ISOLATION, IDENTIFICATION AND CONTROL OF FUNGI ASSOCIATED WITH POSTHARVEST FRUIT ROT OF MANGO, USING PLANT EXTRACT (Vernonia amygdalina Del.) IN WUKARI, TARABA STATE.

BY

ISAIAH FELIX GWATANA (UR201500821).

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES,

FACULTY OF PURE AND APPLIED SCIENCES.

IN PARTIAL FULFILMENT FOR THE REQUIREMENT FOR THE AWARD OF A BACHELOR OF SCIENCE DEGREE IN BIOLOGICAL SCIENCES.

FEDERAL UNIVERSITY WUKARI, TARABA STATE.

SEPTEMBER, 2019.

4

DECLARATION

I, ISAIAH FELIX GWATANA with matric number UR201500821 do declare that the work in this project report titled “Isolation, identification and control of fungi associated with postharvest fruit rot of mango, using plant extract (Vernonia amygdalina Del.) in Wukari, Taraba State” was carried out by me in the Department of Biological Sciences, Faculty of Pure and Applied Sciences, Federal University Wukari. Information derived from literature has been duly acknowledged in the body of the work and list of references provided. No part of this project report has been previously presented for another degree or diploma in this or any institution.

Isaiah Felix Gwatana ______________________

Name of student Signature/Date

CERTIFICATION

This is to certify that this project work titled, “Isolation, identification and control of fungi associated with postharvest fruit rot of mango, using plant extract (Vernonia amygdalina Del.) in Wukari, Taraba state.” was carried out by ISAIAH FELIX GWATANA (UR201500821) in the Department of Biological Sciences, Faculty of Pure and Applied Sciences under supervision.

Mr. Kyugah Jacob Tersur _________________________

Supervisor Signature/Date

Iliya Ezekiel (Ph.D) _________________________

Head of Department Signature/Date

____________________________ _________________________

External Examiner Signature/Date

DEDICATION

This work is dedicated to God Almighty for his love and protection upon my life.

ACKNOWLEDGEMENT

My deepest gratitude goes to God Almighty for the guidance, good health and strength granted to me in making this work a success.

My profound gratitude goes to my supervisor Mr. Kyugah Jacob Tersur for the unlimited time and effort he took in reading and correcting this work, and his contributions to see that this research work is a success. May God Almighty reward you abundantly sir.

My profound gratitude also goes to the Head of Department, Iliya Ezekiel (Ph.D) and all the staffs of Biological Sciences Department for the knowledge and encouragement given to me. May God Almighty bless you.

My sincere appreciation goes to my beloved parent, Mr. Isaiah David and Mrs. Mercy Isaiah for giving me the love, advices, and financial support I needed. May God almighty enrich you with his blessings and grant you long life.

My special appreciation goes to my brothers and sisters; Grace Isaiah, Godwin Isaiah, Faith Isaiah, Favour Isaiah, and Goodluck Isaiah. May God bless you.

My profound gratitude also goes to the microbiology technologist, Mr. Benedict Longgul and biology technologist, Mr Murtar for their time and effort to see that the practical aspect of this work was a success.

I also want to appreciate some of my friends; Benjamin Felix, Peace Luka, Yakubu Terpase Stephen, Abraham Queen, Ajiri Ovie Philip, Elijah Daniel Burba, Taiwo Alade, Elijah Dio, Danjuma Friday, Genesis Pius and my colleagues in the department for their support throughout my academic journey.

TABLE OF CONTENTS

TITLE PAGEDECLARATIONiCERTIFICATIONiiDEDICATIONiiiACKNOWLEDGEMENTivTABLE OF CONTENTSvLIST OF FIGURESixLIST OF PLATExLIST OF TABLExiABSTRACTxiiCHAPTER ONE11.0 Introduction11.1 Statement of the Problem21.2 Justification of the Study31.3 Significance of the Study31.4 Aim and Objectives3CHAPTER TWO52.0 LITERATURE REVIEW52.1 Scientific Classification of Mango (Mangifera indica L.)62.2 The Origin, History and Spread of Mangoes Around the World62.3 Habitat82.4 Propagation of Mango82.5 Morphology of Mango Fruit82.6 Production and Uses92.7 Fungi112.8 Most Common Fungal Diseases of Mango, their Causal Agents and Symptoms112.8.1 Powdery Mildew122.8.2 Anthracnose122.8.3 Die-back132.8.4 Scab132.8.5 Black-banded132.8.6 Sooty Mould or Sooty Blotch142.8.7 Phoma Blight142.9 Climatic Conditions in Relation to Mango Diseases142.10 Mango Resistance in Relation to Fruit Rot Diseases152.11 Bitter Leaf Plant (Vernonia amygdalina Del.)162.12 Scientific Classification of Bitter Leaf17CHAPTER THREE183.0 MATERIALS AND METHOD183.1 Study Area183.2 Source of Samples203.3 Medium for the Isolation and Identification203.4 Characterization and Identification of Isolates203.5 Pathogenicity Test213.6 Collection and Preparation of Plant Extract213.6.1 Sample Collection213.6.2 Extraction Process213.6.3 Concentration of Extracts223.6.4 PDA-Extract Medium Preparation223.7 Statistical Analysis23CHAPTER FOUR244.0 Results244.1 Pathogenicity test result264.2 Effect of Plant Extracts on Mycelial Growth of Fungal Isolates with time and concentration28CHAPTER FIVE325.0 Discussion, Conclusion and Recommendation325.1 Discussion325.2 conclusion335.3 Recommendations33References34

LIST OF FIGURES

Fig. 1: Map of Nigeria showing the location of Wukari (study area)……………………..............19

Fig. 2: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on radial growth of Rhizopus stolonifer in wukari………………………………………….………………………29

Fig. 3: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on Rhizopus spp. in Wukari………………………………………………………………………………………....30

Fig. 4: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on Aspergillus spp. in Wukari…………………………………………………………………………………....31

LIST OF PLATE

Plate I: Image of mango fruits after the introduction of the isolated fungi…..…………...………27

LIST OF TABLE

Table 1: Showing result on identification of the isolates…………………………………………25

ABSTRACT

Mango is one of the most popular fruits grown throughout the tropical and subtropical regions of the world. It is one of the most desirable fruit in the international market because of its delicious taste and high caloric value. However, postharvest diseases reduce fruit quality and cause severe losses of Mango. The study was carried out to identify and control the fungal pathogens associated with fruit rot of Mango (Mangifera indica L.) using plant extract (Vernonia amygdalina Del.) in Wukari town. A total of two hundred (200) Mango fruits were randomly sampled from four (4) different market in Wukari, Taraba State. The diseased Mango were cultured on a potato dextrose agar (PDA) for 5-7 days in the laboratory, Federal University Wukari. The result indicated that Rhizopus stolonifer, Rhizopus spp. and Aspergillus spp. were responsible for Mango fruit rot in Wukari, and the control measures were taken by the use of plant extract. 50g of powdered plant extract (Vernonia amygdalina) were mixed in 500ml of ethanol to obtain bitter leaf ethanoic solution. Concentrations of (20%, 40%, 60%, 80% and 100%) were used. The result showed that in Aspergillus spp. and Rhizopus stolonifer, 60% concentration of the ethanoic extract were more effective (100.00 and 80.00 % inhibition respectively). In Rhizopus spp. 20% and 40% concentrations were both more effective (100 % inhibition). The concentrations were found to be effective in controlling the radial growth of the fungi as compared with the control (untreated medium)

CHAPTER ONE1.0 Introduction

Mango (Mangifera indica L.), locally known as Mangoro (Hausa), Mangolo (Igbo) and Mangoro (Yoruba) in Nigeria is an erect, branched, medium to large-sized tree. The leaves are alternately arranged, evergreen or nearly evergreen (Muhammad and Amusa, 2005). It has a wide crown and inflorescence with numerous flowers (Ugese et al., 2012; Shri, 2013). The tree is cultivated in many tropical and sub-tropical regions of the world. Mango fruits vary in shape, size, colour, texture and flavor (Nelson, 2008). Mango is known worldwide as “king of fruits”. It belongs to the genus Mangifera consisting of numerous species of tropical fruiting trees in the flowering plant family Anacardiaceae. It is a large branched, perennial tree, which attains great heights with symmetrical rounded canopy, approximately 30-40 feet tall. Flowers appear in large terminal inflorescence producing fruits. The skin of the fruit may be green, yellow, or red, depending upon the variety of the fruit (Nelson, 2008; Fowomola, 2009; Shri, 2013). The name 'Mango' was derived from Tamil word 'mangkay' or 'man-gay' (Shri, 2013; Martins, 2014). The tree is native to Asia particularly eastern India, Burma, and the Andaman Islands where it spread to the East Africa in the 10th century. The Portuguese introduced it to West Africa in the 16th century (Morton, 1987). Mango came to Nigeria in the 20th century through itinerant merchant missionaries and colonialist and is currently the integral part of indigenous cropping system (Ugese et al., 2012). The guinea and Sudan zones of Nigeria are credited with producing greater percentage of the fruit in the country. Earlier studies indicated that Agricultural Research Institute in Zaria was the center where improved mangoes varieties were obtained (Avav and Uza, 2002). Mangoes are grown in eighty-five countries worldwide. Sixty-three of such countries provided more than 1000 metric tons in 1999. Developing countries account for about 98% of the total world production (Yusuf and Salau, 2007). Despite lack of encouragement to large scale production of tropical fruits, Nigeria occupies 8th position in world ranking of mango producing countries as at 2002. This suggests the potential of tropical fruits in the country.

Mango fruits in Wukari metropolis are under threat by fungi pathogens. Diseases has rendered its production non attractive to both farmers and home gardeners in the region. The high moisture content of mango fruit makes them highly susceptible to the attack of pathogen. Major loss of harvested mango fruit is caused due to fungi. The disease caused during pre-harvest of fruit also responsible for the lowering the quality and market value of the fruit. Post spoilage of fruit may be due susceptibility of fruit to the fungal growth. Susceptibility of fruit to the attack of fungi is varying according to season, cultivar, mechanical, injuries and handling at the time of harvesting as well as by postharvest treatment and storage condition (Derbyshrine and Shipway, 1978 and Dennis, 1983).

1.1 Statement of the Problem

Mango production in West Africa has an important socio-economic impact on farm households and on West African countries economies. Mangoes play an integral part in rural household lives not only by being rich nutrient source but also by serving as common goods that are consumed casually. The fruit is very sweet and is consumed fresh, or transformed into juices, jams, dried fruit and so on.

However, the growth of mango production in west Africa is dangerously threatened by attack of diseases such as fungi which are causing not only losses amounting up to 80% of total production in the field (Entomological society of Nigeria, 1991), but also losses in market shares in Europe and other parts of the world where damaged fruits are destroyed or returned to exporting countries. And also it effects on health of consumers. Some of the diseases attacking mango fruits are Anthracnose, mango scab, powdery mildew and so on.

In light to all these, the current study sought to isolate, identify and provide solutions on how to control this fungal pathogen that causes fruit rot disease of mango. The use of plant extracts such as bitter leaf has been found to be effective in the control of these fungi associated with mango fruit rot disease of mango and also found to be harmless to humans, beneficial organisms and the environment.

1.2 Justification of the Study

Mango fruit is one of the most popular fruit in the tropical region and it is increasingly being consumed in the developed countries. It is not only important for it sweetness, but it is also important to the body as it helps to promote brain health, care of the eyes, fight cancer, strengthens the immune system, aid in digestion and so on. And also it can serve as source of income to the country. However, mango fruits production and consumption has reduced due to fungal activities on them, which also affect the health of human beings.

1.3 Significance of the Study

This research study however, will help to identify the fungal pathogens that are responsible for causing fruit rot disease of mango and recommend how those pathogens can be controlled so as to increase the production and reduce health risk in the community.

1.4 Aim and Objectives

The aim of this study is to determine the fungal pathogens causing mango fruit rot disease of mango fruits and how it can be controlled using plant extract.

The objectives of this study are:

1. Collection of mango fruits and isolation of the fungi associated with fruit rot of mango in Wukari.

2. Identification of fungal pathogens causing fruit rot of mango in Wukari.

3. Control of the fungal pathogens found in mango fruit using plant extract (Vernonia amygdalina).

CHAPTER TWO2.0 LITERATURE REVIEW

Mango (Mangifera indica L.) is a member of the plant family, Anacardiaceae (Cashew family or poison ivy family). The species name of mango is Mangifera indica which means “an Indian plant bearing mangoes”. The cultivated mango is probably a natural hybrid between Magnifera indica and Magnifera sylvatica. There are two classes of cultivars, indo china and West Indian. The over 1000 known mango cultivars are derived from two strains of mango seed, mono-embryonic (single embryo) and poly-embryonic (multiple embryo).

The productivity of mango is affected by pre-harvest and postharvest diseases, which reduce the fruit quality and cause severe losses, because they leave them as unmarketable fruits. The mango tree and especially the fruit is a host to a number of pathogens which cause rotting before and after harvest, like Colletotrichum responsible for mango anthracnose. Among the various diseases, anthracnose is the most serious disease, widely distributed all over mango growing regions in the world. Pre-harvest losses may be due to cultural practices like irrigation, fertilizer addition, pruning, biological factors like pre-harvest infection by fungi, Botryodiplodia theobromae causing stem rot, pests like fruit flies and mango stone weevil invading at the time of flowering and ripening and environment factors like soil condition, texture, nutrients present, presence of inorganic pollutants. Post-harvest losses in mangoes might be due to the perishable nature of the fruit, during harvesting due to improper physiological maturity, time and techniques of harvesting, and during packaging and transport due to inefficiency, abrasions and compression, storage and marketing

2.1 Scientific Classification of Mango (Mangifera indica L.)

Mango belongs to the Kingdom: Plantae; Subkingdom: Tracheobionta; Superdivision: Spermatophyta; Division: Magnoliophyta; Class: Magnoliopsida; Subclass: Rosidae; Order: Sapindales; Family: Anacardiaceae; Genus: Mangifera; Species: Mangifera indica L.

Source: United States Department of Agriculture (USDA).

2.2 The Origin, History and Spread of Mangoes Around the World

The history of mango began thousands of years ago on the Indian sub-continent. The mango is the national fruit of India, Pakistan and the Philippines. It is also the national tree of Bangladesh. Not only is it one of the most highly prized fruits of South Asia. There is consensus among the historians and horticulturists that the cultivated mango has originated in India. Vavilov (1926) has suggested Indio-Burma region as the centre of origin of mango based on the observed level of genetic diversity. Mukherjee (1951) considered origin of genus Mangifera probably in the South-East Asia but the origin of cultivated Mango in the Assam-Burma region. Extensive comparison of the anatomy and morphology of several modern day species of the genus Mangifera with the fossil samples reinforced the view that North-East India is the centre of origin of mango genus, from where it has spread into neighboring areas of South-East Asia and then slowly to the whole world. Mangoes have been cultivated in South Asia for thousands of years and reached Southeast Asia between the fifth and fourth centuries B.C. The earliest mention of mango, Mangifera indica, that means “the great fruit bearer,” is in the Hindu scripture dating back to 4000 B.C. The wild mango originated in the foot hills of the Himalayas of India and Burma and about 40 to 60 of these tree still grow in India and South east Asia. Over the year’s mango groves have spread to many parts of the tropical and sub-tropical world. As the mango became cultivated, as early as 2000 B.C. its flavor, size and texture developed. The explorers who tasted the mango were enchanted with its aromatic qualities and ambrosial flavor and introduced the fruit to other tropical countries. As the mango adapted to new locales, new varieties evolved and many names were bestowed upon it such as “apple of the tropics, “King of fruit” and “Fruit of the Gods”. After its domestication in India more than 4000 years ago, traders, travelers and rulers have taken mango for plantation in different subtropical regions of the world over the last 25,000 years. During 4-5th centuries B.C. the Buddhist monks took mango to Malaya Peninsula and East Asia. Mango was first introduced in china from India during middle of the 7th Century A.D. when Chinese traveler Hwen T’sang returned from India to china with the mango. Further in the 10th Century A.D. the Persians carried it to East Africa (Purseglove, 1969). The 14th century Moroccan traveler Ibn Battuta reported it at Mogadishu. The cultivation of mango began slowly moving westward with the spice trade. The Portuguese, who landed in Calcutta in 1498, were the first to establish a mango trade. English word mango originated from Malayalam “manga” and Tamil “mangai”. The Portuguese were fascinated by the fruit on their arrival in Kerala and introduced it to the world as “Mango”. The Portuguese introduced grafting on mango trees to produce extraordinary varieties likes Alphonso. Alphonso is named after de Albuquerque, a nobleman and military expert who helped to established the Portuguese colony in India. The traveling mango then hitched a camel ride from Persia and caravanned to the African continent about the year 1000. Spanish explorers brought mango to South America and Mexico in the 1600‟s. The first attempt to introduce the mango into the US came in 1833 to Florida. A very small percentage of fresh mangoes available commercially in the US are grown in South Florida and Southern California. During 16th century AD the Portuguese have taken it to west Africa and Brazil. After becoming established in Brazil, the mango was carried to the west Indies being first planted in Barbados about 1742 and later in the Dominican Republic. It reached Jamaica about 1782 and early in the 19th century it reached Mexico from Philippines and the West Indies (Morton, 1987). Mango reached Miami in 1862 or 1863 from the west Indies and it is believed seedling was poly-embryonic and from “No. 11” parent (Litz, 2009). In same decade, about 40 varieties of Mangoes from India were initially planted in 1875 in North Queensland Australia after post-European colonization (Morton, 1987).

2.3 Habitat

The mango (Mangifera indica) belongs to the dicotyledonous family- Anacardiaceae. There are several species of genus Mangifera that bear edible fruit. It is native tropical Asia and has been cultivated in the Indian subcontinent for over 4000years and is now found naturalized in most tropical countries. The mango tree grows on a wide range of soils (optimal pH 5.5-7.5), optimum growth temperature (24-27oC) and a wide range of rainfall (annual 400-3600mm) conditions.

2.4 Propagation of Mango

Mango is propagated by seed and various vegetative methods. The genetic quality of a mango seedling depends on the embryo type of the seed. Poly-embryonic seeds will usually produce three to ten seedlings from each seed, most of which will come true to type with the tree they came from. Poly-embryonic seeds also contain one embryo that is genetically different from the parents; i.e., this embryo will produce an off-type seedling. In contrast to poly-embryonic seeds, mono-embryonic seeds produce only one seedling for each seed that is always genetically different from the parents. For this reason, most mono-embryonic varieties are propagated by grafting onto poly-embryonic rootstocks.

2.5 Morphology of Mango Fruit

Mango fruits are ovoid-0blong drupe, green when young, on ripening yellow, seed solitary. There is great variation in the form, size, colour and quality of the fruits. These drupes may be nearly round, oval, ovoid-oblong, or somewhat kidney v-shaped, often with a break at the apex, and are usually more or less lop-sided. They range from 21/2 to 10 inch (6.25-25cm) in length and from a few ounces to 4 to 5ibs (1.8-2.26kg). The skin is leathery, waxy, smooth, fairly thick, aromatic and ranges from light- or dark-green to clear yellow, yellow-orange, yellow and reddish-pink, or more or less blushed with bright- or dark-red or purple-red, with fine yellow, greenish or reddish dots, and thin or thick whitish, grey or purplish bloom, when fully ripe. Some have a “turpentine” odour and flavour, while others are richly and pleasantly fragrant. It is extremely juicy with a flavour range from very sweet to sub acid to tart. It may have along one side a beard of short or long fibers. Within the stone is the starchy seed, mono-embryonic.

2.6 Production and Uses

Mangoes, ‘the apples’ in the Orient, are appreciated as the choicest of the indigenous fruits. They are the six most important fruit produced in the world behind oranges, bananas, grapes, apples and plantains (FAO, 2001). During the past two decades, the world production of mango was estimated to be increased by a 100%, with more than 25 million metric tons in 2000 (FAO, 2001). At the same time, mango production has increased significantly outside the traditional growing regions of Asia such as in Africa, the western hemisphere, and Australia (Mukherjee, 1997). In 2016, global production of mangoes was 46.5 million tons per year, led by India with 40% (19 million tons) of the world total. China and Thailand were the next largest (FAOSTAT 2016). Nigeria occupies 8th position in world ranking of mango producing countries as at 2002.

Mango have long been recognized as more than just edible ripe fruit. The edible uses of the fruit include non-ripe fruit, seed, and processed products such as achars, chutneys, preserves, etc. The fruit is eaten for its nutritional value, its medicinal value, and for its pleasant flavor. The fruit and its by-products are used for animal fodder, and the timber is used for canoe building and making charcoal. Today mango and its flavor are added to many products, such as fruit juices, ice creams, wines, teas, breakfast cereals, muesli bars, and biscuits. Mango are predominantly grown for their fruit, which is mostly eaten ripe as a dessert fruit. Mature green mangos are also eaten fresh or as pickles. Green eating varieties are distinguished from others by their sweet, non-starchy, non astringent flavor at the green-mature stage of fruit development. Mature green eating mangoes are eaten in several ways throughout the world. In Thailand they are sliced or grated in fresh salad, pickled (ma mung dong), soaked in water and sugar (ma mung chaien), salted and dried (ma mung khem), sliced in vinegar or fish sauce (ma mung pla wa arn), or eaten as a crunchy fruit. In many places, for example Samoa, the fruits are eaten green because someone else will eat them if one waits for ripening or because fruit fly larvae are not yet developed. Fresh mangoes are processed and preserved in to a wide range of products including pulps, juices, frozen slices, dried slices, pulp (fruit leather), chutneys, jams, pickles, canned in syrup, and sliced in brine. Mangoes are a highly nutritious fruit containing carbohydrates, proteins, fats, minerals, and vitamins, in particular vitamin A (beta carotene), B1, B2, and vitamin C (ascorbic acid). As the fruit ripens, concentrations of vitamin C decrease and glucose, fructose, and sucrose concentrations increase. Mangos make a significant seasonal contribution to diet of many Pacific islanders that primarily have a starch-based diet.

Mango purees and essences are used to flavor many food products such as drinks, ice creams, wines, teas, breakfast cereals, muesli bars, and biscuits. In parts of India the seed is eaten as a boiled or baked vegetable or ground into a starchy flour. Alcoholic beverages made from mangos include wines and liquors made in Australia and India. Specialty teas are occasionally flavored with fragrant mango flowers. In addition to mango’s food value, it has also been used for its medicinal value. In India, a drink made from unripe mango fruit is used as a remedy for exhaustion and heat stroke. Half-ripe fruit eaten with salt and honey is used for a treatment of gastro-intestinal disorders, bilious disorders, blood disorders, and scurvy. Ripe mangos are a rich source of vitamin A, and are used to treat vitamin A deficiency such as night blindness. Diabetes has been treated with a drink made from the infusion of fresh mango leaves. Dried mango seed ground into flour is used to treat diarrhea. Diarrhea and throat disorders are treated by gargling bark extracts mixed with water. In India, fruit sap has been used to treat the pain of bee and scorpion stings. Many of the traditional Indian medicinal uses of mango involve eating unripe fruit. It should be noted that unripe fruit contains a lot of the toxic sap that when eaten in excess can cause throat irritation, indigestion, dysentery, and colic. Livestock eat on fallen fruit. Seeds and by-products of processing fruit have been used to feed cattle, poultry, and pigs.

2.7 Fungi

A fungus is a eukaryote that digests food externally and absorbs nutrients directly through its cell walls. Most fungi reproduce by spores and have a body (thallus) composed of microscopic tubular cells called hyphae. Fungi are heterotrophs and, like animals, obtain their carbon and energy from other organisms. Fungi can be biotrophs, saprotrophs (saprophytes, saprobes) and necrotrophs. Fungi were once considered to be primitive members of the plant kingdom, just slightly more advanced than bacteria. Fungi are an important group of plant pathogens, most plant diseases are caused by fungi, but fewer than 10% of all known fungi can colonize living plants (Knogge, 1996).

2.8 Most Common Fungal Diseases of Mango, their Causal Agents and Symptoms

Over 140 fungi (70 diseases), about 12 nematodes and a dozen of phanerogamic parasites and epiphytes are associated with this fruit (Prakash and Srivastava, 1987). Mango is subject to a number of diseases at all stages of its development. Some of these diseases cause heavy loss and are limiting factor in mango cultivation in some regions.

2.8.1 Powdery Mildew

Powdery mildew of mango is a devastating disease of mango. It was first recorded on mango in 1914 in Brazil (Briton Jones, 1923, Uppal et al., 1941). The characteristic symptom of the disease is the white superficial, powdery fungal growth On inflorescence, stalks of the inflorescence, new leaves and young fruits resulting in dropping of flower sand young fruits. New leaves are attacked mostly on their underside, but in advanced cases both sides of the leaves are attacked (Prakash and Srivastava, 1987). The symptoms are restricted to the area of the central rib and such leaves curl and are distorted (Palti, et al., 1974). Histopathological studies revealed that mildew fungus has globular haustoria and due to the type of its conidial germination it should be referred to Erysiphe polygoni (Uppal, 1937 and PaIti, et al., 1974). As no description of the perfect stage of mango mildew fungus was given, the name of the conidial stage Oidium mangiferae Berthet is preferred (Uppal et al., 1941).

2.8.2 Anthracnose

Anthracnose is a common, destructive and wide spread disease. It causes blossom blight, peduncle blight, leaf spot, twing blight, wither tip, fruit russeting or staining and fruit rot (Singh, 1960; Prakash and Srivastava, 1987). The severity of the disease may vary according to prevailing weather conditions (Prakash and Raoof, 1985c). Mango fruit is attacked by this organism at two stages in their development. (a) young fruits, a week or two old, and (b) older fruits, on which black spots are produced. Initially the spots are usually round but later grow, coalesce and form large irregular blotches and cause extensive rotting. The latent infection is carried from the field and develops further during storage. Healthy fruits develop infection after coming in contact with diseased fruits (Sohi et al., 1973; Prakash and Srivastava, 1987). Anthracnose is caused by Colletotrichum gloeosporioides which has been considered a conidial stage of Glomerella cingulata (Small, 1926).

2.8.3 Die-back

Die back is known to be prevalent in India and other mango growing countries. It is posing a great threat to the profitable cultivation of mango (Prakash and Raoof, 1979,1985, 1989). In a recent survey die back was found one of the most important diseases of mango in Orissa (Misra and Prakash, 1988). The disease is most conspicuous during the season following main rains (Prakash and Raoof 1985). It is characterized by dying back of twigs from top downwards. Discoloration and darkening of the bark at a certain distance from the tip is the external evidence of the disease. Such dark patches are generally seen on young green twigs and hardly distinguishable in older branches.

2.8.4 Scab

Mango scab was discovered in Florida by Ruehle (1943) but it is probable that it was present there for a number of years prior to its discovery. Scab is caused by the fungus Elsinoe mangiferae Bitan. & Jenk. (Bitancourt and Jenkins, 1946). The scab fungus (E. mangiferae) attack leaves, twigs, panicles, blossoms, stems and fruits. It forms blotches on the bark of stems and spots on the mango fruit.

2.8.5 Black-banded

The occurrence of disease On mango is recorded by Massee from Poona (Saccardo, 1906). Disease is noticed on the midribs and veins of leaves, twigs and branches of mango as black velvety fungal growth. The incidence of this disease is very low on the main branches. It is considered appropriate to name it as a black banded disease. The fungus is confined to the upper layer of bark (Reddy et al., 1961). The disease is caused by Rhinocladium corticolum, Massee. Subramanian (1956).

2.8.6 Sooty Mould or Sooty Blotch

Sooty mould or Sooty blotch on mango is very common wherever honey dew or sugary substance secreting insects viz. mango hopper, scales, coccids and mealy bugs are found (Prakash and Srivastava, 1987). The disease is characterized by the presence of a black velvety thin membranous growth of black mould on the leaf lamina. Sooty mould is caused by the fungus Meliola mangiferae Earle (Butler and Bisby, 1931; Uppal et al., 1935; Yamamoto, 1940; Hansford and Thirumalachar, 1948). Capnodium mangiferae Cke. & Brown (Vaheeduddin, 1953); Capnodium ramosum Cke. (Butler and Bisby, 1931; Uppal et al., 1935).

2.8.7 Phoma Blight

It is a widespread disease of mango. Initially the lesions are minute, irregular, yellow to light brown, scattered over the leaf lamina. As the lesions enlarge, their colour changes to brown to cinnamon, and these become irregular. Fully developed spots are characterized by dark margin and dull grey necrotic centers. The disease is caused by the fungus Phoma glomerata (Corda) Wall. and Hochapf.

2.9 Climatic Conditions in Relation to Mango Diseases

The impacts of climate change can be positive, negative or neutral, since these changes can decrease, increase or have no impact on diseases, depending on each region or period. These impacts will also be observed on plants and other organisms as well as on other agroecosystem components. The environment can influence host plant growth and susceptibility; pathogen reproduction, dispersal, survival and activity; as well as host-pathogen interaction. The classic disease triangle establishes the conditions for disease development, i.e., the interaction of a susceptible host, a virulent pathogen and a favorable environment. The analysis of the potential impacts of climate change on plant diseases is essential for the adoption of adaptive measures, as well as for the development of resistant cultivars, new control methods or adapted techniques, in order to avoid more serious losses (Chakraborty, 2004; Chini, 2005).

The powdery mildew (PM) caused by Oidium mangiferae is a common problem of mango. (Misra and Prakash, 1988) found that predominance of susceptible cultivar 'Dashehari', high wind velocity for 3-4 days with maximum temperature around 35°C, minimum temperature around 17°C, relative humidity of minimum 23.4-25.5 per cent and maximum 73.383.9 per cent (means dryer climate) are conducive for the rapid spread of mildew pathogen in Kakori-Malihabad mango belt of U.P. They further found that maximum temperature of 35°C play crucial role in the epidemic of PM. If maximum. temperature does not reach 35°C during the latter half of March in northern plains, the epidemic of powdery mildew does not assume (Misra and Prakash, 1997).

Anthracnose of mango is an important disease of mango. it is found that the optimum temperature for infection of anthracnose is around 25°C. Moist conditions, frequent rains with temperature around 30-350C favour the disease development (Misra and Prakash, 1997). Infection progresses faster in wounded tissues, and in ripe fruits (Prakash et al., 1996; Misra and Prakash, 1997).

Fruit rots, which is favoured by higher humidity and rainfall may decrease due to less rain pattern. Therefore, it can be said that the change in climate will be having varied effect. However, adverse climate may harm host, make it weak and may predispose plant for various diseases.

2.10 Mango Resistance in Relation to Fruit Rot Diseases

The plant naturally has own defense mechanisms upon parasite infection. These are considered as defensive barriers to plant pathogen. Dynamic and ongoing evolutionary battles host-parasite have resulted in the utilization of highly specific and sophisticated attack strategies by the pathogen and equally elaborate defense responses by the host (Prusky, 1998). A successful pathogen must have the capability to overcome host defenses and initiate to attach under the physiological conditions prevalent (Prusky, 1998).

Plant cells contain preformed proteins, namely polygalacturonases inhibitor proteins, that can specifically and effectively inhibit polygalacturonases of fungal origin (Prusky, 1998). This has been proved in vitro by Labavitch et al. (1998) with extracts of many sorts of plant tissue, including a large assortment of fruits.

Fruit can restrict fungal colonization by (i) preformed barriers where deposition of phenolic polymers occurs, or (ii) by preformed antifungal compounds (Prusky, 1998). Preformed antifungal compounds tend to concentrate in the outer layers of the fruit hosts. In mango fruit, a mixture of antifungal compounds consisting of 5-12-cis-heptadecenyl resorcinol and 5-pentadecenyl resorcinol was found at fungi toxic concentration in the peel of unripen mango fruit that were resistant to Aternaria alternata (Droby et al., 1986, 1987). Also in this case, 5 substituted resorcinol occurred at sub fungi toxic levels in ripening fruit at the same time as symptoms of decay appeared in inoculated fruit (Prusky, 1998). The concentration of these compounds decreased faster during ripening in disease susceptible cultivars than in resistant cultivars (Prusky and Keen, 1993).

2.11 Bitter Leaf Plant (Vernonia amygdalina Del.)

Bitter leaf (Vernonia amygdalina) is derived from the leaves of a small ever-green shrub found all over Africa belonging to the family Asteraceace. It is well known as a medicinal plant for diabetes and fever (Adenuga et al., 2010). Vernonia amygdalina commonly called bitter leaf is the most widely cultivated species of the genus Vernonia which has about 1,000 species of shrubs (Muanya, 2015). It is vegetatively cultivated by seed or stem cutting at an angle of 45° and it is popular in most of West Africa countries including Nigeria, Cameroon, Gabon and Congo Democratic Republic. It is locally abundant in sloughs, home gardens and even all over places in the Southern part of Nigeria (Bonsi et al., 1995 and Burkill, 1985). Although most popularly used for food, it has also, been traditionally used for its medicinal properties. Bitter leaf is an important medicinal plant which has hypoglycemic and anti-diabetic properties (Kigigha, et al., 2015; Owen et al., 2011). Moreover, the extract has been found to mitigate chemical toxicity (Ikeh et al., 2014). Scientific studies show that bitter leaf is rich in bioactive compounds such as alkaloids, saponins, terpenes, flavanoids, phenolic acids that have various medicinal properties including treatment of microbial infections.

2.12 Scientific Classification of Bitter Leaf

The plant is scientifically classified as belonging to the Kingdom: Plantae; Phylum: Tracheophyta; Class: Magnoliopsida; Order: Asterales; Family: Asteraceae; Genus: Vernonia; and species: Vernonia amygdalina Del.

Source: Arctos plants.

CHAPTER THREE3.0 MATERIALS AND METHOD3.1 Study Area

The study was conducted in Wukari Town. Wukari is a Local Government Area in Taraba State as shown in (Figure 1). it is in the Southern Senatorial District Headquarters of Taraba State alongside Ibi, Donga, Ussa, and Takum Local Government Areas. It is located in north region of Nigeria at southern guinea savanna with coordinate’s latitude 7051’N-7.8500N and longitude 9047’E-9.7830E covering an area of 4,308km2 and having a population of 241,546 based on 2006 census, annual precipitation of 1205mm and it has average temperature of 26.80. A lot of agricultural products such as yams, maize and fishes can be found in Wukari town because of the people there are predominantly farmers. Taraba state is situated in the north eastern parts of Nigeria and lies within the Northern guinea savannah belt with annual rainfall of about 1201mm and varying temperature between 26.80c and 29.80c.

Fig. 1: Map of Nigeria showing the location of Wukari (study area).

3.2 Source of Samples

Two hundred (200) mango fruits were collected from four (4) different markets within Wukari Town from May-June, 2019. Simple random sampling technique was employed. All the samples collected from the markets were placed in a sterile polythene bags separately and labelled according to their names. The samples were taken to the Biological Sciences Laboratory, Federal University Wukari for further study.

3.3 Medium for the Isolation and Identification

The growth medium that was used for this study is Potato Dextrose Agar (PDA) (Smith and Onons, 1983). Potato powder was dissolved with distilled water in a conical flask and placed on a heater for 30 minutes to dissolve completely (3.9g per 100mil). It was autoclaved at 1210c for 15 minutes. The prepared medium was allowed to cool for 30 minutes, and after it has cooled, Potato Dextrose Agar (PDA) medium was poured aseptically into the bottom-half of the petri-dishes untilled solidification. After it has solidified, the slants were used for the growth of the fungi.

Small pieces, measuring 2mm2 each of infected tissue were cut off from the mango fruits with the help of sterile sharp knife. Pieces of diseased fruits were washed with distilled water and then dried using sterile filter paper. The pieces were separately transferred to sterilize Petri-dishes containing potato dextrose agar (PDA) medium and incubated at 280c for 5 days. This was followed by sub-culturing on PDA for 5-7 days, then sub-cultured were repeated on a new set of PDA plates, until pure culture of isolates were obtained.

3.4 Characterization and Identification of Isolates

The wet mounts of isolates in lacto phenol in cotton blue were examined microscopically and identified based on their colonial morphology, mycelia structure, spores and associated structures according to Alexopoulos et al. (2002).

A small portion of mycelia were introduced on a clean glass slide with an inoculating needle and then teased. Two drop of lacto phenol blue were added, covered with cover slip and observed under the microscope for hyphen nature and disposition of mature fruiting structure. The different characteristics of fungal isolates were recorded.

3.5 Pathogenicity Test

Healthy mango fruits were surface sterilized with 90% ethanol, and incisions were made on them using sterile 4mm cork borer, use to cut pellets of agar containing the culture of fungal mycelia of isolates. These fungi were then inoculated into the hole created on the healthy mango fruit in a laminar flow chamber. The inoculated wound was sealed with petroleum jelly. The inoculated fruits were placed in a clean polythene bag (one fruit per bag) each moistened with wet balls of absorbent cotton wool to create a humid environment and incubated at 280c for 3 days of incubation period. The causal agents were re-isolated from the infected fruits and compared with the original isolates (Akinmusire 2011).

3.6 Collection and Preparation of Plant Extract3.6.1 Sample Collection

Fresh leaves of bitter leaf were collected within Wukari Town. The leaves were washed thoroughly using tap water to remove impurities and air dried aseptically and allowed to dry at room temperature. The dried leaves were grinded into powdered form using a sterile mortar and pestle.

3.6.2 Extraction Process

The grinded Vernonia amygdalina was extracted using the process adopted by Abalaka (2012). 50g of the of the leaf extract was soaked for 24 hours in 500ml of ethanol. The extract was filtered using whatman No1 filter paper. This process was repeated to obtain a clear ethanol extract of plant.

3.6.3 Concentration of Extracts

Applying the techniques of (Mukherjee et al., 2011). The ethanol extract was diluted in ethanol to obtain various concentrations of the extracts as follows: 100ml of the extract was not diluted to obtain 100 percent concentration of the extract. Also, 80ml of the extract was diluted with 20ml of ethanol to obtain 80 percent concentration. The dilution procedure was continued to obtain lower concentrations of the extract as follows; 100, 80, 60, 40 and 20.

3.6.4 PDA-Extract Medium Preparation

Following the procedures of (Mukherjee et al., 2011). PDA culture media in conical flasks were sterilized in an autoclave at temperature of 1210C for 15 minutes. After autoclaving, about 20ml of the medium was poured in each 9cm sterilized Petri dishes. 2ml from each of the concentration of the extract (20, 40, 60, 80 and 100%) was dispensed into the Petri dish and agitated thoroughly with the melted PDA forming Potatoes Dextrose Leaf Extract Agar (PDLA). Mycelial discs were prepared using a cork borer (4mm diameter) from the tip of the isolates. One disc of the isolates was placed at the centre of a Petri dish after solidification of the PDLA. Each treatment was replicated for all concentrations of the extract. The medium without plant extract served as control. All the plates were incubated at 270C. Radial growths of test organisms were measured every 24 hours starting from the 4-6days after inoculation (a total of 3 days), with the aid of a meter rule. Percentage pathogen growth inhibition was calculated with respect to radial growth of pathogen on control plates (without leaf extracts) as reported by (Terna et al., 2016) as: .

Where, PRG = Percentage Radial Growth Inhibition of fungus; Pc = Radial growth of fungus in control plate; P = Radial growth of fungus in the presence of Vernonia amygdalina leaf extracts.

3.7 Statistical Analysis

The analysis of data was performed with Microsoft excel 2016(Windows XP) for the plotting of the bar charts. Descriptive analysis of Percentage pathogen growth inhibition for the fungal isolates were performed on the collected data.

CHAPTER FOUR4.0 Results

Three fungi isolates were identified. Rhizopus stolonifer, Rhizopus spp. and Aspergillus spp. are the fungi pathogens responsible for mango fruit rot in Wukari L.G.A., Taraba State (the study area). These pathogens are responsible for disease-related to the reductions of fruit quality and cause severe losses in yield of the fruit. The isolates were identified using the mycological atlas at the library of Department of Microbiology, University of Jos (Unijos), Nigeria. And characterized based on their colony morphology on PDA medium and microscopic characteristics.

Table 1: Showing result on identification of the isolates.

Fungi isolated

Colonial morphology of fungi on PDA

Microscopic characteristics

Rhizopus stolonifera

Colonies light grey, growing rapidly and filling the petri dish with dense cottony mycelium, producing mass of sporangia.

A bundle of sporangiophores was formed. Sporangiophores smooth-walled, aseptate, light brown, simple, arising in group of 3-5 from stolons opposite rhizoids. Sporangia globose and sub-globose with some flattened base (white at first turning black afterwards) and many spores.

Rhizopus spp.

Colonies of Rhizopus spp. grow very rapidly, fill the petri dish, and mature in 4 days. The texture is typically cotton-candy like. From the front, the colour of the colony is white initially and turns grey to yellowish brown in time. The reverse is white to pale.

Sparsely septate broad hyphae, sporangiophores, rhizoids, sporangia and sporangiopores are visualized. Sporangiophores are brown in colour. Sporangia are located at tip of the sporangiophores, the have flattened bases, sporangiopores is round to ovoid in shape.

Aspergillus spp.

Appeared cottony when young but later turned black completely or dark brown and look powdery.

The head darkly coloured, resulting in colonies that are black, dark brown. The spherical vesicle bears phialides over its entire surface and thus gives rise to a mop like head of conidia.

4.1 Pathogenicity test result

The isolates that were introduces into the fresh healthy mango fruits causes the mango to rot and there is reduction in weight of the fruit. And the re-isolated fungi from the fruits were viewed under the microscope and confirmed to be the same as the previous isolates (Rhizopus stolonifer, Rhizopus spp. and Aspergillus spp.).

Plate I: Image of mango fruits after the introduction of the isolated fungi.

4.2 Effect of Plant Extracts on Mycelial Growth of Fungal Isolates with time and concentration

All concentrations of ethanol plant extracts suppressed the mycelial growth of the three tested pathogens; the effect was proportional to the concentration of the extract. The highest inhibition for Rhizopus stolonifer and aspergillus spp. was at 60% concentration. However, the 20% and 40% concentrations of the extract gave the highest inhibition for Rhizopus spp. Statistically, there was significant difference in inhibition of radial growth in all the fungal isolates at all concentrations compared to the control (PDA medium containing the isolates without treatment), which have 0% inhibition on the fungi. The highest inhibition percentage was observed on Rhizopus spp. and Aspergilus spp. with 100% inhibition. (Fig. 3 and 4).

Fig. 2: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on radial growth of Rhizopus stolonifer in Wukari.

Fig. 3: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on Rhizopus spp. in Wukari.

Fig. 4: A bar chart showing the effect of ethanol extract of Vernonia amygdalina on Aspergillus spp. in Wukari.

CHAPTER FIVE5.0 Discussion, Conclusion and Recommendation5.1 Discussion

Generally, most fungi are considered pathogenic which could cause infections and allergies (Monso et al., 2004). Aspergillus spp. are known to produce several toxic metabolites, mycotoxin which is very important toxin worldwide because of the hazard it poses to plant and animal health (Peraica et al., 1999; and Petzinger and Weidenbach, 2002). Rhizopus stolonifer is one of the most important pathogenic fungi with serious postharvest damages in a variety of fruits. The fungus has a strong hydrolytic enzyme system and basically destroys cell walls (Tang et al., 2012).

The result obtained showed that the leaf extracts at their different level of concentrations tested were effective in inhibiting the growth of fungi pathogens of mango fruit. It was observed that the effect of bitter leaf extract proved to have antifungal effect in inhibiting the pathogens, this suggests that the plant contain active compounds/phytochemical that affect the growth of Rhizopus stolonifer, Rhizopus spp. and Aspergillus spp. hence agreeing with the report of (John et al., 2016), who reported that Vernonia amygdalina was effective against the mycelial growth of Fusarium oxysporum (32.26%), Rhizopus stolonifer (22.58%) and Geotrichumcandidum (45.16%). (Blake, 1985) also reported that the presence of certain allele-chemicals in plant extracts account for their detrimental effects on microbial cell division, cell elongation and nutrient uptake. (Harborne, 1989; Wiendenfield and Roder, 1991; Singh et al., 2003) also reported that such chemicals either dissolve the cytoplasm or render it inactive, and are also capable of penetrating the microbial walls and distorting routine microbial metabolic processes.

The result indicated that Vernonia amygdalina suppressed the growth of fungi at low concentration of the extract (20% and 40%) which correspond with the report of (Sangoyomi et al., 2010) who reported that extracts of Vernonia amygdalina were able to inhibit mycelial growth of fungi at low concentrations. Although, (Banso et al., 1999; and Martins et al., 2015) also observed that test extracts at increasing concentrations significantly inhibited the radial growth of fungal pathogens, the increase in fungal growth inhibition with increased extract concentrations could be linked to the buildup of toxic materials which tend to inflict more inhibitory reactions on affected fungal cells in higher concentrations.

5.2 conclusion

This work has identified three fungi to be responsible for the fruit rot disease of mango. Plant materials previously reported as good agro chemicals have also been found capable of reducing the effects of fungal in agricultural produce. Bitter leaf used in this study is capable of inhibiting fruit rot disease of mango at low concentrations, since they are strongly effective for suppressing the mycelia growth of the fungal pathogens causing this infection.

5.3 Recommendations

Therefore, it is recommended that fungicides containing low concentrations of bitter leaf extracts should be employed to inhibit the growth of fungi in mango, so as to reduce the prevalence of the infection and exposure of the plant to toxic chemicals. This will ensure good quality of mango and reduce the health hazard in Wukari.

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20%

DAY 1DAY 2DAY 356.6755.5653.3340%

DAY 1DAY 2DAY 352.225048.8960%

DAY 1DAY 2DAY 38075.5666.6780%

DAY 1DAY 2DAY 366.6763.3361.11100%

DAY 1DAY 2DAY 363.3356.6754.44

Concentration

% of inhibition

20%

DAY 1DAY 2DAY 310010010040%

DAY 1DAY 2DAY 310010010060%

DAY 1DAY 2DAY 37067.7865.5680%

DAY 1DAY 2DAY 367.7864.4461.11100%

DAY 1DAY 2DAY 371.1166.6763.33

Concentration

% of inhibition

20%

DAY 1DAY 2DAY 372.2266.6762.2240%

DAY 1DAY 2DAY 357.7855.5653.3360%

DAY 1DAY 2DAY 310095.5692.2280%

DAY 1DAY 2DAY 363.3361.1157.78100%

DAY 1DAY 2DAY 388.8986.6778.89

Concentration

% of inhibition