Faculty of Bioscience Engineering Academic year 2011 – 2012

CAUSES OF POSTHARVEST LOSSES OF MANGO AND SHEA FRUITS

IN NORTHERN GHANA, WEST AFRICA

Kassim SAEED

Promoter: Prof. dr. ir. Monica Höfte

Master’s dissertation submitted in partial fulfillment of the requirements for the degree of Master of Nutrition and Rural Development,

main subject: Human Nutrition

COPYRIGHT

“All rights reserved. The author and the promoters permit the use of this Master’s Dissertation for

consulting purposes and copying of parts for personal use. However, any other use fall under the

limitations of copyright regulations, particularly the stringent obligation to explicitly mention the source

when citing parts out of this Master’s dissertation.”

Ghent University, 20th January, 2012

Promoter The Author

Prof. dr. ir. Monica Höfte Kassim Saeed

Faculty of Bioscience Engineering Academic year 2011 – 2012

CAUSES OF POSTHARVEST LOSSES OF MANGO AND SHEA FRUITS

IN NORTHERN GHANA, WEST AFRICA

Kassim SAEED

Promoter: Prof. dr. ir. Monica Höfte

Master’s dissertation submitted in partial fulfillment of the requirements for the degree of Master of Nutrition and Rural Development,

main subject: Human Nutrition

i

ABSTRACT

Pre and Post-harvest diseases caused by fungal pathogens may develop on fruits and other products either

on the field or between harvesting and consumption. The threat of post-harvest disease influences the way

most horticultural crops are handled especially after harvest. Therefore the correct identification of the

causal pathogen is necessary for proper treatment of fruits, management and control strategies of

pathogenic fungi.

The primary aim of the present study was to contribute to the knowledge of postharvest losses of mango

(Mangifera indica) and shea (Vitellaria paradoxa) fruits in Northern Ghana where shea tree crops and

among other trees are widely distributed in the natural savanna vegetation as well the cultivation of

mangos. These trees are of not only economic importance but also as a source of food and nutrition, and

livelihood to the people of northern Ghana. The objective of this study was to isolate and identify

specific pathogens causing post harvest diseases of shea fruits and mangos.

Healthy, matured but unripe mango and shea fruits were collected from Tamale in the Northern region,

Wa and Kaleo in the Upper west region of Ghana. Isolations and identifications of the pathogens was

carried out in Ghent university, Belgium.

In the current study, pathogenic fungi including three members of Botryosphaeriaceae were identified

using molecular technique (DNA sequence analysis : ITS 4 and ITS 5 and the 5.8S rDNA) and

morphological characteristics (conidia). These Botryosphaeriaceae members are Lasiodiplodia sp.,

Botryosphaeria sp., Pseudofusicoccum sp., and the rest includes Colletotrichum gloeosporioides, Phoma

sp., Pseudofusicoccum sp., Phomopsis sp., and Pestalotiopsis sp. However Pseudofusicoccum sp. was

identified on both mango and shea fruits. Botryosphaeriaceae members were all identified on mango

whiles Phomopsis sp., Phoma sp. and Pestalotiopsis sp. were identified on shea fruits. To the best of my

knowledge, this is the first report of using both molecular method (DNA sequence analysis) and

morphological characteristics (conidia) to identify fungal pathogens of mango and the shea fruits in

Northern Ghana.

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WORD OF THANKS

I owe a debt of appreciation to many people who in diverse ways aided the process that culminated in the

preparation of this dissertation. My utmost gratitude goes to the all mighty Allah (God) for his guidance

and protection granted me during this study and throughout my stay in Belgium.

The largest share of my appreciation goes to Prof. dr. ir. Monica Höfte, the head of the

Phytopathology Department, Faculty of Bioscience Engineering, University of Ghent for her

relentless supervision of this work and providing me with research materials.

I extend my profound gratitude to the coordinator of Human Nutrition and Rural Development,

ir. Anne-Marie De Winter and also Marian Mareen and to all supportive staff of the Faculty who inspired

and guided me during my life as a student in the University and Belgium.

My heartfelt appreciation also goes to Ilse Delaere, the lab assistant of the Phytopathology department

whose enormous and crucial involvement in the practical work lead to the success of this study. Mention

must also be made to all the researchers in the Phytopathology lab for their help, friendliness and respect

showed me during the laboratory work for this study.

I will like to express my sincere thanks to Veronic Lewyllie, Katrien Deltombe and Tom Anaert for their

unprecedented love, care, material and financial support offered me for the duration of my studies in

Belgium.

My special thanks also goes to Tineke, my love friend, for the encouragement and editing of this report.

My earnest thanks and gratitude goes to my mum, family, numerous friends and relations whose moral

support provided the necessary zeal and strength for the accomplishment of this task but to those whose

names have not been mentioned, I am grateful to you all.

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TABLE OF CONTENTS

ABSTRACT I

WORD OF THANKS II

LIST OF TABLES V

LIST OF FIGURES VI

LIST OF ABBREVIATIONS VII

CHAPTER 1 : INTRODUCTION 1

1.1 INTRODUCTION 1

1.2. Overview of the study area 2

1.3 Rational of the study 3

CHAPTER 2 : LITERATURE REVIEW 5

2. SHEA AND MANGO 5

2.1 The Shea tree (Vitellaria paradoxa) 5

2.1.1 Uses and importance of the shea tree 8

2.1.2 Production of shea nuts 12

2.1.3 Pest and diseases of Vitellaria 13

2.2 Mango (Mangifera indica) 16

2.2.1 Uses of mango 18

2.2.2 Production of Mango 19

2.2.3 Pest and diseases of mangoes 20

CHAPTER 3 : MATERIALS AND METHOD 29

3.2 Sample collection 29

3.3 Media preparation 30

3.4 Isolations of fungal pathogens 30

3.5 Identification of postharvest fungi 31

3.6 Amplification of rDNA ITS 33

CHAPTER 4 : RESULTS 36

4.1 Sample collection and isolation 36

4.2 PCR (polymerase chain reaction) analysis 38

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4.4 DNA ITS sequences analysis 38

4.5 Cultural and growth characteristics 40

Characteristics on PDA and matheur media of the mango isolates 40

Characteristics on PDA and matheur media of shea isolates 43

CHAPTER 5 : DISCUSSION 47

MANGO ISOLATES 47

SHEA ISOLATES 50

DIFFICULTIES 52

CHAPTER 6 : CONCLUSION AND RECOMMENDATION 54

LIST OF REFERENCES 56

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LIST OF TABLES

Table 2. 1 World production estimates for some selected tree seed crops 13

Table 2. 2 Diseases of shea fruits 14

Table 2. 3 Fresh mango exports from Ghana, 2005-2008 (in tones) 19

Table 2. 4 Diseases of mangoes caused by pathogens (fungi and nonfungal agents) 21

Table 4. 1 Various Groups of isolates of mango and shea fruits sorted 37

Table 4. 2 Identified host pathogen of isolate from nucleotide blast on NCBI website of mango and shea

fruits groups after consensus with BioEdit 39

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LIST OF FIGURES

Figure 2. 1 (A)Small grey dots show areas of shea trees (Vitellaria paradoxa) in West Africa. (B) Map of

Northern Ghana 6

Figure 2. 2 Shea trees (A) and shea fruits (B) 7

Figure 2. 3 Bark of vitellaria (A). Leaves of vitellaria with fruits (B) 8

Figure 2. 4 Shea butter: yellow (A) and white (B) 11

Figure 2. 5 Shea nuts with shells 12

Figure 2. 6 Pestalotia leaf spots symptom on Vitellaria. paradoxa at Bole, Ghana 15

Figure 2. 7 Fruits abortion caused by the larva 15

Figure 2. 8 Regions of Ghana (written in green- A). Ecological zones of Ghana (B) 17

Figure 2. 9 Mango fruit (A). Mango tree with fruits (B) 19

Figure 2. 10 (A)Mango anthracnose symptoms on mangos, (B) the “tear stain” effect, (C) linear necrotic

regions lending an alligator-skin effect, often associated with cracking of the epidermis 23

Figure 2. 11 Disease Cycle of anthracnose 24

Figure 2. 12 (A) Stem end rot covering almost the entire fruit. (B)Stem end rot spreading from the end of

the stem 25

Figure 2. 13 Disease cycle of stem end rot of mango 26

Figure 3. 1 Map showing regions where fruits were collected 29

Figure 3.2 (a) blotted tissues on sterile tissue paper; (b)plated tissues; (c) fungal growth of mango tissues

on Mathuer and PDA media after 6 days of incubation; (d) fungal growth of shea tissues after 6 days of

incubation 30

Figure 3. 3 Erlenmeyer flasks at 10th day of incubation with the growth suspension 32

Figure 3. 4 (A) 1% agarose ‘slab’ gel before UV illumination. (B) Fragments from PCR of the rDNA ITS

region and 5.8S rDNA 33

Figure 4. 1 Patterns and length of bands shown by the isolates 38

Figure 4. 2 Cultural characteristics on PDA, matheur medium and morphology of conidia of mango

isolates. 42

Figure 4.3 Cultural characteristics on PDA, matheur medium and morphology of conidia of mango

isolates. 44

Figure 4. 4 Spores of some groups of the isolated pathogens earlier identified microscopically 45-46

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LIST OF ABBREVIATIONS

CBEs Cocoa Butter Equivalents

CBRs Cocoa Butter Replacers

CRIG Cocoa Research Institute of Ghana

DNA DeoxyriboNucleic Acid

IF Infra Red

INERA Institut de l’Environnement et de Recherches Agricoles

ITS Internal Transcribed Spacers

FAO Food and Agricultural Organization

GCMB Ghana Cocoa Marketing Board

GNA Ghana News Agency

GSS Ghana Statistical Service

GoG Government of Ghana

NCBI National Center for Biotechnology Information

NMIMR Noguchi Memorial Institute for Medical Research

PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar

PDB Potato Dextrose Broth

UE/R Upper East Region

UNSSCN United Nation System Standing Committee on Nutrition

USAID United States Agency for International Development

UV Ultra Violet

UW/R Upper West Region

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CHAPTER 1 : INTRODUCTION

1.1 Introduction

The Food and Agricultural Organization (FAO) describes the loss of foods in the post-harvest system as

not new since it has always been a problem for humankind. Post-harvest losses of fruits and vegetables

are more severe in developing countries than in well developed countries. The recent rapid increasing

populations in the poorest countries in which there is already food scarcity requires ways and means to

conserve mankind's food supply in order to alleviate hunger and malnutrition. Meanwhile, some far-

sighted individuals have been drawing attention to the problem of post-harvest losses for many years.

Estimates of production losses in developing countries are hard to tell, but some put losses as high as 50

percent, or half of what is grown (FAO, 1980). Reduction in this wastage, if economically can be avoided,

would be of great significance to growers and consumers alike.

Minimizing postharvest losses is very essential because it will ensure food adequacy both in quantity and

quality to every inhabitant on this earth and some projections are that world population will rise from to

8.3 billion in 2025. Global production of vegetables reached up to 486 million tons, while that of fruits

reached 392 million tons (Panhwar, 2006). Fruits are highly perishable products and their quality can be

affected by post-harvest practices such as handling, transportation, storage and marketing (Naureen et al.,

2009). Decrease in post-harvest losses can therefore minimize cost of production, improve trade and

distribution, enhance food security especially in developing countries, lower the price for consumers and

as well increase farmers income.

Ghana, a developing country with agriculture as the main stay of the economy, undoubtedly faces

postharvest losses especially in highly perishable crops. Postharvest diseases and poor postharvest

handling practices can be major causes of postharvest losses. This remains a serious challenge facing the

country as it still struggles with developmental issues most importantly in the agricultural sector.

Mangoes and shea fruits are among those that are affected and sometimes results in substantial losses.

This does not only leads to economic loss but also has a direct negative impact on food security and

nutrition of individuals.

Fruits can play a major role in combating food insecurity, reducing malnutrition and undernutrition

particularly in developing countries where there is food scarcity and absolute poverty. Fruits play an

important part in the diet of individuals, hence its importance in nutrition cannot be over emphasized.

They contain essential minerals and vitamins which are needed for a healthy life. Deficiency in some

important nutrients from fruits can result in adverse health effects.

2

According to the Ghanaian Journal (2011), GH¢700,000 or about 327,000 euro is lost annually due to

losses (some caused by disease pathogens) and inadequate post harvest management particularly of

perishable produce. Ghana loses about 20 to 50 per cent of fruits, vegetables, roots and tubers and about

20 to 30 per cent of cereals and legumes annually, which has resulted in the country experiencing food

insecurity.

With the exception of direct economic concerns, diseased fruits can present a potential health risk. Some

fungal genera such as Pencillium, Aspergillus, Alternaria and Fusarium are known to produce

mycotoxins under certain favourable conditions. Mycotoxin is a substance produced as secondary

metabolite by fungi that grow on fruits, vegetable, and cereals in the field or in storage. Some important

mycotoxins includes aflatoxins, ochratoxins, patulin and alternaria toxins. The greatest risk of mycotoxin

contamination takes place when diseased produce (fruits, vegetables) happens to be used in processed

food or animal feed because some of them are highly stable during processing and can reach the

consumer (Barkai-Golan and Paster, 2008).

UNSSCN (2004) reports that there is an incredible increase in the number of people who are

undernourished. Again, there are information of 33% of African population being suffering from chronic

malnutrition, food insecurity and getting less than the recommended calorie intake of 2100kcal

(Boussarde et al., 2005). In order to reduce food insecurity, eliminate hunger and malnutrition in

developing countries like Ghana, alternative control measures to reduce post harvest losses of fruits and

other sources of nutritious food needs to be identified.

1.2. Overview of the study area

Ghana is a coastal country located in the west coast of Africa endowed with natural resources. It shares

boundaries with Burkina Faso to the North, Ivory Coast to the West, Togo to the East and to the South by

the Gulf of Guinea. It has a coastline of more than 565 km and covers a total land area of about 238,500

km². Ghana is divided into 10 administrative regions and 138 districts. There exist three ecological zones:

the sandy coastline; the densely forested of the middle belt and the Western part; and the savannah in the

North. Ghana has a population of about 21.9 million with agriculture predominantly traditional and

remains the main sector of employment (FAO, 2009). The type of weather in Ghana is tropical,

determined by the harmattan - wind from Sahara - and the monsoon from the South Atlantic and the

average annual temperature is about 26ºC, though temperatures can reach 40 ºC in dry months (GSS et

al., 2004).

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The Northern sector of Ghana covers about 2/3 of the total land area and lies in the dry Sudan and Sahel

savannahs of West Africa where rainfall is very low, inconsistent and unpredictable (Fobil, 2010) and

administratively, it comprises the Upper West, Upper East, Northern Regions with Wa, Bolgatanga and

Tamale as their regional capitals respectively (figure 2.1 B). The shea tree (Vitellaria paradoxa), mango

(Mangifera indica), baobab (Adansonia digitata) and Dawadawa (Parkia filicondia) are among the most

common, economically and nutritionally important tree crops found in the three regions are (Siaw, 2001;

GoG, 2005). Some of these trees also occurs naturally in the savanna vegetation of Northern Ghana. The

main economic activities mostly found in the northern sector is agriculture and is generally subsistence.

The annual distribution of rainfall is unimodal, thus comes once a year, in northern Ghana.

The Northern Region is the biggest region in Ghana and shares boundaries to the north by both Upper east

and Upper west regions. The natural vegetation is Guinea savannah. The maximum day temperatures

between November and February can reach 33° C to 37° C and minimum night temperatures vary

between 20° C and 22° C.

The Upper West region is located in the north-western part of Ghana and it shares borders to the north

and west by Burkina Faso, south by Northern region and to the east by the Upper East region. Food

production is poor which sometimes result in seasonal food insecurity. The vegetation is generally semi-

savannah with light undergrowth and scattered shrubs. The climate is tropical with an average minimum

temperature of 22.6 ºC and maximum of 40 ºC.

1.3 Rational of the study

The Northern part of Ghana especially the Northern and Upper east regions face problems of acute and

chronic malnutrition, undernutrition especially among young children (FAO, 2009). These regions are

also stricken with poverty for decades coupled with problems of food insecurity. A report of 2005 by the

Government of Ghana (GoG) reveals an inequality of poverty which indicates that five out of ten regions

in Ghana had more than 40% of their population living in poverty in 1999 while the worst affected being

the three northern savannah regions (Upper West, Upper East and Northern Regions). The report further

discloses that nine out of ten persons in the Upper East, eight out of ten in the Upper West, seven out of

ten in the Northern region and five out of ten in the Central and Eastern regions were classified as poor in

1999 (GoG, 2005).

4

Problems such as food insecurity can be caused by periods of long drought, but also postharvest losses of

perishable crops such as mangoes and shea fruits are a contributing factor. Postharvest losses can be due

to diseases, poor infrastructure and inadequate postharvest practices and thus can lead to substantial losses

of these fruits to almost nothing during the lean season (period of low food availability) further

compounding the problem of frequent food shortages, poverty, diseases bringing about malnutrition and

undernutrition problems. More so, the rainy season in the North is unimodal (comes once in year)

compared to the southern part where is bimodal (twice). This further increases the risk of food shortages

because the already available ones are not preserved well particularly the fruits due to postharvest losses.

Mango (Magnifera indica) and shea fruits (Vitellaria paradoxa) are among the important crops not only

nutritious, but also economically important to the people of northern part of Ghana.

Not much attention has been paid to the prevention and minimizing postharvest losses of these perishable

crops especially the shea and mango fruits due to diseases caused by some pathogens leading to high

losses. In spite of the local, national, nutritional and economic importance of these fruits, little is known

about both pre and postharvest diseases of the fruits (mango and shea fruits) and needs more research and

that is why this dissertation seeks to explore. lf therefore these losses can be reduced by appropriate

prevention strategies of the disease through further research and good postharvest practices, it will help

improve the food security situation and food availability in Northern Ghana and subsequently reduce the

undernutrition and malnutrition problems. The huge economic loss to the country will also be minimal.

AIM AND OBJECTIVE OF THE STUDY

Aim

- To contribute to the knowledge of the causes of postharvest losses of shea fruits and mangoes in

Northern Ghana and the country as whole in order to improve food security, improve nutrition, reduce

hunger and poverty in Northern Ghana

Objective

- To identify and isolate specific pathogens causing postharvest diseases of mangoes and shea fruits

5

CHAPTER 2 : LITERATURE REVIEW

2. Shea and Mango

2.1 The Shea tree (Vitellaria paradoxa)

The shea tree recently has gained importance as an economic crop because of the soaring demand for its

butter, both domestically and internationally. In respect of the need to find replacements for the expensive

cocoa products, and to make best use of the economic exploitation of the enormous shea resource in

Ghana, the Cocoa Research Institute of Ghana (CRIG) started scientific research into the cultivation and

processing of shea nuts (Fobil, 2010).

The shea tree also known as Vitellaria paradoxa, widely known under its synonym Butryospermum

paradoxum belongs to the Sapotaceae family. It is considered a monotypic genus with two subspecies

stretching nearly 5,000 km across the African savanna (Marnaz et al., 2004). The classification history

and synonymy of the shea tree followed a very twisted evolution since the oldest specimen was first

collected by Mungo Park on May 26, 1797 before finally arriving at the name Vitellaria with subspecies

paradoxa and nilotica (Fobil, 2010).

The shea tree in nature grows in the wild and occurs in East Africa, ranging through southern Sudan,

northern Uganda, western border of Ethiopia, and northeastern corner of the Democratic Republic of

Congo. Vitellaria again ranges from eastern Central African republic to the west within a few kilometers

of the Atlantic coast of Senegal (Maranz et al., 2004). It can be found in 18 countries (Benin, Ghana,

Chad, Burkina Faso, Cameroon, Central African Republic, Ethiopia, Guinea Bissau, Cote D’Ivoire, Mali,

Niger, Nigeria, Senegal, Sierra Leone, Sudan, Togo Uganda, Congo and Guinea) across the African

continent of 500 - 750 kilometer wide and extends to about 5000 kilometer long across these countries

(Moore, 2008) (figure 2.1 A).

The Food and Agricultural Organization (FAO, 1988a) reports that shea tree in Ghana occurs widely in

the Guinea savannah and less in the Sudan Savannah. The tree can be found over almost the entire area of

Northern Ghana, covering about 77,670 km² in West Dagomba, South Mamprusi, West Gonja, Tumu,

Lawra, Wa and Nanumba with East Gonja having the densest (figure 2.1 B). Meanwhile there is sparse

shea tree cover found in Brong-Ahafo, Ashanti, and Eastern and Volta regions in the south of the country

6

Figure 2.1 (A) Small grey dots show areas of shea trees (Vitellaria paradoxa) in West Africa. White dots are shea varieties in East Africa – Source: Peace

Corps Ghana, 2008. (B) Map of Northern Ghana. Large black dots indicates regional capitals and small black dots district capitals.

Shea tree usually grows to an average height of about 15m long with profuse branches and thick waxy

and deeply fissured bark (figure 2.3 A) that makes it fire resistant (Ezema et al., 1992) although on rare

occasions they have been recorded to grow up to 25 meters (Moore, 2008). The leaves are green and are

arranged spirally, mostly in dense clusters at the tips of branches (Nikiema & Umali, 2007).

In general it is accepted that the tree reaches maturity and produce fruits between 3 to 5 years while other

people argue it takes 15 or 20 years to fruit (McNally, 2008). On the contrary, some writers as Nikiema &

Umali (2007) states that, the shea tree (Vitellaria) begins flowering at 10–25 years and maturity is

reached at 20–45 years. They further emphasized that its life span is between 200 to 300 years. Vitellaria

(shea tree) starts to bear fruits at maturity and flowering begins by early November, with picking and

gathering lasting from April to August every year. When the fruits ripe, they fall under the tree by their

own weight and are collected and picked by hand (Fobil, 2010).

Shea tree (figure 2.2 A) is a relatively versatile species and its optimal growth conditions is reflected in its

large geographical distribution. It has favourable conditions under which growth will be fastest and

healthiest. Vitellaria flourish on dry, sandy soils that have a good humus cover, but can be found on a

range of soil types. Its extensive and relatively shallow rooting system makes it tolerable to extended dry

seasons which can last up to 8 months as well as the infrequent droughts experienced in the savannah

zone. On the other hand, its shallow rooting system does not provide much anchorage and makes them

particularly susceptible to being blown over in strong rainstorms which regularly occur in the wet season

A B

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Figure 2. 2 Shea trees (A) and shea fruits (B) – Source: field survey, July 2011, Ghana

(Moore, 2008). Their availability and existence are also affected by bushfires, felling of trees for firewood

and poor farming practices.

Shea fruits (figure 2.2 B) serve as an important source of food for many organisms and other animals

including birds, bats, elephants, sheep and pigs (Marnaz et al., 2004; USAID 2004). The fruits also

contribute to food security in northern Ghana, mainly for the rural poor, especially since their ripening

happen together with the lean season of food production (Fobil, 2010). The value of the shea tree to the

economy of Ghana became more significant in the early 1970s when it was announced that its vegetable

fat can be use in cocoa butter equivalent (CBEs) production and in the cosmetic and pharmaceutical

industry (Moore, 2008). Therefore the Ghana Cocoa Marketing Board (GCMB) in collaboration with the

CRIG took on the job to research into the plant (Vitellaria paradoxa). But the slow growth and late

maturation has discouraged the Vitelleria in plantations (Nikiema & Umali, 2007).

B A

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Figure 2. 3 (A) Fissured Bark of Vitellaria. - Source: Nikiema & Umali, 2007. (B) Leaves of vitellaria with fruits - Source: field July, 2011

2.1.1 Uses and importance of the shea tree

Apart from the crucial role the shea fruit play in its highly nutritional capacity, food security and

availability during the lean season (period of low food availability) in northern Ghana, it has many other

uses, for instance the shea butter extract from the nuts has local, national and international uses.

The production of shea nut and processing of the butter serves as one of the main sources of employment

for the rural women and children who are engaged in gathering it. Shea butter is an important edible oil

for the people of northern Ghana and much of Western Africa, being the most essential source of fatty

acids and glycerol in their diet (Saul et al., 2003). It is also useful in the pharmaceutical and cosmetic

industries as an important raw material and a precursor for the manufacture of soaps, candles, and

cosmetics (Adomako, 1985).

Shea butter (figure 2.4) is applied as a tame for the treatment of sprains, dislocations and the relief of

minor aches and pains. Some applications include its use as an anti-microbial agent for promotion of

rapid healing of wounds, as a pan-releasing agent in bread baking and as a lubricant for donkey carts

(Marchand, 1988). In a traditional setting, shea butter of low quality is used as an illuminant (or fuel, in

lamps or as candles).

A B

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2.1.1.1 Nutritional and health benefits

The growing importance of the shea butter has frequently overshadowed other uses of the tree particularly

the value of the fruit pulp. Though the nuts being an essential export commodities, its fruit pulp

nevertheless is widely consumed. It also plays a major role in the local economy and diet as well as

occupies an important period of time in the annual local dietary cycle (Maranz et al.,2004).

The edible part of the shea fruit is extremely nutritious and has important nutrients for the human body.

The fruit provides important source of food for communities and rural poor particularly at the period of

food shortages, hunger and other catastrophes. In addition to providing health benefits, it also provides

some income (FAO, 2007; Okafor ,1985).

Fobil (2010) describes that the green fruit has a fleshy edible pulp, which contains 0.7-1.3g protein and

41.2g carbohydrate and is sweet. The pulp is also a rich source of ascorbic acid and has 196.1mg/100g

compared with 50mg/100g in oranges. The iron and calcium content compares very well with raspberries:

1.93mg/100g as against 0.92mg/100g for iron, and 36.4mg/100g as against 26mg/100g for calcium

(Fobil, 2010).

Furthermore, FAO estimates that B vitamins are also present and the sugar content varies from 3-6%,

equally distributed between glucose, fructose and sucrose. These nutrients and mineral composition are in

no doubt very important to the diet and growth of an individual hence its befitting role in nutrition (FAO,

1988b).

Okullo et al., (2010) also analysed the mineral composition and reported that, the fruit pulp contains

significant and adequate amount crude proteins, crude fibre, Vitamin C and nutrients (Ca, K, Mg, Na

and Fe) and is equivalent to other edible fruits like mango and stated that it must therefore be promoted in

human nutrition. They further explain that the Na/K ratio (0.14-0.35) of the pulp also makes the shea

fruit an important resource for managing high blood pressure which is an emerging non-communicable

disease in most developing countries (Okullo et al., 2010). The reason is that low consumption of K and

Ca stimulates Na-induced hypertension and an increase intake of K may reduce blood pressure by

increasing urinary sodium excretion. Calcium intake lowers the risk of osteoporosis and gives strong

bones (Van Camp et al., 2009). Again deficiency in Fe can result in anemia especially in children and

pregnant women. These potentials are a good news in improving the health and nutritional status of

individuals and communities with these health problems.

Moreover, a similar study on the nutrient composition of the shea fruit pulp by Maranz et al., (2004)

showed many shea tree fruit pulp had potassium more than 1000mg/100g and has a rich source of calcium

10

(141mg/100g) compared with marula (35mg/100g) and mango (10mg/100g).They also found out that

copper is the only mineral that was deficient and phosphorous, zinc and magnesium are comparable to

marula.

The shea butter (figure 2.4) is an extract from the nuts and constitutes the greatest fraction of oil intake in

most homes in the Northern and Upper regions of Ghana (USAID, 2004).

Lastly, a decoction of young leaves taken from the flora of shea tree is used as vapour bath to treat

headaches and migraines in Ghana. The leaves in warm water form a foamy opalescent liquid, with which

a patient’s head is bathed (CRIG, 2002).

2.1.1.2 Industrial/international uses

The significance of the shea tree to the economy of Ghana grew to be even more important in the early

1970s when it was made known that it was one of only six plant species whose vegetable fat can be used

in the manufacture of Cocoa Butter Equivalents (CBEs) in chocolate as well as being a valued ingredient

in the pharmaceutical and cosmetics industries (Official Journal of the European Communities, 2000).

The shea tree at present produces the second most important oil crop in Africa after palm oil, but as it

grows in areas unsuitable for palm, it takes on primary importance in West Africa and in regions where

yearly rainfall is less than 1000mm (Poulsen,1981). However, it goes down in popularity in the urban

areas within these regions due to the pungent odour it emits, when it becomes rancid (Ayeh, 1981b).

Vitellaria also has a great, untapped capacity for producing abundant quantities of sap that can constitute

an important source of raw material for the gum and rubber industry (Fobil, 2010). According to Hall et

al., (1996), the shea nut (figure 2.5) out of which the shea butter is produced has numerous industrial

applications, but the bulk of kernels (nuts) (about 95%) provide an important raw material for Cocoa

Butter Replacers (CBRs), and are used for manufacturing chocolate and other confectionery. They

emphasized that its minor uses includes cosmetics and pharmaceuticals. The Unilever (UK), Arhus

(Denmark), Fuji Itoh and Kaneka-Mitsubishi (Japan) and Karlsham (Sweden) shares the export market for

CBRs.

Chalfin (2004) clarified that shea butter is usually less expensive than cocoa butter and also adds

durability to the chocolate, making it less crumbly and more uniform. Moore (2008) explained that the

ongoing debate of mass significance to the shea butter industry is about the levels of purity desirable to

officially call it chocolate. Whiles some countries like the United States, France, Belgium do not permit

11

Figure 2. 4 (A) Yellow shea butter and (B) white shea butter. Source : (http:/www.honesttownbeauty.com/upload/Sheabutter

_large_3.jpg)

any CBRs in chocolate production, others like Denmark, Sweden, Switzerland do allow up to 5 percent

of CBR content and other Eastern European countries allow up to 15 percent of CBR content (Vermilye,

2004).

Despite the fact that, France and Belgium do not allow shea butter as a CBR, they do allow its use as

CBR for cakes or sweets (Hall et.al., 1996). Shea nuts are also more and more being exported for the

utilization in the cosmetics industry as a constituent in lotions, makeup, baby ointments, hair care

products and soaps (Akosah-Sarping, 2003). Nonetheless, only about 20% of shea nuts are processed

prior to exportation . The reason is, it is less expensive for industries to purchase unprocessed nuts and

produce shea butter from them in their mechanized processing plants as opposed to buying processed

nuts already in shea butter form (Moore, 2008).

2.1.1.3 Indigenous/Local use

Hall et al. (1996) have since reported about the French explorer Roger Caillie who travelled across

Western Africa in 1830 describing the traditional shea butter uses : “the indigenous people trade with it,

they eat it and rub their bodies with it; they also burn it to make light; they assure me that it is a very

beneficial remedy against aches and pains and sores and wounds for which it is applied as an unguent”.

Caillie’s statement clearly shows that the traditional application of shea butter have not changed much

since 1830. Regardless of being increasingly substituted by commercially produced lotions in many

communities, shea butter is traditionally used as a skin and hair moisturizer and for protection against the

sun (Ezema & Ogujiofor, 1992).

A B

12

Figure 2. 5 Shea nuts with shells – Source: Ghana business News (2011)

Shea butter has a high melting point (between 32-45°C) close to body temperature. An attribute that

makes it suitable as a base for ointments and medicines. It also used to treat horses internally and

externally for girth galls and other sores (Fobil, 2010). The healing properties and effects of shea butter

are thought to be somewhat attributable to the existence of allantoin, a substance known to trigger the

growth of healthy tissue in ulcerous wounds (Wallace-Bruce, 1995). It is also traditionally smeared on

pregnant women during childbirth, on new born babies and adolescents because of its relieving effects

(Moree, 2008).

2.1.2 Production of shea nuts

FAO (2006) approximate that the main exporting countries Ghana - world’s largest producer of nuts

between 2000 and 2003 - Benin, Burkina Faso, Togo exports about 650,000 tons of shea nuts annually.

Shea nuts are exported either by the nut themselves after the roasting process, or the nuts are processed

into shea butter within the country of origin, and then exported (Boffa, 2000).

In 1998 Africa earned 10.5 million dollars from 56,000 tons of shea nuts exported, of which 60% came

from Ghana (Akrofi and Amoah, 2009). Even though tree seed crops such as cocoa and cashew are

widely produced and exported, the estimates includes 7 out of the 19 producer countries and the figures

may not be extremely reliable, but they do highlights on the relative importance of shea (table 2.1)

(Maranz et al.,2004).

13

Table 2. 1 World production estimates for some selected tree seed crops (Maranz et al., 2004)

Tree Botanical name Product Metric tones

Cocoa Theobroma cacao L. Beans 2,830,742

Cashew Anacardium occidentale L. Nuts 1,698,895

Shea Vitellaria paradoxa Nuts 647,500

Pistachio Pistacia Vera L. Nuts 548,759

Kola Cola spp. Nuts 217,500

2.1.3 Pest and diseases of Vitellaria

A number of pests and diseases are reported to attack the shea plant causing its growth retardation which

affects the yield of fruit production and nuts quality. Damage to the leaves is caused by larvae Cirina

butryospermi, Loranthus spp. and Anarcridium moestum. Larvae of Mussida nigrioella and Ceratiris

silverstrii infest the pulp of mature fruits (Orwa et al., 2009).

Most of the diseases affecting the fruit begins on the field and could even sometimes be infected before

dropping. The fruits are usually not harvested but picked or collected after dropping on the ground which

could cause wounds making it more susceptible for pathogenic infestation. If picking is delayed the fruit

become infected. The pulp (0.7-1.3g of protein and 41.2g of carbohydrate) is sugary and makes it very

sweet (Fobil, 2010) which attracts a lot of pathogens and can enter the kernel and infect the nut. Some of

these pathogens ought to be investigated and that is one of the purpose of this research study. This is

because there is little information and knowledge about the diseases of shea plant in Ghana despite its

local and national importance (Akrofi and Amoah, 2009).

Tapinanthus (African mistletoe), a plant parasite genus is known to be the most important cause of tree

mortality in the northern limit of the savannah in Burkina Faso and Mali. Parasites in addition reduce the

growth of the distal ends of the branches, affects wood quality and increases susceptibility to attack by

pathogens. Infestation is also reported to affect fruit production (Orwa et al., 2009).

Leaf spot is a disease that affect the shea tree and Pestalotia is found to be one of the causes of leaf spot

disease (table 2.2). Akrofi and Amoah (2009) reported in their research that a range of fungi was isolated

from the infected leaves but only Pestalotia spp. was found to be the causal pathogen of leaf spot of

14

Vitellaria paradoxa. They further explains that infected trees of the shea were not killed but are inactive.

Little is known about the pre or postharvest diseases of shea fruits and that is one of the objectives this

study intends to look at. Fusicladium butyrospermi is a microorganism that can also cause leaf-mosaic

disease (table 2.1) of shea tree (Orwa et al., 2009).

Akrofi and Amoah (2009) mentioned the diseases (leaf spot) can have adverse effect on yield and can be

due to the reduction in photosynthetic capacity and efficiency of infected leaves. They explained that as

leaf spot increases in plants, effective leaf surface area decline implying that seriously infected leaves add

very little to the growth of the plant and hence the negative impact on nut yield observed in Vitellaria

paradoxa.

Moreover, it is reported that during kernel (nut) development in trees, nuts serve as high-energy sinks,

drawing some of the photosynthates from leaves on nut-bearing terminals (Davis and Sparks, 1974). A

similar response may be occurring in V. paradoxa and this may perhaps account for the reduction in yield

of the infected trees.

PESTALOTIOPSIS

Pestalotiopsis sp. which belongs to the same family Amphisphaeriaceae as Pestalotia also causes of

diseases of leaf spot, scabby fruit canker, tip blight, needle blight, gray blight on a range of plants

(Pirone, 1978). It has been identified to be the causal organism scab diseases in guava in Hawaii (Keith et

al., 2006). They are also thought to be opportunistic pathogens that affect stressed plants (Coyier et al

1987). Meanwhile Pestalotia and Pestalotiopsis is not yet known whether they are distinct genera of not

and are somewhat similar in their morphology (Maharachchikumbura et al., 2011).

Table 2. 2 Diseases of shea tree (Orwa et al., 2009)

Disease Pathogen Family name

Leaf spot Pestalotia heterospora

Amphisphaeriaceae

Leaf - mosaic Fusicladium butyrospermi Venturiaceae

15

Figure 2. 6 Pestalotia leaf spots symptom on Vitellaria. paradoxa at Bole, Ghana - Source:

Akrofi and Amoah 2009

Figure 2. 7 Fruits abortion caused by the larva - Source: Institut de l’Environnement et de Recherches Agricoles (INERA)

2.1.3.1 Symptoms of diseases (leaf spot)

The leaf spot diseases (figure 2.6) attacks the leaves of the shea fruit. The spots are brown and are spread

on the leaves and reduces the effective leaf surface area. This affects the photosynthesis of the plant and

thus fruit production.

Insects larvae of Salebriasp, Pyralidae, Lepidoptera also cause fruits abortion on the plant making the

fruit not reaching its maturity.

16

There is not much available information on the control of the shea tree diseases since there is also limited

information about the diseases. The tree however grows naturally in the wild but is mostly found in

human inhabitation. However there is ongoing research on how to propagate the tree or domesticate it.

Some few information on the control strategies by INERA (Institut de l’Environnement et de Recherches

Agricoles) in Burkina Faso includes girdling and pruning.

This is done by cutting away the bark and cambium in a ring around an infected branch. This will

interrupt the circulation of nutrients from the infected parts to unaffected parts of the branch.

Pruning (cutting away unwanted or excessive branches) can also be done as a means of controlling

mistletoe and the improvement of shea tree’s fruit production. When pruned, it will prevent the mistletoe

parasite from climbing the tree.

2.2 Mango (Mangifera indica)

Mango belong to family Anacardiaceae and the genus Mangifera. There are several species of genus

Mangifera that bear edible fruit. The majority of the fruit trees (figure 2.9 left) that are commonly known

as mangos belong to the species Mangifera indica. Mangifera originates from tropical Asia, with larger

number of species found in Borneo, Java, Sumatra, and the Malay Peninsula. The most-cultivated

Mangifera species, M. indica (mango), originates from India and Myanmar (Bally, 2006).

Mango has turned out be naturalized, adapted and is cultivated throughout the tropics and subtropics. Its

spread and naturalization has occurred in line with the spread of human populations, and as such, mango

plays a significant role in the diet and cuisine of many diverse cultures. There are over 1,000 named

mango varieties throughout the world, which is a indication of their value to humankind (Bally, 2006).

Mangifera is a perennial, evergreen, and branching tree which can grow to a height of 30-40 m tall. The

trees can flower after 5-7 years but grafted and propagated trees which are generally smaller can flower

after 3-4 years (Ploetz et al, 1994). Mango can grow on a wide range of soils (optimal pH 5.5–7.5.) and

are relatively tolerant to drought. They are also tolerant to low temperatures (0 °C - frost), optimum

growth temperatures (24–27 °C) and a wide range of rainfall (annual 400–3600 mm) conditions (Bally,

2006). Its adaptation throughout the world makes its existence evident in Ghana especially in the northern

part of Ghana where rainfall is once a year followed by a period of drought and the mango flourishes very

well in these regions.

17

Even though the mango trees are all over Ghana, commercial productions are found in two typical agro-

ecological zones (figure 2.8 B). They are the Northern Ghana around Tamale and Southern Ghana around

Greater Accra, Eastern and Volta Regions (FAO, 2009).

The Ghana News Agency (GNA) reported that, the country has immense comparative advantage for the

cultivation of grafted mango because most of the lands of the coastal savannah, northern Ashanti, the

transitional zones (figure 2.8 A) of Ashanti and Brong Ahafo regions, the northern Volta region, and the

whole of Northern, Upper East and Upper West regions are suitable for mango production that meet

international quality specification (figure 2.8 A). These areas have abundant moisture and hot

temperatures that are favourable for large-scale production of mango. As a tree crop that flourishes best in

areas of moderate rainfall and high light intensity, the savannah areas are the most excellent for mango.

GNA further highlighted that agronomists have argued that, Ghana with a better comparative advantage

in Africa with regards to precipitation, soil and proximity could become an important producer within a

few years if the nation pays attention to that industry (GNA, 2008).

Figure 2. 8 (A) Regions of Ghana (written in green) – Source: Nutrition Country profile 2009. (B) Ecological zones of Ghana

A B

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2.2.1 Uses of mango

2.2.1.1 Nutritional and health benefits

Mangos are a extremely nutritious and contain carbohydrates, proteins, fats, minerals, vitamins: vitamin

A (beta carotene), B1, B2, and vitamin C (ascorbic acid) (Bally, 2006).These nutrients no doubt play a

crucial role in human nutrition thus the health of the individual. For instance, deficiency in vitamin A can

lead to reversible night blindness and keratinization of normal mucous tissue of the eye, lungs skin and

other ectodermic tissues. Lack of vitamin B1 can cause beri-beri (oedema and heart hypertrophy). Again

deficient in vitamin C which is a vitamin for humans and primates results in scurvy (Van Camp et al.,

2009). Mangos also make important seasonal contributions to the diet of many countries in the tropics

especially African countries that primarily have a starch (carbohydrates)-based diet.

Ripe mangos fruits are rich sources of vitamin A and are used to treat vitamin A deficiencies such as

night blindness. Also drinks made from the infusion of fresh mango leaves has been used to treat diabetes

and dried mango seed ground into powder is used to treat diarrhea. Diarrhea and throat disorders are

treated by bubbling the bark extracts mixed with water (Bally, 2006).

Some other uses of the mango includes its use in agro forestry and environmental practices such as

livestock shelter, home gardens, fence post, wind breaks and animal foods. Other uses include: flavorings

in which its puree is used to give flavor to many foods such as drinks, ice cream, wines, teas etc., honey

(from its nectar), making leaf vegetables from boiled young leaves and used for tannin/dye (Martin et al.

1998).

Naturland reports the importance of mango that ripened fruits are eaten fresh everywhere, and used to

make juice or marmalade, dried and made into candy (figure 2.9 left). Also all leftovers from the fruits

can be used as animal feed. For instance, the young leaves are very good as cattle feed, because they

have a protein content of 8-9% and a high Ca content as well. The bark and leaves of mango trees can

also be used as a dye for cloth. The wood from mango trees is highly suitable for making charcoal which

is widely used in rural areas in Africa as a source of fuel (Naturland, 2001).

19

Figure 2. 9 (A) Mango fruit . (B) Mango tree with fruit - Source: Bally, 2006

2.2.2 Production of Mango

According to GNA (2008), the major producing countries of mangos includes India, Mexico, Pakistan,

Brazil, Indonesia, China, Haiti, the Philippines, Madagascar and Tanzania. The production of mango

globally currently stands at about 25 million tons of fresh fruits and 290,000-processed mango pulp, puree

and juice. Africa out of this produces only 2.5 million tons (about 10%) of fresh fruits and 11 per cent of

processed mango. Ghana's current production is reported to have increased from about 1,200 tons in 2007

to about 2,000 in 2008 (Table 2.3). The varieties of mangoes that are grown in Ghana include Kent,

Keitt, Palmer, Haden, Tommy Atkins, Irwin, Sensation, Julie, and the local variety (GEPC, 2005).

Table 2. 3 Fresh mango exports from Ghana, 2005-2008 (in tones) – Source: Ghana Export Promotion Council, 2005

2004 2005 2006 2007 2008

227 772 366 1071 2000

A B

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2.2.3 Pest and diseases of mangoes

2.2.3.1 Pests and control

Several insects live in/on and feed on the mango tree but only a few are known to be harmful to the

mango and therefore considered as pest. There are a range of pest and diseases that affects mango. Some

pest may cause damage to the fruits while on the tree (pre-harvest) while others cause damage to the fruits

after harvest (postharvest). Fruit production, and tree growth, can therefore be severely affected by insect

pests and diseases. These pests can usually be adequately managed in commercial orchards.

There are species of scale insects that are known to be pests of mango, including Phenacaspis dilatata, P.

cockerelli, Ceroplastes rubens, and Aulacaspis tubercularis. When they infest, the areas turn pale green

or yellow and eventually die. The insects attack every part of the tree and are often a serious pest in the

nursery. Two main known species Penicillaria jocosatrix and Chlumetia euthysticha are larvae tip borers

and bore into and kill the young developing flushes (Bally, 2006).

Cotton scales, mealy bugs, cicadas and black flies (create honey dew) are the most serious pest of

Mangifera and are all sucking insects that live on the leaves, young buds and shoots. They cause a lot of

damage but they all have natural enemies (ladybird larvae, wasps, spiders) and other types are parasitic

fungi e.g. with cicadas and black flies to combat and minimize their effects (Naturland, 2001).

The control of scale insects can be done with a ‘winter-spraying’, using paraffin oil (white oil) shortly

before the larvae hatch from their eggs. The paraffin oil is sprayed on as a 3 % water emulsion. The worst

damage occurs during blossoming, therefore plantation should be checked regularly around such times in

order to make up the brew and spray it early enough. Mealy bugs can be controlled by wrapping smooth

plastic bands around the trunk because mealy bugs lay their eggs on the ground next to the trunk. Should

they infest the tree, a solution of 1% soft soap (potassium soap) with 1 % pure alcohol is quite effective

(Naturland, 2001).

2.2.3.2 Mango fruit diseases

Mangoes are climacteric fruits which are characterized by a sudden surge in respiration rate which

usually occurs after harvest known as climacteric rise. This means the fruit shows an increase in carbon

dioxide and in ethylene production during ripening in which starch is transformed to sugar. There is also

change in skin colour, development of aroma and flavor. Postharvest losses and decay of mango fruits can

be traced to infections that occur either between flowering and maturity of fruit or during harvesting and

21

subsequent storage of fruits. Furthermore, mango fruits are susceptible to wound and damage during

postharvest handling (lenticels spotting, sap burn), temperature injury (heat and chilling injury) and

mechanical damage which may occur during transportation, and in pack houses (brushing, abrasion,

pressure and impact damage) (Johnson et al., 1995). Mangoes are also susceptible to pre and postharvest

diseases just like any other fresh produce which can result in considerable losses (Govender, 2005). Pre

and postharvest infections of mangoes are largely caused by fungal pathogens such as Colletotrichum

gloeosporioides which causes anthracnose diseases and stem-end rot diseases caused by Botrysphaeria

spp. (Lonsdale, 1993a). These are the most severe postharvest diseases of mangoes and infect the mature

fruits. The mango powdery mildew caused by Oidium mangifera is also one of the severe diseases of

mangoes and affects mango leaves, panicles, and young fruits (Nelson, 2008) and the bacterial black spot

diseases caused by Xanthomonas campestris pv. Mangifera indicae (Stovold, 2004).

Table 2. 4 Diseases of mangoes caused by fungal pathogens (Ploetz et al, 1994)

Disease Host pathogen

Anamorph Teleomorph

Anthracnose Colletotrichum gloeosporioides Glomerella cingulata

Altenaria rot Alternaria alternate

Gray leaf spot Pestalotiopsis mangifera

Pink disease Erythricium samonicolor

malformation Fusarium subglutinans

Powdery mildew Oidium mangifera

Scab Elsinoe mangifera

Stem-end rot Lasiodiplodia theobromae Botryosphaeria spp.

Dithiorella spp. Botryosphaeria spp.

Phomopsis mangifera

Pestalotiopsis mangifera

ANTHRACNOSE

According to Bailey and Jeger (1992), Colletotrichum species are among the most successful plant fungal

pathogen, attacking a very wide range of plants growing in both temperate and tropical environments

causing damage to plant leaves, stems, roots, flowers, and fruits. They are highly specific to individual

tissues. Anthracnose caused by the fungal pathogen Colletotrichum gloeosporioides is the most wide-

spread diseases of mangoes (Bailey & Jeger, 1992). Though losses do occur on the field, postharvest

22

losses to this disease are very important and serious (Ploetz, 2003). Anthracnose disease poses a great

deal of challenge not only to those who are engaged in the international commerce of the fruit (Ploetz,

2003), but also to local consumption because of the vital role it plays in food security particularly to most

people in developing countries especially in Northern Ghana.

Nelson (2008), describes Colletotrichum gloeosporioides as an ubiquitous fungi which is the asexual

stage of the pathogenic fungus and is responsible for many diseases (anthracnose) on many tropical fruits

such as banana, avocado, legumes, coffee, papaya and among others. Conidia (produced in acervuli) and

ascospores (produced in and are released from the perithecia) are the two main inoculums (Bailey and

Jeger, 1992). Conidia and ascospores in young acervuli and perithecia are enclosed in a moist hydrophilic

mucilaginous material known as the spore matrix. These reproductive structures when matured under dry

conditions, the matrix forms a crusty deposit which bind a lot of spores together. The spread of spores

from young acervuli happens in water droplets and distribution of dry spores masses from older acervuli

and ascospores from perithecia can be carried out by wind (Bailey and Jeger, 1992).

Symptoms

The causal agent of anthracnose is Colletotrichum gloeosporioides which is a major and important fungal

pathogen of mangoes. It has been reported that major losses occur from flowering till fruit set and also

after harvest. Anthracnose is very common in wet, humid, warm weather environment and favor

infections in the field. Warm, moist temperatures further aids postharvest anthracnose development in

mangoes (Nelson, 2008).

Nelson (2008) explains the symptoms of anthracnose occurs on the leaves, petioles, twigs, flower clusters

and fruits on mango (figure 2.10). On the leaves, wounds begin as small, angular, brown to black spots

that can expand to form extensive dead areas and can drop off during dry weather. On flowers, the

symptoms are small black or dark-brown spots which can extend and kill flowers before fruit production

severely decreasing yield.

Ripe fruits affected by anthracnose develop sunken, major, dark brown to black decay spots (figure 2.10)

before or after picking and fruits can drop from trees prematurely thus causing pre harvest infection of the

fruit. The fruit spots can finally enter deep into the fruit, resulting in extensive fruit rotting. The majority

of green fruit infections remain latent and largely undetectable until ripening. Fruits that appear healthy at

harvest can develop significant anthracnose signs rapidly upon ripening (Nelson, 2008).

23

Figure 2.10 (A) Mango anthracnose symptoms on mangos , (B) the “tear stain” effect , (C) linear necrotic regions lending an alligator-skin effect,

often associated with cracking of the epidermis (right) – Source: Nelson, 2008

A second symptom type on fruits consists of a “tear stain” symptom (figure 2.10 B), a linear necrotic

regions on the fruit that may or may not be associated with superficial cracking of the epidermis,

exhibiting an “alligator skin” effect and even causing fruits to develop wide, deep cracks in the epidermis

that extend into the pulp (figure 2.10 C). Lesions on stems and fruits may produce prominent, pinkish-

orange spore masses under wet conditions (Nelson, 2008).

According to John et al., (1995) and Arauz (2000), postharvest infections of anthracnose show a rounded

black to brown lesion with a rounded border on the fruit surface. Lesions are normally confined to the

peel but in severe situations, the fungus can enter the pulp. In the advanced stages of the disease, the

fungus produces acervuli and lots of orange or pink masses of conidia in the lesions (Freeman et al., 1998;

Arauz, 2000).

Disease Cycle (Anthracnose)

In the field, the pathogen infects non-wounded and intact immature green fruits. Conidia produced by

pathogen on branch terminals, mummified inflorescences, flower bracts and leaves are the most important

sources of inoculums (Dodd et al., 1991). Conidia are disseminated by rain splash and infection needs

free moisture (Jeffries et al., 1990). Spores germinate and then form appressoria. Whiles appressoria age,

they happen to melanized and strengthens the appressorium which makes penetration of the cuticle easy

by infection pegs that the appressoria produce. Melanized appressoria presence and frequency of

occurrence is reported to have been used to foretell when infection is possible and anthracnose control

A B C

24

Figure 2. 11 Disease Cycle of anthracnose – Source: Arauze, 2000

measures that are needed (Dodd et al., 1991). The fungus by means of its appressoria enter and stays in

the sub cuticlar until postharvest stage (Dickman et al., 1983).

Fruits that reaches approximately 4 cm (1.6 inches) in diameter after infection possess fruit’s natural

defense mechanisms that protect it from anthracnose by inducing the fungus into a quiescent period. The

natural defense mechanisms breaks down when the fruit softens during the ripening process, and latent

infections of anthracnose develop into black lesions (figure 2.11) that rot the whole fruit in days (Bally,

2006). When the climacteric period of the fruit sets in, lesions begin to develop. Fruit-to-fruit infections

of anthracnose does not occur but postharvest anthracnose is a monocyclic disease (Arauz, 2000).

25

Figure 2.12 (A) Stem end rot covering almost the entire fruit (B) Stem end rot spreading from the end of the stem – Source:.

Haggag, 2010 (Agric and Biological Journal of North America)

A B

STEM-END ROT DISEASES

Stem-end rot diseases can be caused by the fungal pathogens Lasiodiplodia theobromae, Phomopsis

mangifera or Dithiorella dominicana (Bally, 2006) and Colletotrichum gloeosporioides. Botryosphaeria

parva formerly known as Dithiorella dominicana however occurs as saprophytes and endophytes in

several host and as pathogen of many woody trees (Johnson et al., 1990; Roux, 1993). Stem-end rot

caused by Botryosphaeria shows itself either during pre and/or post-harvest and is the telemorph of the

fungus (Jacobs, 2002). When the mango is infected, it produces a lot of dark grey-black, fluffy mycelium

on either oatmeal or PDA (Potato Dextrose Agar) (Johnson et al., 1992; Dodd et al., 1997). Though

anthracnose is one of several fruit diseases that affect pre‐ and post‐harvest quality (Ploetz, 2003),

stem‐end rots caused by several different fungi, in particular Lasiodiplodia theobromae can be severe

where anthracnose is prevalent but well controlled.

Symptoms

Blossom blight occurs as a result of the colonization of the blossom by the pathogen. The pathogen

colonizes the tissue in favourable conditions causing twig die-back and extensive cankers of stems and

trunks. The fungus stays dormant on the fruit until fruit begins to ripe (Johnson et al.,1992; Lonsdale,

1993b). At the peduncle and pedicel tissues, rot takes place and in severe cases covers the entire body of

the fruit. Brown soft decomposition starts at the stem end of the mango and quickly spreads throughout

the entire fruit body. A straw coloured fluid from the stem-end and steel-grey mycelium may cover up the

surface of the fruit (Govender, 2004).

26

Figure 2. 13 Disease cycle of stem end rot of mango - Source: Govender, 2004

In extreme cases, the decay manifests itself in a similar way only that it does not start at the peduncle and

pedicel tissues but rather covers the whole body of the mango fruit (figure 2.12). In the orchard, the

fungus also passes on the disease to the fruit but remains dormant until fruit begins to ripe after harvest

when it recommences activity and colonizes fruit body giving rise to stem-end rot (Lonsdale, 1993b).

Affected fruits may split open as they collapse and straw-coloured fluid comes out of the splits in the side

of the fruits. Ploetz et al. (1994) indicates that, Botryosphaeria spp. easily spread from affected to healthy

neighboring fruit bring about considerable and substantial losses due to decay at the end of export chain.

In some instances, Colletotrichum gloeosporioides are associated with stem-end rot (Govender, 2004).

Disease cycle

According to Govender, (2004) Botryosphaeria stays alive throughout the winter season in a form of

black pycnidia and perithecia in wart-like stroma on living, dead cankered limbs of tree or mummified

fruits and injuries and breaks in the cuticle of the fruits necessitates infection. Ascopores are freed by the

perithecia during spring rains and the conidia produce within the pycnidia comes out. They carried by

rain splash to other parts of the trees and the dispersion of the conidia may go on throughout the rainy

season. Fruit becomes fairly infected early, but rotting does not develop until the fruit reaches its maturity

(figure 2.13).

27

Symptoms of postharvest development that takes place in this way is termed as stem rot or soft brown rot

(Govender, 2004). However, both symptoms have been found to be caused by the same fungal pathogens

(Jacobs, 2002). Blossoms blight symptoms can also occur on the inflorescence (Johnson et al.,1992;

Lonsdale, 1993b). The spectrum of pathogens that causes fruit rot disease is influenced and favoured by

any condition that decreases tree vigour, drought, high temperatures, winter injury, nutrition level of host,

sunscald and poor pruning practices as well as low or unbalanced nutrition (Govender, 2004; Ploetz et al,

1994). It takes 3-7 days for symptoms of stem-end rot to appear after harvest at 25 ºC but can be held up

for 7-12 days at 13 ºC. Stem-end rot fungi have also been found to be associated with twig and branch

dieback (Ploetz et al, 1994).

2.2.3.3 Control of mango disease

Proper identification of the pathogen causing postharvest disease is vital to the choice of a suitable disease

control strategy. For this reason the importance of this study to correctly isolate and identify pathogens

for better pre and postharvest control strategies of mango fruits. The control of postharvest diseases of

mangoes will be effective if pre-harvest management programmes are regularly carried out properly to

control diseases in mangoes. Ippolito and Nigro (2000) notes that pre-harvest control is essential in

providing some level of control to previously established infections such as quiescent and developing

ones. In the past, pre-harvest control of mango infections depended largely on copper fungicidal field

sprays while postharvest treatments included dips in hot water at 55ºC for 5 minutes with fungicides such

as prochloraz often incorporated in water (Pelser and Leser, 1989; Korsten et al., 1992).

Oosthuyse (2000) indicate that recent control of pre-harvest infection measures include the use of copper-

based sprays, benomyl (Benlate) and propiconazole (Tilt). A hot dip in a suspension of benomyl

(100g/100L) or hot dip in prochloraz (180ml/1L) emulsion is a post harvest treatment of mango.

Nelson (2008) describes the integrated management of mango diseases(Anthracnose, stem-end rot etc) to

consist of five steps: Pre-harvest control involves (i) Site selection thus choosing a hot, dry area, and

avoiding wet areas since anthracnose are most prevalent in humid environment , (ii) cultivar selection

(choosing resistance varieties to manage anthracnose) example Ah Ping, Fairchild, Gouveia, Harders,

Keitt, Momi K, Pope, and Rapoza which were recommended for Hawaii based on their horticultural

properties, (iii) cultural practices in the field which includes sanitation by pruning trees annually and

removing falling debris from the ground, plant spacing to reduce epidemics and intercropping (inter-

planting mango with other types of trees that are not hosts of mango anthracnose will hinder epidemics,

(iv) the use of fungicides sprays in the field to control anthracnose on susceptible mango cultivars and (v)

28

careful handling of fruits after harvest to prevent mechanical damage will avoid the invasion of pathogens

through wounds and injury on fruits.

Irradiation by exposing fruits to shortwave Infra red (IR) and ultra violet (UV) can be used to reduce

postharvest decay but have not been successfully put into practice. This is because of the impacts on the

fruit quality factors such as scalding and changes in taste (Moline et al.,1999). In addition biological

control can also be used to control the mango diseases.

Post harvest treatment of mangoes can slow down or minimize symptom development if refrigeration is

kept at 10 °C, but no chilling of fruits before they are ripe else there may be chilling injury, hot water dip

may be applied for 15 minutes at about 49–55 °C depending on the variety. Vapour heat, forced - air dry

heat can be used for 3–6 hours at various temperatures, depending on variety and lastly heated fungicide

dips (aqueous) but products and temperatures may vary (Nelson, 2008).

29

CHAPTER 3 : MATERIALS AND METHOD

3.2 Sample collection

Unripe but matured mango and shea fruits, were collected from Wa municipal, Kaleo district in the Upper

West region and Tamale metropolis in Northern region of Northern Ghana in August 2011. Shea tree

leaves were also collected from Upper west region (UW/R).

In UW/R, mango fruits were collected from the market (in Wa) and from different trees on the same farm

(in Kaleo) whiles the shea fruits were collected from a tree in the Kaleo district. In the Northern region,

mango fruits were taken from Tamale market whiles the shea fruits were sampled from trees at Zoozugu

in the Tamale metropolis. No mangoes were collected from farm in the Northern region because they

have already been harvested at that period. No fruits were sampled from the Upper East region due to the

fact that the region is geographically similar to Upper West region. The varieties of mango fruits collected

were keitt, kent and the local mango (sometimes called Indo China). The fruits were kept at room

temperature (23 -26ºC for two days before they were wrapped in envelopes and brought to Belgium

(Ghent). Some fruits were ripe at the arrival in Belgium. The fruits were stored in a fridge at 4 ºC in the

phytopathology laboratory of Ghent University till the beginning of the analysis.

Locations of sample

Figure 3. 1 Map showing regions where fruits were collected

30

Figure 3. 2 (a) blotted tissues on sterile tissue paper; (b)plated tissues; (c) fungal growth of mango tissues on mathuer and PDA

media after 6 days of incubation; (d) fungal growth of shea tissues after 6 days of incubation

3.3 Media preparation

Mathuer medium, potato dextrose agar (PDA), and potato dextrose broth (PDB) were used. The following

components for each medium were measured and mixed thoroughly in 1000ml distilled water in a 1liter

glass bottle and autoclaved for 21 minutes at 121 ºC. Agarose gel (1%) was also used.

Mathuer medium - bacto peptone (2.0g), glucose (2.80g), KH2 PO4 (2.72g), MgSO4 .7H2O (1.2 g) and

agar agar(15 g)

Potato Dextrose Agar (PDA, Difco) medium – PDA (39 g)

Potato Dextrose Broth (PDB) medium – PDB (24 g)

3.4 Isolations of fungal pathogens

Healthy tissues , diseased tissues, and tissue segments of advancing margins of lesions of fruits (mango

and shea fruits) were cut with the help of a scalpel knife. Leaf tissues of the shea tree were also

excised.The tissues were surface sterilized in sodium hypochloride solution of 1% (which sterilizes the

excised tissues) for one minute. They were rinsed twice at each time for one minute in a sterile distilled

water and blotted dry with a sterile tissue paper. They were then plated on both Matheur and PDA media

under a laminaire flow which blows sterile air. One or two excised tissues of the mango and shea were put

on each medium of Matheur and PDA. They were incubated for 6 days at room temperature (21-25 oC)

under 12 hour light and dark conditions (figure 3.1).

A total of 25 petri dishes of Matheur and PDA media were plated for mango tissues (13 mathuer, 12

PDA) and 12 for shea tissues (6 Matheur, 6 PDA). The plates were coded as; MxUy or MxNy (where M-

mango, x-plate number, U or N-upper west or northern region, y-exact location where fruit was sampled

from) for the mango plates and SxUy or SxNy (where S-shea fruits) for the shea plates.

(a) (b) (c) (d)

31

PURIFICATION

After some days, the plates were inspected to observe the growth of pathogens on the media. Some

growth was better observed on matheur than on PDA media and vice versa. This could be that some

pathogens can grow better on Matheur or PDA medium. Subsequently, young mycelium emerging from

the tissues were sub cultured on fresh Matheur and PDA media with the help of a sterile loop to obtain

pure isolates of the pathogens. They are checked and transferred on to new Matheur and PDA media

weekly and kept at room temperature (21-25 oC) to maintain cultures at all times for correct identification

of fungi pathogens.

3.5 Identification of postharvest fungi

After three consecutive times of purifying the plates, they were inspected again to for growth and purity.

Some plates exhibited similarities in morphological growth of fungi for mango and shea isolates plates

and were therefore divided into groups. Four different groups (group 1 to 4) with similar growth

morphologies of isolates within each group were identified for the mango as well as four different groups

(group 1 to 4) with similar morphologies of isolates within each group were identified for the shea. They

were grouped according to the growth shown (colony characteristics). Purification was done for the

various groups to ensure pure isolates were obtained at all the times.

Temporary slides from each group of mango and the shea isolates were made after carefully selecting the

purest isolates and observed under light microscope. Germinating spores and mycelia were observed

under the microscope to be Colletotrichum spores from some slides prepared from the mango groups and

Pestalotiopsis from the shea slides of the shea group were identified with reference to Barnett and Hunter

(1998). Some unidentified spores were also observed from both groups (mango and shea). To confirm the

observations above and to know which species of fungi they were, their DNA (deoxyribonucleic acid)

were extracted for sequencing.

SET UP

Before the DNA extraction, pure isolates were further selected from the various groups resulting in a total

of eight isolates with codes: M12Nm, M19Us, M29Us, M24Us, SGUk, S7Nz, S20Uk and S12Nz.These

isolates were grown on liquid potato dextrose broth (PDB) in Erlenmeyer flasks sealed with cotton and

aluminum foil. They were incubated in dark condition at room temperature (21-25 ºC) for 7 to 10 days.

Parafilm was used to further secure the cotton seal and aluminum foil. With the help of a sterile cork

borer, plugs of young mycelium of the isolates were taken and transferred into a sterile 150ml

32

Figure 3. 3 Erlenmeyer flasks at day 10 of incubation with the growth

suspension

Erlenmeyer flask containing 25ml of liquid PDB. The Erlenmeyers were prepared by sealing a 150 ml

flat bottom with a cotton plug and covered with aluminum foil and autoclaved for 21 to 30 min at 121 ºC.

DNA EXTRACTION

On the 10th day, mycelia mats of the 8 isolates grown in the Erlenmeyers (figure 3.2) were pat dry

between sterile filter paper. They were then ground to a fine powder together with liquid nitrogen using a

mortar and pestle. During handling the samples should not be allowed to thaw. The powder were

collected in tubes kept frozen in liquid nitrogen for the extraction of their DNA. Further extraction

procedures were carried out with a commercially available DNA-extraction Kit (DNeasy Plant Mini

Kit,QIAGEN) and (Wizard®SV Genomic DNA Purification System) following the manufacturer’s

instructions.

DNA YIELD (CONCENTRATION) MEASUREMENT

The final extract (genomic DNA) yields were read using Nanodrop spectrophotometer. The Nanodrop®

ND-1000 spectrophotometer allows fast and accurate measurement of DNA concentration and quality.

The spectrophotometer was first blanked with 2µL sterile water and re-blanked with buffer and then 2µL

extract of each of the sample was loaded on the pedestal. After measurements of the absorbance values,

the samples were stored at (-20 ºC) before PCR (Polymerase Chain reaction) analysis.

33

Figure 3. 4 (A) 1% agarose ‘slab’ gel before UV illumination. (B) Fragments from PCR of the rDNA ITS region and

5.8S rDNA . Numbers 1-8 : isolate M12Nm, M19Us, M29Us, M24Us, S7N, S20U, S12N and SGU respectively; M: molecular

marker (MassRulerTM DNA ladder Mix)

3.6 Amplification of rDNA ITS

Prior to the running of the PCR, a master mix was made (PCR reactions) comprising 25µL volume

containing 2 µL genomic DNA (5-10 ng/ µL), 2.5 µL 10X PCR buffer (QIAGEN), 5 µL Q-solution

(QIAGEN), 0.5 µL dNTPs (10nM, Fermentas GmbH), 1.75 µL of each primer (ITS4 and ITS5) (10 µM),

0.15 µL Taq polymerase (5units/ µL) and 11.3 µL sterile highly purified water. These constituents for the

master mix were multiplied by the number of samples (8). An extra sample was made to correct for

pipetting errors. Amplifications of the nuclear rDNA ITS region included the 5.8 S ribosomal DNA was

carried out with primers ITS4 and ITS5 (White et al., 1990). PCR was then later run in a flex cycler

(Analytikjena, Belgium) with a first denaturation step at 94 ºC for 10min followed by 35 cycles at the

following conditions: 1min at 94 ºC, 1min at 55 ºC and 1min at 72 ºC with a final extension step at 72 ºC

for 10min.

ELECTROPHORESIS

In order to verify if amplification of rDNA occurred in the PCR, electrophoresis was carried out which

separated the fragments. A 1% agarose gel stained with ethidium bromide with wells made in it (by a

comb placed in it during the solidification) was formed. The gel was placed in the electrophoresis

machine containing 0.5% TAE (Tri/Acetate/EDTA) - buffer and the DNAs loaded with buffer (5 µL for

each isolates) were carefully pipetted into the wells in the slab gel. The MassRulerTM DNA ladder Mix

was also pipetted into ends of the wells in the slab gel. The machine was turned on at 100V for 20

minutes after which slab gel was then viewed under the UV- transilluminator (enhanced by the staining

ethidium bromide) and photographed (figure 3.3).

M 1 2 3 4 5 6 7 8 M A B

34

PCR PRODUCTS (DNA) PURIFICATION

Before sequencing, the PCR products (DNA) were purified again. This purification step is to purify single

or double-stranded DNA fragments from the PCR and other enzymatic regions. This was carried out with

a commercially available kit protocol (QIAquick PCR Purification Kit Protocol), following the protocol

provided by the manufacturer. Their concentrations (DNA yields) were measured again with Nanodrop®

ND-1000 spectrophotometer.

Sequencing of the purified PCR products (DNAs) were conducted for both strands with primers ITS4 and

ITS5. A total volume of 10 µL including a specified volume of highly sterile water (of 20ng/ µL

concentration) of the purified DNAs were each provided in a 1.5ml tubes labeled with a barcode.4 µL of

each of the primer solution (ITS4 and ITS5) were added resulting in 16 samples in total (8 samples added

with ITS4 and 8 samples added with ITS5). The lids of the tubes were closed tightly and put in an

envelope and sent to LGC Genomics (Berlin, Germany) for sequencing. Direct sequencing was then

performed for the purified PCR products carried out for both strands with primers ITS4 and ITS5.

Consensus sequences were created with BioEdit 7 version and the nucleotides blast were done on the

national center for biotechnology information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

DILUTION FACTOR

Due to the fact that the PCR products (DNA) concentrations and volume were more than the required (10

µL and concentration of 20 ng/µL) needed for sequencing after purification, there was a need for the

dilution to get the desired volume. However, total length of bands (fragments) that measured beyond

1000bp after reference to the MassRulerTM DNA ladder Mix were diluted twice of the required

concentration (2*20 ng/µL).

First, the volume of the PCR products was determined as:

Vpcr = Vtot seq / [Cpcr ÷ Cseq]

Secondly, the volume of highly purified water was determined as:

Vw = ( Vtot seq - Vpcr )

35

Therefore total volume required(10 µL) for sequencing Vtot seq= ( Vw + Vpcr )

Vpcr - volume of PCR products (DNA) required for sequencing

V tot seq - total volume required for sequencing

Vw - volume of water to be added

Cpcr - concentration of PCR products required after purification (ng/ µL)

Cseq - concentration of required for purification (ng/ µL)

MICROSCOPIC EXAMINATION OF THE ISOLATES

After direct sequencing and the nucleotide blast of the consensus sequences on NCBI website in which

pathogens were finally identified, slides were then prepared again for the various isolates of the selected

groups and viewed under the microscope to further distinguish and confirm how the spores and mycelia

growth looks like. This was done to cross check earlier identifications of some pathogens with the

microscope after reference to Barnett and Hunter (1998) before the molecular technique was carried out

to verify the observations.

36

CHAPTER 4 : RESULTS

4.1 Sample collection and isolation

A total of 18 mango, 50 shea fruits and some leaves were collected from both Upper west and Northern

regions. 5 (kiett) mango fruits were collected from the farm in Kaleo, 2 (kent) from market and 6 (indo-

china) from a tree in secondary school in Wa in the Upper west region. Three (kent) mango fruits were

sampled from Tamale market in Northern region. Thirty shea fruits were taken from trees in the wild in

Kaleo from Upper west and 24 shea fruits from trees in the wild from Zoozugu in the Northern region.

Some leaves of shea tree were collected from Upper west region. A total number of thirty isolates were

obtained and separated into eight different groups. Four groups for the mango isolates and four for the

shea isolates as Lisiodiplodia theobromae (5 isolates), Botryosphaeria sp. (3 isolates), Pseudofusicoccum

sp. (3 isolates), Colletotrichum gloeosporioides (3 isolates) for the mango fruits collection, and Phoma sp.

(4 isolates), Pseudofusicoccum sp. (4 isolates), Phomopsis sp. (4 isolates), Pestalotiopsis sp. (4 isolates)

for the shea fruits and leaves. They were sorted into the various groups due to the similarity in

morphology (especially cultural characteristics) observed.

Pestalotiopsis sp. in this present study was initially identified microscopically after reference to (Barnett

and Hunter, 1998) and was identified and confirmed by ITS sequence analysis. The pathogen was isolated

from shea fruits (green but matured) collected in Tamale (Northern region) and leaves sampled from the

Kaleo (Upper west region). But the identification for ITS analysis was made on the isolation from shea

fruits from Northern region though isolations from the leaves were sorted into the same group due to their

indistinguishable cultural characteristics. Also the initial microscopic identification of Pestalotiopsis was

from the leaves collected from the Upper west region before sequence analysis from the fruit. The table

below shows the various groups of isolates for the mango and shea fruits which were seen to be

morphologically the same because of their growth of colonies.

37

Table 4.1 Various Groups of isolates of mango and shea fruits sorted

Mango isolates

Lasiodiplodia

theobromae

Botryosphaeria sp. Pseudofusicoccum sp. Colletotrichum gloeosporioides

M7Um

M14Nm

M20Um

M26Us

M12Nm*

M22Us

M23Us

M19Us*

M29Us*

M17Us

M2Uf

M26Uf

M21Us

M24Us*

* Isolates selected for DNA extraction and identification. MxUy = Mangos from the Upper west region, MxNy = mangos from

the Northern Region. m = market, f = farm, s = school. U=Upper west region, N=Northern region.

Shea isolates

Phoma sp. Pseudofusicoccum sp. Phomopsis sp. Pestalotiopsis sp.

SeUk

SAUk

S2Uk

SGUk*

S8Nz

S2Nz

S7Nz*

S3Nz

S4Nz

SFUk

S1Nz

S20Uk*

S10Nz

S6Nz

S12Nz*

S13Uk

* Isolates selected for DNA extraction and identification. SxUk = shea fruits from Kaleo (Upper west region), SxNz = shea fruits

from Zoozugu (Tamale in the Northern region). k= Kaleo, z = Zoozugu.U=upper west region, N=Northern region.

One pure isolate was carefully selected from each group of the mango and shea fruits isolates for further

analysis (see table 4.1). One pure isolate was selected because they have similar morphology and

assumed that they were all the same. Secondly due to time constraints all the isolates DNA could not have

been extracted and sequenced for identification.

Isolates were obtain from healthy, unripe but matured mango and shea fruits though only a few mango

fruits taken from the market (Wa and Tamale) showed little sign of symptoms of disease. The fungi were

maintained and renewed regularly on fresh PDA and matheur media kept at room temperature (21-25oC)

for additional studies.

Botryosphaeria sp., Pseudofusicoccum sp., Colletotrichum gloeosporioides are Botryosphaeriaceae

members that were identified from mangoes in Upper west region and Lisiodiplodia thoebromae on

mangoes from Northern region. Phomopsis sp., Phoma sp. were identified on shea fruits from the Upper

west region and Pseudofusicoccum sp., Pestalotiopsis sp. from shea fruits collected in Northern region.

38

Figure 4. 1 (A) Patterns and length of bands shown by the isolates. Numbers 1-8 : isolate M12Nm, M19Us, M29Us, M24Us,

S7Nz, S20Uk, S12N and SGUk respectively; (B) M: molecular marker (MassRulerTM DNA ladder Mix) and Massruler DNA

ladder mix reference.

Pseudofusicoccum sp. were identified on some fruits on mango and shea fruits from the two regions.

Pestalotiopsis sp. was isolated on both shea fruits and leaves but identified on fruits from Zoozugu in the

Northern region. Lasiodiplodia sp. was only identified on keitt cultivar of mango and the

Botryosphaeriaceae members were identified only on the local cultivar (sometimes called indo-china)

though they were isolated on the other cultivar as well but belongs to the same group.

4.2 PCR (polymerase chain reaction) analysis

Electrophoresis which was carried out to confirm the occurrence of the amplification of the DNA samples

(isolates) by PCR showed almost equal length of bands except for some two isolates with different

patterns of bands The isolates M29Us and S7N had their bands with a total length above 1000bp with

reference to the MassRulerTM DNA ladder Mix. The rest of the six isolates (M12Nm, M19Us, M24Us, S20U,

S12N and SGU) had their bands with length varying from 550 to 600bp with reference to the DNA ladder

mix as well (figure 4.1).

4.4 DNA ITS sequences analysis

The direct sequencing of the purified PCR products which was conducted on both primers (ITS4 and

ITS5) and the consensus sequences (nucleotides) created with BioEdit blasted on NCBI website gave

identities of the sequences. The ITS sequences (ITS 4 and ITS 5) for each isolates using the BiodEdit

M 1 2 3 4 5 6 7 8 M A

B

39

were cut/trimmed both at the beginning and at the end, reversed compliments, aligned, and consensus

created. The consensus were then blasted on NCBI website for identification. The sequences (ITS4 or

ITS5) were also blasted separately and the same identification were obtained as the consensus.

The identifications of the sequences for the mango isolates includes M12Nm - Lasiodiplodia

theobromea, M19Us - Botryosphaeria sp., M29Us - Pseudofusicoccum sp., and M24Us - Colletotrichum

gloeosporioides, while the identities and coverage for the shea isolates are SGUk- Phoma sp.,S7Nz-

Pseudofusicoccum sp., S20Uk-Phomopsis sp. and S12Nz-Pestalotiopsis sp. Meanwhile Pseudofusicoccum

sp. appeared on both the mango and shea fruits isolates (table 4.1). The percentage maximum identities of

the ITS sequences from the consensus nucleotide blast on NCBI were 99%, 100%, 100%, 100% for

mango isolates and 100%, 99%, 99%, 99% for the shea isolates respectively for the above stated

pathogens.

Table 4. 1 Identified host pathogen of isolate from nucleotide blast on NCBI website of mango and shea

fruits groups after consensus with BioEdit

Code Pathogen Source Host Location GenBank code*

M12Nm Lasiodiplodia

theobromae

fruit Mangifera indica Tamale (NR) GQ469934.1

M19Us Botryosphaeria sp. fruit Mangifera indica Wa (UWR) DQ480360.1

M29Us Pseudofusicoccum sp. fruit Mangifera indica Wa (UWR) GQ471807.1

M24Us Colletotrichum

gloeosporioides

fruit Mangifera indica Wa (UWR) HM575266.1

SGUk Phoma sp. fruit Vitellaria paradoxa Kaleo(UWR) FJ985695.1

S7Nz Pseudofusicoccum sp. fruit Vitellaria paradoxa Zoozugu(NR) JF966392.1

S20Uk Phomopsis sp. fruit Vitellaria paradoxa Kaleo(UWR) EF432292.1

S12Nz Pestalotiopsis sp. fruit Vitellaria paradoxa Zoozugu(NR) HQ607806.1

*GenBank codes are the closest match and obtained from NCBI site of the nucleotide blast. NR- Northern region UWR- Upper

west region.

The GenBank codes obtained from the various isolates are the closest match from the NCBI website.

GQ469934.1(Lasiodiplodia theobromae) strain CMW28626 identified on Terminalia species.

40

DQ480360.1 (Botryosphaeria sp.) NR-2006-M76 internal transcribed spacer associated with Garcinia

species. GQ471807.1 (Pseudofusicoccum sp.) JWM-2009a strain CMW26824 associated with native

Terminalia spp. HM575266.1 (Colletotrichum gloeosporioides) isolate CM0705 associated with

anthracnose on citrus. FJ985695.1 (Phoma sp.) 27AT1704 identified in nests of fungus-growing ants.

JF966392.1 (Pseudofusicoccum sp.) strain CMM1450 associated with mango Dieback. EF432292.1

(Phomopsis sp.) B17A isolated from Garlic mustard (Alliaria petiolata) and Black mustard (Brassica

nigra). HQ607806.1 (Pestalotiopsis sp.) ATT035 identified from communities in gardens of fungus-

growing ants (Hymenoptera: Formicidae).

4.5 Cultural and growth characteristics

Typical growth, cultural and morphological characteristics were revealed by the various selected isolates

from the groups of mango and shea fruits. Their growth (especially the mycelia growth), morphology

and cultural characters (including colours of the colony) on PDA (potato dextrose agar) and matheur

media were different when observed after a period of 7 days under room temperature of 21- 25 oC.

Characteristics on PDA and matheur media of the mango isolates

On the PDA and matheur plates, the majority of mango isolates first develop grayish to whitish zonate

colonies in which some later develops colour and acervuli. The colours gradually becomes darker as

fungi grow old with time (figure 4.2). Colletotrichum gloeosporioides (M24Us): on PDA, colonies are

grayish and production of aerial mycelia ranges from thick mat colonies to sparse tufts compared to the

growth colonies on matheur media (Ploetz et al, 1994). On matheur media, colonies appear pale white

with production of aerial mycelia ranging from sparse to thick mat with sclerotia in light orange colour

around the sparse mycelia at the center. Conidia described by Barnett and Hunter (1998) are hyaline,

unicellular, ovoid or oblong to falcate to parasitic (figure 4.2 A) and the acervuli are disc – shaped or

cushion-shaped, waxy, with typically dark, spines or setae at the edge or among conidiophores though not

seen under the microscope during microscopy. Rate of colony growth on both PDA and matheur are

almost the same at 7 days.

Pseudofusicoccum sp. (M29Us) : colonies on PDA are mostly purplish and is smoky gray at the centre.

The purplish mycelia are moderately appressed . The aerial mycelia are slightly fluffy and less dense at

the centre and thick towards the edges with mycelia almost reaching the lid of the Petri dish. But on

matheur media, colonies are completely white. The production of aerial mycelia seem to be of thick wavy

mats to sparse. The Conidia are ellipsoid to rod-shape, either straight or slightly bent and their apices are

round, smooth with granular content hyaline. They have thin wall, covered with a persistent mucus layer

41

and are unicellular, (Palvic et al., 2008). Growth of colonies on PDA media appears faster than on

matheur (figure 4.2 B).

Botyrosphaeria sp. (M19Us) : PDA media have colonies that are smoky gray to light gray. The mycelia

are dense and short fluffy in the middle. Aerial mycelia production range from thick to sparse and

appressed. Similar characteristics is observed on mathuer media only that colony growth is slower as

compared to PDA media. The Conidia are ellipsoid to rod-shaped, hyaline with granular content, and

either straight or slightly curved. Apices are round, have thin wall and posses one cell ( figure 4.2 C).

Lasiodiplodia theobromae (M12Nm) : colonies on PDA media are dark gray to black, and fluffy mycelia.

Aerial mycelial production is sparse to thick fluffy covering the surface of the entire media. Aerial

mycelia production on PDA media is relatively the same due to the fast growth of mycelia. On mathuer

medium colonies are white to dark gray and fluffy but less dense compared to colonies on PDA. Aerial

mycelia production on matheur is somewhat thick to sparse. Conidiophores are aggregated in stromatic

body and short. Conidia are matured with thick wall and 2-celled, dark brown colouration, and ovoid to

elongate (figure 4.2 D).

42

M24Us

M29Us

M19Us

M12Nm

Figure 4. 2 Cultural characteristics on PDA, matheur medium and morphology of conidia of mango isolates. (A)

Colletotrichum gloeosporiodes (M24Us)(Bar-50µm); (B) Pseudofusicoccum sp. ( M29Us) (Bar-20µm); (C) Botryosphaeria sp.

(M 19Us) (Bar-20µm); (D) Lasiodiplodia theobromae (M 12Nm) (Bar-50µm)

PDA medium Matheur medium Conidia/mycelia

A

B

C

D

50µm

20µm

20µm

50µm

43

Characteristics on PDA and matheur media of shea isolates

Most of the isolates on PDA and matheur plates media of the shea also initially develops grayish to white

colonies except some few isolates ( Phoma sp., Pseudofusicoccum sp.) on PDA media (figure 4.3).

Pestalotiopsis sp. (S12Nz): colonies are dark grayish-brown to white-gray on PDA media. Aerial mycelia

productions starts from concentrated tufts in the center and to sparse. Matheur media exhibits quite the

opposite. Colonies are dark olive-green in the center to pale white. Production of aerial mycelia is

sparse from the center to thick fluffy. Conidia are dark, fusiform, have 6 cells which are hyaline and thick

walls. They have pointed end cells, ellipsoid to fusiod with 2 or more hyaline and 2 or 3 apical

appendages (figure 4.3 A) (Barnett and Hunter, 1998). Rate of colony growth on PDA is slightly faster

than matheur with variations.

Phoma sp. (SGUk) : colonies are dark brownish and concentrated at the center and some patches at the

edges on PDA media. Aerial mycelia is from dense to sparse and has slow growth. On matheur media

colonies are light brown to gray with dark brown sclerotia at the center. Aerial mycelia growth is

uniform. They have dark brown pycnidia and ostiolate. Also have a curved wide beak piercing the

epidermis ( figure 4.3 B). Conidia are small, unicellular with hyaline and is ovoid to elongate (Barnett and

Hunter, 1998). Colony growth on matheur is quicker than on PDA and varies.

Phomopsis sp. (S20Uk): colonies are deep gray to whitish - gray on PDA media. Mycelia are appressed

and their production is dense from the center to sparse. Colonies on matheur media are almost similar to

PDA only that colony growth is slower than PDA and aerial mycelia not dense compared to PDA media.

Conidia are hayaline, unicellular of two types – ovoid to fusoid alpha conidia and filiform, curved or bent

beta conidia ( figure 4.3 C ) (Barnett and Hunter, 1998).

Pseudofusicoccum sp. (S7Nz): colonies on PDA media smoky gray and dense at the center. Aerial

mycelia growth ranges from thick fluffy to sparse tufts towards the edges. But colonies on matheur media

are white to less dense towards the edges. Mycelia production are thick wavy at the center to sparse tufts.

Conidia are ellipsoid to rod-shape, either straight or slightly bent and their apices are round, smooth with

granular content hyaline and have thin wall and are unicellular but there were no sporulation. Pycnidia is

dark and globose (figure 4.3 D). Growth of colonies are slower on matheur media than PDA media.

44

S12Nz

SGUk

S20Uk

S7Nz

Figure 4. 3 Cultural characteristics on PDA, matheur medium and morphology of conidia of mango isolates. (A)

Pestalotiopsis sp. ( S12Nz) (Bar-20µm); (B) Phoma sp. (SGUk ) (Bar-100µm); C Phomopsis sp. (S20Uk) (Bar-20µm); (D)

Pseudofusicoccum sp. (M12Us) (Bar-100µm).

PDA media Matheur media Conidia and/or mycelia

D

C

B

A

20µm

100µ

100µm

20µm

45

Pestalotiopsis sp.

x100 x60 x40

Phoma sp.

x40

Lasiodiplodia theobromae(unmature spores)

x60 x40

46

Botryosphaeria sp.

x60 x60 x100

Pseudofusioccum sp.

x60 x60

Colletotrichum gloeosporoides

x60

Figure 4. 4 Spores of some groups of the isolated pathogens earlier identified microscopically. Groups are: Pestalotiopsis

sp., Phoma sp., Lasiodiplodia theobromae, Botryosphaeria sp., Pseudofusioccum sp., Colletotrichum gloeosporoides

47

CHAPTER 5 : DISCUSSION

As far as the literature is concerned, there is very little information about fungal pathogens causing

diseases of mango and the shea fruits in Northern Ghana especially in the Northern region and Upper

west region. Some fungal pathogens are known to infect (pre and postharvest infection) and cause serious

disease in fruits which sometimes results in fruit losses.

In the present study, pathogenic fungi were isolated and identified from mature but unripe mango fruits,

shea fruits and shea leaves. The pathogens identified in this study are Lasiodiplodia sp.,

Botryospahearia sp., Pseudofusicoccum sp., Colletotrichum gloeosporioides for mango isolates and

Pestalotiopsis sp., Phoma sp., and Phomopsis (Diaporthe sp.) for shea isolates. However

Pseudofusicoccum sp. was isolated and identified on both mango and shea fruits. These pathogenic fungi

were identified based on primarily ITS sequences analysis and conidial morphology with reference to

Barnett and Hunter (1998) and not specifically on colony characteristics though they may be true for

some of the isolates. These pathogens are known to cause various diseases on a wide range of host plant

including mango and the shea trees. Some pathogens may be host specific whiles others are not. These

identified pathogens represents the various groups of isolates that were sorted earlier based on colony

characteristics that were similar.

Mango isolates

Among the isolated pathogens, Lasiodiplodia theobromae., Pseudofusicoccum sp., and Botryospahearia

sp. are members of the Botyosphaeriaceae family. Lasiodiplodia theobromae was isolated and identified

from mango fruits collected from market in Tamale (Northern region), Botryospahaeria sp. and

Pseudofusicoccum sp. were identified and isolated from mangoes collected from tree in Wa (Upper west

region). These isolates represents three different groups for the mango when sorted based on similar

growth in colony characteristics. Each group comprise of isolates exhibiting similar culture

characteristics from which the purest plate was selected irrespective of the location of sample.

Botryosphaeriaceae members have been reported to be linked with several host tree plants that acts as

pathogens (Crous et al., 2006) and have also been established to be the causal organisms of cankers, die

back on stems and stem-end rot diseases in mangoes (Sakalidis et al., 2011; Costa et al., 2010 ).

Meanwhile Lasiodiplodia theobromae and Botryosphaeria sp. causes Stem-end rot diseases in mangoes

and Lasiodiplodia theobromae is known to survive in hot regions (Sakalidis et al., 2011; Ploetz et al,

1994). But L. theobromae produced the largest lesions at 30 °C when mango fruit were co-infected

together with Neofusicoccum parvum (Sikadilis et al., 2011). The isolation of these pathogens in this

48

study from mango fruits taken from Northern Ghana where temperatures ranges between 33°C to 37°C

(maximum day temperatures) in Northern region and 22.6 °C to 40 °C in the Upper west region which

could be a reflection of the reports stated before about Lasiodiplodia sp. prevailing in hot environments

which in this case Northern Ghana is no exception. These regions are among the hottest part of Ghana.

Some few observation in the field is that mango sold in the market are sometimes left to stand in the

scorching sun. This may facilitate the development of stem-end rot disease caused by Lasiodiplodia sp.

Despite the mango fruits collected were not ripe but matured, these fungal pathogens (Lasiodiplodia

theobromae and Botryosphaeria sp.) yet were isolated. This might be that they found their way into the

plant tissue through seeds, lenticels or stomata and infect stems and fruits or even enter via wounds

(Smith et al., 1996). Again is possible they may have established asymptomatically in the field in which

the disease will later manifest itself due to stress or upon ripening of the fruits (Johnson, 2008). This

means the fungi can be in their quiescent phase and waiting to be disturbed by some stress on the host

such as water, temperature which may possibly trigger the pathogen life cycle.

Adansonia digitata (baobab tree) is among the eight species of Adansonia sp. found naturally in the

savanna vegetation of Northern Ghana (Siaw, 2001) where Mangifera indica (mango) and Vitellaria

paradoxa (shea tree) are commonly found and also cultivated. Baobab is reported to be a host of the

Pseudococcoidae family (Orwa et al., 2009) and seven new species of Botryosphaeriaceae including

Pseudofusicoccum sp. (Pseudofusicoccum adansoniae, P. ardesiacum, P. kimberleyense) from

Adansonia gibbosa (one of the Adansonia sp.) have been described by Palvic et al., (2008) in Western

Australia. Moreover, a study on the biogeographical on baobabs by Baum et al., (1998) discovered that A.

gibbosa in Australia is closely related to A. digitata found in Africa than those found in Madagascar. The

study also presented a fascinating view of the unusual distribution of the trees between Australia and

Africa to have occurred after the division of Gondwana. Further studies carried out in the Kimberley

region of Western Australia on canker associated fungi on mangoes also identified the same

Pseudofussicoccum sp. as those demonstrated by Palvic et al., to be among the potential pathogens or

endophytes (Sakalidis et al., 2011). The isolation and identification of Pseudofusicoccum sp. in this study

on mango fruits sampled from Northern Ghana (Upper west) where A. digitata is also commonly found

may perhaps confirm the colonizing ability of members of Botryosphaeriaceae on a wide range of

woody hosts in different environment (Slippers and Wingfield, 2007), and a reflection of their potential of

infecting both mango trees and fruits (Sikadilis, 2011).

49

A lot of literature and studies on the fungal pathogen Colletotrichum gloeosporioides which causes

anthracnose on a range of crops have also been isolated and identified in this study. It has been reported

to be found in all mango producing areas worldwide and is most significant in places with high rainfall

and humidity (Ploetz et al, 1994). It has also been indicated to cause damage to most plant parts (roots,

leaves, stems, flowers, fruits) in both temperate and tropical environment and can affect fruits of all ages

(Nelson, 2008; Ploetz et al., 1994; Bailey, 1992). The isolation and identification of this ubiquitous

pathogen (Colletotrichum gloeosporioides) from mango fruits collected from a tree in the Upper West

region in Northern Ghana is found in the tropics of Africa and is in line to most of the literatures stated

above about where they can be found to infect their hosts. Colletotrichum sp. was also isolated and

identified among other fungi from mango leaves and panicles showing die back symptoms and fruit with

stem-end rot in northeastern Brazil (Costa et al., 2010).

Despite the fact that the pathogen was isolated from green, unripe but matured mango fruit did not

suggest that the pathogen could not have find its way in/on the fruit. Colletotrichum species are known

for their ability to infect fruits at any stage and remains latent until favourable conditions returns. It is

explained by Nelson (2008) that many green fruits infections stays quiescent until the unset of ripening

hence fruits that seems healthy at harvest may develop significant anthracnose symptoms quickly upon

ripening. Such could be the case in the present study though fruits were collected green and transported

to Belgium, climacteric conditions may have set in, and the fruits could show symptoms later on.

Mechanical damage on fruits may also aid easy penetration of pathogens and infection of fruits.

The conidia characteristics of some of the isolated pathogens of mango makes them quite easy to be

identified from one another though phylogenic analysis was not performed. For instance, conidia of

Lasiodiplodia sp. allows relatively easy identification of the conidia among the other conidia pathogens.

More so, the presence of the 2 cells at maturity, which has dark brown colouration and thick brown wall

makes Lasiodiplodia theobromae easier to be differentiated from the rest. Conidia of Pseudofusicoccum

sp.,and Botryosphaeria sp. were less distinguishable especially those of Pseudofusicoccum sp. and

Botryosphaeria sp. due to their overlapping conidial shapes and colours. Spores of Colletotrichum

gloeosporioides were not seen and could be that they not ready for sporulation at the time of microscopy.

Nevertheless there were more distinct differences in their colony characteristics, growth rate on PDA and

matheur media (Figure 4.2).

50

Shea isolates

Pestalotiopsis sp. identified in this study have also been described as an ubiquitous pathogens found in

both tropical and semi tropical plants and isolated as entophyte from stems, leaves, flowers and fruits

from tropical and rainforest plants by (Metz et el., 2000). It has been isolated as one of the pathogens on

mangoes in Brazil by Costa et al. (2010) and the pathogen Pestalotiopsis mangiferae is reported to cause

gray leaf spot in mangoes (Ploetz et al., 1994). The shea and mango trees found in Ghana is undoubtedly

found in tropics and can also be a host and also susceptible to this pathogen as mentioned in various

literatures. It is therefore not surprising in the current study that this pathogen have also been isolated.

Leaf spots, needle blight, tip blight, and gray blight disease symptoms found on hardy ornamentals have

been reported to be caused by Pestalotiopsis sp. (Pirone, 1978) but Pestalotia sp. have also been isolated

from leaves of the shea tree that cause leaf spot on shea trees in Ghana (Akrofi and Amoah, 2009). Some

studies by Patel et al. (1950) indicated that Pestalotia causes symptoms on green fruits and rarely on

leaves. This was quite not the case in the present study. Pestalotiopsis sp. was isolated on both fruits and

leaves of the shea plant which is directly in consonance with the studies conducted by Amoah and Akrofi

(2009), Keith et al. (2006), and Metz et al. (2000). Furthermore scab diseases have been demonstrated to

be caused by Pestalotiopsis sp. on guava fruits (Psidium guajava) in Hawaii (Kieth et al., 2006). This

may be an indication of the colonizing ability of the pathogen on various plant parts (leaves and fruits).

However there are confusion on the taxonomy of the genera (Pestalotia and Pestalotiopsis) and it is not

yet known if they are even different genera (Maharachchikumbura et al., 2011) but have been linked to

cause a number of diseases in plants.

Though fruits and leaves of Vitellaria collected were initially healthy before transporting them to

Belgium, the isolation of the Pestalotiopsis sp. could be an evidence that the pathogen is opportunistic

that affects stressed plants or weak pathogen invading aged leaves (Metz et al., 2000) or may be that it has

the ability to also infect and remain latent on their host and manifest itself during favourable conditions.

This is because fruits and leaves that were detached from their parent tree might have gradually been

losing some of its natural defense mechanism when it was detached from the parent plant.

Phomopsis sp. have been associated to also cause leaf spots, cankers, root rots, fruit rots, diebacks,

blights, decay and wilts on a broad range of plant hosts including a number of economically important

hosts worldwide (Santos and Philips, 2009). For example Phomopsis mangifera is linked to also cause

stem-end rot and dieback in mango (Costa et al., 2010 ; Ploetz et al., 1994). Although there are no

previous reports of Phomopsis sp. isolations from shea tree, however it has been isolated and identified

51

from fruits sampled from the Upper West region in the present study. Mango and shea trees as stated

earlier are among the economic and important trees found in the vegetation in Northern Ghana and

mango fields are sometimes found in close proximity to the shea trees, thus the mango trees could be a

source of inoculums. The dynamics of this source of inoculum needs further studies. There are numerous

arguments on the taxonomic history of Phomopsis/Diaporthe regarding the use of the names telemorph

and an morph states (Santos et al., 2010). But according to Udayanga et al. (2011), Phomopsis is the

most often used for diseases causing organisms as described above than Diaporthe hence the use of

Phomopsis in this study. Again it could be that the mango fields which are near to the shea tree

population could also serve as a source of inoculums to the shea tree. Some Phomopsis species have been

reported to even cause health problems in humans and other mammals, though it has not been recognized

as such on the shea fruits but could be a major threat to health should this findings proves to be true by

further studies. They are also sometimes saprobes and plant pathogens (Sutton et al., 1997; Garcia-Reyne

et al., 2011). This is because is potential producer of mycotoxin called phomopsin in chestnut

(Osmonlieva et al., 2001).

Phoma sp. and Pseudofusicoccum sp. are among the fungi pathogen isolated and identified from the shea

tree. Phoma sp. was isolated from fruits in the Upper west Region whiles that of Pseudofusicoccum sp.

was isolated from shea fruits in the Northern region. As mentioned before little is known about the

pathogens of the shea tree in Ghana and Phoma sp. has not yet been established to cause infection on shea

tree. However Phoma sp. causes phoma blight in mango. It can only be observed on matured or old

leaves which could probably be that is also a weak pathogen that invades stress plants. It causes spot that

are fully developed and are characterized by dark margin and dull necrotic centre (Medina and Garcia,

2002). Though is associated to cause infection of mango leave, its isolation on shea fruit is possible since

it is not host specific (Boumediane,2010).

The shea tree (Vitellaria paradoxa ) and baobab tree (Adansonia digitata) are among the trees that co -

exist naturally in the savannah vegetation of Northern Ghana. As stated by Orwa et al.(2009) that

Adonsonia digitata serves as a host to the Pseudococcoidae family and the isolation of Pseudofusicoccum

sp. from Adonsonia gibbosa in Australlia by Palvic et al.(2008) may be clear indication that the

Pseudofusicoccum sp. identified on both the shea and mango may perhaps have come from the Adansonia

digitata. This finding however is not conclusive and needs further studies since no reports have been

made from Adansonia digitata specifically in Ghana.

52

Conidia characters of some shea isolates makes them easier to be distinguished from the others. For

example the conidia of Petalotiopsis sp. makes it completely distinct from the others. Their conidia have

5 cells , have 2 to 3 apical appendages and are hyaline, posses three median cells ranging from light

brown to dark brown. Phomopsis sp. and Phoma sp. on the other hand have conidia that makes them also

distinguishable from each other. The acervuli of the Phoma sp. allows easier identification as well

conidia of the Pseudofusicoccum sp. of the mango isolates as described by Palvic et al. (2008) from the

Adansonia sp. Again their colony characteristics and growth rate were quite different from each other on

PDA and matheur media (figure 4.3). Most growth rate of isolates on PDA seems to be faster than on

matheur with some few exceptions for instance the growth of Phoma sp. on matheur was faster than on

PDA. This could be that PDA has the required nutrients that enables better growth of most fungi than

matheur (figures 4.2 and 4.3).

In addition, the differences in the growth rate of the mycelia of the various isolates (mango and shea) on

both matheur and PDA media could be affected by temperature since cultures of both media were kept

under room temperature (21-25 oC). Some pathogens may show better growth on particular media under

certain conditions than others. For instance, a study by Kieth et al. (2006) revealed that some Pestalotia

sp. have optimum growth at 26 oC on PDA media. But mycelia growth of Lasiodiplodia sp. (figure 4.2 A)

on both media showed an extra ordinary growth at the same temperature. Temperatures in Northern

Ghana is between 22 – 36 oC and this could be an indication that the pathogen is able to infect effectively

at those temperatures. Another study on Lasiodiplodia theobromae produced large lesions at 30 oC

during an inoculation studies by Sakalidis et al. (2011).

The level of concentrations when measured, and electrophoresis of the PCR products of mango and shea

isolates somehow confirms that the amplification of the ribosomal DNA did went well though efficiency

of the amplification was not verified.

Difficulties

Some difficulties did come up when comparing both ITS sequences results from nucleotide blast

search. For instance the choice of Phomopsis or Diaporthe sp. on the GenBank data base. This leads to

support the complexities the in redefining them as mentioned by Udayanga et al. (2011). There were also

quite difficulties encountered in describing the colony characteristics due to the overlapping in growth of

the mycelia of some isolates on both media. This may buttress the problems faced in identifying and

describing species of fungal pathogens based only on morphological characters. Finally it was not easy to

take pictures of the spores of all the isolates because some take longer time to sporulate.

53

54

CHAPTER 6 : CONCLUSION AND RECOMMENDATION

Most frequently, anytime matters relating to food availability and accessibility comes up, the foremost

reaction often is, and to a large extent will always be to produce more food resulting in both private and

public interventions in agriculture and related sectors aiming at increasing food production. On the other

hand, as essential as maximizing food production to meet food for availability and accessibility is, it can

never be the way out in itself to ensuring food security until measures are put in place to also ensure and

guarantee food preservation, protection and availability at all times and in times of need. The difficulty

is not that adequate amount of food is not produced, but enough is not conserved due to poor post harvest

practices, inadequate information about important pathogens causing pre and post harvest losses hence

poor and insufficient control strategies. In order to conserve enough food to guarantee food availability in

Ghana, it is important to pay greater attention to minimizing postharvest losses by researching into

potential pathogens that are capable of causing serious disease on important and economic crops such

Mangifera and Vitellaria.

This study have isolated and identified a number of potential pathogens (Lasiodiplodia sp.,

Botryospahaeria sp., Pseudofusicoccum sp., Colletotrichum gloesporioides , Pestalotiopsis sp., Phoma

sp., and Phomopsis.) from mango and shea fruits from Northern and Upper west regions of Northern

Ghana. Most of these pathogens have also been reported from other parts of the world to cause various

diseases on a wide range of host which could result in food losses. The study also revealed that certain

pathogens that were not previously reported on Mangifera and Vitellaria in Northern Ghana needs

serious and urgent attention. Pathogenic fungi such as Pseudofusicoccum sp., Phoma sp., and Phomopsis

sp. have also been revealed to be associated with the shea tree in addition to Pestalotiopsis sp., and

Pseudofusicoccum sp. have been reported for the first time on mango in Northern Ghana according to this

studies.

Moreover, the findings in this study have demonstrated the likelihood of cross infection of some

pathogens from one host to another. Notable among them is the possibility of Pseudofusicoccum sp. on

mango and shea trees that might have come from Adansonia digitata (baobab) which is known to be the

host of Pseudococcoidae family. Both baobab and shea trees exist together in the same environment in

the savanna of northern Ghana. Furthermore the isolation of Phoma sp., and Phomopsis sp. from shea

tree could also emanate from the mango since they are both found in proximity to each other in the

savanna vegetation. Phoma and Phomopsis sp. have been mentioned to be linked to mango and may

serve as a source of inoculums for the shea tree.

55

It is essential to find out the infection pathway of the pathogenic fungi since understanding of the

infection pathway can lead to good management and postharvest handling practices. These are key to

minimize inoculums and infections of the host thus reduction of diseases on crops and associated health

problems posed by some pathogens such as Phomopsis sp. More work is needed to be done to explain the

relationship between native host of some pathogens such as Pseudofusicoccum sp. on Adansonia digitata

that are associated to both mango and the shea tree. Also further studies is required to verify the gene

flow, whether the fungi are moving from the native host into the crops or vice versa. These pathogens if

not controlled properly will not only pose an economic risk but also health risk and food shortages in the

northern part of the country.

56

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