ABSTRACT
Mycological investigations of the array of fungal pathogens associated with postharvest rots of fruits (Persea americana, Annona muricata, Citrus sinensis, Carica papaya and Lycopersicon esculentum) in Akwa Ibom State, and the potentiality of aqueous and ethanolic leaf extracts of Heinsia crinita, Lasienthera africanum, Gongronema latifolium and Piper guineense as bio-control/preservatives on the fresh fruits were conducted using standard mycological techniques. The phenotypic and genotypic identifications of the fungal pathogens isolated from fruit samples were carried out using standard cultural, morphological and molecular methods respectively. The pathogenicity, mycotoxins production of the isolates, antifungal efficacies of the extracts on mycelial radial growth and spore germination, qualitative phytochemistry and Fourier Transformation Infrared Spectroscopy (FT-IR) of the extracts were determined using standard mycological, agar well/diffusion, Chemical and Atomic Absorption Spectroscopy (AAS) techniques. The phylogenic relationships among the fungal species were also constructed. Fourteen fungal genera identified were Talaromyces, Lasiodiplodia, Trichoderma, Pichia, Penicillium, Mucor, Moniliella, Geotrichum, Candida, Aspergillus, Rhizopus, Absidia, Fusarium and Purpureocillium. The pathogenicity tests revealed that the fungal isolates were able to cause rots at varying degrees. The most rapid rots were caused by R. oligosporous, P. kudriavzevii and A. niger within 24 hours, while A. aculeatus and M. suaveolens were slower in initiating rots (5th day). These fungal isolates produced mycotoxins in different range of concentrations. The highest concentration of ochratoxin was produced by A. niger (10.5ppb) and the lowest was by A. parasiticus (0.1ppb). The highest concentration of aflatoxin was seen in A. nomius (15.6ppb) and the lowest inA. aculeatus and A. carbonarius(0.3 ppb). Phytochemical screening of the extracts revealed the presence of alkaloids, tannins, saponins, cardiac glycosides in varied concentrations. The FT-IR analysis of the extracts revealed groups of carboxylic acids, amines, esters, ketones, and aromatics with absorption bands ranging from 534.3 to 3416cm-1. The extracts exhibited inhibitory effects on the fungal radial mycelial growth and spore germination with ethanolic extracts of G. latifolium being the most potent and aqueous extracts of H. crinita being the least effective (inhibiton zones of 21.0mm and 8.0mm respectively). The regression values (R2) of the plant extracts concentration/inhibitory zones ranged from 0.75 to 1.0. The extracts at the 750mg/ml concentrations were able to preserve the fresh fruits up to 14 days. The dendrogram showed great similarities among the fungal isolates, with T. koningiopsis being related to P. lilacinum and P. kudriavzevii closely related to G. candidum. This study establishes the array of mycotoxigenic fungi associated with post-harvest fruit rots and efficacies of these plant extracts as biocontrol of fruit spoilage organisms due to the bio-active secondary metabolites and chemical functional groups present in the plant extracts.
TABLE OF CONTENTS
TITLE PAGE
Title
Page -
- - - - -
- i
Certification -
- - - - -
- ii
Declaration
Page - - - - -
- - iii
Dedication
-
- - - - -
- iv
Acknowledgements
- -
- - - - -
v
Table
of Contents - - -
- - - - vii
List
of Tables - - - -
- - - xii
List
of Figures - - - -
- - - xv
List
of Plates - - - -
- - - xvii
Abstract
-
- - - - -
- xxi
CHAPTER
ONE: INTRODUCTION - -
- - 1
1.1 FRUITS AND MICROORGANISMS - -
- 1
1.2
DEFINITIONS - - - -
- - 1
1.2.1
Fungal Pathogens - -
- - -
1
1.2.2
Post – harvest Rots - -
- - - - 2
1.3
DISEASE MANAGEMENT -
- - - 4
1.4
MEDICINAL PLANTS - -
- - - 5
1.5
JUSTIFICATION OF THE STUDY - - -
- 7
1.6 OBJECTIVES
OF THE STUDY - - - - 8
CHAPTER TWO: LITERATURE REVIEW - - - 9
2.1 ECONOMIC IMPORTANCE OF FRUITS - - -
9
2.1.1
Persea
americana (Mill) Fruits - - -
- 9
2.1.2
Citrus
sinensis (Osbeck) Fruits - - -
- 10
2.1.3
Carica
papaya (L) Fruits - - -
- - 12
2.1.4
Annona
muricata (L) Fruits - - - -
- 13
2.1.5
Lycopersicon
esculentum (Mill) Fruits - - -
14
2.2 FUNGAL
PATHOGEN ASSOCIATED WITH SPOILAGE OF
FRUITS - - - - 14
2.2.1 Fungal Pathogen Associated with Spoilage of Persea
americana (Mill) Fruits - -
- - - 14
2.2.2 Fungal
Pathogens Associated with Spoilage of Citrus
sinensis
(Osbeck)
Fruits - - - - -
- 15
2.2.3 Fungal Pathogens Associated with Spoilage of Carica
papaya(L) Fruits - - - -
- - 16
2.2.4 Fungal Pathogens Associated with Spoilage of Annona
muricata(Linn) Fruits -
- - - - 17
2.2.5 Fungal Pathogens Associated with Spoilage of Lycopersicon
esculentum (Mill) Fruits - -
- - - 18
2.3 DETECTION
AND ISOLATION OF FRUIT SPOILAGE
ORGANISMS -
- - - - -
18
2.4
MEDICINAL PLANTS - -
- - - 19
2.4.1
Gongronema
latifolium (Benth et Hook) - - -
19
2.4.2
Heinsia
crinita (Afz) G. Taylor - - -
- 20
2.4.3
Lasienthera
africanum (P. Beauv) - - -
- 21
2.4.4
Piper
guineense (Schumach) - -
- - 21
2.5
MYCOTOXINS - - - -
- - 22
2.5.1
Aflatoxins - - - -
- - 22
2.5.2
Ochratoxin - - - -
- - 23
2.5.3
Critinin - -
- - - - 23
2.5.4
Ergot Alkaloids - - - -
- - 23
2.5.5
Patulin
-
- - - - -
23
2.5.6
Fumonisins, Trichothecenes and
Zearalenone - - 24
2.6 TOXIC PHENOMENA ASSOCIATED WITH
MYCOTOXINS 24
CHAPTER THREE:
MATERIALS AND METHODS - - 26
3.1
STUDY AREA - - - -
- - 26
3.2
MATERIALS -
- - - - -
28
3.3
COLLECTION OF SAMPLES - -
- - 28
3.4
METHODOLOGY - - - -
- - 31
3.4.1 Sterilization of Glass Ware - -
31
3.4.2
Preparation and Sterilization of Media - - - 31
3.5 ISOLATION
AND IDENTIFICATION OF FUNGAL
PATHOGENS
FROM THE FRUITS - - -
31
3.6
PATHOGENICITY TESTS - - -
- - 32
3.7 DETECTION OF MYCOTOXINS PRODUCTION -
32
3.8 PREPARATION
OF AQUEOUS AND ETHANOLIC
LEAF
EXTRACTS - - - - -
- 33
3.9 PHYTOCHEMICAL
SCREENING OF THE LEAF
EXTRACTS
- - - - - -
- 33
3.9.1
Test for Alkaloids - -
- - - - 34
3.9.2
Test for Tannins - -
- - - - 34
3.9.3
Test for Saponins - -
- - - - 34
3.9.4
Test for Flavonoids - -
- - - - 35
3.9.5
Test for Cardiac Glycosides - - -
- - 35
3.9.6
Test for Anthraquinones - -
- - - 36
3.9.7
Test for Resins - - - -
- - 36
3.9.8
Test for Deoxy-Sugar - - -
- - - 36
3.9.9
Test for Protein - -
- - - - 37
3.9.10
Test for Phlobatanins - -
- - - 37
3.10 FOURIER TRANSFORMATION INFRA-RED
SPECTROSCOPY
ON LEAF EXTRACTS - - - 37
3.11 PARTITIONING OF THE CRUDE LEAF EXTRACTS - 38
3.12
ANTIFUNGAL ASSAY - -
- - - 38
3.12.1 Determination of Inhibitory Activity of Leaf
Extracts on Fungal
Growth Using Agar Dilution Method - -
- 38
3.12.2
Preparation of the Fungal Spores -
- - - 39
3.12.3 Determination of Inhibiting
Activity of Leaf Extracts on Fungal
Spore
Germination using Agar-well Diffusion Method -
39
3.12.4 Use of the Leaf Extracts as Bio
Control/Preservative agents - 40
3.13 MOLECULAR CHARACTERIZATION 40
3.13.1
DNA Extraction and Purification - - - - 40
3.13.2 DNA
Amplification using Eppendorf Thermal Cycler -
42
3.13.3
PCR Condition - - -
- - - 42
3.13.4 Gel
Electrophoresis of gDNA and PCR Products -
43
3.13.5
PCR Product Purification - -
- - - 43
3.13.6 DNA
Sequencing of ITS 4 and ITS 5 Genes - -
- 43
3.14 STATISTICAL ANALYSIS - - - - - 44
CHAPTER FOUR: RESULTS AND DISCCUSSION - - 45
4.1 CULTURAL,
MORPHOLOGICAL AND MICROSCOPIC CHARACTERISTICS OF FUNGALISOLATES FORM
DISEASED P.
americana, C. sinensis,C. papaya, A. muricata
AND
L. esculentum FRUITS - - -
- - 45
4.2 FUNGAL
PATHOGENS ISOLATED FROM DISEASED
FRUIT
SAMPLES - - - - -
- 77
4.3 APPEARANCE
OF FUNGAL ISOLATES ON DISEASED
FRUITS
- - - - -
- - 85
4.4 PATHOGENICITY
TESTS OF FUNGAL ISOLATES ON
POST-HARVEST
FRESH HEALTHY FRUITS - - 97
4.5 CONCENTRATION
OF AFLATOXIN AND OCHRATOXIN
IN
DISEASED FRUIT SAMPLES - - - -
115
4.6 CONCENTRATION
OF AFLATOXIN PRODUCED BY
FUNGAL
ISOLATES IN DISEASED FRUIT SAMPLES - 117
4.7 CONCENTRATION
OF OCHRATOXIN PRODUCE BY
FUNGAL
ISOLATES IN DISEASED FRUIT SAMPLES - 120
4.8 PERCENTAGE YIELDS OF PLANT EXTRACTS -
124
4.9 PHYTOCHEMICAL
CONSTITUENTS OF PLANT LEAF
EXTRACTS
- - - - - -
- 127
4.9.1 Phytochemical Constituents of
Ethanolic Extracts of Plants - 127 4.9.2 Phytochemical Constituent of Aqueous
Extracts of Plants - 128
4.10 FOURIER
TRANSFORMATION INFRA-RED ANALYSIS
OF
EXTRACTS - - - - -
- 133
4.11 ANTIFUNGAL ACTIVITIES OF LEAF EXTRACTS - 147
4.11.1 Antifungal Activities of Leaf Extracts on Radial Growth of
Fungal
Isolates - - -
- - - 147
4.11.2 Antifungal Activities of Leaf Extracts on Spore Germination
ofFungal
Isolates - - - - -
- 147
4.12 USE
OF LEAF EXTRACTS AS BIO-PRESERVATIVES - 163
4.13 MOLECULAR CHARACTERIZATION - -
- 165
4.14
DISCUSSION -
- - - - -
170
4.15
CONCLUSION - - - -
- - 173
REFERENCES
-
- - - - -
175
Appendix
-
- - - - -
185
LIST
OF TABLES
TITLE PAGE
Table
3.1 DNA Cocktail Mix - -
- - - 42
Table
3.2 PCR Condition - -
- - - 42
Table 4.1a Cultural,
Morphological and Microscopic characteristics
of
fungal isolates from diseased fruit samples -
55
Table 4.1b Cultural,
Morphological and Microscopic characteristics
of fungal
isolates from diseased fruit samples -
63
Table 4.1c Cultural,
Morphological and Microscopic characteristics
of fungal
isolates from diseased fruit samples -
72
Table 4.1d Cultural,
Morphological and Microscopic characteristics
of fungal
isolates from diseased fruit samples -
76
Table 4.2 Fungal
pathogens isolated from Diseased P.
americana
Fruits -
- - - - 80
Table 4.3 Fungal
pathogens isolated from Diseased C.
sinensis
Fruits -
- - - - 81
Table 4.4 Fungal
pathogens isolated from Diseased C.
papaya
Fruits -
- - - - 82
Table 4.5 Fungal
pathogens isolated from Diseased A.
muricata
Fruits -
- - - - 83
Table 4.6 Fungal
pathogens isolated from Diseased L.
esculentum
Fruits -
- - - - 84
Table 4.7 Appearance
of fungal isolates on the diseased
P. americana fruit - -
- - - 90
Table 4.8 Appearance
of fungal isolates on C. sinensis fruit
Samples
-
- - - - 91
Table 4.9 Appearance
of fungal isolates on C. papaya fruit
Samples
-
- - - - 92
Table 4.10 Appearance
of fungal isolates on A. muricata fruit
samples -
- - - - 94
Table 4.11 Appearance
of fungal isolates on L. esculentum
fruit
samples -
- - - - 95
Table 4.12a Pathogenicity
Tests of the fungal isolates on
P. americana fruit sample - -
- - 100
Table 4.12b Pathogenicity
Tests of the fungal isolates on
P. americana fruit sample - -
- - 101
Table 4.13a Pathogenicity
Tests of the fungal isolates on
C. sinensis fruit sample - -
- - 103
Table 4.13b Pathogenicity
Tests of the fungal isolates on
C. sinensis fruit sample - -
- - 104
Table 4.14a Pathogenicity
Tests of the fungal isolates on
C. papaya fruit sample - -
- - 106
Table 4.14b Pathogenicity
Tests of the fungal isolates on
C. papaya fruit sample - -
- - 107
Table 4.15a Pathogenicity
Tests of the fungal isolates on
A. muricata fruit sample - -
- - 109
Table 4.15b Pathogenicity
Tests of the fungal isolates on
A. muricata fruit sample - -
- - 110
Table 4.16a Pathogenicity
Tests of the fungal isolates on
L. esculentum fruit sample - -
- - 112
Table 4.16b Pathogenicity
Tests of the fungal isolates on
L. esculentum fruit sample - -
- - 113
Table 4. 17 The occurrence and concentration of Aflatoxin
and
Ochratoxin in diseased fruit samples -
116
Table 4. 18 The occurrence and concentration of Aflatoxin
produced by
fungal isolates from diseased fruit -
119
Table 4. 19 The occurrence and concentration of Ochratoxin
produced by
some fungal isolates from diseased fruit 122
Table 4. 20 Percentage yield of plant extracts - -
126
Table 4.21 Phytochemical
constituents of Ethanolic extracts of plants 129
Table 4.22 Phytochemical constituents of Aqueous extracts of plants 130
Table 4.23 Frequency Absorption Bands of H. crinita - - 135
Table 4.24 Frequency Absorption Bands of L. africanum - 138
Table 4.25 Frequency Absorption Bands of G. latifolium - 141
Table 4.26 Frequency Absorption Bands of P. guineense - 144
Table 4. 27a Antifungal activities of Ethanolic leaf extracts
of P. guineense on radial growth of fungal
isolates - 149
Table 4. 27b Antifungal activities of Aqueous leaf extracts
of P. guineense on radial growth of fungal
isolates - 150
Table 4.28 Antifungal
activities of Ethanolic leaf extracts of
P. guineense on fungal spore germination
- -
151
Table 4.29 Antifungal
activities of Ethanolic leaf extracts
G. latifolium on fungal spore germination - -
152
Table 4.30 Antifungal
activities of Ethanolic leaf extracts of
H. crinita on fungal spore germination - -
153
Table 4.31 Antifungal
activities of Ethanolic leaf extracts of
L. africanum on fungal spore germination
- -
154
Table 4.32 Antifungal
Activities of Aqueous leaf extracts of
P. guineense on fungal spore germination
- -
155
Table 4.33 Antifungal
Activities of Aqueous leaf extracts of
G. latifolium on fungal spore germination - -
156
Table 4.34 Antifungal
Activities of Aqueous leaf extracts of
H. crinita on fungal spore germination - -
157
Table 4.35 Antifungal
Activities of Aqueous leaf extracts of
L. africanum on fungal spore germination
- -
158
Table 4.36 Regression
Coefficient of antifungal activities of
Ethanolic
leaf extracts on Fungal Spore Germination- 159
LIST OF FIGURES
TITLE PAGE
Figure 2.1 Toxic Phenomena Associated with Mycotoxins
- 25
Figure 3.1 Map
of Akwa Ibom State, Nigeria, showing the three
Senatorial
districts -
- - - 27
Figure
4.1 Lasiodiploidia theobromae -
- - - 49
Figure
4.2 Trichoderma koningiopsis -
- - - 53
Figure
4.3 Rhizopus oligosporus - -
- - 56
Figure
4.4 Aspergillus niger -
- - - 58
Figure
4.5 Aspergillus carbonarius -
- - - 62
Figure
4.6 Moniliella suaveolens - -
- - 65
Figure
4.7 Mucor racemosus -
- - - 67
Figure
4.8 Geotrichum candidum -
- - - 69
Figure
4.9 Absidia corymbifera - -
- - 70
Figure 4.10 Graph of Ochratoxin level in diseased
fruits -
123
Figure 4.11 Frequency
absorption bands of Heinsia crinita
(Aqueous) 136
Figure 4.12 Frequency
absorption bands of Heinsia crinita
(Ethanolic) 137
Figure 4.13 Frequency absorption bands of L. africanum (Aqueous) 139
Figure 4.14 Frequency absorption bands of L.
africanum (Ethanolic) 140
Figure 4.15 Frequency absorption bands of G. latifolium (Aqueous) 142
Figure 4.16 Frequency absorption bands of G.
latifolium (Ethanolic) 143
Figure 4.17 Frequency absorption bands of P. guineense (Aqueous) 145
Figure 4.18 Frequency absorption bands of P.
guineense (Ethanolic) 146
Figure 4.19 Relationship
between concentration of P. guineense
and
inhibitory
zones as exhibited by T. verruculosus
- 160
Figure 4. 20 Relationship
between concentration of P. guineense
and
inhibitory
zones as exhibited by L. theobromae - 160
Figure 4.21 Relationship
between concentration of P. guineense
and
inhibitory
zones as exhibited by P. lilacinum 160
15
Figure 4.22 Relationship
between concentration of G. latifolium
and
inhibitory
zones as exhibited by L. theobromae - 160
Figure 4.23 Relationship
between concentration of G. latifolium
and
inhibitory
zones as exhibited by A. niger - -
161
Figure 4.24 Relationship
between concentration of G. latifolium
and
inhibitory
zones as exhibited by A. sclerotiorum
- 161
Figure 4.25
Relationship between
concentration of H. crinita and
inhibitory
zones as exhibited by P. citrinum -
161
Figure 4.26 Relationship
between concentration of H. crinita
and
inhibitory
zones as exhibited by A. parasiticus - 161
Figure 4.27 Relationship
between concentration of H. crinita and
inhibitory
zones as exhibited by C. tropicalis - 162
Figure 4.28 Relationship
between concentration of L. africanum
and
inhibitory
zones as exhibited by T. verruculosus
- 162
Figure 4.29 Relationship
between concentration of L. africanum
and
inhibitory
zones as exhibited by F. culmorum - 162
Figure 4.30 Relationship
between concentration of L. africanum
and
inhibitory
zones as exhibited by M. racemosus - 162
Figure 4.31 Phylogenetic
Tree (Dendrogram) showing the level of
similarities
among the fungal isolates. - - 169
LIST OF PLATES
TITLE PAGE
Plate
1a: Piper guineense Leaves _ -
_ - 29
Plate
1b: Heinsia crinita Leaves -
- - -
29
Plate
1c: Lasienthera africanum Leaves -
- - 30
Plate
1d: Gongronema latifolium Leaves -
- - 30
Plate 2a: Talaromyces
verruculosus (front view of young culture) 47
Plate 2b:
T. verruculosus (reverse view of a young culture) - 47
Plate
2c: T. verruculosus (front view of an older culture) - 47
Plate
2d: T. verruculosus (reverse view of an older culture) - 47
Plate 3a: Lasiodiplodia
theobromae (front view of a young culture) 48
Plate 3b: Lasiodiplodia
theobromae (back view of a young culture) 48
Plate 3c: Lasiodiplodia
theobromae (front view of an older culture) 48
Plate 3d: Lasiodiplodia
theobromae (back view of an older culture) 48
Plate 3e: photomicrograph of hyphal network of L. theobromae 48
Plate 4a: Trichoderma
koningiopsis (young culture, white in colour,
zonated)
-
-
-
52
Plate 4b: Trichoderma
koningiopsis (older culture, turning green) 52
Plate
5a: Pichia kudriaavzevii (front view of plate) - - 54
Plate
5b: Pichia kudriaavzevii (back view of plate) - - 54
Plate
6a: Rhizopus oligosporus (front view of plate) - - 54
Plate
6b: Rhizopus oligosporus (back view of plate) - - 54
Plate
7a: Aspergillus niger (front view of plate) - -
57
Plate
7b: Aspergillus niger (back view of plate) - -
57
Plate 7c: Photomicrograph of sporangiophores and
sporangium
of
A. niger - -
- - 57
Plate
8a: F. culmorum (front view) -
- - -
60
Plate
8b: F. culmorum (back view) -
- - 60
Plate
9: Fusarium solani -
- - 60
Plate
10a: Aspergillus parasiticus (front view) - - -
60
Plate
10b: Aspergillus parasiticus (back view) - - -
60
Plate
11a: Aspergillus carbonarius (front view) - - -
61
Plate
11b: Aspergillus carbonarius (back view) - - -
61
Plate
12a: Mucor racemosus (front view)
- - -
66
Plate
12b: Mucor racemosus (back view) - -
- 66
Plate
13: Geotrichum candidum -
- - -
66
Plate
14a: Candida tropicalis (culture on plate) -
- 71
Plate
14b: Candida tropicalis (photomicrograph) - -
71
Plate
15a: Candida utilis (culture on plate) -
- - 71
Plate
15b: Candida utilis (Photomicrograph) -
- - 71
Plate
16a: Aspergillus aculeatus (front view) -
- - 74
Plate
16b: Aspergillus aculeatus (back view) -
- - 74
Plate
17a: Aspergillus nomius (front view) -
- - 74
Plate
17b: Aspergillus nomius (back view) -
- - 74
Plate 18a: Purpureocillium
lilacinum (front view of young culture) 74
Plate 18b: Purpureocillium
lilacinum (reverse view of young culture) 74
Plate 18c: Purpureocillium
lilacinum (front view of older culture) 75
Plate
19a: Candida pseudotropicalis -
- - -
75
Plate
19b: Photomicrograph of Candida pseudotropicalis - 75
Plate
20a: Diseased P. americana fruit from Uyo market -
93
Plate 20b: Diseased
P. americana fruit from Ikot Ekpene
market 93
Plate 21a: Diseased C.
sinensis fruit from Ikot Ekpene market - 93
Plate
21b: Diseased C. sinensis fruit from Eket market - - 93
Plate
22a: Diseased C. papaya fruit from Uyo market - -
93
Plate 22b: Diseased C.
papaya fruit from Ikot Ekpene Market - 93
Plate
23a: Diseased A. muricata fruit from Uyo market - - 96
Plate 23b: Diseased A.
muricata fruit from Ikot Ekpene market
96
Plate 24a: Diseased L. esculentum fruit from Uyo market 96
Plate 24b: Diseased L. esculentum fruit from Eket market 96
Plate 25a: P.
americana fruits inoculated with L.
theobromae - 102
Plate
25b: P. americana fruits inoculated with A. nomius -
102
Plate 26a: P.
americana fruits inoculated with T.
koningiopsis - 102
Plate 26b: P. americana
fruits inoculated with T. verruculosus
- 102
Plate
27a: P. americana fruits inoculated with G. candidum -
102
Plate
27b: uninoculated P. americana (Control) -
- 102
Plate
28a: C. sinensis inoculated with A.
aculeatus - - 105
Plate
28b: C. sinensis inoculated with A.
niger - - - 105
Plate
29a: C. sinensis inoculated with C.
utilis - - - 105
Plate
29b: C. sinensis inoculated with L.
theobromae - - 105
Plate
30a: C. sinensis inoculated with T.
verruculosus - - 105
Plate
30b: Uninoculated C. sinensis (Control) - -
- 105
Plate
31a: C. papaya inoculated with A.
niger - - - 108
Plate
31b: C. papaya inoculated with G.
candidum - - 108
Plate
32a: C. papaya inoculated with L.
theobromae - - 108
Plate
32b: C. papaya inoculated with P.
lilacinum - - 108
Plate
33a: C. papaya inoculated with F.
solani - - - 108
Plate
33b: Uninoculated C. papaya (Control) - -
- 108
Plate
34a: A. muricata inoculated with A.
parasiticus - - 111
Plate
34b: A. muricata inoculated with P.
lilacinum - - 111
Plate
35a: A. muricata inoculated with L.
theobromae - - 111
Plate
35b: A. muricata inoculated with A.
aculeatus - - 111
Plate
36a: A. muricata inoculated with A.
nomius - - 111
Plate
36b: Uninoculated A. muricata (Control) -
- 111
Plate
37a: L. esculentum inoculatedwith M.
racemosus - 114
Plate
37b: L. esculentum inoculated with R.
oligosporus - 114
Plate
38a: L. esculentum inoculated with A.
niger - - 114
Plate
38b: L. esculentum inoculated with P.
kudriavzevii - 114
Plate 29a: L.
esculentum inoculated with G.
candidum 114
Plate
29b: Uninoculated L. esculentum (Control) - 114
Plate 40a: Maceration
of Dried Powered Leaves in Maceration Tanks 125
Plate 40b: Initial filtration of crude extract - -
- 125
Plate 40c: Concentration
of Plant Extracts Using a Rotary Evaporator 125
Plate 40d: Dried Ethanolic plant extracts - -
- 125
Plate 40e: Dried aqueous plant extracts - - -
- 125
Plate 40f: Reconstituted extracts ready for use - - -
125
Plate
41a: Frothing Test -
- - - 131
Plate 41b: Test for free Anthraquinone - - -
- 131
Plate
41c: Cardiac glycoside - -
- - 131
Plate 41d: Test for combined Anthraquinone - -
- 131
Plate
41e: Test for Alkaloids - -
- - 131
Plate
41f: Test for Flavonoids - -
- - 131
Plate
42a: Extract Partitioning setup - -
- - 132
Plate
42b: Extract Partitioning - -
- - 132
Plate
42c: Extract Partitioning - -
- - 132
Plate 43 Spore
suspension production in a water
bath
with a shaker -
- - - 148
Plate 44a – 44f: Fungal spore germination inhibition
(Zones
of inhibition) - - - -
148
Plate 445a and 445b: Unprotected and
Protected C. papaya -
164
Plate 46a and 46b: Unprotected and protected
L. esculentum - 164
Plate 47a and 47b: Unprotected
and Protected C. sinensis - 164
Plate 48: Genomic
DNA (gDNA) for the 20 samples on
1.5%
Agarose gel -
- - - 167
Plate 49: Gel
electrophoresis showing PCR product for
the
20 samples on 1.5% Agarose gel - -
168
20
CHAPTER 1
INTRODUCTION
1.1
FRUITS AND MICROORGANISMS
The association
between fruits, microorganism and humans have been long and interesting and may
have developed before recorded history (Willey et al., 2008). Nutrients needed for growth, repair and control of
body processes are usually obtained from fruits since these fruits contain
mineral elements, vitamins and sugar (Hawksworth, et al, 2005). It is normal for these fruits to be consumed raw as
this is the best way of obtaining their valuable nutrients. There are various
ways of using fruits in our diet. This include preparation of juices,
production of wine, marmalades, jams and making of salads. (Hobbs, 1998;
Kimball, 1999). There are also medicinal properties attributed to some fruits.
According to Nakamura and Miyoshi (2006), eating of Carica papaya may reduce the risk of some types of cancer, while
Eno et al., (2000) demonstrated that
the fruit juice of C. papaya can
lower the blood pressure in their study of mice. Citrus sinensis when taken as an infusion has shown the ability to
lower fevers, stop headaches and stabilize heart palpitations. The juice from C. sinensis hastens removal of metabolic
waste from the body. Vitamin C, a major component found in C. sinensis helps to boost the body immune system thus helping the
body to fight infections.
1.2
DEFINITIONS
1.2.1 Fungal pathogens: These are fungi which are able to invade
and cause infection of an intact, formerly healthy tissue. Secondary fungal
pathogens are those fungi which may not invade a healthy tissue, but can cause
spoilage after the tissue has been damaged by some physical or physiological
causes.
1.2.2 Post
harvest rots: These are spoilage or damage to plants/fruits which occur
after the fruits had been harvested from the tree/field. The causes of the rot
might have set in while the fruit was on the tree, but rot or decay only shows
up after the fruits have been harvested especially as the fruit begins to ripen
in the market places, in storage, before consumption. Post-harvest rots or
spoilage are sometimes referred to as “Market Diseases” of the affected fruits.
The causes of post-harvest rots are many and varied.
It could be due to the presence of heavy load
of the spoilage bacteria and fungi, heat among the stored fruits, pressure as
the fruits are piled on top of each other during transportation, or bruises and
cuts from poor harvesting and handling procedures. Post- harvest rots cause a
lot of loses to farmers and fruit sellers and poses potential health hazards to
consumers of such fruits.
This study is concerned with post-harvest
rots caused by fungal pathogens and how to intervene in controlling them to
help reduce loss to farmers and sellers, and reducing attendant health risk of
eating fungal contaminated fruits.
During the
sequence of fruit handling from post-harvest handling, transport, storage,
marketing, to the final consumption, microorganisms can affect the fruit
quality as well as human health (Nagy and Olson, 2007). Spoilage refers to any
change in the condition of food in which the food becomes less palatable or
even toxic. These changes may be accompanied by alteration of taste, smell,
appearance or texture. The surfaces of fruits harbour large numbers of both
yeasts and moulds although the yeast lack mechanism to invade the plant tissue
and are therefore secondary rather than primary agents of spoilage. Some of the
fungi responsible for spoilage are true plant pathogens in that they invade and
can cause infection of an intact, formerly healthy
23
tissue. Others
are saprophytic and can only become established after the fruit has been
damaged by some physical or physiological cause. Since fruits are harvested
locally, there are always bruises and cuts and according to Krogh (1992) most
of the fruits displayed in the market have cuts and bruises which aid penetration
of microbes. Owing to their high nutritional value, particularly sugar and low
pH, fruits serve as good substrate for micro-organisms whose activities
constitute the most important causes of
fruit rots.
The high
concentration of various sugars, minerals, vitamins and amino acids also
provide a good platform for the successful growth and survival of saprophytic
fungi. Also, due to the low pH in fruits, most of the spoilage organisms
associated with fruit rots are usually fungi (Jay, 2005). Numerous microbial
defects of agricultural crops are characterised by the type of microorganisms
responsible for the deterioration. Different genera of fungal pathogens have
been implicated in the spoilage of fruits by various workers. These include: Alternaria, Aspergillus (black rot of citrus fruits), Botrytis (grey mould rot of citrus fruits), Fusarium (brown rots of citrus fruits and pineapple), Geotrichum (sour rots of citrus fruits),
Penicillium (blue and green mould rot
of citrus fruits, brown rot of pineapple fruits), Colletotrichum (brown to black spot – Anthracnose – of citrus
fruits, avocados and paw-paw), Diaporthe
(stem end rots of citrus fruits), Botrydodiplodia
(ripe rots of paw-paw) (Oram et al.,
2005; Adisa and Fajola, 2002). Rhizopus, Fusarium (soft rot of tomato,
pawpaw), Candida (watery rot of
tomato, pawpaw and soft rot of pineapple) (Effiuvwevwere, et al., 2005; Onuegbu, 2002). Aspergillus,
Colletotrichum, Rigidoporus, Candidia, Rhizopus
(deterioration of sour-sop) (Okwulehie and Alfred, 2010; Nweke and Ibiam,
2012).
Some of the fruit fungal pathogens are also
human pathogens and may result in food infection or food poisoning if consumed
with the fresh fruits. According to Prasad
24
(1992), besides the huge losses in
income to fruit marketers, consumption of spoilt fruits causes serious health
hazards such as salmonellosis, gastroenteritis, staphylococcal
gastro-enteritis, botulism, aspergillosis, etc. Some fungal pathogens produce
mycotoxins which have various health implications when consumed with the
fruits. Mycotoxins are secondary fungal metabolites, toxic to humans, animals
and plants (Ismaiel and Papenbrock, 2015). Among the hundreds of known
mycotoxins, aflatoxin, citrinin, patulin, penicillic acid, tenuazonic acid,
ochratoxin A, cytochalasin, fumonisin are associated with plant produces
(Ismaiel and Papenbrock, 2015). Aflatoxins were detected from infected paw-paw
fruits, both before and after autoclaving fruits for 15mins at 1210C
(Baiyewu et al., 2007). The majority
of the mycotoxins are produced by fungi in the genera Aspergilus, Penicillium and Fusarium.
1.3 DISEASE MANAGEMENT
Disease management in fruits and vegetable
crops worldwide is heavily dependent upon the application of synthetic
fungicides or chemicals for pathogen control. For example, the shelf life of
orange fruits can be doubled if the fruit is coated with polyethylene/wax
emulsion (Morton, 1987). However, restrictions on fungicide use and widespread
emergence of pathogen resistance has increased global demand for more
sustainable production systems and driven research towards alternative disease
control strategies. Biological control, which includes elicitors of host
defence, microbial antagonists and natural products, offers an attractive
alternative to synthetic pesticides (Elmer et
al., 2005).
Over the last two decades, biological control
of plant pathogens has emerged as a viable disease control strategy (Harman,
2000; Elad and Stewart, 2004) Numerous factors are responsible for increasing
interest in biological control including the negative effects of fungicides on
human health (White, 1998), increased regulatory
25
restrictions (Janisiewicz and
Korsten, 2002), traceability protocols for crop protection practices, nil
residue tolerance in some export markets, continued interest in organics
pathogen resistance to commonly used fungicides (Rosslenbroich and Stuebler,
2000) and a lack of replacement products.
Inhibition of spoilage and/or human pathogenic
fungi on fruits by extracts of medicinal plants and their subsequent
application as bio preservatives will be a good alternative to chemical
compounds and fungicides used in fruit preservation.
1.4
MEDICINAL PLANTS
Medicinal plants have been of age long
remedies for human diseases because they contain components of therapeutic
value (Nostro et al., 2000). Some of
them are also used for prophylactic purposes. Increasing interests in herbal
remedies have been incorporated into orthodox medicinal plant practice.
Diseases that have been managed traditionally using medicinal plants include
malaria, epilepsy, infantile convulsion, diarrhoea, dysentery, fungal and
bacterial infections (Sofowora, 1996). Numerous Nigerian medicinal plants are
used traditionally and have been shown to possess biological activities against
diseases. Such medicinal plants include Gongronema
latifolium “Utasi”, Heinisa crinita
“Atama” Lasienthera africanum
“Editan” and Piper guineense “Adusa”
(Odukoya, et al 2006, Edet et al., 2010; Udoh, et al., 2013; Ebong et al.,
2014).
Piper
guineense (Schumach) is a plant
belonging to the family Piperaceace.
It is commonly referred to as African black pepper. It has more than 70 species
throughout the tropical and subtropical region of the world. It is known by the
Igbo as Uziza;
Yoruba
as ‘Iyere’ and Ibibio as ‘Adusa’ (Edet et
al., 2010; Udoh et al., 2013;
Ebong et
al., 2014). The
leaves of piper guineense are considered aphrodisiac, appetitive,
26
carminative and eupeptic. P. guineense has been used for treating
cough, bronchitis, rheumatism, infertility in women, heart problems and asthma.
Lasienthera
africanum (P. Beauv) (Editan) belongs to the family Icacinaceae and order
Celestrales. L. africanum is consumed
as vegetables in the South-Eastern State of Nigeria. It is believed to have
cooling effects on the body, “purifying effects” and prevent internal bleeding.
L. africanum is of medicinal
importance and has been therapeutically used as antacid, laxative and analgesic
(Sofowora, 1993; Adegoke and Adebayo-tayo, 2009). The leaves of this plant have
antiplasmodic, laxative, antipyretic, antiulcerogenic, anti-diabetic and
antimalarial activities (Okokon et al., 2009).
The leaf extract has been reported to contain alkaloids, terpenes, saponins,
tannins, flavonoids, anthraquinones and cardiac glycosides (Okokon et al., 2009).
Gongonema
latifolium (Benth et Hook) (utasi) is a climber with woody, hollow glabrous
stem and greenish yellow flower. It is a perennial edible shrub, belonging to
the family Asclepiadaceae. It is known as ‘Utazi’ in South Eastern and as
‘Madumaro’ in South Western part of Nigeria. G. latifolium is widely employed in Nigeria for various medicinal
and nutritional purpose. Reports on the inhibitory effects of G. latifolium on micro-organisms, its
hypoglycemic, cardio-protective, anti-inflammatory, hypolipidemic and
antioxidative properties have been scientifically documented (Eleyinmi, 2007;
Edet et al, 2009). The leaves and
stem of G. latifolium are also traditionally used for the
management and treatment of diabetes, hypertension, cough, typhoid fever and
malaria.
Heinsia crinita [(Afz) (G. Taylor)]
(Bush apple) belongs to the family Rubiaceae
(Etukudo, 2003). H. crinita is common across the tropical
region from Guinea to Western Cameroun and Equatorial Guinea and across the
Congo basin to East and
South Central Africa, Akwa Ibom, Calabar (Ajibesin et al, 2008). In Efik, it is called
27
“Atama”, while the Yorubas and
Igalas called it “Tonoposho” and “Fumbwa”, respectively. In Southern Nigeria,
particularly in Akwa Ibom, the leaves of H.
crinita are used for cooking vegetable soup. The leaves are also used for
the treatment of hypertension, infertility, sore throat, catarrh, abscess,
craw-craw and head lice in children (Etukudo, 2003; Ajibesin et al, 2008).
Fourier
Transformation Infrared (FT-IR) Spectrophotometer is a rapid, valuable,
reliable and sensitive tool for the characterization and identification of
chemical functional groups present in compounds or plant samples based on the
measurement of vibration of a molecule excited by infrared radiation in the
wave length range of 400 to 4000cm-1.
The molecular
method, especially polymerase chain reaction (PCR), is the most important and
sensitive technique presently available for the detection of plant pathogens.
PCR allows the amplification of millions of copies of specific DNA sequences by
repeated cycles of denaturation, polymerization and elongation at different
temperatures using specific oligonucleotides (Primers), deoxy-ribonucleotide
triphosphates (dNTPs) and a thermostable taq DNA polymerase in the adequate
buffer.
1.5
JUSTIFICATION OF THE STUDY
Fruits have high
nutritional value as they are rich in sugars, minerals, vitamins and other
biotic molecules. They help in repair and control of body processes. They also
serve as a good source of income for the people. However, there is growing
concern in the microbiology safety of fruits. The contamination of fresh fruits
with plant and human pathogens can cause considerable economic losses for the
industry, apart from being the origin of food borne diseases. The use of fungicides
as pathogen control or preservatives is being discouraged due to their negative
effects on human health. Some
28
edible medicinal plants locally
available in Akwa Ibom State can be used to interfere efficiently with the
growth of food borne pathogens on fresh fruits and thus can be used as bio
preservatives to inhibit the growth and colonization of fungal pathogenic and
spoilage organisms. Furthermore many plant pathogens are now resistant to most
of the synthetic fungicides currently in use. There is therefore the need for
novel sources of antifungal agents, to replace the ineffective fungicides.
Local edible medicinal plant extracts may be possible sources.
1.6
OBJECTIVES OF
THE STUDY
This study aims at
assessing the antifungal activities of aqueous and ethanolic leaf extracts of
four edible indigenous medicinal plants of Akwa Ibom origin:
Gongronema latifolium
“Utasi”, Heinsia crinita “Atama”,
Lasianthera africanum
“Editan
and Piper guineense “Adusa” against
fungal pathogens associated with postharvest rots of some fruits in Akwa Ibom
State.
Specific objectives include:
1.
to isolate, characterize and identify fungi
associated with post-harvest rots of some fruits in Akwa Ibom State.
2.
to determine the pathogenicity of the fungal
isolates.
3.
to test for mycotoxins production and
concentration in the diseased fruits and in the fungal isolates.
4.
to find out the qualitative phytochemical
constituents and chemical functional groups of the aqueous and ethanolic leaf
extracts of Gongronema latifolium, Heinsia crinita, Lasienthera africanum and Piper
guineense.
5.
to evaluate the antifungal activity of the
aqueous and ethanolic leaf extracts of G.
latifolium, H. crinita, L. africanum and P. guineense on spore germination and radial growth of the fungal
isolates.
6.
to assess the bio control/preservative potential
of the leaf extracts of G. latifolium, H. crinita, L. africanum
and P. guineense on fresh fruits.
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