BIOCONTROL POTENTIAL OF LACTIC ACID BACTERIA AGAINST SPOILAGE FUNGI OF TOMATO FRUITS

ABSTRACT

 The antagonistic effect of Lactobacillus plantarum and Lactobacillus fermentum against spoilage fungi of tomato fruits were demonstrated. Lactobacillus plantarum and Lactobacillus fermentum were isolated from five different yoghurt samples and three different Ogi samples by plating on MRS Agar plates. Spoilage fungi of tomato fruits were isolated on PDA and were identified as Aspergillus niger, Rhizopus Stolonifer, Mucor spp. Using their colonial morphology and microscopic characteristics. The inhibitory potential of Lactobacillus plantarum and Lactobacillus fermentum  against isolated spoilage fungi of tomato was determined using the Agar well diffusion techniques. The results showed that after 24hours of incubation, Aspergillus niger was inhibited by Lactobacillus fermentum with zone of inhibition 12mm; Rhizopus stolonifer was inhibited by Lactobacillus Plantarum with zone of inhibition of 8mm; Mucor spp. was only inhibited by Lactobacillus plantarum with zone of inhibition 7mm. Furthermore, all the bacteria isolates showed an optimum growth temperature of 25 and 37⁰C and optimum growth pH of 5 and 6. The inhibitory potential exhibited by Lactobacillus plantarum and Lactobacillus fermentum showed that they are highly effective against eliminating spoilage fungi of tomato fruits. Thus, they have a biocontrol potential against fungi spoilage of tomatoes. Further studies should be conducted to determine the specific metabolites responsible for the antifungal activity and methods designed so that they can be used as bioaerosols or used in modified atmosphere packaging of tomato so as to prolong the  shelf life of tomato fruits.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents v

List of Tables vii

List of Figures viii

Abstract ix

CHAPTER ONE

1.1 Introduction 1

1.2 Aims and objective 4

CHAPTER TWO

2.0 LITERATURE REVIEW 5

2.1 Biocontrol 5

2.2 Lactic acid Bacteria: A  biocontrol agent 7

2.3 Fungal Pathogens of tomato fruit spoilage 11

CHAPTER THREE

3.0 MATERIALS AND METHODS 15

3.1   Materials 15

3.2   Methods 15

3.2.1 Sample collection 15

3.2.2 Media preparation 16

3.2.3   Isolation of Lactic acid Bacteria from Yoghurt and Ogi 16

3.2.4   Isolation of Fungi from spoilt tomato fruits. 16

3.2.5 Isolation of pure cultures of Lactic acid Bacteria and Fungi 16

3.2.6 Identification of fungi isolates. 17

3.2.7 Identification of Lactic Acid bacteria 17

3.3 Microscopy 17

3.4 Biochemical tests 19

3.5 Determination of the effects of pH on the growth of Lactic

acid bacteria isolates.   20

3.6 Determination of the effects of Temperature on the growth

of Lactic acid bacteria isolates. 21

3.7 Determination of the biocontrol efficacy of Lactobacillus

plantarumAnd Lactobacillus fermentum metabolites against

Rhizopus stolonifer, Aspergillus niger, and Mucor spp. from

spoilt tomato fruits. 21

CHAPTER FOUR

4.1 Results 22

CHAPTER FIVE

5.1 Discussion 33

5.2 Conclusion 35

5.3   Recommendations 36

References 37

Appendices

LIST OF TABLES

Table  Titles Pages

1: Isolated Number of fungal pathogens associated with tomato spoilage. 13

2:. Morphological and biochemical characteristics of Lactic acid bacteria

isolates in ogi and yoghurt sold in Umuahia,Abia state. 24

3:. Physiological characteristics of isolates from ogi and yoghurt based

on pH 25

4: Physiological characteristics of isolates from ogi and yoghurt

based on temperature. 26

5: Colonial Morphology and microscopic characteristics of fungal isolates 31

6: Antagonistic activity of Lacticobacillus plantarum and Lactobacillus 

fermentum against isolated spoilage fungi of tomatoes fruit. 32

LIST OF FIGURES

Figures    Titles               Pages

1:  Effects of temperature on the biomass of the Lactobacillus spp. (Ya and Yb) 27

2:  Effects of temperature on the biomass of the Lactobacillus spp. (Oa and Ob) 28

3:  Effects of pH on the biomass of the Lactobacillus spp.(Ya and Yb) 29

4:  Effects of pH on the biomass of the Lactobacillus spp.(Oa and Ob) 30

CHAPTER ONE

1.1  INTRODUCTION

Plant disease cause severe crop loses and make agriculture dependent on adequate    disease control. Managing and controlling plant disease efficiency is important for crop growers, environmentalists, legislators, policy makers and implementers. Disease management strategies primarily depend on sanitary practices and well timed pesticides applications. (Deahl et al., 1993). Many farmers heavily depend on Agro chemical and mainly rely on fungicides. This fungicide can prevent infection but not all have curative activity. Therefore the interval between spraying is usually short in addition to the appearance for more aggressive isolates and that are no longer inhibited by chemical protestants, hence the burden on the environment is high. Subsequently, plant pathogens are responsible for large amounts of chemicals fungicide applied annually exacerbating control strategies (Deahl et al., 1993; freytag et al., 1994). To cope with these problems and due to the increase of public concern about adverse effects of Agrochemicals on food safety and environment. There are need to stimulate the search for control strategies that are more durable and preferable based on natural products. Therefore alternative approaches that can be incorporated into integrated pests managements of plant disease .preservation of foods by natural and microbiological methods can be a satisfactory approach solving economic losses due to microbial spoilage of raw material and food products to reduce the incidence of food borne illness and meet the food requirement of the growing world population ( Dehne and Schellander, 2012).

Since many people are concerned about the safety of chemicals, preservations are questioned with regard to their safety, the potential application of microbes as bio-control to fungal spoilage pathogens in foods and healthcare have received increasing attention in recent years.

Biocontrol agents which includes effective micro organisms and microbial products and organic fertilizer have been attracting attention a alternatives to chemical  agents (Fravel et al., 2005)

Many species  of  Bacillus including Bacillus cereus ,Bacillus subtilis and Bacillus mycoides are known to suppress several pathogens belonging to the genera Rhizoctonia, Sclerotina, Fusarium, Phytophthora as stated by Fiddaman and Rossall,(1994).Also some species of lactobacillus including Lactobacillus plantarum and Lactobacillus fermentum all can act as biocontrol against certain fungi pathogens such as Aspergillus, Rhizopus and Mucor. (Fowoyo and Uzoma, 2014)

Tomato is a fleshy fruit, it is one of the most widely   grown vegetable food crop in the world, secondly only to the potato with world production about 152.9 million ton (S74.1 billion) according to ACSMF (2005).Tomatoes are eaten directly either as raw vegetable or consumed in a variety of processed products like ketch-up, sauce, chutney, juice, diced soup, paste puree etc. it is a rich source of vitamin A and C and also contain minerals like iron, phosphorus .Tomatoes are highly susceptible to fruit decay from the field through post harvest handling. Post-harvest decays result in wounds bruised tissue and fruit softening (Maul et al. 2000). Tomato plant is attached from many serious diseases under green house and field condition. The spoilage of fresh fruit and vegetable is generally due to physiological disorder and microbial spoilage caused by fungi and bacteria. Several important disease of tomato reduces crop yield and the most devastating plant pathogens are fungi  (Agrios, 2005)

Microbial spoilage of fruits and vegetables is known as rot which consists of colour change, loss of texture and often odor. Fungal pathogens cause devastating losses of crops and post harvest fruits throughout the world (Wang et al., 2008).Organisms that cause food spoilage – mold, yeast and bacteria are always present in air water and soil. Fungi are the most important and prevalent pathogens infecting a wide range of fruits and causing destructive and economically important losses of fruits during storage, transportation and marketing (Sommer et al., 1992) . Different fungal species such as Alternaria, Fusarium, Penicillium, Mucor, Rhizopus, Aspergillus, Geotricuim, Phytophtora and Botrytis have been implicated as spoilage organisms (Sommer et al., 1992). However,Aspergillus niger, Rhizopus stolonifer andMucor spp have being isolated from spoilt tomatoes (Ogunbawo et al., 2014). Aspergillus niger has been reported to cause disease called black mold on certain fruits and vegetable such as grapes, tomatoes, onion and peanuts (Sharma, 2012).Rhizopus stolonifer is a fungus known to be a causal organism of soft rot of fruits and vegetable (Agrios, 1997) .Mucor sppare primarily post harvest rots but also occur on ripe fruits in the field. Infected fruits are initially slightly discolored, gradually turning pale brown. The tissue rapid softens and collapse and under humid conditions the fruits is soon covered with a dense, fluffy white mycelium which bears stiff black headed sporangia. Other common diseases of tomato caused by fungi areSeptorialeaf spot caused by the Septotria lycoperici. Early blight caused by the fungus Alternaria solani.Late blight caused bythe fungus Phytophtora infestans (Maul et al., 2000) Fusarium and Verticulum wilts are also quite common. Lactic acid bacteria have well been established as organisms that produce inhibitory compounds against bacteria and fungi. The antimicrobial activity of lactic acid bacteria is primarily attributed to a wide variety of active antagonistic metabolites that includes organic acid (lactic, acetic formic,propionic , butyric,hydroxyl-phenyllactic acid and phenyllactic acid) or antagonist compounds (carbon dioxide, ethanol, hydrogen peroxide, fatty acids,acetone, diacetyl). Bacteriocins (Nisin, Reuterin, Reutericyclin, Pediocin, Lactin,Entrocin Etc) Muhialdin et al.,2011; Reis et al., 2012). The production of this metabolic counterreacts the growth of spoilageorganisms present in tomatoes (Schnurer and Magnusson, 2005).

1.2            AIMS AND OBJECTIVE

Ø To isolate pathogenic fungi responsible for tomatoes fruits spoilage.

Ø To isolate Lactic acid bacteria from Yoghurt and Ogi.

Ø To determine the antagonistic efficacy of Lactic acid bacteria

Ø To Determine the Biocontrol potential of Lactobacillus plantarum against fungi spoilage of tomatoes.

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