ISOLATION AND CHARACTERISATION OF MICROORGANISMS ASSOCIATED WITH ROT DISEASES OF FRUIT, STEM AND LEAF OF CARICA PAPAYA J

By

ONIHA, MARGARET IKHIWILI

Presented To

Department of Biological Science

Fruit, stem and leaf rot is a major constraint to Carica papaya production. Twenty-one papaya plant samples showing soft rot symptoms and comprising of fruits, stems and leaves were collected from the Covenant University Papaya Research Plantation for microbial investigation. The back, first layer after the back, inner layer and the seeds from four papaya fruits, as well as stems (4) and leaves (4), showing symptoms of sooty rot and white fluffy rot, were also analyzed. Following sample preparation and serial dilutions, three dilutions of each sample were plated out in duplicates on nutrient agar, potato dextrose agar and papaya fruit agar using the direct inoculation, streak, pour plate and spread plate methods. Pure cultures of each observed microorganism was purified using sub-culturing techniques, then cultural, microscopic and biochemical characteristics were used for identification of specific isolates. Koch’s postulates for the determination of the microbial etiology of diseases were used to determine the causal agent of the soft rot of papaya fruit. A total of ten microorganisms, Aspergillus niger, Aspergillus flavus,  Aspergillus fumigatus,  Mucor spp,  Penicillium spp,  Alternaria spp,  Colletotrichum gloeosporioides, Bacillus spp, Staphylococcus spp and Pseudomonas spp, belonging to five fungi and three bacteria genera were isolated. Papaya fruit agar supported the growth of A. niger, A. fumigatus, A. flavus, Mucor spp, Bacillus spp and Staphylococcus spp but not Pseudomonas spp. The most frequently isolated bacteria from the soft rot samples was Staphylococcus spp and was isolated from 85.7%, 42.9% and 71.4% of the fruits, stems and leaves respectively, while Pseudomonas spp was the least frequently isolated bacteria from all the plant parts. The most frequently isolated fungi from the soft rot fruits was Mucor spp while A. niger was the most frequently isolated from the stem (57.1 %) and leaf (42.9%). Colletotrichum gloeosporioides was isolated from the fruits, but not from the soft rot infected stems and leaves. The most frequently isolated bacteria from the sooty rot and white fluffy rot samples were Pseudomonas spp (75%) and Bacillus spp (62.5%) respectively, while Penicillium spp was the most frequently isolated fungus both from the sooty rot (56.3%) and white fluffy rot (43.8%) samples. Pathogenicity tests presumptively identified Mucor spp as the most important pathogen associated with soft rot of papaya fruit.

Title Page -  -  -  -  -  -  -  -  -  -  - i
Certification -  -  -  -  -  -  -  -  -  - ii
Dedication -  -  -  -  -  -  -  -  - iii
Declaration -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  - iv
Acknowledgement -  -  -  -  -  -  -  -  - v
Table of Contents -  -  -  -  -  -  -  -  -  - vii
List of Tables -  -  -  -  -  -  -  -  -  -  - x
List of Plates -  -  -  -  -  -  -  -  -  -  - xi
List of Figures -  -  -  -  -  -  -  -  -  -  -  - xii
List of Photomicrographs -  -  -  -  -  -  -  -  - xiv
Abstract -  -  -  -  -  -  -  -  -  - xv

CHAPTER ONE
INTRODUCTION
111  Statement Of Problem -  -  -  -  -  -  -  - 4
112  Research Objectives -  -  -  -  -  -  -  -  - 4

CHAPTER TWO
LITERATURE REVIEW
21  Classification -  -  -  -  -  -  -  - 5
22  Origin and Distribution -  -  -  -  -  -  - 6
23  Botanical Description -  -  -  -  -  -  -  - 9
24  Types of Papaya -  -  -  -  -  -  -  -  -  -  - 12
25  Growth habit and Soil -  -  -  -  -  -  -  -  - 14
26  Pollination -  -  -  -  -  -  -  -  - 15
27  Propagation -  -  -  -  -  -  -  -  -  - 16
28  Harvesting -  -  -  -  -  -  -  -  -  - 16
29  Nutritional value -  -  -  -  -  -  -  -  -  -  - 17
210  Economic Importance -  -  -  -  -  -  -  -  - 17
211  Industrial Importance -  -  -  -  -  -  -  - 18
212  Chemical Compounds -  -  -  -  -  -  -  -  - 18
213  Toxicity and Allergy -  -  -  -  -  -  -  -  - 19
214  Health Benefits -  -  -  -  -  -  -  - 20
215  Insect Pests and Nematodes -  -  -  -  -  -  -  - 24
216  Diseases of Papaya -  -  -  -  -  -  -  -  - 28

CHAPTER THREE
MATERIALS AND METHODS
31  Culture Media -  -  -  -  -  -  -  - 34
312  Reagents and Indicators -  -  -  -  -  -  -  - 34
313  Laboratory Wares -  -  -  -  -  -  -  -  - 34
32  The Papaya Farm -  -  -  -  -  -  -  -  -  - 34
321  Raising of Nursery -  -  -  -  -  - 34
322  Tagging and maintenance of the nursery and farm -  -  -  -  -  -  - 35
323  Collection of samples -  -  -  -  -  -  -  -  - 35
3 24  Documentation of disease symptoms -  -  -  -  -  - 35
325  Processing of samples -  -  -  -  -  -  -  -  - 36
326  Culture techniques -  -  -  -  -  - 37
33  Sterilization of glassware -  -  -  -  -  -  - 38
331  Preparation of media -  -  -  -  -  -  -  -  - 38
332  Preparation of Papaya fruit agar -  -  -  -  -  -  -  - 38
333  Isolation of microorganisms -  -  -  -  -  - 39
334  Procedure for subculturing -  -  -  -  -  -  -  -  - 40
335  Preservation of isolates -  -  -  -  -  -  -  - 40
336  Identification and characterization of isolated strains -  -  -  -  - 40
34  Biochemical tests -  -  -  -  -  -  -  -  -  - 42
35  Identification of mould isolates -  -  -  -  -  -  - 45

CHAPTER FOUR
Results -  -  -  -  -  -  -  -  -  - 47

CHAPTER FIVE
Discussion -  -  -  -  -  -  -  -  -  - 89
Recommendation and Conclusion -  -  -  -  -  -  -  -  - 93
Contribution to Knowledge -  -  -  -  -  -  -  - 97
References -  -  -  -  -  -  -  -  -  - 98
Appendices -  -  -  -  -  -  -  -  - 107


LIST OF TABLES

Table 1: Shows production of papaya by region -  -  -  -  -  -  -  - 8
Table 2: Showing fruit characteristics of papaya cultivars in major producing countries -  - -  - 13
Table3: Chemical components of per 100g orange-fleshed papaya fruit -  -  - 23
Table 4: Shows sample types and distribution -  -  -  -  -  -  - 48
Table 5: Shows cultural and microscopic characteristics of fungi isolated from papaya
samples -  -  -  -  -  -  -  -  -  -  - 49
Table 6a: Shows cultural and microscopic characteristics of bacteria isolated from papaya
samples -  -  -  -  -  -  -  -  -  -  - 50
Table 6b: Shows the biochemical characteristics of bacteria isolated from the papaya
samples -  -  -  -  -  -  -  -  -  -  - 51
Table 7: Shows bacterial isolates from sooty rot and white fluffy rot of papaya samples -  -  - 52
Table 8: Shows fungal isolates from sooty rot and white fluffy rot of papaya samples -  -  -  - 53
Table 9: Shows the bacterial isolates from a total of 33 samples -  -  -  - 54
Table 10: Showing fungal isolates from a total of 33 samples -  -  -  - 55

LIST OF PLATES
PAGES
Plate 1: Shows Aspergillus niger on Potato Dextrose Agar plates  -  -  -  -  - 73
Plate 2: Shows Aspergillus flavus on Potato Dextrose Agar plates  -  -  -  - 74
Plate 3: Shows Staphylococcus spp on Nutrient Agar plate -  -  -  -  - 75
Plate 4: Shows Bacillus spp on Nutrient Agar plate -  -  -  -  -  -  -  - 76
Plate 5: Shows Mucor spp on reisolation from papaya fruit sample of pathogenicity test -  - 88

LIST OF FIGURES

Figure 1: Shows papaya major producing countries -  -  -  -  - 8
Figure 2: Shows the frequency of occurrence (%) of bacterial isolates from with soft rot of
papaya samples -  -  -  -  -  -  -  -  -  -  - 56
Figure 3: Shows the frequency of occurrence (%) of fungal isolates from papaya with soft rot of
papaya samples -  -  -  -  -  -  -  -  -  -  - 57
Figure 4: Shows the frequency of occurrence (%) of bacterial isolates from papaya with white
fluffy rot -  -  -  -  -  -  -  -  -  -  - 58
Figure 5: Shows the frequency of occurrence (%) of fungal isolates from papaya with white
fluffy rot -  -  -  -  -  -  -  -  -  -  - 59
Figure 6: Shows the frequency of occurrence (%) of bacterial isolates from papaya with sooty
rot -  -  -  -  -  -  -  -  -  -  -  - 60
Figure 7: Showing the frequency of occurrence (%) of fungal isolates from papaya with sooty
rot -  -  -  -  -  -  -  -  -  -  -  - 61
Figure 8: Shows the frequency of occurrence (%) of fungal isolates from papaya with soft rot,
sooty rot and white fluffy rot -  -  -  -  -  -  -  -  - 62
Figure 9: Shows the frequency of occurrence (%) of bacterial isolates from papaya with soft rot,
sooty rot and white fluffy rot -  -  -  -  -  -  -  -  -  - 63
Figure 10: Showing frequency of occurrence of bacterial isolates from 33 samples -  -  -  -  - 64
Figure 11: Showing frequency of occurrence of fungal isolates from 33 samples -  - 65
Figure 12: Showing the frequency of occurrence (%) of fungal species from papaya with rot
symptoms using papaya fruit agar  -  -  -  -  -  -  -  -  - 66
Figure 13: Showing the frequency of occurrence (%) of bacteria species from papaya with rot
symptoms using papaya fruit agar  -  -  -  -  -  -  -  -  -  - 67
Figure 14: Showing ripe papaya sample with dark whitish coloration and sunken lesion -  -  - 68
Figure 15: Showing ripe papaya with soft rot symptoms -  -  -  -  -  - 69
Figure 16a and 16b: Showing unripe papaya fruits showing sooty rot and white fluffy rot
respectively -  -  -  -  -  -  -  -  -  - 70
Figure 17: Showing papaya stem with sooty rot and white fluffy rot  -  -  -  - 71
Figure 18: Showing papaya leaf sample with sooty rot and white fluffy rot  -  -  -  - 72
Figure 19: Showing the frequency of occurrence (%) of fungi species isolated from healthy
papaya fruits used for pathogenicity test -  -  -  -  -  -  - 86
Figure 20: Showing brown fluffy aerial growth from infected papaya fruit -  -  -  -  -  - 87