Abstract

 

Pesticides are widely used in preventing and controlling the diseases and pests of crop, but at the same time pesticide residues have brought serious harm to human’s health and the environment. Hence their elimination from contaminated environmental sites is highly needful and bioremediation is the most convenient option because of its cost-effective and ecofriendly nature.

In this study, the ability of wood rot fungi to degrade pesticides in artificially contaminated medium was investigated to determine whether the fungi would be suitable for the bioremediation of the pesticides in soil. For bioremediation of Dichlorvos and Lambdaccyhalothrin. pesticides, fungal species isolated from decaying wood materials and identified using macroscopic and microscopic features were screened for pesticide degrading ability. Initially, the fungal isolates were inoculated into mineral salts medium with Dichlorvos and Lambdaccyhalothrin. pesticides as the sole source of carbon and incubated for 7days to assay the level of tolerance of these fungal isolates. Although all the fungal isolates were able to grow on pesticide modified-agar plates with high level of tolerance, only one isolate exhibited the highest tolerance for pesticides with great growth on the plates. The fungus with the best tolerance ability was identified by molecular sequencing. Blast result and phylogenetic analysis of nucleotide sequencing suggested that the isolate was closely related to Fusarium solani with 77% sequence identity. Screening of the isolate for its efficiencies in degrading Dichlorvos and Lambdaccyhalothrin. was carried out based on enrichment technique and spectrometric analysis. The fungal isolates were inoculated with each of the two pesticides at a concentration of 150 ppm for 20days. The biodegradation rate of the two pesticides on liquid media was determined using UV spectrophotometer. The results showed that the Fusarium solani isolate had a high efficiency to degrade Dichlorvos showing 66.7% degradation and Lambdaccyhalothrin. with 50.2% removal after the degradation period. Enzyme production (laccase, manganese peroxidase and lignin peroxidase activities) was quantified. Laccase activity ranged from a minimum of 1.62U/ml after 7days to a maximum of 1.90U/ml after 21days while MnP activity ranged from 0.051U/ml to 0.066U/ml. Maximum MnP activity was produced after 21days. LiP activity of the isolate ranged from 0.2 to 0.705U/ml with highest being produced after 21days. The results suggested that the test isolates have the ability to degrade different groups of pesticides, supported by the capacity for expression of a range of extracellular enzymes. Due to their high biodegradation activity, the fungus isolated from this work merit further study as potent biological agents for the remediation of soil or water contaminated with the pesticide Dichlorvos and Lambdaccyhalothrin.

 TABLE OF CONTENTS

Title Page                                                                                                                                i

Declaration                                                                                                                             ii

Certification                                                                                                                          iii

Dedication                                                                                                                            iv

Acknowledgements                                                                                                              v

Table of Contents                                                                                                                 vi

List of Tables                                                                                                                        ix

List of Figures                                                                                                                       xi

Abstract                                                                                                                                xiii

CHAPTER 1: INTRODUCTION

1.0       Introduction                                                                                                               1

1.1       Aims and Objectives                                                                                                  7

CHAPTER 2:  LITERATURE REVIEW

2.1       Overview of Pesticides                                                                                              8

2.2       Classification of Pesticides                                                                                       12

2.3       Microbial degradation                                                                                               14

2.4       Fungal involvement in biodegradation of pesticides                                                 15

2.5       Soil contamination                                                                                                    16

2.6       Environmental concerns regarding the contamination with pesticides                     17

2.7       Environmental factors affecting biodegradation by fungi                                   19

2.7.1    Temperature, oxygen and nutrient availability                                                     19

2.7.2    Water availability                                                                                                  20

2.8       Biodegrading capabilities of Fungi                                                                       21

2.9.      Effect of pesticide on soil microbes and plants             28

2.10     Principles of Pesticide Biodegradation                                                                 36

 

CHAPTER 3

3.0       MATERIAL AND METHODS

3.1       Study Site                                                                                                             46

3.2       Chemicals                                                                                                              46

3.3       Sample Collection                                                                                                 46

3.4       Isolation of Fungi from Cellulosic portion of wood                                             46

3.5       Identification and Characterization of Fungal Isolates                                        47

3.6       Viability of fungal Isolates to pesticide containing medium                    47

3.7       Determination of the ability of Fungal Isolate to grow in pesticides

 (dichlorvos and lamdaccyhalothrin) in Liquid Culture                                      48

3.8       Molecular Identification and characterization of isolate with best

Biodegradation Ability                                                                                         48

3.8.1    DNA extraction                                                                                                    49

3.8.2    Gel electrophoresis                                                                                               50

3.8.3    PCR reaction: amplification of DNA                                                                   50

3.8.4    DNA Sequencing                                                                                                 51

3.9       Biodegradation Assay of pesticides by Fusarium solani                                     51

3.10     Gas Chromatography of pesticides degradation by Fusarium solani                  52

3.11     Determination of the Minimum Inhibitory concentration                                    54

3.12     Analysis of Crude enzymes activities involved in the degradation                     54

           

CHAPTER 4: RESULTS AND DISCUSSION                                                                                 

4.0       Results            and discussion                                                                                         57

4.1       Isolation and characterization of the fungal isolates                                            57

4.2       Viability of fungal Isolates to Pesticides containing medium                              59

4.3       Growth of the Fungal Isolate in pesticide containing medium                            68

4.4       Molecular Analysis of Fusarium solani                                                               73

4.5       Biodegradation of Pesticides by Fusarium solani                                                76

4.5.1    Biodegradation determined by Gas Chromatography                                                      79

4.6       Results on Minimum inhibitory concentration                                                      84

4.7       Crude Enzyme Activities                                                                                      87

CHAPTER 5:   CONCLUSION AND RECOMMENDATIONS                             89

References                                                                                                            96

Appendices                                                                                                           110

 

 

 

LIST OF TABLES

TABLE	TITLE	PAGE
4.1	Macroscopic and Microscopic description of fungal Isolates	58
4.2a	Adaptation of Fusarium solani  to dichlovos containing medium.	60
4.2b	Adaptation of Fusarium solani to Lambdaccyhalothrin containing medium	61
4.2c	Adaptation of Cladosporum specie to dichlovos containing medium	62
4.2d	Adaptation of Cladosporum specie to Lambdaccyhalothrin containing medium	63
4.2e	Adaptation of Penicillium specie to dichlovos containing medium	64
4.2f	Adaptation of Penicillium specie to Lambdaccyhalothrin containing medium	65
4.2g	Adaptation of Aspergillus fumigates to dichlovos containing medium	66
4.2h	Adaptation of Aspergillus fumigates to Lambdaccyhalothrin containing medium	67
4.3.1a	Biodegradation assay of Fusarium solani in PDA amended with pesticides	69
4.3.1b	Biodegradation assay of Cladosporum specie in PDA amended with pesticides	70
4.3.1c	Biodegradation assay of Aspergillus fumigates in PDA amended with pesticides	71
4.3.1d	Biodegradation assay of Penicillium specie in PDA amended with pesticides	72
4.4	Nucleotide sequence of Fusarium solani	74
4.5a	Percentage degradation of Lambdaccyhalothrin by Fusarium solani	77
4.5b	Percentage degradation of Dichlorvos by Fusarium solani	78
4.6a	MIC of Dichlorvos on the Isolates	85
4.6b	MIC of Lambdaccyhalothrin on the Isolates	86
4.7	Results of Crude enzyme activity of Fusarium solani	88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

FIGURE	TITLE	PAGE
4.4	Appearance of Fusarium solani on X400 objective	74
4.5a	Control result of Dichlovors degradation using Gas Chromatography	80
4.5b	Result of Dichlovors degradation by Fusarium solani using Gas Chromatography	81
4.5c	Control result of Lambdacyhalothrin degradation using Gas Chromatography	82
4.5d	Result of Lambdacyhalothrin degradation by Fusarium solani using Gas Chromatography	83