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EFFECTS of BOTANICALS and BIOCONTROL AGENTS on GROWTH and AFLATOXIN PRODUCTION by Aspergillus Flavus INFECTING MAIZE in SOME PARTS of NIGERIA

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EFFECTS OF BOTANICALS AND BIOCONTROL AGENTS ON GROWTH AND AFLATOXIN PRODUCTION BY Aspergillus flavus INFECTING MAIZE IN SOME PARTS OF NIGERIA BY OKECHI, OGECHUKWU CALISTA PG/Ph.D./09/54408 DEPARTMENT OF MEDICAL LABORATORY SCIENCES FACULTY OF HEALTH SCIENCES AND TECHNOLOGY COLLEGE OF MEDICINE UNIVERSITY OF NIGERIA ENUGU CAMPUS OCTOBER, 2014 TITLE PAGE EFFECTS OF BOTANICALS AND BIOCONTROL AGENTS ON GROWTH AND AFLATOXIN PRODUCTION BY Aspergillus flavus INFECTING MAIZE IN SOME PARTS OF NIGERIA BY OKECHI, OGECHUKWU CALISTA PG/Ph.D./09/54408 A THESIS SUBMITTED TO THE DEPARTMENT OF MEDICAL LABORATORY SCIENCES, FACULTY OF HEALTH SCIENCES AND TECHNOLOGY, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) IN MEDICAL LABORATORY SCIENCES (MEDICAL MIROBIOLOGY), COLLEGE OF MEDICINE, UNIVERSITY OF NIGERIA ENUGU CAMPUS SUPERVISOR: PROFESSOR N.F. ONYEMELUKWE OCTOBER, 2014 i DEPARTMENT OF MEDICAL LABORATORY SCIENCES COLLEGE OF MEDICINE UNIVERSITY OF NIGERIA Telegrams NIGERSITY, ENUGU ENUGU CAMPUS HEAD OF DEPARTMENT NIGERIA OUR REF:……………………UN/CM/MLS/B2 Tel. YOUR REF: ……………… DATE: …………… CERTIFICATION Mr / Mrs / Miss OKECHI OGECHUKWU CALISTA a Ph.D student of the Department of Medical Laboratory Sciences, College of Medicine University of Nigeria, Enugu Campus, majoring in MEDICAL MICROBIOLOGY has satisfactorily completed the requirement for the research work. The results embodied in the work have not been submitted in part or full to any Diploma or Degree of this in any other University. Supervisor’s Name: PROF N .F. ONYEMELUKWE Signature: _____________________________________ ii DEDICATION To Almighty God and my loving mother Mrs. Caroline Nwamaka Okechi. iii ACKNOWLEDGEMENTS My profound gratitude goes to Almighty God for making this study a reality. Special thanks go to my supervisor Prof (Mrs) N. F. Onyemelukwe that made the completion of this study a reality. I am indebted to my Honey, Pharm Calistus Dozie Nwakile for his encouragement, guidance and generosity towards the realization of this study. My special gratitude goes to all my colleagues and laboratory supervisors for their encouragement and support; especially Mrs. Blessing Eluke, Mrs. Oluchi Okeh, Mrs Ngozi Ezeodili, Augustina Onovo, Obioma Omeje, Mrs Attah and Ceey in the Department of Medical Laboratory Sciences, University of Nigeria, Enugu Campus. My special thanks go to the staff of Emmanuel Research Centre, Uwani, Enugu. I also extend my gratitude to the staff of Institute of Human Virology, University of Benin Teaching Hospital, Benin who in one way or the other encouraged me during the course of the study especially in the pre-treatment and post-treatment data analyses. My thanks go to my academic adviser Barr (Dr) P.A Achukwu for his loyalty in the course of this study. I am indebted to R-Biopharm AG, Darmstadt, Germany, for their marvelous kind support in supplying the RIDASCREEN®Aflatoxin Total Enzymeimmunoassay kit for the quantitative analysis of aflatoxins. I continue to say a very big thank you again and May the Good Lord reward you abundtantly. Amen. iv 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 v 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 vi 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 216 vii LIST OF TABLES Tables Title Page 3.1: Distribution According to Northern and Southern Maize Varieties 70 3.2: Distribution according to number of maize samples collected. 73 3.3: Distribution of maize batches sampled between 2011 and 2013 75 4.1: Overall incidence of mycoflora from eighteen different maize batches analysed. 94 4.2: Overall incidence of bacteria flora from eighteen different maize batches analysed. 110 4.3: Bacterial inhibition zone diameter using EO of Cymbopogon citratus (lemon grass) 118 4.4: Inhibition of growth of Geotrichum candidum by some edible plant extracts and essential Oils (EOs). 120 4.5: Bacteria inhibition zone diameter by Moringa Oleifera methanolic extracts. 122 4.6: Growth inhibition M (mm) of Aspergillus flavus on Cymbopogon citratus 124 4.7: Percentage growth inhibition ((M %) Of Aspergillus flavus on Cymbopogon citratus 125 4.8: Growth inhibition M (mm) of Aspergillus niger on Cymbopogon citratus 127 4.9: Percentage growth inhibition M(%) of Aspergillus niger on Cymbopogon citratus 128 4.10: Growth inhibition M (mm) of Aspergillus flavus on Ocimum gratissimum 130 4.11: Percentage Growth Inhibition M (%) OF Aspergillus flavus ON Ocimum gratissimum 131 4.12: Growth inhibition M (Mm) of Aspergillus niger on Ocimum gratissimum 133 4.13: Percentage growth inhibition M (%) of Aspergillus niger on Ocimum gratissimum 134 4.14: Growth inhibition M (mm) of Aspergillus flavus on Annona muricata 136 4.15: Percentage growth inhibition M (%) of Aspergillus flavus on Annona muricata 137 4.16: Growth inhibition M (mm) of Aspergillus niger on Annona muricata 139 4.17: Percentage growth inhibition M(%) of Aspergillus niger on Annona muricata 140 4.18: Growth inhibition M (mm) of Aspergillus flavus on Moringa oleifera. 142 4.19: Percentage growth inhibition M (%) of Aspergillus flavus on Moringa Oleifera 143 4.20: Growth inhibition M (mm) of Aspergillus niger on Moringa oleifera 145 4.21: Percentage growth inhibition M (%) of Aspergillus niger on Moringa oleifera 146 4.22: Total natural aflatoxin levels in maize batches collected from different parts of Nigeria 148 4.23: Total natural aflatoxin (AF) (ng/kg) reduction according to maize specification using eo of Cymbopogon citratus (lemon grass) 150 4.24: Total natural aflatoxin (AF) (ng/Kg) reduction according to maize specification using EO of Ocimum gratissimum (scent leaf) 152 viii LIST OF TABLES Contd. Tables Title Page 4.25: Total natural aflatoxin (AF) (ng/kg) reduction according to maize specification using methanolic extract of Moringa oleifera 154 4.26: Total natural aflatoxin (AF) (ng/kg) reduction according to maize specification using Methanolic extract of Annona muricata 156 4.27: Total natural aflatoxin (AF) (ng/kg) reduction according to maize specification using co-cultures of fungi isolated 162 4.28: Total natural aflatoxin (AF) (ng/kg) distribution according to maize specification using co-cultures of bacteria 164 4.29: Distribution according to % reduction with some extracts from edible plants in northern maize varieties in Nigeria 166 4.30: Distribution according to % reduction with some extracts from edible plants in southern maize varieties in Nigeria 168 4.31: Biocontrol effect of aflatoxin (AF) on growth of A.
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    Joardar et al. BMC Genomics 2012, 13:698 http://www.biomedcentral.com/1471-2164/13/698 RESEARCH ARTICLE Open Access Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability Vinita Joardar1*, Natalie F Abrams1, Jessica Hostetler1, Paul J Paukstelis2, Suchitra Pakala1, Suman B Pakala1, Nikhat Zafar1, Olukemi O Abolude3, Gary Payne4, Alex Andrianopoulos5, David W Denning6 and William C Nierman1 Abstract Background: The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Results: Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25–36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T.
  • Classification of Aspergillus, Penicillium

    Classification of Aspergillus, Penicillium

    available online at www.studiesinmycology.org STUDIES IN MYCOLOGY 95: 5–169 (2020). Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species J. Houbraken1*, S. Kocsube2, C.M. Visagie3, N. Yilmaz3, X.-C. Wang1,4, M. Meijer1, B. Kraak1, V. Hubka5, K. Bensch1, R.A. Samson1, and J.C. Frisvad6* 1Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; 2Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; 3Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, P. Bag X20, Hatfield, Pretoria, 0028, South Africa; 4State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3, 1st Beichen West Road, Chaoyang District, Beijing, 100101, China; 5Department of Botany, Charles University in Prague, Prague, Czech Republic; 6Department of Biotechnology and Biomedicine Technical University of Denmark, Søltofts Plads, B. 221, Kongens Lyngby, DK 2800, Denmark *Correspondence: J. Houbraken, [email protected]; J.C. Frisvad, [email protected] Abstract: The Eurotiales is a relatively large order of Ascomycetes with members frequently having positive and negative impact on human activities. Species within this order gain attention from various research fields such as food, indoor and medical mycology and biotechnology. In this article we give an overview of families and genera present in the Eurotiales and introduce an updated subgeneric, sectional and series classification for Aspergillus and Penicillium. Finally, a comprehensive list of accepted species in the Eurotiales is given. The classification of the Eurotiales at family and genus level is traditionally based on phenotypic characters, and this classification has since been challenged using sequence-based approaches.