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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 21daysThe 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.


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


1.0       Introduction                                                                                                               1

1.1       Aims and Objectives                                                                                                  7


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




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









Macroscopic and Microscopic description of fungal Isolates



Adaptation of Fusarium solani  to dichlovos containing medium.



Adaptation of Fusarium solani to Lambdaccyhalothrin containing medium



Adaptation of Cladosporum specie to dichlovos containing medium



Adaptation of Cladosporum specie to Lambdaccyhalothrin containing medium



Adaptation of Penicillium specie to dichlovos containing medium



Adaptation of Penicillium specie to Lambdaccyhalothrin containing medium



Adaptation of Aspergillus fumigates to dichlovos containing medium



Adaptation of Aspergillus fumigates to Lambdaccyhalothrin containing medium



Biodegradation assay of Fusarium solani in PDA amended with pesticides



Biodegradation assay of Cladosporum specie in PDA amended with pesticides



Biodegradation assay of Aspergillus fumigates in PDA amended with pesticides



Biodegradation assay of Penicillium specie in PDA amended with pesticides



Nucleotide sequence of Fusarium solani



Percentage degradation of Lambdaccyhalothrin by Fusarium solani



Percentage degradation of Dichlorvos by Fusarium solani



MIC of Dichlorvos on the Isolates



MIC of Lambdaccyhalothrin on the Isolates



Results of Crude enzyme activity of Fusarium solani



















Appearance of Fusarium solani on X400 objective



Control result of Dichlovors degradation using Gas Chromatography



Result of Dichlovors degradation by Fusarium solani using Gas Chromatography



Control result of Lambdacyhalothrin degradation using Gas Chromatography



Result of Lambdacyhalothrin degradation by Fusarium solani using Gas Chromatography









APPENDIX                                                   TITLE                                                           PAGE


Absorbance and OD reading of the isolate in Lambdaccyhalothrin



Absorbance and OD reading of the isolate in Dichlorvos



Percentage degradation of the pesticide Lambdaccyhalothrin by Fusarium solani during the 20days degradation study



Percentage degradation of the pesticide Dichlorvos by Fusarium solani during the 20days degradation study



Photomicrograph of Fusarium solani at X400 magnification



Photomicrograph of  Damping off of Fusarium solani at X400 magnification



General Gas chromatography result of Fusarium solani against study pesticides











1.0       Introduction

Certain modern advances in agriculture only give short-term gains and often lead to ecological degradation that are no longer sustainable by man (Ramakrishnan, 2007). Increasing environmental awareness has resulted in regulatory measures that aim to remedy past mistakes and protect the environment from future contamination and exploitation. These measures are intended to protect the environment and protect human health. Some of the contaminants of concern are chemicals coming from pesticides. They are diluted locally by manufacturers or are mixed with other chemicals to obtain the formula required for local conditions. Unfortunately, these compounds continue to persist naturally. These chemicals persist in the soil and sediments despite their continued use from where they can be introduced directly into the food chain.  These contaminants can pose severe health problems once they find their way into drinking water wells (Mitra et al., 2001). Pesticides are the solution for the ever increasing need to increase quantity and quality of agricultural products. Pesticides also play important role as a suppressor of disease vectors and pests affecting the health and welfare of the world. Due to the high consumer expectations and the ever increasing world population, the uses of pesticides cannot be over emphasized (Ingram et al., 2005). Although each pesticide is intended to kill a specific insect, the vast majority of these chemical agents penetrates other regions where they are not intended especially air, water, and sediment and soil surfaces. Pesticide can easily contaminate air, soil and water when they run out of fields during rain, escape from storage tanks, and when not properly disposed of, especially when sprayed with air.

They are important in agriculture, horticulture and forestry and cause toxic effects to some unwanted organisms. However, they are not always so selective and can therefore affect other organisms, including man. Residues from these pesticides often lodge in surface and groundwaters, sewage and soils. One important reason for the appearance of such residues is the unsatisfactory management of the chemicals, especially residues remaining after usage, at farms, machine stations and in society in general.

Environmental pollution refers to the contamination of the physical, chemical as well as the living organisms found in the environment resulting in alteration of natural environmental processes (Fuleker, 2009). Pollution involves the introduction into the environment of contaminants harmful to humans, plants, animals, and organisms, or cause damage to the environment. These pollutants are only termed hazardous or contaminants when the quantity exceed certain natural levels (Jilani, 2013).

Pesticides have a negative effect beside their beneficial effect. Pesticides are chemicals with harmful effects on both human beings and environment, pesticides are substances employed in the prevention, repelling, mitigation or destroying pest's (insects, fungi, and weeds), that compete for food supply, adversely affect comfort, or endanger human health. Hundreds of millions of people are affected by pesticides each year in sub-Saharan Africa, and many more are indirectly exposed to food, water, household dust, spray slip and pollution in homes. Humans come in contact with these chemicals in a variety of ways such as inhalation, ingestion, and skin contact with pesticides (found in environmental media such as soil, water, air and food). Exposure to pesticides causes severe and chronic health problems (Mitra et al., 2001). Pesticides used for agricultural purposes are discharged in the environment and come into direct or indirect human contact. There have been reported surge in cancer, sterility amongst men and female, kidney diseases, endocrine disorders, suppression of immune system as well as neurological and behavioural disorders particularly among children resulting from pesticide poisoning. (Wang et al., 2006; Baxter and Cummings, 2008). Human health hazards are varying with the extent of exposure.

Mild human health problems from misuse of pesticides include mild headaches, fever, skin rash, blurred vision and other neurological disorders, while severe human health problems include stroke, blindness and even death (Niewiadomska, 2004).

Pesticide pollution to the local environment also affects the lives of birds, wildlife, domestic animals, fish and livestock. Improper usage of these pesticides in uncontrolled amounts not only disturbs soil conditions but also destroys the healthy pond of bio-control agents commonly associated with plants (Reddy and Matthew, 2001).

There are many methods to clean the soil of pesticides such as chemical treatment, evaporation and incineration. Chemical treatment and volatility are potentially complex due to the production of large amounts of acids and alkalis, which must then be removed. The most reliable physico-chemical method of combustion for the destruction of these compounds has met with fierce public opposition due to its toxic emissions and its high economic costs (Kearney, 1998).

Overall these physicochemical cleaning technologies are expensive and inefficient (Kearney, 1998; Nerud et al., 2003).

Removal of pesticides and their residues from contaminated soil has become an environmental priority, and both physicochemical and biological degradation processes have been studied. Although chemotherapeutic and physical therapies are faster than biological therapies, they are generally more destructive to the affected soil, more permeable, more energetic, and more expensive than biodegradation process (Foght et al., 2001).

Considering the biodegradable processes, many bacteria and white-rot fungi (WRF) are reported to enhance the degradation process in pure cultures (Hay and Focht, 2000; Kamanavalli and Ninnekar, 2004). Some of these pesticides or residues are dangerous and they persist in the environment. Cleaning toxic waste bases on farms using traditional waste disposal methods such as incineration and landfilling is often very expensive (Reddy and Matthew, 2001). Due to the scale of this problem and the lack of a reasonable solution, a quick, cost-effective, environmentally responsible cleaning method is greatly needed.

The use of microorganisms to decompose toxic organophosphates is an efficient, economical approach that has been successful in laboratory studies.

Biodegradation can be defined as a spontaneous procedure in which microorganisms are used to convert or reduce the toxicity of environmental pollutants into less toxic or nontoxic forms, thus reducing their effect on the environment (Talley, 2005). The removal of organic wastes by microbes leads to environmental cleanup.

There are three outcomes of biodegradation:

a.       A minor change in an organic molecule, leaving the main structure still intact.

b.      Fragmentation of a complex organic molecule in a way the fragments could be re-assembled to yield the original structure.

c.       Complete mineralization, which involves complete breakdown of organic molecule into its inorganic form.

Microorganisms used in biodegradation can be aerobic and anaerobic (Gray, 2004; Moharikar et al., 2005). Some have been isolated and genetically engineered for effective biodegradable capabilities, including the ability to reduce regenerated pollutants, ensure better survival and colonization, and achieve improved degradation rates in polluted areas (Gavrilescu and Chisti, 2005).

The most important way for pesticides to decompose and disperse in ecosystems is through microbial transformation (Rajasankar et al., 2013). Zheng et al. (2012) demonstrated the presence of microbial decomposition as a promising way to clean chloroacetamide herbicide from polluted environment.

The goal of biodegradation is to reduce the level of contaminant to a minimum undetectable, nontoxic or acceptable level, i.e. to completely mineralize organopollutants to carbon dioxide within the limits set by the regulatory agent (Pointing, 2001). The complete breakdown of the compounds is essential from an environmental point of view because it prevents absolute toxicity (Gan and Koskinen, 1998).

The use of bioremediation to remove contaminants is generally less expensive than the equivalent physiochemical methods. This technology provides the ability to treat polluted soil and groundwater at the site without the need for excavation (Balpa et al., 1998; Kearney, 1998), which requires little energy input and protects the soil structure (Hohener et al., 1998). The most attractive feature of bioremediation is the reduced impact on natural ecosystems, which must be more acceptable to the general public (Zhang and Qiao, 2002).

Fungi have many benefits that can be used in biodegradable systems. The nature of these fungi which are composed of lignin-degrading systems may reduce the range of insoluble chemicals including lignin or very different or highly toxic environmental pollutants (Barr and Aust, 1994). Microbial growth habits are also favorable for allowing rapid colonization of substrates, and hybrid extension activates the infiltration of soil contaminants in ways that other organisms cannot do (Reddy and Matthew, 2001). It can enhance physical, mechanical and enzymatic interactions with the surrounding environment (Maloney, 2001). In addition, these fungi use cheap and abundant lignocellulose products as nutrients. They can tolerate a wide range of environmental conditions notably temperature, pH and humidity (Maloney, 2001) and do not require conditioning prior to a particular contaminant because their degradation system is triggered by nutrient deficiencies (Bar & Aust, 1994).

Pesticides cause serious health hazards to lifestyles because they cause rapid fat solubility and bioacumulation in non-target organisms (Reddy and Matthew, 2001). The main forms of pollution are direct applications to agricultural crops, accidental leaks during transportation and production, as well as effluents from tanks treated for livestock ectoparite control (Baxter and Cummings, 2006).

The effects of pesticides can be analyzed from two different perspectives: environmental and public health. The first occurs when pesticides are introduced into food chains, for example:

a) Phytoplankton and zooplankton (indicators of water pollution) create a change in population decline;

b) Producing oncology, neurotoxic and the fertility and reliability of their offspring (vertebrates, fish, waterfowl, insects and mammals);

c) The presence of pesticides in the environment has caused organisms considered to be vectors of diseases to acquire resistance (e.g. malaria, dengue and scabies), instead of decreasing the number of other beneficial insects (such as pollen);

d) Altering biochemical cycles by reducing macro and microbiota,

1.1 Aims of the study

To determine the ability of wood rot fungi to degrade the pesticides, dichlorvos and lambdaccyhalothrin.

Specific  Objectives

a.       To isolate and identify fungal species from decaying wood.

b.      To adapt the fungal isolates to grow in pesticides containing solid medium.

c.       To determine the ability of the fungus to degrade pesticides in liquid medium.

d.      To characterize the isolate with the highest capacity to degrade the pesticides.

e.       To analyze Gas Chromatography of degradation products after growth in pesticide contaminated liquid medium.

f.       To grow F. solani in liquid medium contaminated with dichlorvos and lambdaccyhallothrin

g.      To determine the minimum inhibitory concentration (MIC) of the pesticides to the isolates.

h.      To determine the activities of lignolytic enzymes in the culture of the isolates.


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