Effects of Botanicals and Biocontrol Agents on Growth and Aflatoxin Production by Aspergillus Flavus Infecting Maize in Some Parts of Nigeria

ABSTRACT

In a comprehensive study to assess the effects of botanicals and biocontrol agents on growth and aflatoxin (AF) production by Aspergillus flavus (A. flavus) infecting maize, a total of 1143 maize samples, collected in eighteen batches of five maize varieties (yellow, white, pop, variegated and mixed) from northern and southern parts of Nigeria were investigated between June 2011 to December 2013. Samples collected from field, 414 (36.2%) and stored batches, 729 (63.8%) were cultured on to mycological and bacteriological media after surface sterilization using 0.1 % sodium hypochlorite solution (NaOCl). Fungal isolates 358 (31.3%) and bacterial isolates 111 (9.71%) were identified using standard methods. Aspergillus niger 72 (20.1%) ranked highest followed by A. flavus 62 (17.3%) amongst the fungal isolates while Staphylococcus aureus 48 (43.2%) followed by Escherichia coli 32 (28.8%) were the most predominant bacterial isolates. Fungal isolates, 220 (61.5%) were more in the field samples than in the stored samples 138 (38.5%) and the difference was statistically significant (p < 0.05). Bacterial isolates were more in stored samples, 75 (67.6 %) than in the field samples 36 (32.4 %) (p < 0.05). A seasonality pattern was observed favouring the rainy season, 245 (68.4 %) than the dry season, 113 (31.6%) for fungal distribution. The reverse was observed for bacteria with 75 (67.6 %) and 36 (32.4%) for the dry and rainy seasons respectively during the study period (p < 0.05). Of the total of 62 (17.3 %) A. flavus isolates, 26 (41.9 %) were found to be aflatoxigenic strains and was more in the field 17 (65.4 %) than in the stored 9 (34.6%) samples (p < 0.05). Generally, maize were more contaminated by fungi from the southern 290 (83.1%) than the northern samples 59 (16.9%) which was statistically significant (p < 0.05). Similar pattern was observed for bacterial isolates with values of 57.7 % and 42.3 % from the South and North respectively though the difference was not statistically significant (p > 0.05). Detection of the total natural aflatoxin (AF) levels in the eigthteen maize batches studied showed a mean range of 2450 to 125300 ng/kg. The highest detectable mean level of AF was from stored samples (125300 ng/kg) which were statistically significant from the highest detectable mean level from field samples (112000 ng/kg) (p < 0.05). This total natural aflatoxin were reduced to various levels using various edible plant extracts (Annona muricata and Moringa oleifera) and essential oils (EOs) (Cymbopogon citratus and Ocimum gratissimum) with a range of percentage reduction ranging from 0 to 91.8 % for Cymbopogon citratus, 7.1 % to 88.3 % for Ocimum gratissimum, 0 to 77.9 % for Moringa oleifera and 23.3 % to 90 % for Annona muricata showing the highest effect. There were statistically significant differences in the percentage reduction amongst these plant treatments (p < 0.05) using ANOVA. Similarly, in in-vitro studies using plate experiments, the various plant extracts also showed significant reductions in counts/ mean clearance zone diameter according to the extract concentration on the various fungi/ bacteria studied. Co-inoculation studies using co-bacteria or co-fungal pairings with A. flavus also showed varying inhibitions. Saccharomyces cerevisiae, Geotrichum candidum and Aspergillus niger significantly inhibited production and detoxification when co-administered with A. flavus. Among the bacteria tested Bacillus subtilis and Pseudomonas aeruginosa showed significant reduction in AF production and detoxification when co-administered with A.flavus. However, these bacteria are potentially pathogenic and their virulence need to be genetically altered before they can be used together with the fungi mentioned above as biocontrol agents. 

TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents v

List of tables viii

List of Figures x

Abstract xi

CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Aims and Objectives 11

CHAPTER TWO

2.0 LITERATURE REVIEW 12

2.1 Definitions of Aspergilus flavus 12

2.1.1 Taxonomy of Aspergillus flavus 12

2.2 Variants Strains of A. flavus 13

2.2.1 Aspergillus flavus var. columnaris 13

2.2.2 Aspergillus flavus S and L strains 13

2.3 Life Cycle of Aspergillus flavus 13

2.4 Maize Production in Nigeria 23

2.4.1 Germplasm collection 25

2.4.2 Maize varieties grown in Nigeria 27

2.4.3 Aspergillus flavus in field maize 28

2.5 Effect of Aflatoxins on Livestock 31

2.6 Pathogenesis of Aflatoxin (AF) 34

2.7 Mechanism of Transmission of Aflatoxins 37

2.8 Aflatoxins used as Biological Weapons 38

2.9 Aflatoxin Infection Process 38

2.10 Clinical Syndrome of Aflatoxicosis 40

2.11 Pathogenic Mechanism of Aflatoxicosis 42

2.12 Detection of Aflatoxin 45

2.12.1 Other types of diagnostic tests 46

2.13 Preventive Measures of Aflatoxins 49

2.13.1 Other ways of preventing aflatoxin contamination in field 51

2.14 Biological Control of Aflatoxins 56

2.14.1 Use of plant extracts in controlling of aflatoxigenic fungi and aflatoxins 57

2.14.2 Plant oils on control of aflatoxigenic fungi and aflatoxins 59

2.15 Insecticidal Control of Aflatoxins on Crops 62

2.16 Use of Microbes as Biocontrol Agents of Aflatoxigenic Fungi 64

2.16.1 Biocontrol of bacteria and phytopathogenic fungi 65

2.16.2 Antagonism used as biocontrol agents 66

CHAPTER THREE

3.0 MATERIALS AND METHODS 68

3.1 Study Area 71

3.2 Study Periods 71

3.3 Laboratory Procedure 76

3.3.1 Isolation of fungi from maize 76

3.3.1.A Mycological studies 76

3.3.1.B Laboratory studies 76

3.3.2.A Bacteriological studies 77

3.3.2.B Cultural techniques 77

3.3.2.C Identification of bacteria 78

3.4 Assay of Natural Occurence of Aflatoxin in Maize Samples. 82

3.4.1 Screening for natural aflatoxin from maize samples 82

3.4.1a Preparation of maize samples for measurement of Aflatoxin 83

3.4.1b Test procedure 83

3.5 Screening of Aspergillus flavus Isolates for Aflatoxigenic Potentials 84

3.5.1 Laboratory experiment to determine the aflatoxin producing ability of A. flavus isolates 84

3.6 Collection and Identification of Plant Materials for Extraction 85

3.6.1 Extraction of EOs and methanolic extracts from the plant leaves 85

3.6.1a Preparation of sample plants for extraction 86

3.6.1b Procedure for methanolic extraction 86

3.6.1c Procedure for extraction of EOs 86

3.7 Antifungal Activity of EOs and Methanolic Extracts of Some Plants 87

3.7.1 To determine the fungiotoxicity of the essential oil (EO) and plant extracts 87

3.8 Antibacterial Activity of EOs and Methanolic Extracts of Some Plants 88

3.8.1 Determination of the bactericidal/ bacteristatic effects of the essential oil (EO) and plant extracts 88

3.9 Use of Botanicals (Phytocontrols) to Inhibit Growth of A. flavus and Aflatoxin (AF) Production 89

3.9.1 Treatment of maize with EOs and methanolic plant extracts methodology 89

3.10 Bioremediation Studies: 89

3.10.1 Preparation of inoculums 90

3.11 Biocontrol Experiments: (In-vitro Plate Assay) 91

CHAPTER FOUR

4.0 RESULTS 93

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS 171

5.1 Discussion 171

5.2 Conclusions 190

5.3 Recommendations 191

References 193

Appendix