ISOLATION, IDENTIFICATION AND CHARACTERISATION OF MICROORGANISM IN DECAYING PAWPAW FRUIT

ABSTRACT

Microorganisms associated with decayed pawpaw fruits were isolated and identified. Following sample preparation and serial dilutions, diluents of each sample were plated out in duplicates on nutrient agar and sabouroud dextrose agar using  pour plate and spread plate methods. A total of seven fungal species, Aspergillus spp, Mucor spp, Penicillium spp, Fusarium spp, Yeast, Monilia spp, and Rhizopus spp were identified. Lactobacillus spp, Escherichia coli, Bacillus spp, Staphylococcus spp, Pseudomonas spp, and Proetus spp were the identified bacterial isolates. In fungi Aspergillus spp had the highest percentage occurrences (100%) while Monilia spp had the least percentage occurrences (28.57%). In bacterial Pseudomonas spp had the highest percentage occurrences of (71.43%) while Lactobacillus spp had the least percentage occurrence of (28.57%).  Results of pathogenicity test showed that four fungal species were able to cause decay in healthy pawpaw fruit, including Aspergillus spp, Penicillium spp, Fusarium spp, Monilia spp. These isolates that cause rot were considered causative agent for spoilage of pawpaw fruits.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                            ii                                                                                                                         Dedication                                                                                                                               iii                                                                                                                              Acknowledgement                                                                                                                  iv                                                                                                                  Table of Contents                                                                                                                   v 

List of Tables                                                                                                                          viii

 Figure                                                                                                                                     ix                                                                                                                        

Abstract                                                                                                                                   x

CHAPTER ONE

INTRODUCTION                                                                                                                 1

1.1.1 Statement of Problem                                                                                                    5

1.1.2 Research Objective                                                                                                        5

CHAPTER TWO

LITERATURE REVIEW                                                                                                    6

2.1 Classification                                                                                                                    7

2.2 Origin and Distribution7

2.3 Botanical Description                                                                                                    10

2.4Types of Papaya                                                                                                           13

2.5 Growth habit and Soil16

2.6 Pollination                                                                                                            17

2.7 Propagation                                                                                                           18

2.8 Harvesting                                                                                                             18

2.9 Nutritional value                                                                                                    19

2.10 Economic Importance                                                                                               20

2.11 Industrial Importance                                                                                               20

2.12 Chemical Compounds                                                                                             21

2.13 Toxicity and Allergy                                                                                               22

2.14 Health Benefits                                                                                                        23

2.15 Insect Pests and Nematodes                                                                                     28

2.16 Diseases of Papaya                                                                                                           31

CHAPTER THREE

3.0 Materials and Methods                                                                                             37

3.1 Sample Collection                                                                                                   37

3.2.1 Media Preparation                                                                                               37

3.2.2 Sample Preparation                                                                                               37      

3.2.3 Isolation of microbial isolate from decayed pawpaw fruits                                          38

3.3.1 Direct placement technique                                                                                       38

3.3.2 Characterisation and identification                                                                            38

3.3.3 Characterisation of fungal isolates                                                                              39

3.4.1 Characterisation of bacteria isolates                                                                          39

3.4.2 Biochemical test                                                                                                   39

3.4.3 Gram staining                                                                                                       39

3.4.4 Catalase test                                                                                                         40

3.4.5 Citrate test                                                                                                               40

3.4.6 Coagulase test                                                                                                      40

3.4.7 Indole test41

3.4.8 Voges Proskeur test                                                                                               41

3.5.1 Sugar fermentation test                                                                                            41

3.5.2 Oxidase test                                                                                                                42

3.5.3 Methyl-red test                                                                                                          42

3.6 Pathogenicity test                                                                                                        42

CHAPTER FOUR

4.0 Results                                                                                                                   44

CHAPTER FIVE

5.0 Discussion                                                                                                                  48

5.1 Conclusion                                                                                                             50

5.2 Recommendation                                                                                                     50

REFERNCES                                                                                                                 51

APPENDIX                                                                                                                  59

LISTS OF TABLE

LIST OF FIGURES

Title                                                                     Page

1               Papaya Major Producing Countries                       9                                                            

CHAPTER ONE

1.0  INTRODUCTION

Fruits are very important and have high dietary and nutritional qualities (Barth et al., 2009). Studies have evaluated the association of fruit and vegetable consumption with the reduction of risk of specific diseases (Hung et al., 2004). Their consumption has dramatically increased by more than 30% during the past few decades (Barth et al, 2009). Fresh fruits and vegetable consumption increased by 25.8% to 32.6%, respectively, and far exceeded the increases observed for processed fruit and vegetable products. It is also estimated that about 20% of all fruits and vegetables produced is lost each year due to spoilage (Barth et al., 2009). Raven, et al., (2005) reported that 20 new human fungal pathogens are documented each year. It is estimated that about 20-25% of the harvested fruits are decayed by pathogens during post-harvest handling even in developed countries (Droby, 2006; Zhu, 2006).

Deterioration of foods generally is attributed to two main causes which are natural degradation due to activities of enzymes and growth of microorganisms (bacteria, molds and yeasts). These microorganisms can result in useful products through their activities particularly during fermentation of foods such as wine and cheese. The negative effects of these microbial activities result in decay, rotting of food and food poisoning hence the basis of microbial food spoilage occurs when these microorganisms release their own enzymes into the foods and absorb the nutrients thereby changing the physical and chemical states of the foods; thus lowering the nutritional value. Bacteria and fungi may also produce waste products which act as poisons or toxins, thus causing the renowned ill-effects (Bakriet al., 2010)

Pawpaw (Carica papaya) is a popular fruit plant grown all over the wetter parts of West Africa, tropical and subtropical regions of the world being one of the most nutritious and cheapest fruits grown and consumed in Nigeria (Baiyewu, 1994). It is usually grown as compound fruit crop or semi-wild fruit crop from discarded seeds (Kutheet al., 1974). Pawpaw (Carica papaya) is a member of the small family “Caricaceae” allied to the  “passifloracea”.which incorporates 35 latex-containing species in six genera, Carica, Cylicomorpha, Jarilla, Jacaratia, Horovitzia and Vasconcellea (Badillo, 2002) with the genus Carica, consisting of only one species. Although opinions differ on the origin of C. papaya, it is likely that C. papaya originates from the lowlands of eastern Central America, from Mexico to Panama (Nakasone et al., 1998). It is a fast growing, short-lived, woody, large herbaceous plant having latex vessels in all its fruits with an upright unbranched stem covered all over with leaf scars (Agnew, 1968; Dick, 2003). It generally only branches when injured (Garret, 1995). The stem is hollow with a light green to a tan brown colour bearing leaves that are large and deeply palmately lobed with the older leaves abscising as new leaves emerge (Morton, 1987). The papaya fruit is melon-like, oval to nearly round, elongated club–shaped and is rich in latex when unripe with a green coloured skin. As it ripens, it becomes light or deep yellow externally and the thick wall of succulent flesh becomes aromatic yellow-orange or various shades of red. The fruit is then juicy, sweetish and some types are quite musky (Morton, 1987). The fruit consist largely of water, sugar, vitamins A and C, protein and ash (Baiyewu, 1994). It is an interesting tree in that the male and female parts exist in different trees. Pawpaw Fruits, apart from being taken as food also have some medicinal importance. It has a mild laxative action and the fruits, leaves and seeds are used medicinally against worm and ulcer (Baiyewu, 1994). Its main medicinal use is as a digestive agent; it is prescribed for people who have difficulty digesting protein and is used to break up blood clots after surgery, which is due to the presence of enzymes papain in the plant’s latex. The latex from the trunk of the tree is also applied externally to speed the healing of wounds, boils and warts. The seed is used to expel worm and the flower may be taken in an infusion to induce menstruation. It has also been reported that annonaceousaceto genus derived from the extract of the twigs of the pawpaw tree may be good chemotherapeutic agents for cancer as these compounds inhibit enzymes necessary for metabolic processes in tumour cells. Increasing interest in medicinal herbs has increased scientific scruting of their therapeutic potentials and safety thereby providing physicians with data to help patients make wise decisions about their uses.

Economically, carica papaya (pawpaw) is the most important species within the Caricaceae, being cultivated widely for consumption as a fresh fruit and for use in drinks, candies and as dried and crsystallised fruit (El Moussaoui et al., 2001). Nutritionally, papaya is a good source of calcium.

Papaya also has several industrial applications due to the proteolytic enzyme called papain hence their use in the production of chewing gums, tenderizing meat and drug preparation for various digestive ailments and for the treatment of gangrenous wounds as well as in the textile and cosmetics industries (Kochhar, 1986; Villegas, 1997). Biochemically, its fruits are good sources of several proteins and alkaloids with application to pharmaceutical, food, industries, e.t.c. (El Moussaoui et al., 2001). The seed is used to expel worms and the flower may be taken in an infusion to induce menstruation (Duke, 1984 and Oduola et al., 1986). Increasing interest in medicinal herbs has increased scientific scrutiny of their therapeutic potentials and safety thereby providing physicians with data to help patients make wise decisions about their use (“O”Hara et al., 1998).

Papaya fruits are beset with problems of field and storage rot. Gutpa and Pathak (1986) identified 22 different fungi in post-harvest decay of papaya fruits which include Aspergillus niger, Aspergillus flavus, Rhizopus oryzae, Fusarium moniliforme. Bacterial pathogens involved with rot of papaya include the species of soft-rotting Erwinia, Pseudomonas, Xanthomonas, Cytophaga and Bacillus (Liao et al., 1987; Lund, 1983). These microorganisms, under the influence of environmental factors (include temperature, hydrogen ion concentration (pH), oxygen, moisture content) pose a serious threat to papaya fruit production. Besides the losses in income to the papaya fruit marketers, the rotten fruits could also cause a health hazard to consumers. Krogh (1992) had earlier reported that most microbes infecting plant tissues often produced secondary metabolites in their hosts, which are known to be hazardous to animals including man. Some of these metabolites include the ergot alkaloids on cereals by Clavisep spp, fumonisin on maize by Fusarium spp, aflatoxins and ochratoxinson several plants produced by Aspergillus spp (Prasad, 1992). Nigeria is the third largest producer of papaya in the world with 703,800 metric tons (FAO, 2010), it’s plentiful and sold all year round as well as being globally consumed by people for multiple benefits hence, the need for preservation and to boost its production. This can be achieved among other things by preventing the occurrence of these diseases through studying the ecology of the microbes.

1.1       STATEMENT OF PROBLEM

Papaya is a useful plant with nutritional, medicinal and health benefits. In spite of all these benefits, the plant is besieged by lots of pathogens both in the field and post-harvest diseases and these diseases result in yield losses thus making its valuable components unavailable. The papaya farm in Covenant University is particularly useful as a source of raw materials for the production of some patented products. This farm is however, infested with pathogens on fruits, stems, and leaves resulting in huge yield losses making the desired raw materials unavailable.

1.2       RESEARCH OBJECTIVES:

• To isolate the microorganisms associated with the rot diseases of fruits, stems and leaves of papaya

• To identify and characterize the isolated microorganisms.

• To determine the pathogenicity of the isolated microorganisms for papaya.

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