolites.

Metabolite + Moringa oleifera

Alkaloid +

Tannins +

Saponins +

Phenolic compounds +

Flavonoids +

Terpenoids +

Glycoside +

Anthraquinones +

Anthocyanodsides +

Nb. + means present.

39
 CHAPTER FIVE

5.0 DISCUSSIONS, RECOMMENDATION AND CONCLUSION

5.1. Discussion

The isolation and distribution of fungi in spoilt banana in Makurdi markets is a

discovery which exposed an array of fungi which are pathogenic to man and animals.

The identified fungal organisms associated with spoilt fruits in the study area include

A.niger,R stolonifer, and colletotrichum spp.suggesting that these fungal organisms

could be responsible for the fruit spoilage. This finding is in conformity with the

works of (S.M Yahaya et al., 2018) and (Jimyoung Lim et al.,2002) which reported

the identification and isolation of A.niger,R.stolonifer and Colletotrichum musae from

banana in Nigeria(kano) and korea.

Literatures indicate that the processes such as harvesting, storing, packing and

transportation of fruits may encounter physical injury that increases the post- harvest

loss and the possibility of fungal contamination. In addition, the problem may be

enhanced from poor management of banana fruits in makurdi markets.(Gultie et

al.,2013).Market conditions that favour conditions contamination can be worsened by

poor hygiene of vendors, poor handling practices and poor environmental conditions

which are unsafe for marketing. The consequence of the problems could be some

increased loss of fruit due to microbial spoilage and existence of human pathogens

(Okojie et al.,2014).

Out of the fungi isolated in this study Colletotrichum musae (wadata market )which

causes a diseases called Brown spot on banana and cause a lesion on the ripe banana

or wounded banana and can be harmful to humans had the highest occurrence (5%)

followed by Rhizopus stolonifer of northbank (4.33%) while Aspergillus niger of

40
wurukum (1.33%) had the lowest frequency of occurence. However, (Muhammad et

al.,2019) reported that Colletotrichum musae was found to be the most frequently

occurring fungi isolated from the samples obtained representing 35.7% in Kasuwar

Daji ,14.25% and 7.14% for C.musae, A.niger and R.stolonifer respectively. These

differences could be attributed to number and type of fruits examined in both studies.

My findings are also in partial concordance with a similar research conducted by

Abdullahi et al.,2016 whose results shows a total of seven (7) species isolated from

banana fruit. However, only two (2) species isolated from this study (colletotrichum

musae and Rhizopus stolonifer) are part of the seven (7) isolates. A. alternata,

C.lunata, Ulocladuim botrytis, Acremonium sp.,Curvularia sp., are exceptions.Also

the result showed C.musae to have the highest percentage occurence (42%).Raut et

al.,2004 and Ranasinghe et al.,2005 reported that, banana suffer from serious post-

harvest loses caused by fungal infection especially C.musae.

Most of the fungal organisms isolated in this study play a pivotal role in the

deterioration of food and feed system and some are able to produce toxic compounds

for humans and animals. R.stolonifer is a significant agent of fruit disease. It is a

threadlike mold and a heterotrophic species; it depends on sugar or starch for its

source of carbon substance for food. It uses food matter, mostly soft fruit, like grapes

and banana, as a food source for growth and, nutrition and reproduction.

Fungal pathogen are causing losses of marketable quality and hygiene of fruits,

resulting in major econmic problem in Nigeria and the world at large. Fruit spoilage

can be prevented using physical (Vinod K, 2009) and chemical methods (Vinod k,

2009), but no efficient strategy has been proposed so far to reduce the microbial

growth. However, the use of plant based extracts as biocontrol sounds effiective in

increassing the shelf life of banana. The moringa extract used in this current study

41
seem to have exhibited antibacteria and antifungal actions against test pathogens due

to the presence of bioactive compounds. These phytochemicals infer strong inhibiting

properties (Akinbode and Ikotun, 2008; Al-Malki and Rabey, 2015). Moringa seed

extracts show some strong antifungal activity against A. niger, R. stolonifer, and

R.solani.This antifungal activity is due to the presence of 30% seed oil present in the

moringa seed (Abalaka et al.,2012). When the seed was ground into a powder during

extract preparation and the whole extraction process, oil was pressed out and released

into the Moringa aqueous extract solution. This resulted in the Moringa seed extract

having a frothy and saponified appearance which might have enhanced its antifugal

efficacy, and also increased the contact time between test pathogens and extract. This

improved contact is due to the Moringa oil being a good absorbent and moisturizer,

which further created a condusive wet environment which enhanced the antifungal

activity (Anwar et al., 2007; Bukar et al., 2010).

5.2 Conclusion

This study has shown that A.niger, R.stolonifer and Colletotrichum musae were

isolated from a spoilt banana fruit. These pathogenic fungi species are associated with

fruit spoilage and are of economic and public health significance. Care should be

taken during handling of these fruits, technology based modern preservative method

such as pasteurization, vacuum packing, radiation and plant based bio-control extracts

are suggested to enhance the quality and shelf life of post-harvest banana fruit.

42
5.3. Recommendation

Based on this research and findings, I recommend that;

Discovering that Moringa oleifera aqueous extracts are sources of secondary

metabolites with antifungal properties, further research should be made and further

developments for its development in agriculture should be considered giving us

broader options in controlling post-harvest diseases.

Proper handling, storage, transportation and hygiene should be brought to awareness

of consumers, retailers, distributors and producers alike. through educative avenue

and initiated by governmental bodies, individuals and NGOs.

Total avoidance of contaminated fruits to prevent implications that may lead to illness

and even death.

43