CRANFIELD UNIVERSITY AHMED ABDEL-HADI MOLECULAR ECOLOGY OF ASPERGILLUS SECTION FLAVI SPECIES: APPROACHES TO UNDERSTAND THE ROLE OF AFLATOXIN GENES IN AFLATOXIN BIOSYNTHESIS CRANFIELD HEALTH APPLIED MYCOLOGY GROUP PhD Thesis Academic year: 2007-2011 Supervisors: Prof. Naresh Magan Dr. David Carter February 2011 CRANFIELD UNIVERSITY CRANFIELD HEALTH Ph.D. Thesis Academic Year 2007-2011 AHMED ABDEL-HADI MOLECULAR ECOLOGY OF ASPERGILLUS SECTION FLAVI SPECIES: APPROACHES TO UNDERSTAND THE ROLE OF AFLATOXIN GENES IN AFLATOXIN BIOSYNTHESIS Supervisors: Prof. Naresh Magan Dr. David Carter February 2011 This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy © Cranfield University 2011. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner. Abstract ABSTRACT This is the first study to integrate and correlate the effect of ecophysiological factors on the life cycle of Aspergillus flavus by carrying out complementary work on gene expression of the aflatoxin gene cluster, with growth, sporulation and phenotypic toxin production. This information was used to understand the role of ecological factors on key biosynthetic genes and examine the use of such information for control of aflatoxin production using RNA interference. Ecological studies showed the profiles for growth, sporulation and aflatoxin B (AFB ) 1 1 production with optimum ranges of water activity (a ) and temperature for AFB production w 1 being identified. A. flavus grew faster at 0.99 a at all temperatures, but optimally at 30-35°C. w The highest amount of asexual conidia was produced at 0.95 a followed by 0.90 a and then w w 0.99 a at all temperatures examined. Interestingly, the partitioning of AFB into biomass, w 1 medium and spores showed that at 0.99 a , about 50% of the mycotoxin was present in the w biomass and the medium, with very little present in the spores. However, as water stress was imposed there was a switch to a significantly higher channelling of AFB (about 45%) into 1 the spores, especially at 0.95 and 0.93 a levels. w A microarray analysis was used to examine the effect of a x temperature interactions on the w relative expression of the aflatoxin gene cluster for the first time using A. flavus NRRL 3357. This showed that under mild stress conditions (20°C/0.99 a ) several of the cluster genes, in w particular aflS and aflJ, were highly induced concomitant with high levels of phenotypic AFB production. Highest amounts of AFB were produced in all conditions where aflS 1 1 expression was elevated. When the ratio between the normalised expression data of the aflS/aflR genes was generated, high ratios were obtained at 25°C and 30°C at 0.99 and 0.95 a and low ratios at25°C and 30°C at 0.90 a . This is in agreement with the AFB production w w 1 profile. Studies were then conducted to discriminate between aflatoxin and non-aflatoxin strains of Aspergillus section Flavi isolated from Egyptian peanuts using PCR and RT-PCR. This was done by examining whether strains had some key aflatoxin genes (aflD, aflM, aflP and aflR). The transcription of aflD was used as a marker to discriminate between aflatoxin and non- i Abstract aflatoxin producers, while aflM, aflP and aflR failed to differentiate between aflatoxigenic and non-aflatoxigenic strains. In this study A. flavus EGP3 did not produce any AFB on YES, 1 however, it produced higher AFB than A. flavus EPG14 (an aflatoxin producer on YES) in 1 stored peanuts. Quantitative real-time PCR was developed for the aflD gene and used to examine the temporal relationship between expression and AFB contamination in stored peanuts over 1 periods of 6 weeks under different a treatments at 25oC. There were significant differences w between aflD gene expression at the three a levels; higher expression at 0.90 a in weeks 1– w w 3, when compared to 0.95. In contrast, in the driest treatment (0.85 a ) none or very low aflD w expression occurred. The populations of A. flavus (CFUs g-1) increased over time with the highest at 0.95 a . Highest AFB production was at 0.90 and 0.95 a after 3–6 weeks storage. w 1 w A had a significant effect on aflR transcription at 0.95 a over the 6-week period, while at w w 0.90 a , only in the last 2 weeks. Correlations between different factors showed that log w AFB xlog CFUs, log AFB x a , and log CFUs x a were statistically significant, while log 1 1 w w CFUs x RQ-PCR and RQ-PCR x a were not. The aflR gene may not have an important role w in the regulation of aflD expression in food matrices (e.g. peanuts). RNA silencing technique was employed to target the mRNA sequence of the aflD gene, to control aflatoxin production. A decrease in aflD mRNA, aflR mRNA abundance, and AFB 1 production (98%, 97% and 97% of the control level) in A. flavus NRRL3357 when treated with siRNA, respectively. There was a significant inhibition of aflD and AFB production in 1 protoplasts of A. flavus EGP9 and AFG production by A. parasiticus NRRL 13005. Changes 1 in AFB production in relation to mRNA levels of aflD showed a good correlation (r=0.88; P 1 =0.00001); changes in aflR mRNA levels in relation to mRNA level of aflD also showed a good correlation (r=0.82; P =0.0001). These detailed studies suggest that stress applied by external abiotic factors has a complex role in the activation of the genes and that their activation may be some kind of stress response leading perhaps to an increased fitness of the producing fungus to withstand the imposed stress conditions. Also, these studies have found a rapid molecular assay for the detection of aflatoxin-producing species in contaminated peanuts, and a good target gene for inactivation, to develop efficient means of aflatoxin control by using RNA silencing technology. ii Acknowledgment ACKNOWLEDGMENTS I would like to express my sincere gratitude to my first supervisor Prof. Naresh Magan for his effective advice, continuous encouragement, and broad discussion during my research work. I am grateful to my second supervisor Dr. David Carter for the guidance and helping me in my molecular work. I am also thankful to Prof. Rolf Geisen (Max-Rubner Institute, Karlsruhe, Germany) for performing microarray analysis for aflatoxin genes expression. I am also grateful to Egyptian Higher Education Ministry and Al-Azhar University, Assuit branch, for financial support and giving me the opportunity to undertake this project. Grateful thanks are given to my colleague Yousef Sultan for supplying with Aspergillus section Flavi strains used in the study. I am also thankful to Dr. Bhatanagar (USDA, New Orleans, USA) for providing with Aspergillus flavus NRRL 3357 To my friends and colleagues at Cranfield University, I owe many thanks for always being there to help and who have given me immeasurable support over the years. Finally, I would like to thank my family for believing in me and supporting me throughout the long period of this study. This thesis is dedicated to them. iii Table of contents TABLE OF CONTENTS ABSTRACT........................................................................................................................ I ACKNOWLEDGEMENTS.............................................................................................. III TALE OF CONTENTS..................................................................................................... IV LIST OF FIGURES............................................................................................................ VII LIST OF TABLES............................................................................................................. XI LIST OF PLATES.............................................................................................................. XIII ABBREVIATIONS............................................................................................................ XIV CHAPTER 1....................................................................................................................... 1 1 LITERATURE REVIEW.......................................................................................... 1 1.1 General introduction............................................................................................... 1 1.2 Mycotoxins............................................................................................................... 3 1.3 Aflatoxins.................................................................................................................. 5 1.3.1 Worldwide limits for aflatoxins in food ………………………………………. 6 1.3.2 Aflatoxins and Peanuts........................................................................................ 8 1.3.3 Aflatoxin biosynthesis......................................................................................... 9 1.4 Environmental factors, fungal growth and mycotoxin production.................... 12 1.4.1 Water relations of fungi....................................................................................... 12 1.4.2 Temperature......................................................................................................... 13 1.5 Analytical methods for the detection of mycotoxins............................................ 17 1.6 Molecular methods.................................................................................................. 19 1.6.1 Polymerase Chain Reaction (PCR)...................................................................... 19 1.6.2 Reverse transcription-Polymerase chain reaction (RT–PCR)............................. 20 1.6.3 PCR in the detection of fungi.............................................................................. 23 1.7 Real time PCR.......................................................................................................... 25 1.8 Microarrays.............................................................................................................. 31 1.9 RNA interference (RNAi)....................................................................................... 32 1.9.1 RNAi mechanism................................................................................................ 34 1.9.2 siRNA delivery………………………………………………………………… 34 1.10 OBJECTIVES........................................................................................................ 37 CHAPTER 2....................................................................................................................... 40 2 ECOLOGY OF ASPERGILLUS FLAVUS IN RELATION TO AFLATOXIN GENE CLUSTER EXPRESSION................................................................................. 40 2.1 INTRODUCTION.................................................................................................. 40 2.2 MATERIALS AND METHODS............................................................................ 42 2.2.1 Fungal strain........................................................................................................ 42 2.2.2 Media................................................................................................................... 42 2.2.3 Adjustment water activity of media for ecophysiological studies....................... 43 2.2.4 Effect of water activity x temperature interactions on growth, sporulation, aflatoxin B production and expression of aflatoxin cluster gene expression............. 43 1 2.2.4 (a) Measurement of growth.................................................................................. 43 2.2.4 (b) Determination of aflatoxin B ......................................................................... 43 1 iv Table of contents 2.2.4 (c) Spore production in relation to environmental factors....................................... 44 2.2.5 Partitioning and distribution of Aflatoxin B in spores, biomass and medium... 46 1 2.2.6 Extraction of RNA and Molecular analyses......................................................... 46 2.2.6 (a) Isolation of RNA from samples...................................................................... 46 2.2.6 (b) Microarray experiments.................................................................................. 48 2.2.7 Statistical analysis............................................................................................. 48 2.3 RESULTS................................................................................................................ 50 2.3.1 Effects of water activity and temperature on growth........................................... 50 2.3.2 Effect of time, temperature, and water activity on aflatoxin B production by A. 1 flavus................................................................................................................................ 50 2.3.3 Effects of water activity,and temperature on asexual sporulation........................ 50 2.3.4 Partitioning of aflatoxin B in spores, biomass and medium................................. 56 1 2.3.5Microarray analysis of aflatoxingene cluster expression in relation to environmental factors..................................................................................................... 56 2.3.6 Expression of specific genes................................................................................ 59 2.4 DISCUSSION.......................................................................................................... 59 CHAPTER 3...................................................................................................................... 67 3 DETECTION OF AFLATOXIGENIC STRAINS BY PCR................................... 67 3.1 INTRODUCTION................................................................................................... 67 3.2 MATERIALS AND METHODS............................................................................ 68 3.2.1. Strains, Media and growth conditions................................................................ 68 3.2.2. Screening for aflatoxin production using coconut cream agar medium............. 68 3.2.3. Aflatoxin B extraction and HPLC analysis......................................................... 68 1 3.2.4 Molecular methods.............................................................................................. 68 3.2.4 (a) Isolation of RNA from the samples............................................................... 68 3.2.4 (b) Aflatoxin genes expression............................................................................. 69 3.2.4 (c) DNA isolation and amplification.................................................................... 69 3.2.5 Studies on peanuts................................................................................................ 73 3.2.5 (a) Aflatoxin B extraction analysis..................................................................... 73 1 3.2.5 (b) RNA extraction and RT-PCR for peanut samples.......................................... 75 3.3 RESULTS................................................................................................................. 75 3.3.1 Screening the ability of the strains for aflatoxin production............................... 75 3.3.2 Analysis of aflatoxin gene transcription.............................................................. 75 3.3.3 Studies on peanuts............................................................................................... 77 3.4 DISCUSSION........................................................................................................... 83 CHAPTER 4...................................................................................................................... 87 4 MONITRING OF NOR-1 (AFLD) GENE OF ASPERGILLUS FLAVUS IN PEANUTS...................................................................................................................... 87 4.1 INTRODUCTION.................................................................................................... 87 4.2 MATERIALS AND METHODS............................................................................. 89 4.2.1 Fungal strain and growth conditions.................................................................... 89 4.2.2 Inoculation of peanut samples.............................................................................. 89 4.2.3 Determination of colony forming units (CFUs)................................................... 90 v