Contribution of intrinsic factors to heat resistance ... - UNSWorks

CONTRIBUTION OF INTRINSIC FACTORS TO HEAT RESISTANCE OF ASCOSPORES OF

BYSSOCHLAMYS

A thesis

submitted to The University of New South Wales

as fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

by

ANH LINH NGUYEN B. Sci. Hons (Food Science and Technology) (UNSW, Australia)

School of Chemical Engineering

The University of New South Wales

Sydney, NSW, Australia

January 2012

PLEASE TYPE

Surname or Family name: NGUYEN

First name: ANH LINH

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

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Abbreviation for degree as given in the University calendar: PhD

School: School of Chemical Engineering Faculty: Engineering

Title: CONTRIBUTION OF INTRINSIC FACTORS TO HEAT RESISTANCE OF ASCOSPORES OF BYSSOCHLAMYS

Abstract 350 words maximum: (PLEASE TYPE)

Byssoch/amys is a fungus that causes spoilage of heat processed fruit products by producing heat resistant ascospores. This thesis investigated heat resistant properties of ascospores of Byssoch/amys .fulva and Byssoch/amys nivea. Heat inactivation of the ascospores at temperatures of 82.5 - 90°C showed activation, shoulder, exponential reduction and tailing phases. The D values of the ascospores increased significantly with culture age over the period of 4 - 24 weeks, and the average increase was 3.5 - 7-fold for B . .fulva strains and 2 - 4-fold for B. nivea strains. Ascospores of B . .fulva were more heat resistant than those of B. nivea.

Ultrastructure of the ascospores examined by electron microscopy comprised a cell wall, a thick intermediate space (IMS) and a dense cytoplasm. Examining the arrangement of materials in the ascospores with differential scanning calorimetry provided evidence of a glassy state. Aging improved the robustness of the ascospore structure whereas heating reduced its integrity. Older ascospores occasionally had more sub-layers in the IMS. Ascospores of B . .fulva bad a more resilient structure and more elaborated outer coating than those of B. nivea.

Flow cytometry revealed heterogeneity in preparations of dormant and heat treated ascospores at different ages. Heating affected the integrity of the ascospore wall, which was manifested as increased autofluorescence, permeability to SYTO 9 and clumping of the ascospores. Sub-populations of activated and sub-lethally injured ascospores mostly appeared after heat treatments.

Proteomic analysis of ascospores by 2D gel electrophoresis showed that less than 4% of their total proteins significantly changed their expression after heating and aging. Most of the changed proteins were metabolic enzymes, which possibly had been pre-synthesised and stored in dormant ascospores. For B. nivea, three proteins were identified as heat shock proteins 60, 70 and general stress protein 39. Latent protein synthesis, denaturation and defragmentation of proteins into peptides possibly caused changes in the protein expressions.

The results showed that population, structural and molecular factors contribute simultaneously and synergistically to the beat resistance of Byssoch/amys ascospores. Data obtained provide a platform for further studies to understand the biological mechanisms of heat resistance of their ascospores and for the food industry to develop more effective heat processes to inactivate these fungi.

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Acknowledgements

I wish to express my utmost sincere gratitude to my university supervisor, Prof Graham

Fleet (School of Chemical Engineering, UNSW, Australia) and my CSIRO supervisors,

Dr Ailsa Hocking and Dr Nai Tran-Dinh (Food and Nutritional Sciences,

Commonwealth Scientific and Industrial Research Organisation, North Ryde, Australia)

for their expert advice, guidance, endless encouragement and support during the course

of this research and the completion of this thesis.

I would also like to thank the two following organisations for their financial support

which really made PhD life less stressful: the University of New South Wales for

granting the University International Postgraduate Award and CSIRO for funding all

research activities that I have carried out at North Ryde facility.

I wish to thank all members of the Food Processing and Microbiology Research Group

at North Ryde for their warm welcoming and sharing their expertise, ideas and

experience since the first day I came. With their encouragement and support, I have

really had a taste of being a professional researcher without losing a work-life balance.

I would also thank several lecturers, researchers, technicians of various research

facilities that I have had opportunities to work with in different parts of my project. I

wish to thank Dr Minoo Moghaddam (CSIRO Health and Molecular Technology, North

Ryde) for her provision of the DSC machine and help in operation and maintenance; Dr

Janet Paterson (School of Chemical Engineering, UNSW) for her advice on the DSC

experiment; Ms Debra Birch and Ms Nicole Vella (Microscopy Unit, Macquarie

University, NSW) for their instruction and help in preparing samples and using electron

microscopes; Dr Alamgir Khan, Dr Charlie Ahn, Ms Veronika Polaskova and Ms Vidya

Nelaturi (Australian Proteome Analysis Facility, Macquarie University) for their help in

experimental guidance and advice with regard to proteomic analysis.

And many thanks to the group of foodie friends, whom I have known since the

undergraduate time, for their effort in keeping me happy.

Finally, I would like to thank my family and my girlfriend back in Vietnam for their

endless love, patience and support throughout this study.

i

Table of Contents

List of Figures .............................................................................................................. v

List of Tables .............................................................................................................. ix

List of Appendices ..................................................................................................... xi

Abstract ......................................................................................................................... 1

Chapter 1 – Introduction ......................................................................................... 3

Chapter 2 – Literature review ............................................................................... 7

2.1 Taxonomy ............................................................................................................. 7 2.2 Morphology .......................................................................................................... 9 2.3 Physiology ........................................................................................................... 10

2.3.1 Temperature ..................................................................................................... 11 2.3.2 Hydrogen ion concentration (pH) and types of acid ....................................... 13 2.3.3 Gaseous atmosphere ........................................................................................ 14 2.3.4 Water activity (aw) ........................................................................................... 15 2.3.5 Important metabolites produced by Byssochlamys .......................................... 15

2.3.5.1 Metabolic enzymes .................................................................................... 16 2.3.5.2 Mycotoxins ................................................................................................ 16

2.3.6 Other environmental factors ............................................................................ 17 2.4 Ecology and food spoilage caused by Byssochlamys species .................... 19

2.4.1 Ecology of B. fulva and B. nivea ..................................................................... 19 2.4.2 Food spoilage caused by B. fulva and B. nivea ............................................... 20

2.5 Heat resistance of ascospores of Byssochlamys species ............................ 22 2.5.1 Heat resistance of Byssochlamys ascospores in thermal processes ................. 23 2.5.2 D and z values of ascospores of B. fulva and B. nivea .................................... 25

2.6 Factors contributing to the heat resistance of Byssochlamys ascospores ..................................................................................................................................... 29

2.6.1 Effects of food/medium composition on heat resistance of Byssochlamys ascospores ................................................................................................................. 29 2.6.2 Effects of ascospore age on heat resistance ..................................................... 31 2.6.3 Contributions of ascospore ultrastructure to heat resistance ........................... 32 2.6.4 Chemical composition and heat resistance of ascospores ............................... 34 2.6.5 Trehalose, glassy state and heat resistance of ascospores ............................... 35 2.6.6 Molecular factors of heat resistance of Byssochlamys ascospores .................. 36

2.7 Concluding remarks ......................................................................................... 37 Chapter 3 – Thermal inactivation of ascospores of Byssochlamys fulva and Byssochlamys nivea during aging ............................................................... 39

3.1 Introduction ....................................................................................................... 39 3.2 Materials and methods .................................................................................... 41

3.2.1 Fungal cultures, growth conditions and ascospore harvest ............................. 41

ii

3.2.2 Heat resistance of ascospores at different ages ............................................... 42 3.2.3 Heat resistance of sequential generations of ascospores surviving heat treatments ................................................................................................................. 43

3.3 Results ................................................................................................................. 43 3.3.1 Effects of age on thermal inactivation of ascospores ...................................... 43 3.3.2 Thermal reduction time (D) of Byssochlamys ascospores ............................... 50 3.3.3 Heat resistance of sequential generations of ascospores surviving heat treatments ................................................................................................................. 54

3.4 Discussion ........................................................................................................... 58 3.4.1 Inactivation kinetics of Byssochlamys ascospores .......................................... 58

3.4.1.1 Heat activation phase ................................................................................ 58 3.4.1.2 Shoulder and tailing phases ...................................................................... 59

3.4.2 Heat resistance and age of Byssochlamys ascospores ..................................... 61 3.4.3 Proposed mechanisms of age-induced heat resistance in ascospores .............. 63 3.4.4 Calculating D values from non-logarithmic survival curves ........................... 63

3.5 Concluding remarks ......................................................................................... 64 Chapter 4 – Electron microscopy investigation of ultrastructure of Byssochlamys ascospores at two different ages and after different heating treatments ................................................................................................... 66


4.2.1 Fungal samples and heat treatment of ascospores ........................................... 67 4.2.2 Reagents used for sample preparation for EM ................................................ 68 4.2.3 Sample preparation .......................................................................................... 68

4.2.3.1 Scanning electron microscopy (SEM) ....................................................... 68 4.2.3.2 Transmission electron microscopy (TEM) ................................................ 69

4.2.4 Dimensions of the ascospores and image processing ...................................... 69 4.3 Results ................................................................................................................. 70

4.3.1 SEM study of Byssochlamys ascospores ......................................................... 70 4.3.2 TEM study of Byssochlamys ascospores ......................................................... 77

4.4 Discussion ........................................................................................................... 83 4.4.1 Multilayered ultrastructure and heat resistance of Byssochlamys ascospores . 83 4.4.2 Effects of heat treatments on the ultrastructure of Byssochlamys ascospores . 85 4.4.3 Effects of age on the ultrastructure of Byssochlamys ascospores.................... 86 4.4.4 Recommendations for future research using electron microscopy .................. 87

4.5 Concluding remarks ......................................................................................... 87 Chapter 5 – Application of Differential Scanning Calorimetry (DSC) to examine the arrangement of material in Byssochlamys ascospores ....... 89


5.2.1 Fungal strains, ascospore harvest and heat treatment ...................................... 90 5.2.2 Preparation of ascospore samples for DSC ..................................................... 91 5.2.3 Differential scanning calorimetry .................................................................... 91

5.2.3.1 Selection of scanning rates ....................................................................... 91 5.2.3.2 DSC analysis of ascospore samples .......................................................... 92 5.2.3.3 Other comparative experiments ................................................................ 92

iii

5.3 Results ................................................................................................................. 93 5.3.1 Screening for the optimal scanning rate .......................................................... 93 5.3.2 DSC analysis of Byssochlamys ascospores ..................................................... 95

5.3.2.1 Comparison of dormant ascospores between ages, species and strains . 100 5.3.2.2 DSC comparisons of ascospores after heat treatments .......................... 102

5.4 Discussion ......................................................................................................... 108 5.4.1 Characteristics of DSC transitions of Byssochlamys ascospores .................. 108 5.4.2 Arrangement of intracellular materials and heat resistance of Byssochlamys ascospores ............................................................................................................... 110

5.4.2.1 Comparisons between species and ages ................................................. 110 5.4.2.2 Comparisons between physiological states induced by heat .................. 110

5.4.3 Possible existence of a glassy state in Byssochlamys ascospores .................. 111 5.4.4 Experimental designs and effects on DSC profiles ....................................... 112

5.4.4.1 Effects of sample preparation methods ................................................... 112 5.4.4.2 Effects of scanning (heating) rates .......................................................... 113

5.5 Concluding remarks ....................................................................................... 114 Chapter 6 – Heterogeneity in ascospore populations of Byssochlamys and its relationship with heat resistance ........................................................ 116

6.1 Introduction ..................................................................................................... 116 6.2 Methodology ..................................................................................................... 117

6.2.1 Fungal strains and ascospore preparation ...................................................... 117 6.2.2 Thermal treatments of Byssochlamys ascospores for flow cytometry ........... 118 6.2.3 Staining of ascospores with fluorescent dyes ................................................ 119 6.2.4 Microscopic examination of SYTO 9-stained ascospores............................. 119 6.2.5 Flow cytometric analysis ............................................................................... 120 6.2.6 Data collection and analysis .......................................................................... 120 6.2.7 Procedure of differentiating groups of ascospores ........................................ 121

6.3 Results ............................................................................................................... 123 6.3.1 Penetration of SYTO 9 into ascospores......................................................... 123 6.3.2 Evaluation of fluorescent dyes for differentiating sub-populations of Byssochlamys ascospores ....................................................................................... 125 6.3.3 Differentiating sub-populations of unstained ascospores .............................. 129

6.3.3.1 Sub-populations of unstained ascospores of B. fulva ............................. 129 6.3.3.2 Sub-populations of unstained ascospores of B. nivea ............................. 132

6.3.4 Differentiating sub-populations of SYTO 9-stained ascospores ................... 135 6.3.4.1 Sub-populations of SYTO 9-stained ascospores of B. fulva .................... 135 6.3.4.2 Sub-populations of SYTO 9-stained ascospores of B. nivea ................... 140

6.4 Discussion ......................................................................................................... 145 6.4.1 Penetration of SYTO 9 into Byssochlamys ascospores and the protective roles of the cell wall of the ascospores ............................................................................ 145 6.4.2 Evaluation of SYTO 9 and PI in differentiating sub-populations of Byssochlamys ascospores ....................................................................................... 146 6.4.3 Fluorescence mechanisms of SYTO 9 in Byssochlamys ascospores ............. 147 6.4.4 Differentiating sub-populations of Byssochlamys ascospores ....................... 148

6.4.4.1 Sub-populations of dormant ascospores ................................................. 148 6.4.4.2 Sub-populations of heat activated ascospores ........................................ 149 6.4.4.3 Sub-populations of heat-inactivated ascospores ..................................... 149

6.5 Concluding remarks ....................................................................................... 151

iv

Chapter 7 – Proteomic profiles of ascospores of Byssochlamys species detected by two dimensional gel electrophoresis during aging and heat treatment ................................................................................................................... 153

7.1 Introduction ..................................................................................................... 153 7.2 Materials and methods .................................................................................. 155

7.2.1 Isolation and preparation of ascospore samples ............................................ 155 7.2.2 Heat treatment of ascospores ......................................................................... 155 7.2.3 Proteomic analysis of ascospores .................................................................. 156

7.2.3.1 Reagents .................................................................................................. 158 7.2.3.2 Protein extraction and quantification ..................................................... 158 7.2.3.3 Two-dimensional (2D) gel electrophoresis ............................................. 160 7.2.3.4 Protein visualisation and gel image analysis.......................................... 161 7.2.3.5 Sample preparation for mass spectrometry ............................................ 161 7.2.3.6 Peptide mass fingerprinting by mass spectrometry ................................ 162 7.2.3.7 Database search and protein identification ............................................ 163

7.3 Results ............................................................................................................... 164 7.3.1 General features of proteome of Byssochlamys ascospores .......................... 164 7.3.2 Proteomic profiles of ascospores following heat treatments ......................... 164 7.3.3 Proteomic profiles of ascospores at two ages ................................................ 169 7.3.4 Protein identification ..................................................................................... 173

7.3.4.1 Identification of proteins from B. fulva ascospores ................................ 173 7.3.4.2 Identification of proteins from B. nivea ascospores................................ 174

7.4 Discussion ......................................................................................................... 179 7.4.1 Proteomes of Byssochlamys ascospores after heat treatments and aging ...... 179

7.4.1.1 Changed proteomic profiles of ascospores after heat treatments ........... 180 7.4.1.2 Changed proteomic profiles of ascospores during aging ....................... 181

7.4.2 Protein identification and functions in Byssochlamys ascospores ................. 182 7.4.2.1 Stress related proteins ............................................................................. 182 7.4.2.2 Other identified proteins and their potential functions in ascospores .... 184

7.4.3 Low percentage of significant matches and poor agreement between estimated MW and pI values .................................................................................................. 185 7.4.4 General assessment of the proteome of Byssochlamys ascospores............... 186 7.4.5 Recommendations for future research ........................................................... 187

7.5 Concluding remarks ....................................................................................... 188 Chapter 8 – General discussion and conclusions ........................................ 190

8.1 Introduction ..................................................................................................... 190 8.2 Age, dormancy and heat resistance of ascospores ................................... 191 8.3 Ultrastructure and heat resistance of ascospores .................................... 192 8.4 Proteins and heat resistance of ascospores ............................................... 194 8.5 Population heterogeneity and heat resistance of ascospores ................. 195 8.6 Recommendations for future research ...................................................... 197 8.7 Conclusions ...................................................................................................... 198

References ................................................................................................................. 200 Appendices ................................................................................................................ 240

v

List of Figures

Figure 3.1. Heat inactivation of B. fulva FRR 2299 ascospores from (A) 4 week, (B) 6

week, (C) 8 week, (D) 12 week and (E) 24 week cultures; inactivation temperatures are

85oC (■), 87.5oC (♦) and 90oC (▲) ................................................................................. 44



85oC (■), 87.5oC (♦) and 90oC (▲) ................................................................................. 45

Figure 3.3. Heat inactivation of B. nivea FRR 4421 ascospores from (A) 4 week, (B) 6


82.5oC (○), 85oC (□) and 87.5oC (◊) ............................................................................... 46



82.5oC (○), 85oC (□) and 87.5oC (◊) ............................................................................... 47

Figure 3.5. Average activation levels of B. fulva FRR 2299 (A), B. fulva FRR 2785 (B),

B. nivea FRR 4421 (C) and B. nivea FRR 6002 (D) ....................................................... 49

Figure 3.6. D values of ascospores of B. fulva FRR 2299 (A) and FRR 2785 (B), B.

nivea FRR 4421 (C) and FRR 6002 (D) at different culture ages and inactivation

temperatures; Temperature: 82.5oC (●), 85oC (♦), 87.5oC (■) and 90oC (▲) ................ 53

Figure 3.7. B. fulva FRR 2299: representative survivor curves of subsequent

generations of 4 week ascospore cultures derived from the ‘tailing’ phase of the parent

and three sequential generations ..................................................................................... 55

Figure 3.8. B. nivea FRR 6002: representative survivor curves of subsequent

generations of 4 week old ascospore cultures derived from the ‘tailing’ phase of the

parent and three sequential generations .......................................................................... 56

Figure 3.9. Comparing D values of 4 week ascospores derived from the parent and

three sequential “tailing” generations of B. fulva FRR 2299 and B. nivea FRR 6002.... 57

Figure 4.1. Scanning electron micrographs of B. fulva FRR 2299 dormant ascospores

and ascus at 4 (A, C) and 24 weeks (B, D) ..................................................................... 70

Figure 4.2. Scanning electron micrographs of B. nivea FRR 4421 dormant ascospores at

4 (A, C) and 24 weeks (B, D).......................................................................................... 71

vi

Figure 4.3. Scanning electron micrographs of 24 week old B. fulva FRR 2299 (A, C, E)

and B. nivea FRR 4421 (B, D) ascospores activated at 75oC for 30 minutes ................. 72

Figure 4.4. Scanning micrographs of 24 week old B. fulva FRR 2299 (A, C, E) and B.

nivea FRR 4421 (B, D) ascospores inactivated at 95oC for 30 minutes ......................... 73

Figure 4.5. Distributions of size measurements of dormant and activated ascospores of

B. fulva FRR 2299 and B. nivea FRR 4421 .................................................................... 76

Figure 4.6. Ultrastructures of ascospores of B. fulva FRR 2299. Sample: 4 week,

dormant (A), 24 week, dormant (B), 24 week, activated (C – D), 24 week, inactivated

(E – F) ............................................................................................................................. 78

Figure 4.7. Ultrastructures of ascospores of B. nivea FRR 4421. Sample: 4 week,


(E – F) ............................................................................................................................. 79

Figure 4.8. Area-to-area ratios of different compartments of B. fulva FRR 2299

ascospores using the whole data set or the top 25% based on length and width ............ 81

Figure 4.9. Area ratios of different compartments of B. nivea FRR 4421 using the

whole data set or the top 25% based on length and width .............................................. 82

Figure 5.1. DSC thermograms of ascospores of B. fulva FRR 2299 scanned at four

heating rates: 2.5 Kmin-1 (A), 5 Kmin-1 (B), 7.5 Kmin-1 (C) and 10 Kmin-1 (D) ........... 94

Figure 5.2. DSC thermograms of ascospores of B. fulva FRR 2299 at 6 and 24 weeks.

Treatment: A – dormant, B – activated at 75oC for 30 minutes, C – inactivated at 95oC

for 30 minutes ................................................................................................................. 96



for 30 minutes ................................................................................................................. 97

Figure 5.4. DSC thermograms of ascospores of B. nivea FRR 4421 at 6 and 24 weeks.


for 30 minutes ................................................................................................................. 98



for 30 minutes ................................................................................................................. 99

vii

Figure 5.6. The cooling segments during DSC analysis of ascospores of B. fulva FRR

2299, B. fulva FRR 2785, B. nivea FRR 4421 and B. nivea FRR 6002 ........................ 102

Figure 5.7. Thermograms of Byssochlamys dormant ascospores prepared in crucibles

with punctured lids ........................................................................................................ 106

Figure 5.8. Thermograms of Byssochlamys dormant ascospores prepared in aqueous

forms without drying ..................................................................................................... 107

Figure 5.9. Thermograms of Byssochlamys dormant ascospores prepared by vacuum

drying ............................................................................................................................ 108

Figure 6.1. Data collecting area on forward scattering (FSC) versus side scattering

(SSC) dot plots for B. fulva FRR 2299 (A), B. fulva FRR 2785 (B), B. nivea FRR 4421

(C) and B. nivea FRR 6002 (D) .................................................................................... 121

Figure 6.2. Procedure to differentiate sub-populations of Byssochlamys ascospores .. 122

Figure 6.3. Penetration of SYTO 9 into ascospores in response to heat treatment at

90oC for B. fulva FRR 2299 (left) or 87.5oC B. nivea FRR 6002 (right) ...................... 124

Figure 6.4. Comparative staining of 4 week ascospores of B. fulva FRR 2299 with

different nucleic acid stains ........................................................................................... 127

Figure 6.5. Comparative staining of 4 week ascospores of B. nivea FRR 4421 with

different nucleic acid stains ........................................................................................... 128

Figure 6.6. Sub-populations of unstained ascospores of B. fulva FRR 2299 at different

ages and after a heat treatment at 90oC for up to 30 minutes ........................................ 130


ages and after a heat treatment at 90oC for up to 30 minutes ........................................ 131

Figure 6.8. Sub-populations of unstained ascospores of B. nivea FRR 4421 at different

ages and after a heat treatment at 87.5oC for up to 30 minutes ..................................... 133


ages and after a heat treatment at 87.5oC for up to 30 minutes ..................................... 134

Figure 6.10. Sub-populations of SYTO 9-stained ascospores of B. fulva FRR 2299 at

different ages and after a heat treatment at 90oC for up to 30 minutes ......................... 136

viii


different ages and after a heat treatment at 90oC for up to 30 minutes ......................... 137

Figure 6.12. Changed abundances of sub-populations of B. fulva ascospores stained by

SYTO 9 after a heat treatment at 90oC; ascospores are 4 weeks old ............................ 139

Figure 6.13. Sub-populations of SYTO 9-stained ascospores of B. nivea FRR 4421 at

different ages and after a heat treatment at 87.5oC for up to 30 minutes ...................... 141


different ages and after a heat treatment at 87.5oC for up to 30 minutes ...................... 142

Figure 6.15. Changed percentages of sub-populations of B. nivea ascospores stained by

SYTO 9 after heat treatment at 87.5oC. Ascospores are 4 weeks old ........................... 144

Figure 7.1. Treatment of Byssochlamys ascospores used for proteome analysis ......... 156

Figure 7.2. Proteome analysis of Byssochlamys ascospores ........................................ 157

Figure 7.3. (A) Positions of significantly changed protein spots of B. fulva FRR 2299 in

response to heat treatment at 90oC; (B) normalised volumes of protein spots changing

with heating time ........................................................................................................... 166

Figure 7.4. (A) Positions of significantly changed protein spots of B. nivea FRR 6002

in response to heat treatment at 87.5oC; (B) normalised volumes of protein spots

changing with heating time ........................................................................................... 168

Figure 7.5. Changed proteomic profiles of B. fulva FRR 2299 ascospores during aging.

(A) Significantly different proteins spots between 4 and 12 weeks; (B) Changed

abundance of selected protein spots at 4 and 12 weeks ................................................ 171

Figure 7.6. Changed proteomic profiles of B. nivea FRR 6002 ascospores during aging.

(A) Significantly different spots between 4 and 12 weeks; (B) Increased abundance of

selected protein spots at 4 and 12 weeks; (C) Decreased abundance of selected protein

spots at 4 and 12 weeks ................................................................................................. 172

Figure 8.1. Relationships between the heat resistance and intrinsic properties of

Byssochlamys ascospores investigated in this thesis ..................................................... 191

ix

List of Tables

Table 2.1. Taxonomy of the genus Byssochlamys and its Paecilomyces anamorphs ....... 9

Table 2.2. Distinctive morphological characteristics of Byssochlamys fulva and

Byssochlamys nivea ......................................................................................................... 10

Table 2.3. Physiological properties (except heat resistance) of Byssochlamys fulva and

Byssochlamys nivea ......................................................................................................... 12

Table 2.4. Major food spoilage cases involving Byssochlamys fulva and Byssochlamys

nivea ................................................................................................................................ 21

Table 2.5. Heat resistance of Byssochlamys fulva ascospores with respect to heating

regimens .......................................................................................................................... 24

Table 2.6. Heat resistance of Byssochlamys nivea ascospores with respect to heating

regimens .......................................................................................................................... 25

Table 2.7. D and z values of ascospores of the species Byssochlamys fulva .................. 26

Table 2.8. D and z values of ascospores of the species Byssochlamys nivea ................. 27

Table 3.1. Thermal reduction times (D) in minutes ± standard deviation (SD) of

Byssochlamys ascospores at different ages and inactivation temperatures; each value is

an average of three experiments ...................................................................................... 51

Table 4.1. Dimensions of Byssochlamys ascospores at different ages and physiological

states; measurements are expressed as average values (mean) or medians of the

distribution of the data sets ............................................................................................. 75

Table 5.1. DSC scanning programs for analysis of Byssochlamys ascospores .............. 92

Table 5.2. Peak temperatures and enthalpy at different scanning rates.......................... 95

Table 5.3. Temperatures of major transitions (peaks and step changes) from the DSC

analysis of dormant ascospores of Byssochlamys species ............................................ 101

Table 5.4. Peak and step change temperatures from the DSC analysis of Byssochlamys

ascospores activated at 75oC for 30 minutes ................................................................. 104

Table 5.5. Peak and step change temperatures from the DSC analysis of Byssochlamys

ascospores inactivated at 95oC for 30 minutes .............................................................. 105

x

Table 6.1. Predicted identities of sub-populations of unstained B. fulva ascospores ... 132

Table 6.2. Predicted identities of sub-populations of unstained B. nivea ascospores .. 135

Table 6.3. Predicted identities of sub-populations of SYTO 9-stained B. fulva

ascospores ..................................................................................................................... 138

Table 6.4. Predicted identities of sub-populations of SYTO 9-stained B. nivea

ascospores ..................................................................................................................... 143

Table 7.1. Reagents used in the proteomic analysis of Byssochlamys ascospores ....... 158

Table 7.2. Search parameters set in the database search program Mascot ................... 163

Table 7.3. Significantly changed protein spots (in terms of normalised spot volumes) of

Byssochlamys ascospores following a heat treatment ................................................... 165

Table 7.4. Significantly changed protein spots (in terms of normalised spot volumes)

between 4 and 12 week Byssochlamys ascospores ....................................................... 170

Table 7.5. Identification of selected proteins extracted from B. fulva FRR 2299

ascospores after heat treatments and at different age .................................................... 175

Table 7.6. Identification of selected proteins extracted from B. nivea FRR 6002

ascospores after heat treatments and at different ages .................................................. 177

xi

List of Appendices

Appendix 3.1. Screened heat resistance of the four Byssochlamys strains selected for

usage in this thesis ......................................................................................................... 240

Appendix 3.2. Proportions (in %) of the activated population at 1 minute out of the

initially added ascospore concentration (5 – 6 log10) .................................................. 241

Appendix 3.3. Average activation levels of the four B. fulva and B. nivea strains being

tested in this experiment; each experiment started with a population of 105 – 106

ascospores/mL ............................................................................................................... 242

Appendix 3.4. Representative ascospores suspensions after glass bead treatment of B.

fulva FRR 2299 (A) and B. nivea FRR 4421 (B) .......................................................... 243

Appendix 5.1. Thermograms of B. fulva FRR 2299 ascospores scanned after one week

conditioning .................................................................................................................. 244


conditioning .................................................................................................................. 245

Appendix 5.3. Thermograms of B. nivea FRR 4421 ascospores scanned after one week

conditioning .................................................................................................................. 246


conditioning .................................................................................................................. 247

Appendix 5.5. Excess water in dense ascospore solutions being removed by drying over

granulated silica gel in a desiccator .............................................................................. 248

Appendix 6.1. Comparative staining of 8 week ascospores of B. fulva FRR 2299 with

SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes .......................... 249

Appendix 6.1. (cont’d) Comparative staining of 12 week ascospores of B. fulva FRR

2299 with SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes ......... 250


SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes .......................... 251



xii



Appendix 6.3. Comparative staining of 8 week ascospores of B. nivea FRR 4421 with

SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes ....................... 254

Appendix 6.3. (cont’d) Comparative staining of 12 week ascospores of B. nivea FRR

4421 with SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes ...... 255


SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes ....................... 256





Appendix 6.5. Subpopulations of unstained 4 week ascospores of B. fulva FRR 2299

following a heat treatment at 90oC for up to 30 minutes .............................................. 259

Appendix 6.5. (cont’d) Subpopulations of 8 week unstained ascospores of B. fulva

FRR 2299 ...................................................................................................................... 260


FRR 2299 ...................................................................................................................... 261

Appendix 6.6. Subpopulations of 4 week unstained ascospores of B. fulva FRR 2785

following a heat treatment at 90oC for up to 30 minutes .............................................. 262


FRR 2785 ...................................................................................................................... 263


FRR 2785 ...................................................................................................................... 264

Appendix 6.7. Subpopulations of 4 week unstained ascospores of B. nivea FRR 4421

following a heat treatment at 87.5oC for up to 30 minutes ........................................... 265

Appendix 6.7. (cont’d) Subpopulations of 8 week unstained ascospores of B. nivea

FRR 4421 ...................................................................................................................... 266

xiii


FRR 4421 ...................................................................................................................... 267


following a heat treatment at 87.5oC for up to 30 minutes ........................................... 268


FRR 6002 ...................................................................................................................... 269


FRR 6002 ...................................................................................................................... 270

Appendix 6.9. Subpopulations of SYTO 9-stained ascospores of B. fulva FRR 2299 at 4

weeks old following a heat treatment at 90oC for up to 30 minutes ............................ 271

Appendix 6.9. (cont’d) Subpopulations of SYTO 9-stained ascospores of B. fulva FRR

2299 at 8 weeks old ....................................................................................................... 272


2299 at 12 weeks old ..................................................................................................... 273

Appendix 6.10. Subpopulations of SYTO 9-stained ascospores of B. fulva FRR 2785 at

4 weeks old following a heat treatment at 90oC for up to 30 minutes ......................... 274

Appendix 6.10. (cont’d) Subpopulations of SYTO 9-stained ascospores of B. fulva

FRR 2785 at 8 weeks old .............................................................................................. 275

Appendix 6.10. (cont’d) Subpopulations of SYTO 9-stained ascospores of B. fulva

FRR 2785 at 12 weeks old ............................................................................................ 276

Appendix 6.11. Changed percentages of ascospore subpopulations of B. fulva FRR

2299 after a heat treatment at 90oC ............................................................................... 277


2785 after a heat treatment at 90oC ............................................................................... 278

Appendix 6.13. Subpopulations of SYTO 9-stained ascospores of B. nivea FRR 4421 at

4 weeks old following a heat treatment at 87.5oC for up to 30 minutes ...................... 279

Appendix 6.13. (cont’d) Subpopulations of SYTO 9-stained ascospores of B. nivea

FRR 4421 at 8 weeks old .............................................................................................. 280

xiv


FRR 4421 at 12 weeks old ............................................................................................ 281


4 weeks old following a heat treatment at 87.5oC for up to 30 minutes ...................... 282


FRR 6002 at 8 weeks old .............................................................................................. 283


FRR 6002 at 12 weeks old ............................................................................................ 284

Appendix 6.15. Changed percentages of ascospore subpopulations of B. nivea FRR

4421 after a heat treatment at 87.5oC ............................................................................ 285


6002 after a heat treatment at 87.5oC ............................................................................ 286

Appendix 7.1. Standard curve of the Bradford assay (A); protein concentrations and

conductivity measurements of triplicate protein extracts (S1 – S3) from Byssochlamys

ascospores (B) ............................................................................................................... 287

Appendix 7.2. Molecular weight (A, C) and isoelectric point (B, D) distribution of

protein resolved on 2D-gels of B. fulva FRR 2299 (top) and B. nivea FRR 4421

(bottom) ascospores ...................................................................................................... 288

Appendix 7.3. Differentially expressed proteins of B. fulva FRR 2299 ascospores

during a heat treatment at 90oC ..................................................................................... 289

Appendix 7.4. Differentially expressed proteins of B. nivea FRR 6002 ascospores

during a heat treatment at 87.5oC .................................................................................. 290

Appendix 7.5. Differentially expressed proteins of B. fulva FRR 2299 ascospores at 4

and 12 weeks ................................................................................................................. 291

Appendix 7.6. Protein spots of B. nivea FRR 6002 on 2D gels that significantly

changed with ascospore ages ........................................................................................ 292

Appendix 7.7. Amino acid sequences of the matched proteins of Byssochlamys

ascospores; matched peptides between the searched proteins and the theoretical proteins

are in red ........................................................................................................................ 293

1

Abstract

Byssochlamys is a fungus that causes spoilage of heat processed fruit products by

producing heat resistant ascospores. This thesis investigated heat resistant properties of

ascospores of Byssochlamys fulva and Byssochlamys nivea. Heat inactivation of the

ascospores at temperatures of 82.5 – 90oC showed activation, shoulder, exponential

reduction and tailing phases. The D values of the ascospores increased significantly

with culture age over the period of 4 – 24 weeks, and the average increase was 3.5 – 7-

fold for B. fulva strains and 2 – 4-fold for B. nivea strains. Ascospores of B. fulva were

more heat resistant than those of B. nivea.

Ultrastructure of the ascospores examined by electron microscopy comprised a cell

wall, a thick intermediate space (IMS) and a dense cytoplasm. Examining the

arrangement of materials in the ascospores with differential scanning calorimetry

provided evidence of a glassy state. Aging improved the robustness of the ascospore

structure whereas heating reduced its integrity. Older ascospores occasionally had more

sub-layers in the IMS. Ascospores of B. fulva had a more resilient structure and more

elaborated outer coating than those of B. nivea.

Flow cytometry revealed heterogeneity in preparations of dormant and heat treated

ascospores at different ages. Heating affected the integrity of the ascospore wall, which

was manifested as increased autofluorescence, permeability to SYTO 9 and clumping of

the ascospores. Sub-populations of activated and sub-lethally injured ascospores mostly

appeared after heat treatments.

Proteomic analysis of ascospores by 2D gel electrophoresis showed that less than 4% of

their total proteins significantly changed their expression after heating and aging. Most

of the changed proteins were metabolic enzymes, which possibly had been pre-

synthesised and stored in dormant ascospores. For B. nivea, three proteins were

identified as heat shock proteins 60, 70 and general stress protein 39. Latent protein

synthesis, denaturation and defragmentation of proteins into peptides possibly caused

changes in the protein expressions.

The results showed that population, structural and molecular factors contribute

simultaneously and synergistically to the heat resistance of Byssochlamys ascospores.

Data obtained provide a platform for further studies to understand the biological

2

mechanisms of heat resistance of their ascospores and for the food industry to develop

more effective heat processes to inactivate these fungi.

3

Chapter 1 – Introduction

Since the studies of Nicolas Appert and Louis Pasteur in the 19th century thermal

processes such as pasteurisation have been widely used by food manufacturers to extend

the shelf life and to ensure the microbial safety of foods (Adams and Moss, 2008; Silva

and Gibbs, 2004). However, some microorganisms are able to survive the thermal

processes commonly employed by the food industry, thereby compromising the

intended goals of food stability and food safety. Such organisms are collectively

described as heat resistant microorganisms (Mouchacca, 2007). Sporulation is a

common strategy used by these microorganisms to resist environmental stresses,

including heating. The best known example is the production of heat resistant

endospores by species of bacteria within the genera Bacillus and Clostridium (Atrih and

Foster, 2002; Gerhardt and Marquis, 1989; Scheldeman et al., 2006). Fungi, including

the yeasts and the filamentous fungi, are not generally recognised as heat resistant

microorganisms although they can produce some forms of spores (Adamek and Rönner,

1996; Hocking and Jensen, 2001; Tribst et al., 2009). Some fungal species produce

ascospores as part of their sexual cycle of reproduction. Although the ascospores of

these species do not have the same heat resistance as bacterial endospores, they do,

nevertheless, have considerably greater heat resistance than vegetative cells (Hocking

and Pitt, 2001) and this property needs considerations when designing heat processes

for certain foods and beverages. The heat resistance of fungal ascospores varies with the

species, but ascospores of species within the genera Byssochlamys, Talaromyces and

Neosartorya are most notable for their increased heat resistance (Dijksterhuis, 2007;

Tribst et al., 2009).

Species within the genus Byssochlamys Westling are considered as being thermo-

tolerant, and the two most important species with respect to food safety and quality are

Byssochlamys fulva and Byssochlamys nivea (Hocking and Pitt, 2001; Tournas, 1994).

Similar to other heat tolerant fungi, the heat resistance of Byssochlamys species is

attributed to the production of ascospores, which are able to survive heat treatments at

85 – 90oC for several minutes (Hocking and Jensen, 2001; Houbraken et al., 2006).

More importantly, sub-lethal temperatures in the range 60 – 80oC can activate the

ascospores and trigger their germination mechanism (Beuchat, 1992; Kikoku et al.,

2009; Splittstoesser et al., 1972). The ability of Byssochlamys ascospores to survive

4

temperatures of 60 – 90oC undermines the efficacy of thermal food processes that

operate within this range. Consequently, species within the genus Byssochlamys have

been frequently associated with the spoilage of heat processed fruit-based products such

as fruit juices and canned fruits (Beuchat, 1998; Splittstoesser, 1978; Tribst et al., 2009).

In addition, both B. fulva and B. nivea produce a range of mycotoxins, especially patulin

which may be deleterious to human health (Puel et al., 2010; Sant’Ana et al., 2010).

Thus, the heat resistant ascospores of B. fulva and B. nivea may cause significant

economic loss to the food industry and pose a potential risk to public health.

Because of their commercial significance, various studies have been conducted over the

years to understand the ecology, taxonomy, physiology and biochemistry of

Byssochlamys species, and these properties have been discussed in reviews by Stolk and

Samson (1971), Beuchat and Rice (1979), Pitt and Hocking (2009) and Samson et al.

(2009). Notably, B. fulva and B. nivea are able to grow at low water activity (0.892 –

0.993), acidic conditions (pH 3.0 – 4.5), with a great tolerance to low oxygen and high

carbon dioxide contents (Beuchat and Rice, 1979; Panagou et al., 2010; Roland and

Beuchat, 1984b). Production and germination of ascospores can also occur in these

unfavourable conditions (Splittstoesser et al., 1969, 1972). The heat resistant properties

of these ascospores have been described by various workers (Bayne and Michener,

1979; Casella et al., 1990; Houbraken et al., 2006; King et al., 1979). These two species

also produce strong pecteolytic enzymes that can disintegrate the tissues of fruits (Rice

and Beuchat, 1978a, b; Rice et al., 1977a; Ugwuanyi and Obeta, 1999). Collectively,

these properties explain why B. fulva and B. nivea are major causes of spoilage of heat

processed fruit products.

Many factors can affect the heat resistance of Byssochlamys ascospores and,

consequently, a wide variation in their heat resistant properties has been reported in the

literature (Bayne and Michener, 1979; Casella et al., 1990; Dijksterhuis, 2007;

Splittstoesser, 1978). Ascospores from different species and strains show significant

difference in their response to time/temperature combinations. High sugar, high salt and

high solids content in foods can increase the heat resistance of the ascospores whereas

the presence of food preservatives make them more susceptible to heat (Beuchat, 1981;

Engel and Teuber, 1991; Hatcher et al., 1979). Acidity can either increase or decrease

their heat resistance, depending on both the concentration of hydrogen ions (pH) and

types of acids present in the heating environment (Splittstoesser, 1978; Splittstoesser et

5

al., 1974b). Notably, the age of the cultures from which the ascospores are harvested, or

ascospore age, can affect significantly their tolerance to heat. A general trend is that the

heat resistance increases with the ascospore age. For example, Casella et al. (1990)

showed that thermal reduction times (D values) at 80 – 90oC of B. nivea ascospores

generally increased with ascospore age between 4 and 16 weeks. Prior to the study of

Casella et al. (1990), Yates and Ferguson (1963) also observed that ascospores from 14

day cultures of B. nivea could tolerate heat treatments better than those from 4 week

cultures.

Although some progress has been made in describing the heat resistant properties of

Byssochlamys species, there is limited knowledge about the mechanisms that underpin

this important characteristic. Additionally, compared with other equally important heat

resistant moulds belonging to the genera Talaromyces and Neosartorya (Dijksterhuis et

al., 2007; Fedorova et al., 2007, 2008), the ultrastructure, chemical composition and

genomics of Byssochlamys species are poorly understood.

In recent times, consumer preference is moving towards more natural, preservative-free,

minimally processed foods and beverages (Sloan, 2010, 2011; Zink, 1997). The new

trend limits the flexibility of food processors in controlling heat resistant

microorganisms and their spores by traditional methods since excessive heating and

food preservatives are no longer desirable. In this context, it is important to have a

better understanding of the inherent mechanisms of heat resistance of Byssochlamys

ascospores, since such knowledge may enable food processors to optimise their

processing systems to produce foods with enhanced quality and safety.

This project aims to investigate the contributions of some intrinsic factors to the heat

resistance of Byssochlamys ascospores. Within this scope, the objectives of the studies

reported in this thesis are:

i. To examine the thermal inactivation of Byssochlamys ascospores with respect to

ascospore age at 4, 6, 8, 12 and 24 weeks and several inactivation temperatures

(Chapter 3)

ii. To compare the ultrastructure of Byssochlamys ascospores at their dormant

stage, and at different ages after heat activation and heat inactivation (Chapter 4)

iii. To apply differential scanning calorimetry to examine the arrangements of

intracellular materials and possible glassy state phases in Byssochlamys

6

ascospores at their dormant stage, and at different ages after heat activation and

heat inactivation (Chapter 5)

iv. To characterise the heterogeneity within the populations of Byssochlamys

ascospores at different ages and after heat treatment, using flow cytometry

(Chapter 6)

v. To examine the proteomic profiles of Byssochlamys ascospores at different ages

and after heat treatment, using two dimensional gel electrophoresis and mass

spectrometry (Chapter 7)

The two species, B. fulva and B. nivea, selected for these studies represent strains that

have been implicated in the spoilage of fruit juices and dairy products in Australia, New

Zealand and Brazil.

The outcomes of this project will:

i. Advance knowledge about the heat resistant properties of Byssochlamys

ascospores, and provide a platform for more detailed studies of their heat

resistance mechanisms

ii. Assist food manufacturers to design more effective thermal processes to control

food spoilage by Byssochlamys species

7

Chapter 2 – Literature review

Byssochlamys species are filamentous fungi that can cause spoilage of heat processed

foods due to production of heat resistant ascospores. Their ascospores and mycelial

outgrowth are often found in spoilt products of fruit juices and canned fruits. Research

so far has studied the physiology of the fungi and some external factors that affect the

heat resistance of their ascospores. However, the mechanisms of heat resistance are not

well understood and requirements for such knowledge are coming from the scientific

community, the food industry and consumers. As a background to the objectives of this

thesis described in Chapter 1, this literature review will assess the currently available

knowledge about various aspects of Byssochlamys ascospores and their heat resistance,

focusing primarily on two species, Byssochlamys fulva and Byssochlamys nivea.

The survey will consider some general information about the taxonomy, biology,

physiology and ecology of the two species with regard to their roles in food spoilage.

This will be followed by a more detailed discussion of the ascospores of B. fulva and B.

nivea, the heat resistance of their ascospores and reported factors that contribute to their

thermo-tolerance. Although the material presented in this Chapter mainly concerns B.

fulva and B. nivea, knowledge of other species and microorganisms will be considered

where relevant.

2.1 Taxonomy

The genus Byssochlamys Westling is classified within the kingdom Eumycota (Fungi),

phylum Ascomycota, class Eurotiomycetes, order Eurotiales and family

Trichocomaceae. Being a member of the Ascomycota group, Byssochlamys species are

capable of sexually reproducing by the production of ascospores borne in a sac-like

structure, or ascus (plural: asci) (Malloch, 1981). Each ascus is produced by crozier

formation and bears eight ascospores (Sautter and Hock, 1982). However, unlike other

ascomycetes, Byssochlamys species are characterised by the lack of true peridial

structures, such as cleistothecia or gymnothecia, covering the asci. Their asci are formed

in globoid clusters loosely entangled in webs of fine white hyphae (Pitt and Hocking,

2009; Stolk and Samson, 1971).

8

Byssochlamys nivea is the type species of the genus, first isolated and described by

Westling in 1909 (Brown and Smith, 1957). Byssochlamys fulva was later isolated from

canned fruits in England in the early 1930s. Many other species were then added to the

genus according to various morphological similarities with the type species (Cain, 1956;

Ram, 1968; von Arx, 1986). Some species of the genus Byssochlamys can reproduce

asexually by production of conidia and chlamydospores. Based on the morphological

similarities of the conidial stage, the genus Paecilomyces has been erected to

accommodate the anamorphs of Byssochlamys (Olliver and Smith, 1933; Samson and

Tansey, 1975).

The taxonomy of the genus Byssochlamys has been revised by many workers (Brown

and Smith, 1957; Samson and Tansey, 1975; Stolk and Samson, 1971). Houbraken et al.

(2006) reported at least nine taxa of Byssochlamys and its anamorphs by examining

their macro-morphological and micro-mophological characteristics. There were seven

taxa of Byssochlamys: B. fulva, B. nivea, B. verrucosa, B. zollerniae, B. spectabilis, B.

divaricata and B. lagunculariae, and two of Paecilomyces: P. dactylethromoplus and P.

maximus (Houbraken et al., 2006).

Recent advances in molecular biology have been applied to the taxonomy of the genus

Byssochlamys and its Paecilomyces anamorphs. Luangsa-ard et al. (2004a) examined

the phylogenetic relationships in Paecilomyces and showed the relatedness between

Paecilomyces variotii and B. fulva and B. zollerniae. Houbraken et al. (2008) later

showed that the anamorphic stage of B. spectabilis, formerly Talaromyces spectabilis,

was also P. variotii by analysing encoding genes of internal transcribed spacers, 5.8S

rRNA, β-tubulin, actin and calmodulin.

Samson et al. (2009) combined morphological characterisation with molecular and

extrolite analysis to revise the taxonomy of Byssochlamys and its anamorphs. This

polyphasic approach has distinguished nine species of the genus Byssochlamys: five of

which reproduce sexually (teleomorph) and four are asexual. The five teleomorphs

accepted are B. fulva, B. lagunculariae, B. nivea, B. spectabilis and B. zollerniae. The

four anamorphs accepted are P. brunneolus, P. divaricatus, P. formosus and P.

saturatus. Table 2.1 summarises the most recently revised taxonomies of the genera

Byssochlamys and Paecilomyces (Houbraken et al., 2006; Samson et al., 2009).

9

Table 2.1. Taxonomy of the genus Byssochlamys and its Paecilomyces anamorphs

Species accepted in Houbraken

et al. (2006)

Species accepted in Samson

et al. (2009)

Teleomorph

Byssochlamys

B. divaricata, B. fulva, B.

lagunculariae, B. nivea, B.

verrucosa, B. spectabilis and B.

zollerniae

B. fulva, B. lagunculariae, B.

nivea, B. spectabilis and B.

zollerniae

Anamorphs

Paecilomyces

P. dactylethromoplus and P.

maximus

P. brunneolus, P. divaricatus,

P. formosus and P. saturatus.

2.2 Morphology

Morphologies of B. fulva and B. nivea and their Paecilomyces anamorphs have been

described by Olliver and Smith (1933) and Olliver and Rendle (1934) and later

reviewed by other researchers (Beuchat and Rice, 1979; Brown and Smith, 1957; Pitt

and Hocking, 2009; Stolk and Samson, 1971). Some morphological features of the two

species are described in the following sections and the differences are highlighted in

Table 2.2.

Both B. fulva and B. nivea grow well on Malt extract agar (MEA) and Czapek yeast

extract agar (CYA), which are frequently used in the characterisation of these species.

Their colonies can cover the Petri dish after 7 days, especially at 30oC. The mycelia of

Byssochlamys are usually sparse, low and produce colourless exudates on the surface

when growing for more than 6 weeks. There are clear differences between B. fulva and

B. nivea with respect to colours of the colonies (Table 2.2).

B. fulva and B. nivea form abundant conidia, asci and ascospores after culture for one

week. When observed under the microscope, asci are seen as single, naked spherical

clusters, each of which contains 8 ascospores, which are hyaline, smooth walled and

usually entangled in webs of hyphae. Conidia are formed on phialides, and are smooth

walled and mostly ellipsoidal with flat ends. The ascospores are refractile and brighter

than the conidia. Mature cultures of B. nivea release free ascospores from asci more

frequently than those of B. fulva. Compared with B. fulva, B. nivea also produces fewer

10

conidia than B. fulva, but it does produce chlamydospores. Ascospores and conidia of B.

fulva are generally larger than those of B. nivea (Table 2.2).

Table 2.2. Distinctive morphological characteristics of Byssochlamys fulva and

Byssochlamys nivea

Feature B. fulva B. nivea

Growth on agar Growing well on MEA and

CYA at 25oC and 30oC

Growing better on MEA than

CYA at 25oC, and similarly at

30oC

Conidial colours Buff to brown; reverse is pale

brown to yellow shades

White to slightly grey; reverse

is pale to mid-brown on CYA

and pale to brownish on MEA

Asci 9 – 12 µm in diameter; rarely

release naturally free

ascospores

8 – 11 µm in diameter; readily

release free ascospores

Ascospores 5 – 7 µm long 4 – 6 µm long

Asexual spores

(conidia,

chlamydospores)

Conidia: 7 – 10 µm long; more

in quantity

Conidia: 3 – 6 µm long; more

diverse in types

Chlamydospores: 10 µm in

diameter

Sources: Beuchat and Rice (1979); Pitt and Hocking (2009); Stolk and Samson (1971)

2.3 Physiology

The physiological characteristics of B. fulva and B. nivea underpin their ability to cause

food spoilage. Previous studies have identified several environmental factors affecting

the physiology of these fungi and their ascospores. The findings so far have shown that

B. fulva and B. nivea share a number of common physiological properties while each

species also possesses some distinctive features. The following section reviews major

physiological properties of B. fulva and B. nivea, including responses to temperature,

pH, gaseous atmosphere, water activity and some chemicals. Production of important

molecules, such as enzymes and toxins, by these two species is mentioned. Table 2.3

summarises the main physiological properties of the two species. The heat resistance of

11

ascospores of B. fulva and B. nivea is also dependent on a similar group of variables,

and this property is reviewed in detail in another section.

2.3.1 Temperature

B. fulva has been reported to produce mycelial growth within the temperature range 6.5

– 43oC (King et al., 1969), with the optimum growth occurring at about 30 – 37oC

(Stolk and Samson, 1971). The corresponding figures for B. nivea are 10 – 40oC with a

similar optimum temperature range for growth (Stolk and Samson, 1971). The growth

responses to temperatures may vary with media, solute and water activity but, generally,

for both species growth is significantly slowed at temperatures below 21oC (King et al.,

1969; Roland and Beuchat, 1984a; Roland et al., 1984a, b). Sub-zero temperatures

between -12oC and -7oC exhibited a fungistatic effect on B. fulva cultures but did not

kill the fungi (Beuchat and Rice, 1979). No growth of B. nivea mycelia was reported at

7 and -30oC (Beuchat and Toledo, 1977).

The growth temperatures of B. fulva and B. nivea presented above have been recently

confirmed by a study of Panagou et al. (2010) using the model of Baranyi and the

cardinal model with inflection. The authors estimated the optimal temperature for

growth is 32.1oC, which is similar for both species. The maximum and minimum

growth temperatures of B. fulva are 46.4oC and 9.1oC, respectively. B. nivea grows

within a slightly narrower temperature range than B. fulva, 43.2oC down to 10.5oC.

The optimal growth temperatures are also suitable for production of ascospores and

patulin. Cultures of B. fulva can produce ascospores after 4 – 7 days when grown at

30oC compared with 8 or 19 days at 25oC and 20oC, respectively (Hull, 1939;

Splittstoesser et al., 1969). Similarly, the 30 – 37oC range results in better production of

patulin and more heat resistant ascospores for B. nivea (Engel and Teuber, 1991; Roland

and Beuchat, 1984b) than lower temperatures. These temperatures also support a faster

recovery and growth of B. nivea cultures that have been heat treated compared with

incubation below 30oC (Casella et al., 1990).

12

Table 2.3. Physiological properties (except heat resistance) of Byssochlamys fulva and

Byssochlamys nivea

Feature B. fulva B. nivea Temperature Growth and sporulation: optimal 32oC,

max 46oC, min 9oC

Fungistatic: -12oC to -7oC

Growth and sporulation: optimal

32oC, max 43oC, min 10oC

Inhibitory: -30oC and 7oC

pH Growth: optimal 3.0 – 3.5, tolerant 2 –

9

Sporulation: optimal: 2 – 3

Activating factors: chloride and nitrate

ions

Growth: tolerant 3.5 – 6.0

Germination: tolerant 3.0 – 6.3,

optimal 4 – 4.5

Activating, germinating factors:

acetate ions

Gases Absolute requirement for O2, require

residual O2 (0.27%)

Tolerate high CO2 (20 – 90%) and

high N2 (99.9%) content

Require residual O2 (0.5%)

Tolerate high CO2 (5 – 90%) and high

N2 (30%) content

Growth inhibited by >70% CO2

Water activity Growth: optimal 0.985, max 0.993,

min 0.893, no growth < 0.88

Optimal temperatures support growth

at lower aw

Growth: optimal 0.984, max 0.992,

min 0.892, no growth < 0.88

Optimal temperatures support growth

at lower aw

SO2 Growth: tolerant: up to 50 ppm, or up

to 250 ppm with sugar

Growth: tolerant: 50 – 75 ppm

Patulin production: tolerant up to 25

ppm

Preservatives Potassium sorbate: up to 250 ppm

Sodium benzoate: up to 250 ppm

Potassium sorbate: up to 400 ppm

Sodium benzoate: up to 1000 ppm

Enzymes Different types of pectinase, protease

and lipase

Possibly pectinase, protease and

lipase

Mycotoxins Byssochlamic acid, byssotoxin A Byssochlamic acid, byssotoxin A,

mycophenolic acid, patulin

Sugar Tolerant up to 20%, inhibitory > 30%

Protect ascospores: 28 – 65%

Pressure Mycelia: killed at 200 – 300 MPa

Ascospores: at least 600 MPa

References: Beuchat and Rice (1979), Beuchat and Toledo (1977), Pitt and Hocking (2009), Splittstoesser

et al. (1974b)

13

2.3.2 Hydrogen ion concentration (pH) and types of acid

Several studies have examined the effects of pH on the physiology of B. fulva (Beuchat

ad Rice, 1979; Splittstoesser et al., 1969, 1970). The optimal pH for growth of B. fulva

is 3.0 – 3.5 while the upper and lower limits for growth are pH 2 and pH 9, respectively

(Beuchat and Rice, 1979; Hull, 1939; Olliver and Rendle, 1934; Tournas, 1994).

Sporulation of B. fulva also occurs in the pH range 2 – 9, with the optimal production of

ascospores in the pH range 2 – 4, depending on the medium used (Hebert and Larson,

1972; Splittstoesser et al., 1969). In addition, pH 2 – 4 is more effective in activating

ascospores than higher pH conditions (Splittstoesser et al., 1970). Splittstoesser et al.

(1972) also found good activation of the ascospores at more acidic condition, pH 1,

whereas pH 1.6 – 2.0 can be detrimental to this property.

There have been fewer studies on the effects of pH on the physiology of B. nivea. The

optimal pH for mycelial growth of B. nivea is unknown but the fungus can tolerate and

grow in conditions of pH 3.5 – 6.0 (Yates and Ferguson, 1963). Germination of the

ascospores occurs over a wide range of pH values, 3.0 – 6.3, and most effectively at pH

4.0 – 4.5 (Yates et al., 1968). Ascospores can be thermally activated at pH 1 – 7 but the

lower extreme exhibits the optimal activating effects (Splittstoesser et al., 1970).

Apart from hydrogen ion concentration (pH), the types of acid and specific anions used

to fortify media have varying effects on the physiology of both B. fulva and B. nivea. So

far, only a few studies have examined these factors and more research is needed to

confirm and expand the results available. Splittstoesser et al. (1969) compared the

production of B. fulva ascospores in media with pH amended by addition of either

NaOH, HCl or tartaric acid. The authors found no significant differences among the

three chemicals in supporting the ascospore generation. However, Splittstoesser et al.

(1972) showed that chloride and nitrate ions were better activating factors for B. fulva

ascospores than phosphate and sulphate. For ascospores of B. nivea, acetate ions were

better activating and germinating factors than phosphate, citrate ions and other metal

cations (Yate, 1973; Yates et al., 1968).

14

2.3.3 Gaseous atmosphere

B. fulva and B. nivea are micro-aerobic microorganisms that can withstand extremely

low oxygen tensions. The ability to grow and function on residual oxygen is quite

specific to these species, which is a selective advantage over other heat resistant moulds

in canned products. There are differences in reported levels of oxygen required by B.

fulva. Olliver and Rendle (1934) observed growth of the species under oxygen tension

as low as 0.068 MPa. Oxygen concentrations of 0.27% or a trace amount in an

atmosphere occupied by 99.999% nitrogen were sufficient to support the growth of B.

fulva (King et al., 1969). Mycelial growth stopped if oxygen was completely removed,

but it resumed in the presence of air, proving an absolute requirement of oxygen by B.

fulva (King et al., 1969).

B. nivea appears to be able to survive at much lower oxygen tension than B. fulva.

Growth was observed under a vacuum pressure of 0.013 MPa (Put and Kruiswijk,

1964). Oxygen concentrations of 1.4 – 5.1% in the headspace of sealed pouches

provided sufficient oxygen to support the viability of B. nivea inocula over 13 month

storage at 21oC. However, the protective effect was greatly reduced at 9.5 – 9.7%

oxygen (Roland et al., 1984b). This contradictory effect of oxygen on fungal growth

could be due to detrimental oxidative changes in ascospores.

Yates and Ferguson (1963) reported an increase of B. nivea biomass in virtually pure

CO2 and N2 atmospheres. However, the experimental procedure of this study may not

have created true anaerobic conditions and residual oxygen may still have been present

to support mycelial growth. More research is needed to establish whether B. nivea is

able to grow under facultative anaerobic conditions.

The growth and physiological activities of B. fulva and B. nivea are also dependent on

the concentrations of other gases such as CO2 or N2. B. fulva can tolerate CO2 at a

concentration as high as 20 – 90% in the presence of at least 0.5% oxygen (Pitt and

Hocking, 2009; Taniwaki et al., 2001). Similarly, B. nivea can survive in 5 – 90% of

CO2 or 30% N2 and 20% oxygen (Yates et al., 1967). However, CO2 concentrations

above 70% can have an inactivating effect on the germination of ascospores whereas N2

does not support fungal development (Yates et al., 1968). The current knowledge cannot

explain fully the responses of Byssochlamys species to different atmospheres. Thus,

15

future studies need to focus on investigating the cellular and biochemical activity of

these species under modified gaseous conditions.

2.3.4 Water activity (aw)

The mycelial growth of B. fulva can occur within the water activity range 0.893 – 0.993,

with optimum growth at about 0.985 (Panagou et al., 2010). For B. nivea, the

corresponding aw ranges for growth is 0.892 – 0.992 and the optimal value is about

0.984 (Panagou et al., 2010; Zimmermann et al., 2011). The growth responses to water

activity can vary with medium, solute and growth temperatures but for both species no

growth is generally detected below an aw of 0.84 (Orth, 1976; Roland and Beuchat,

1984a; Valík and Piecková, 2001).

Effects of aw on ascospore germination and patulin production have been reported

predominantly for B. nivea rather than B. fulva. Germination of B. nivea ascospores can

occur at water activity range 0.90 – 0.99, with longer lag time at lower aw values

(Zimmermann et al., 2011). The lower limit of water activity for the germination of B.

nivea ascospores was estimated to be 0.84 (Orth, 1976). Reduced water activity of 0.91

– 0.99 with sugar or sodium chloride could also assist the survival and germination of B.

nivea ascospores exposed to heat and cold conditions (Beuchat, 1981; Beuchat and

Toledo, 1977).

Patulin production of B. nivea occurred at water activity between 0.959 – 0.992, the

lower extreme of which was often associated with cultures grown at temperatures that

were closer to the optimal range 30 – 37oC (Roland and Beuchat, 1984b). The minimum

aw for patulin production at 21, 30 and 37oC were 0.978, 0.968 and 0.959, respectively.

For B. fulva, the water activity range for patulin production is not available. However,

the species has been reported to produce patulin in Czapek-Dox broth containing 0.5%

sucrose and in fruit juices with contents of soluble solids between 4.8 – 18.3% (Rice et

al., 1977b).

2.3.5 Important metabolites produced by Byssochlamys

The extracellular metabolites of Byssochlamys species comprise relatively diverse

chemicals and, generally, have two major impacts on food quality (Houbraken et al.,

16

2006; Samson et al., 2009). One major group of metabolites consists of various lytic

enzymes that can degrade food constituents leading to food spoilage. The second group

is mycotoxins which can affect food safety.

2.3.5.1 Metabolic enzymes

Byssochlamys species produce pectolytic enzymes which can degrade pectins in foods.

Olliver and Rendle (1934) were the first to report the secretion of pectin-degrading

enzymes by B. fulva. This property was confirmed in later studies where a diversity of

pectolytic enzymes was found, including protopectinase, polygalacturonase, pectin-

esterase and pectate lyase (Beaven and Brown, 1949; Chu and Chang, 1973;

Kotzekidou, 1991; Put and Kruiswijk; 1964; Reid 1950; Ugwuanyi and Obeta, 1999).

High biosynthesis and activity of polygalacturonase were required to maintain a good

fungal growth, especially in sugar-limited environments where this enzyme could

transform pectin into more utilizable forms for fungal metabolism (Rice and Beuchat,

1978a, b; Rice et al., 1977a).

Apart from pectinases, Byssochlamys species have been reported to produce other

classes of enzymes, including a rennin-like protease called byssochlamyopeptidase A

(Chu et al., 1973), lipase (Ku and Hang, 1994), α-amylase (Doyle et al., 1998) and

vanillyl-alcohol oxidase (Furukawa et al., 1999a, b). So far most of the studies on

enzyme secretion have been limited to B. fulva and research is needed to determine

whether such properties are also found in B. nivea.

2.3.5.2 Mycotoxins

B. fulva and B. nivea produce several mycotoxins in media and fruit juices, including

mycophenolic acid (Puel et al., 2005), patulin (Puel et al., 2007) and byssochlamic acid

(Samson et al., 2009). B. fulva has been also reported to produce byssotoxin A (Frisvad

and Thrane, 1995; Kramer et al., 1976) but its prevalence in food is poorly documented

due to uncharacterised chemical structure (Houbraken et al., 2006).

Among the compounds above, patulin is the most documented mycotoxin associated

with Byssochlamys species. Patulin is produced by B. nivea and less frequently by B.

fulva in fruit juices (Houbraken et al., 2006; Rice, 1980; Rice et al., 1977b). However,

17

patulin is unstable and, therefore, not detected consistently in dairy products (Taniwaki

et al., 2001). Toxicological studies have shown that patulin is mutagenic, neurotoxic,

genotoxic, immunotoxic and able to cause gastrointestinal effects in mice (Puel et al.,

2010). Because of its toxicity, the residual patulin concentration in foods for human

consumption is recommended to be below 50 ppm (Delage et al., 2003; Drusch and

Ragab, 2003).

The optimal condition for patulin production by B. nivea in apple juice was reported to

be 21oC and aw of 0.978 after 20 days of incubation. The toxin yield was lower when

the mould grew at higher temperatures and lower water activities (Roland and Beuchat,

1984a, b; Sant’Ana et al., 2010). Food preservatives such as SO2, sorbate and benzoate

also suppressed the synthesis of patulin (Roland et al., 1984a).

The genetic mechanisms of patulin production by Byssochlamys species have recently

been described. Two genes, 6msas and idh, coding for respective enzymes, 6-

methylsalicylic synthase and isopoxydon dehydrogenase, which are involved in patulin

biosynthesis have been identified in various B. nivea strains but not in all the tested B.

fulva strains (Dombrink-Kurtzman and Engberg, 2006). Such differences might explain

the frequent detection of patulin secretion from B. nivea but not from B. fulva (Puel et

al., 2007). However, more research is required to confirm the absence of these two

genes in B. fulva and whether it causes the inability to produce patulin.

2.3.6 Other environmental factors

In addition to the effects of temperature, water activity and gaseous atmosphere, other

food processing factors can affect the physiology of B. fulva and B. nivea. Some of the

most studied factors are sulphur dioxide (SO2), other chemical preservatives and sugar.

Sulphur dioxide has inhibitory effects on mycelial growth, ascospore germination and

patulin production in B. fulva and B. nivea. Inhibition by SO2 is higher in an acidic

environment (pH 3) where the molecule occurs mostly as the undissociated (H2SO3)

form (Gillespy, 1940, 1946). The minimal concentration of SO2 to stop growth of B.

fulva mycelium is about 50 ppm (King et al., 1969). For B. nivea, the corresponding

figure is 50 – 70 ppm (Roland and Beuchat, 1984b; Roland et al., 1984b). However, the

fungi can tolerate a much higher concentration of SO2, up to 450 ppm, in the presence

18

of 10% sugar (King et al., 1969). Germination of the ascospores is prevented at high

SO2 concentrations such as 300 ppm (Beuchat, 1976). The use of 90 ppm SO2 in

combination with thermal processes increases the efficacy of the treatments in

inactivating ascospores tenfold (King et al., 1969). A SO2 concentration of 25 ppm is

sufficient to stop patulin production (Roland et al., 1984a).

Other common food preservatives such as benzoate and sorbate compounds are also

inhibitory against Byssochlamys species but the effects varied with species. Potassium

sorbate and sodium benzoate stop growth of B. fulva at a similar concentration of 250

ppm (King et al., 1969), but 400 ppm and 1000 ppm, respectively are needed to prevent

growth of B. nivea (Beuchat, 1976; Roland and Beuchat, 1984b; Roland et al., 1984a).

The inhibition by these preservatives is enhanced when combined with heating

(Beuchat, 1981).

The two less common preservatives, dimethyldicarbonate and diethylpyrocarbonate,

have been shown to stop Byssochlamys cultures from growing at minimum

concentrations of 50 ppm and 600 ppm, respectively (Beuchat, 1976; van der Riet and

Pinches, 1991; van der Riet et al., 1989).

Sugar can affect the behaviour of B. fulva and B. nivea in different ways depending on

concentration. Up to 20% sucrose (w/v) is favourable for mycelial growth, sporulation

and germination while sugar contents above 30% hinder those activities (Hatcher et al.,

1979; Hull, 1939; Olliver and Rendle, 1934). High sugar contents from 28% to 65% can

protect their ascospores against environmental stresses including heating (Splittstoesser

and Splittstoesser, 1977; Splittstoesser et al., 1974b).

Effects of fungicidal chemicals other than food preservatives on Byssochlamys species

have been reported less frequently. Olliver and Rendle (1934) observed that 2.2 gL-1 of

ammonia or 0.5% (v/v) of acetyldehyde inhibited growth of B. fulva. Asci of B. fulva

had a remarkable resistance to various strengths of antiseptic solutions, such as

formaldehyde, Lysol, sodium hypochlorite and mercuric chloride (Hull, 1939).

High pressure processing can affect the physiology of B. fulva and B. nivea. Their

mycelia are inactivated by treatments at 200 – 300 MPa (Butz et al., 1996). Short

treatments, such as 10 seconds or 15 minutes depending on the applied pressure, at the

pressure range 400 – 600 MPa can activate the ascospores (Dijksterhuis and Teunissen,

2004; Ferreira et al., 2009). Longer treatments or pressures above 600 MPa have

19

detrimental effects on the viability of the ascospores (Chapman et al., 2007a; Hocking et

al., 2004; Maggi et al., 1994; Palou et al., 1998) probably because they are activated and

lose their resistance to the high pressure (Black et al., 2007).

2.4 Ecology and food spoilage caused by Byssochlamys species

2.4.1 Ecology of B. fulva and B. nivea

Many ecological surveys have been conducted to identify the occurrence of B. fulva and

B. nivea in the environment and how they might contaminate foods. B. fulva and B.

nivea are widely distributed in the soil, particularly in vineyards, orchards and fields

where fruit is grown (Jesenská et al., 1992, 1993; Luangsa-ard et al., 2004b; Piecková et

al., 1994; Splittstoesser et al., 1971; Ugwuanyi and Obeta, 1991; Wahid et al., 1997;

Yates, 1974). Although the moulds may not grow in the soil environment, their

presence may cause contamination of fruits and fruit products with Byssochlamys

ascospores. In fact, fruits that are frequently in contact with soil are more likely to carry

B. fulva and B. nivea ascospores into final products (Hocking and Pitt, 1984; Hull,

1939; Tournas, 1994). The contamination occurs when fruits are grown close to or

harvested from the ground, or when ascospores are carried in rain splash (Tribst et al.,

2009). Fruits most susceptible to Byssochlamys contamination are passionfruit,

strawberries, grapes, pineapples and mangoes (Cartwright and Hocking 1984; Hocking

and Pitt, 1984; Obeta and Ugwuyanyi, 1995; Splittstoesser et al., 1971; Ugwuanyi and

Obeta, 1991). Some fruits that are less commonly associated with Byssochlamys

spoilage are tomatoes, sugarcane, oranges and apples (Ayesha and Viswanath, 2006;

Kotzekidou, 1997; Kutama et al., 2010; Swanson et al., 1985).

Contamination with Byssochlamys ascospores may arise from contact with damaged

fruits and processing equipment which subsequently serves as a carrier of the moulds

across batches of fruits and products. Olliver and Rendle (1934) isolated B. fulva from

wooden trays, baskets and factory containers. Hull (1939) and King et al. (1969)

detected B. fulva and B. nivea in mummified plums, raspberry refuse, baskets for fruit

collection and grape-processing waste pomace. Wash water, soiled surfaces of storage

20

containers and hand-picking were also potential means of transferring the ascospores

(Splittstoesser et al., 1974a).

B. nivea, but not B. fulva, has been occasionally found in dairy products, such as UHT

milk, raw milk, pasteurised skim-milk, cream, ice-cream and cheese (Aydin et al., 2005;

Engel, 1991; Engel and Teuber, 1991), and unusual foods, such as cucumber brine

(Yates and Ferguson, 1963) and palm wine (Eziashi et al., 2010). The occurrence of B.

nivea in these products suggests a higher tolerance to hostile conditions than B. fulva.

However, the prevalence of B. nivea in dairy products requires more research.

2.4.2 Food spoilage caused by B. fulva and B. nivea

B. fulva and B. nivea are a common cause of spoilage of heat processed fruit juices,

canned fruits and fruit purees (Andersen and Thrane, 2006; Beuchat, 1998; Hocking and

Jensen, 2001; Splittstoesser, 1978; Tribst et al., 2009). Such occurrences can be related

to the presence of these organisms in soil and their production of heat resistant

ascospores, tolerance to acidic and low oxygen conditions, and the secretion of strong

pectinases (Table 2.3). Some of the major published food spoilage cases involving B.

fulva and B. nivea are summarised in Table 2.4.

The first report of processed fruit spoilage by a Byssochlamys species was in England in

the early 1930s and involved B. fulva in canned fruits (Olliver and Smith, 1933; Hirst

and McMaster, 1933; Hull, 1939). Since then B. fulva species have been isolated from

spoilt fruit products in many regions such as Australia (Budnik and Hocking, 1981;

Cartwright and Hocking, 1984; Pitt and Hocking, 2009; Richardson, 1965; Spurgin,

1964), Brazil (Salomão et al., 2008), Canada (Yates, 1974), Greece (Kotzekidou, 1997),

India (Ayesha and Viswanath, 2006) and Nigeria (Kutama et al., 2010; Obeta and

Ugwuanyi 1995).

The spoilage cases involving B. nivea species have been recorded in many regions

including Australia (Pitt and Hocking, 2009), Canada (Yates and Ferguson, 1963),

Germany (Eckardt and Ahrens, 1977a; Engel, 1991; Engel and Teuber, 1991), Greece

(Kotzekidou, 1997) and Holland (Put and Kruiswijk, 1964). The moulds have recently

been reported in fruit products in Turkey (Aydin et al., 2005), India (Ayesha and

Viswanath, 2006) and Nigeria (Eziashi et al., 2010; Kutama et al., 2010).

21

Table 2.4. Major food spoilage cases involving Byssochlamys fulva and Byssochlamys nivea

Species Products Region Year References

B. fulva Canned fruits (strawberries, plums, raspberries, gooseberries, loganberries)

England 1931 – 1934 Hirst and McMaster (1933), Olliver and Rendle (1934)

Canned strawberries Australia 1964 – 1965 Richardson (1965), Spurgin (1964)

Canned apple-grape drink, canned blackberries, cherry pie filling, fruit drink, fruit pudding, grape concentrate, fruits collected from orchards

USA 1967 – 1969 King et al. (1969), Maunder (1969), Splittstoesser et al. (1971)

Canned strawberries Canada 1974 Yates (1974)

Fruit based baby gel product (containing passionfruit), mixed fruit (orange, grape, passionfruit) juices, grape juice, fruit juice jelly

Australia 1980 – 2009 Budnik and Hocking (1981), Cartwright and Hocking (1984), Pitt and Hocking (2009)

Canned tomato paste Greece 1997 Kotzekidou (1997)

Under-processed sugarcane juice in tetrapaks India 2006 Ayesha and Viswanath (2006)

Apple concentrate Brazil 2008 Salomão et al. (2008)

Bottled raphia palm wine Nigeria 2010 Eziashi et al. (2010)

B. nivea Processed strawberries The Netherlands 1963 Put and Kruiswijk (1964)

Apple drink, canned blackberries USA 1965 – 1969 Maunder (1969)

Tinned strawberries Germany 1977 Eckardt and Ahrens (1977a)

Fresh cheese, whole milk, silage and raw milk Germany 1991 Engel and Teuber (1991)

Canned tomato paste Greece 1997 Kotzekidou (1997)

Cheese, fruit yoghurt, ice cream with fruits and fruit juices Turkey 2005 Aydin et al. (2005)

Under-processed sugarcane juice in tetrapaks India 2006 Ayesha and Viswanath (2006)

Bottled raphia palm wine Nigeria 2010 Eziashi et al. (2010)

22

Both species were found to cause spoilage of fruit products, especially grapes, in the

USA (Denny and Brown, 1969; Maunder, 1969; Splittstoesser et al., 1974a).

Interestingly, there was a geographical distinction between the prevalence of the two

species. B. fulva was common in samples of fruit, vegetation and soil obtained from

various orchards and vineyards on the east coast of the USA (Splittstoesser et al., 1971).

In contrast, B. nivea was predominant in samples of soil, grapes, grape-processing lines

and waste pomace in vineyards on the west coast (King et al., 1969). However, there

has been no research into the specific distribution of B. fulva and B. nivea in the USA.

2.5 Heat resistance of ascospores of Byssochlamys species

Ascospores of Byssochlamys species are highly heat resistant, rendering many

commercial thermal processes ineffective. Other structures of the moulds such as

hyphae, chlamydospores and conidia are heat sensitive and eliminated by heating at

65oC for 10 minutes (Houbraken et al. 2006; Splittstoesser et al., 1970, 1972). The Food

and Agricultural Organisation of the United Nations has recommended a heating

regimen of approximately 100oC for up to 60 seconds for fruit juices if Byssochlamys

species are likely to be present (Bates et al., 2001). However, fruit juices under those

treatments can develop thermally induced off-flavours and lose significant nutrient

value (Awuah et al., 2007; Henry and Heppell, 2002; Polydera et al., 2003). Thus, in

practice, milder processes such as 89 – 90oC for 12 – 14 seconds have been

preferentially used by the food industry (Cartwright and Hocking, 1984). Unfortunately,

ascospores of B. fulva and B. nivea cannot be effectively inactivated by those less severe

treatments.

Several studies have investigated the heat resistance of Byssochlamys ascospores using

different methods and conditions. In general, reports on the heat resistance can be

divided into two categories: resistance to various time – temperature combinations or

thermal reduction times (D) and temperature resistance coefficients (z).

23

2.5.1 Heat resistance of Byssochlamys ascospores in thermal processes

Responses of ascospores of B. fulva and B. nivea to various heating regimens are

summarised in Tables 2.5 and 2.6, respectively. The heat resistance of the ascospores is

based on their survival after various heat treatments as reported in the literature.

Ascospores of B. fulva are more heat resistant than those of B. nivea. Processes of at

least 80oC for 20 minutes are required to inactivate B. nivea ascospores (Engel and

Teuber, 1991; Sant’Ana et al., 2009) whereas minimal treatments at 86oC for 53

minutes are needed for effective inactivation of B. fulva (Michener and King, 1974).

Use of lower temperatures for longer time is not practical in modern food production

(Splittstoesser et al., 1971). Moreover, ascospores of the two species can be activated

and outgrow following treatments at 70 – 80oC for about 20 – 60 minutes (Splittstoesser

and Splittstoesser, 1977; Splittstoesser et al., 1970, 1972). Thus, application of a heat

shock at 70 – 80oC has been used in screening and enumeration of heat resistant moulds

in food products (Beuchat, 1992; Beuchat and Pitt, 2001).

The heat resistance data in Tables 2.5 and 2.6 show some inconsistency in responses of

the two species to heat treatments which can be attributed to strain variations, heating

and counting methods, and the food or medium matrix in which the organisms were

present (Dijksterhuis, 2007; Splittstoesser and Splittstoesser, 1977).

24

Table 2.5. Heat resistance of Byssochlamys fulva ascospores with respect to heating

regimens

Heating regimen# Heating medium Lethality* Reference

70 – 80oC, 20 – 60 min

Homogenates of food products

A Beuchat and Pitt (2001)

87 – 88oC, 30 min Canned/bottled fruits NL Olliver and Smith (1933)

89oC, 30 min Saline NL Olliver and Rendle (1934)

84 – 88oC, 30 min Fruit syrups NL “

100oC, 2 – 3.5 min Live steam L “

85oC, 20 min Tomato paste NL Kotzekidou (1997)

85oC, 30 min Citrate buffer (pH 3) L Gillespy (1938)

77oC, 10 min (twice) spaced by 46oC, 30 min

Citrate buffer (pH 3) L “

88oC, 40 min – 95oC, 1.5 min

Strawberry syrup L Richardson (1965)

87.8oC, 81.4 min – 92.2oC, 13.7 min

Reconstituted grape juice

L King et al. (1969)

86.7oC, 118 min – 92.2oC, 6.8 min

Grape juice concentrate

L “

Up to 80oC, 60 min Grape juice homogenates

NL Splittstoesser et al. (1971)

85oC, 60 – 180 min Fruit juices NL Splittstoesser et al. (1974b)

86oC, 53 min Grape juice L Michener and King (1974)

Heat at 95oC, hold at 93oC for 30 sec

Clarified apple juice NL (1.7) Sant’Ana et al. (2009)

Heat at 96oC, hold at 94oC for 30 sec

Clarified apple juice L (4.8) “

* denotes survivability of moulds after heat treatment: A – activation, L – lethal, no detectable colonies,

NL – non-lethal, detectable colonies; numbers in brackets are log reduction of survival counts

25

Table 2.6. Heat resistance of Byssochlamys nivea ascospores with respect to heating

regimens

Heating regimen Heating medium Lethality* Reference

70 – 80oC, 20 – 60 min

Homogenates of food products

A Beuchat and Pitt (2001)

75oC, 7 hr Grape juice L Beuchat and Toledo (1977)

77.5oC, 20 min Ringer's solution NL Engel and Teuber (1991)

85oC, 20 min Tomato paste NL Kotzekidou (1997)

88oC, 45 min 30% sucrose (pH 4) NL Yates and Ferguson (1963)

87.5oC, 10 min Heat processed strawberries

NL Put and Kruiswijk (1964)

90 – 92oC, 10 min Heat processed strawberries

L “

80.5oC, 20 min – 90oC, 20 sec

Ringer's solution L (6) Engel and Teuber (1991)

90oC, 20 sec – 92oC, 12 sec

UHT milk, ice cream L (6) “

* denotes survivability of moulds after heat treatment: A – activation, L – lethal, no detectable colonies,

NL – non-lethal, detectable colonies; numbers in brackets are log reduction of survival counts

2.5.2 D and z values of ascospores of B. fulva and B. nivea

D value is defined as the amount of time required to inactivate 90% of a microbial

population and z value is the temperature increment required to change D values by a

factor of 10 (Adam and Moss, 2008). Some D and z values for the ascospores of B.

fulva and B. nivea that have been reported in different heating media are presented in

Tables 2.7 and 2.8, respectively. In general, B. fulva ascospores have higher D and z

values, and, therefore, are more heat resistant than B. nivea. For ascospores of B. fulva,

the typical D90 value is 1 – 12 minutes (Bayne and Michener, 1979) and the z value is 6

– 7oC (King et al., 1969). For B. nivea, the working D85 value is 0.6 – 1.2 minutes and

the z value is 4.2 – 5.8oC (Quintavalla and Spotti, 1993). Ascospores of B. fulva and B.

nivea are comparable with those of other heat resistant moulds. Ascospores of

26

Neosartorya fischeri have reported D90 values of 4.4 – 6.6 minutes and those of

Talaromyces flavus have D90 values of 2 – 8 minutes (Dijksterhuis and Samson, 2006;

King and Halbrook, 1987; Kotzekidou, 1997). A recent study reported that ascospores

of the new B. spectabilis species have D85 values of 47 – 75 minutes, making this

species one of the most heat resistant moulds (Houbraken et al. 2006).

Table 2.7. D and z values of ascospores of the species Byssochlamys fulva

D value Heating medium References

D85 = 26.18 – 59.78 min Clarified apple juice Sant’Ana et al. (2009)

D86 = 13 – 14 min Grape juice Michener and King (1974)

D87.8 = 4.8 – 11.3 min Grape juice King et al. (1969)

D87.8 = 1.6 min Grape juice with 250 ppm SO2

“

D90 = 1 – 12 min Chemically defined medium, clarified apple juice

Bayne and Michener (1979), Sant’Ana et al. (2009)

D92 = 27 – 37.5 min Distilled water Casella et al. (1990)

D92 = 3.27 – 3.97 min Clarified apple juice Sant’Ana et al. (2009a)

D95 = 1.10 – 2.52 min Clarified apple juice “

z value (oC)

6 – 7 Grape juice King et al. (1969)

7.1 Clarified apple juice Sant’Ana et al. (2009)

10 – 13.9 Grape juice (12oBrix) Hatcher et al. (1979)

27

Table 2.8. D and z values of ascospores of the species Byssochlamys nivea

D value Heating medium Reference

D75 = 60 min Grape juice Beuchat and Toledo (1977)

D80 = 6.6 – 5.7 min Grape juice (17oBrix) Quintavalla and Spotti (1993)

D84 = 36 – 46 sec Ringer's solution, UHT

milk, ice cream

Engel and Teuber (1991)

D85 = 0.6 – 1.2 min Grape juice (17oBrix) Quintavalla and Spotti (1993)

D86 = 12 – 18 sec Ringer's solution, UHT

milk, ice cream


D88 = 33.8 – 49.5 sec

Sucrose or salt solutions

with potassium sorbate

and sodium benzoate

Beuchat (1981), Casella et al.

(1990)

D90 = 2.7 – 4 sec Ringer's solution, UHT

milk, ice cream


D92 = 1.4 – 1.9 sec Ringer's solution, UHT

milk, ice cream “

z value (oC)

4 – 6.1 Potato dextrose agar, MEA Casella et al. (1990)

4.2 – 5.8 Grape juice (17oBrix) Quintavalla and Spotti (1993)

6 – 7 Cream (10% w/v fat

content)


6 – 8 Ringer's solution “

Some studies have reported difficulties in calculating the D values of ascospores of B.

fulva and B. nivea because of the complexity of their death kinetics. Particularly, the

thermal inactivation curves of the ascospores of B. fulva and B. nivea do not always

follow the first order semi-logarithmic pattern. The curves are characterised by a

shoulder phase prior to the accelerating inactivation (King et al., 1979) followed by a

tailing phase towards the end of the treatments (Bayne and Michener, 1979). These

phases are often excluded from the calculation of D values, thereby underestimating the

heat resistance of the ascospores.

28

The tailing phenomenon can be attributed to the presence a subpopulation of more heat

resistant ascospores (Bayne and Michener, 1979; Splittstoesser et al., 1989). Heat shock

proteins may induce a greater heat resistance in the ascospores, thereby causing the

tailing (Casella et al., 1990). Ascospores may escape the heat inactivation by adhering

to the inner wall of the heating test tubes, thereby reducing the heat transfer and

inactivation energy. This strategy has been observed in the thermal treatments of

conidia of Aspergillus niger (Fujikawa and Itoh, 1996; Fujikawa et al., 2000). Tailing

often occurs with a small group of surviving organisms whose population may be at the

lower detection limit of the plate count methods. The high variability in counting results

around this marginal level may cause tailing in the survival curves (Cerf, 1977).

There are contradictory explanations about the shoulder phase. Splittstoesser and

Splittstoesser (1977) associated the shoulder with neither activation nor clumping of B.

fulva ascospores. Casella et al. (1990) suggested that the shoulder phase resulted from a

small group of genetically resistant ascospores and heat shock proteins. Clumping

during heat treatments can increase the heat resistance of bacterial spores, causing both

tailing and shoulder (Cerf, 1977; Furukawa et al., 2005). Asci comprising similarly

resistant ascospores can be more heat resistant due to non-uniform heat penetration into

each cell (Hatcher et al., 1979). More research is needed to systematically examine the

shoulder and tailing phases, and identify their causes. It is particularly important to

know if they are related to genetically resistant ascospores or normal distribution of heat

resistance or uncharacterised mechanisms.

Models of the inactivation kinetics have been applied to ascospores of B. fulva and B.

nivea in order to account for the shoulder and tailing phenomena. King et al. (1979)

modified a model that had been developed for bacterial spores (Alderton and Snell,

1970) to determine non-logarithmic (only shoulder) death rate of B. fulva ascospores.

This approach linearised the thermal death curves by plotting survival counts and time

on logarithmic scales so that parameters analogous to D and z values could be calculated

(Bayne and Michener, 1979). Recently, a model based on the Weibull distribution

frequency has been adopted from bacterial spores to determine thermal destruction

kinetics of B. fulva ascospores in fruit juices (Sant’Ana et al., 2009). This model

incorporates the shape of the survival curves in the calculation of decimal reduction

ratio via shape parameters (Mafart et al., 2002). This approach is able to fit downward

concavity (shoulder) or upward concavity survival (biphasic, tailing) curves. Therefore,

29

more research is still required to understand the complex inactivation kinetics of

Byssochlamys ascospores so that new models customised for dealing with shoulder and

tailing phases can be developed.

2.6 Factors contributing to the heat resistance of Byssochlamys ascospores

Despite the importance of the heat resistance property in contributing to food spoilage,

little is known about the mechanisms behind the survival of Byssochlamys ascospores

during heat treatments. Previous studies on heat resistant microorganisms suggest that

thermotolerance is a function of many factors rather than a single element (Atrih and

Foster, 2001). Many factors contributing to the heat resistance of Byssochlamys

ascospores have been reported. So far, most of the well-defined factors, such as

compositions of foods and heating media, are extrinsic to the ascospores (Beuchat and

Rice, 1979). However, knowledge about intrinsic factors involved in heat resistance is

sparse and still not fully understood. Some intrinsic factors that are reviewed here are

the age, ultrastructure, intracellular configuration and genetics of the ascospores.

2.6.1 Effects of food/medium composition on heat resistance of Byssochlamys

ascospores

The heat resistance of Byssochlamys ascospores varies according to the composition of

the matrix or medium in which they were suspended (Table 2.5 – 2.6). Consequently,

food composition affects the responses of the ascospores to heat treatments.

Sugars, predominantly sucrose and natural sugars in fruit juices, help with the activation

and protect ascospores against thermal inactivation. The protective effects of sugar

depend on factors such as its concentration, soluble solids in fruit juices and pH. Media

with sucrose concentrations above 5% generally activate ascospores of B. fulva more

quickly and effectively than water (Splittstoesser and Splittstoesser, 1977). Sugar

contents up to 20% can increase the heat resistance of ascospores of B. fulva and B.

nivea (Beuchat, 1981; Eckardt and Ahrens, 1977b; King et al., 1969). Similar protection

for the ascospores occurs in fruit juices with high concentrations of soluble solids

including sugar, for instance 5 – 52oBrix, although the effectiveness is reduced in acidic

30

condition (Splittstoesser et al., 1974b). In addition, high sugar concentrations such as

60% do not support the germination and outgrowth of the ascospores (Beuchat and

Toledo, 1977). The protective effects of sugars on the heat resistance of ascospores have

also been reported for Saccharomyces cerevisiae (Splittstoesser et al., 1986),

Neosartorya fischeri (Rajashehara et al., 1996), Talaromyces flavus (Beuchat, 1988;

Scott and Bernard, 1987) and Eurotium herbariorum (Splittstoesser et al., 1989).

Sodium chloride (salt) also protects Byssochlamys ascospores against heat treatments

over a concentration range 3 – 12% (Beuchat, 1981). The adaptive resistance with

increased salt concentration is common for bacteria and their spores, and has been

related to the reduced water activity (den Besten et al., 2010; Jagannath et al., 2005).

The protection of both salt and sugar may involve reduced water activity, dehydration

and immobilisation of intracellular molecules but further studies are needed to confirm

such mechanisms (Beuchat, 1981).

Acidity of the heating media generally increases the heat sensitivity of microorganisms

(Hocking and Jensen, 2001). B. fulva ascospores survived heat treatment best at pH 5

whereas lower pH (3.6) or higher pH (6.6 – 6.8) resulted in much greater inactivation

(Bayne and Michener, 1979). However, the heat sensitivity of the ascospores was

greatly influenced by the types of acids present. Solutions of 0.05M citric, malic and

tartaric acids exhibited protective effects on B. fulva ascospores whereas 0.05M

fumaric, lactic, succinic and acetic acids reduced significantly the survival ability

(Splittstoesser and Splittstoesser, 1977). The group of protective acids also induced a

clear shoulder phase on the survival curves of the ascospores (Splittstoesser et al.,

1974b). Moreover, the D90 values of B. fulva ascospores in fruit juices were reduced by

half to six times when the products contained 0.1 – 0.5% of fumaric or lactic acids and

had pH 3.0 – 3.5 (Splittstoesser et al., 1974b). The antimicrobial effects of organic acids

depend on the proportion of undissociated acid molecules which is higher at lower pH

values. The inactivating effects of acetic, fumaric, lactic and succinic acids may be due

to their lower level of dissociation, which is indicated by higher acid dissociation

constant pKa, than that of citric, malic and tartaric acids. The pKa values of citric, malic

and tartaric acids are 3.09, 3.4 and 3.22, respectively, whereas the corresponding figures

of acetic, fumaric, lactic and succinic acids are 4.76, 4.54, 3.86 and 4.19 (Reich, 2005).

31

The heat resistance of Byssochlamys ascospores decreases significantly in the presence

of chemical preservatives. Sulphur dioxide at a concentration of 90 ppm reduced the

D87.8 values of B. fulva asci by half, from 8.8 to 4.5 minutes, compared with no SO2

treatment (King et al., 1969). Potassium sorbate and sodium benzoate at 500 – 1000

ppm each worked synergistically with heating to decrease the D80 values of B. nivea

ascospores by approximately 1 – 2 and 3 – 4 minutes, respectively (Beuchat, 1981).

2.6.2 Effects of ascospore age on heat resistance

The age of the cultures from which ascospores (ascospore age) are harvested can affect

their heat resistance, however not all ascospores are produced at the same time, causing

variations in their physiological properties (Dantigny and Nanguy 2009). A general

trend is that heat resistance increases with ascospore age. However, very few studies

have examined the effects of age on the heat resistance of Byssochlamys ascospores.

The most thorough research on this topic was conducted by Casella et al. (1990), who

investigated ascospores of one B. nivea strain prepared from cultures grown on MEA

and CYA at 4, 8, 12 and 16 weeks. The inactivation kinetics of the ascospores reported

by Casella et al. (1990) followed a non-linear logarithmic pattern and exhibited various

phases including, in the order of heating time, activation, shoulder, exponential

reduction and tailing. The shoulder was prominent at inactivation temperatures of 80 –

85oC whereas tailing was more frequently found at 87 – 90oC. The authors associated

the shoulder with a balance between activation and inactivation and the tailing stages

with a small group of intrinsically more heat resistant ascospores and heat shock

proteins.

For inactivation temperatures of 80 – 87oC, Casella et al. (1990) found that D values

increased significantly with ascospore age. However, the effect of age was less apparent

at temperatures above 88oC and the inactivation of ascospores at all ages became

practically instantaneous at temperatures of 95oC or higher (Casella et al., 1990).

Prior to the study of Casella et al. (1990), Yates and Ferguson (1963) had observed that

14 day cultures of B. nivea could withstand more severe heat treatments than the 4 day

samples. However, the role of ascospore age was not clear in this study because

Byssochlamys species usually take about one week or longer to start producing

ascospores (Pitt and Hocking 1997). Thus, the 4 day samples used in the study of Yates

32

and Ferguson (1963) could have comprised predominantly heat sensitive structures

including immature ascospores.

Age-induced heat resistance has been reported for other heat resistant moulds. For N.

fischeri, ascospores from 25 day cultures could tolerate a heat treatment (82oC, 60

minutes) better than those from 11 day cultures (Conner et al., 1987). Ascospores from

42 day samples were in turn more heat resistant than those from 25 day cultures

(Conner and Beuchat, 1987). Similarly, ascospores of T. flavus increased their heat

resistance when cultured up to 30 days but this resistance did not increase with further

incubation up to 58 days (Beuchat, 1988).

Williams (1936) investigated the effects of aging from 5 to 30 days on the heat

resistance of spores of Bacillus and Clostridium species, and showed no relationship

between the two parameters. More recently, the aging phenomenon has been reported

for endospores of Clostridium difficile and been associated with super-dormant spores

(Rodriguez-Palacios and LeJeune, 2011). Rodriguez-Palacios and LeJeune (2011) also

found that heating at 63oC reduced the counts of 1 week cultures after 30 minutes but

actually increased those of 20 week spores by 30% after 15 minutes. Super-dormant

spores of Bacillus species, which are prepared by selective germination, are more wet-

heat resistant than just dormant ascospores because of their lower core water content

(Ghosh et al., 2009).

Ascospore age can undermine the efficacy of heat treatments because food products

may harbour ascospores for a significant period of time prior to processing. Although

the effect of age on heat resistance has been documented, there is limited information on

this topic for B. fulva and B. nivea except the study of Casella et al. (1990). Research on

other heat resistant moulds has also failed to provide satisfactory explanations about the

mechanisms involved.

2.6.3 Contributions of ascospore ultrastructure to heat resistance

For both bacterial and fungal species, ultrastructure has an essential role in maintaining

dormancy of spores and rendering them resistant to environmental stresses. Dormancy

and the resultant heat resistance of the spores are achieved predominantly by their

multilayer structure and hydrated cytoplasm. The structure of spores usually comprises

33

three major compartments, consisting of a cell-wall, cortex or cortex-like layers and a

cytoplasm where most materials essential for growth are located (Driks, 1999, 2003).

The cell walls of spores are often significantly thicker than those of vegetative cells.

They can be made of more than one layer and are relatively impermeable and, along

with the cell membrane, prevent the loss of moisture and important intracellular

molecules, as well as preventing the entry of unfavourable chemicals (Atrih and Foster,

2002; Setlow, 2006; Sussman and Douthit, 1973; Warth, 1985). The low moisture

condition inside the spores can stabilise molecules and hinder chemical reactions,

protecting proteins and nucleic acid materials from denaturation (Atrith and Foster,

2002; Gerhardt and Murrell, 1978; Orsburn et al., 2008).

Despite its potential importance to the heat resistance, only a few studies have examined

the ultrastructure of Byssochlamys ascospores. Put and Kruiswijk (1964) used a light

microscope to examine the morphology of B. nivea ascospores found in spoilt canned

strawberries. In their photomicrographs, the ascospores exhibited a distinct coat, an

extremely dense central region and a translucent space sandwiched between these two

compartments. However, the authors did not provide a more detailed description of the

microscopic findings. Later Partsch et al. (1969) used transmission electron microscopy

to identify distinctive features of conidia and ascospores of a strain of B. fulva. They

found that the ascospores had a thicker cell wall (0.2 – 0.3 µm) than the conidia (0.1 –

0.2 µm). In addition, the ascospores had an unusually thick intermediate space (1 – 2

µm) that was absent in the heat sensitive conidia. The thick cell wall and intermediate

space were suggested to be important factors in the heat resistance of the ascospores.

Using scanning electron microscopy, Hatcher et al. (1979) showed a building up of an

outer layer of the asci of B. fulva when comparing samples from 30 and 90 day cultures.

Their result suggested a critical link between ascospore ultrastructure, age and heat

resistance properties.

The multilayered ultrastructure observed in Byssochlamys ascospores is generally

similar to that of other heat resistant moulds. Ascospores of N. fischeri have a thick cell

wall and intermediate space enclosing a dense cytoplasm. Interestingly, the cell wall

comprised four layers including a thick outer cell wall (0.1 – 0.2 µm) (Conner et al.,

1987). Ascospores of T. macrosporus also showed the three major compartments and a

relatively thick multilayered cell wall (Dijksterhuis et al., 2007).

34

In summary, information on the ultrastructure of ascospores of Byssochlamys species is

limited to only a few studies. This needs to be extended to provide more data about the

effects of age on their ultrastructure and changes to this ultrastructure during heat

inactivation.

2.6.4 Chemical composition and heat resistance of ascospores

Chemical composition has a crucial role in determining the dormancy and heat

resistance of microbial spores (Dijksterhuis et al., 2002; Setlow, 2006). Only one study

(Banner et al., 1979) so far has investigated the chemical composition of Byssochlamys

ascospores. Banner et al. (1979) compared the chemical composition of ascospores from

two B. fulva strains with different heat resistance. They found that the ascospores of

both strains had an unusual lipid composition and contained a high proportion of fatty

acids with backbones longer than 20 carbons. However, the heat resistant ascospores

had more total fatty acid, and a higher percentage of fatty acids longer than 18 carbons

than the heat sensitive samples. Moreover, there were no significant differences

between two strains in terms of other components such as carbohydrates, amino acids

and minerals. Thus, the authors concluded that proteins, minerals and carbohydrates

were not heat resistant factors of Byssochlamys ascospores. The fatty acids could

contribute to the heat resistance since they might be involved in membrane transport

systems and rigidity of the cell wall (Fisher et al., 1972). However, no research has been

done since then to elucidate the roles of fatty acids in the heat resistance of

Byssochlamys ascospores.

Despite the conclusions drawn by Banner et al. (1979) about carbohydrates and

proteins, many studies have showed that specific groups of these compounds are related

to thermotolerance. Trehalose, in particular, is one of the most common stress protectors

in fungal cells (Dijksterhuis et al., 2002; Thevelein, 1984). This sugar interacts with

various families of heat shock proteins, protecting cells from environmental stresses

such as dehydration and high temperatures (Tereshina, 2005). The importance of

trehalose and proteins will be discussed in the next sections.

35

2.6.5 Trehalose, glassy state and heat resistance of ascospores

Trehalose is a ubiquitous disaccharide in fungi (Thevelein, 1984). Trehalose contents in

fungal spores are widely variable, ranging from 2.6% to 35% of dry weight (van Laere,

1989). Trehalose has not been reported in B. fulva or B. nivea but it has been found in

ascospores of other heat resistant moulds, such as N. fischeri, having 6.1 µg of trehalose

per mg of dry ascospores (Conner et al., 1987) and T. macrosporus having 9 – 17%

trehalose on a wet cell basis (Dijksterhuis et al., 2002). The fact that trehalose is found

to accumulate at high concentrations in fungal spores suggests its important role during

fungal dormancy.

The major function of trehalose in fungal spores is as an energy reserve (Elbein, 1974;

Sussman, 1976; Tereshina, 2005). A rapid release of the intracellular trehalose often

signals germination (Dijksterhuis et al., 2002, 2007), injury or death of fungal spores

(Mandels et al., 1965). Additionally, trehalose protects cells against many adverse

environmental conditions including heat stress, water stress and oxidative stress.

Accumulated trehalose has been associated with enhanced stress resistances in conidia

of Emericella nidulans (Fillinger et al., 2001) and vegetative cells of Saccharomyces

cerevisiae (Elliott et al. 1996; van Dijck et al., 1995) and Schizosaccharomyces pombe

(Soto et al., 1999).

The protective actions of trehalose generally involve three major properties: the

inactivity in chemical reactions, its ability to replace water in interactions with

biomaterials, and the ability to form a glassy state (Luo et al., 2008). First, trehalose is

chemically and thermodynamically stable at elevated temperatures, at low pH and in

reactions with proteins and, therefore, it does not participate in reactions leading to

degradation of other molecules (Teramoto et al., 2008). Second, trehalose can replace

water in hydrogen bonding with molecules, such as proteins (Allison et al., 1999;

Klimov and Thirumalai, 1997) and membranous phospholipids (Bogaart et al., 2007;

Crowe et al., 1984, 1996), stabilising their structures and preventing denaturation by

dehydration. Although other carbohydrates can replace water, trehalose is more

effective because of the larger hydrated volume which allows it to substitute for more

water molecules (Sola-Penna and Meyer-Fernandes, 1998).

A glassy state is a special arrangement of material molecules at low moisture content,

which is in semi-solid and stable structure but still maintains a certain degree of

36

flexibility of the fluid state (Leopold et al., 1994). The glassy state mechanism has been

a popular hypothesis to explain the heat resistance of fungal ascospores because it

creates an intracellular environment protecting biological materials against chemical

and mechanical denaturation (Dijksterhuis, 2007; Dijksterhuis et al., 2007). The low

moisture content inside the ascospores is favourable for a glassy state because

polyhydroxy sugars such as trehalose, sucrose, mannitol and erythritol can substitute

water in hydrogen bonding with intracellular molecules and protect them (Allison et al.,

1999; Dougan et al., 2011; Izutsu and Kojima, 2002; Sola-Penna and Meyer-Fernandes,

1998). A glass-like structure has been observed inside endospores of Bacillus species

and has been associated with the heat tolerance of these cells (Leuschner and Lillford,

2003; Stecchini et al., 2006). Despite suggestive results from other species, the presence

of a glassy state in Byssochlamys ascospores has not been examined and further

research is needed to investigate such a structure and how it might affect the heat

responses of the fungi.

2.6.6 Molecular factors of heat resistance of Byssochlamys ascospores

The genomes of B. fulva and B. nivea species have not been fully sequenced. So far

only genes that are needed for taxonomy and detection of those two species have been

sequenced, including 18S rDNA (Luangsa-ard et al., 2004a), 28S rRNA (Untereiner et

al., 2002), 5.8S rDNA, ITS1, ITS2 (Houbraken et al., 2008; Samson et al., 2009), β-

tubulin (Nakayama et al., 2010; Puel et al., 2007), actin (Houbraken et al., 2008) and

calmodulin (Samson et al., 2009). Some genes encoding for important enzymes are also

sequenced, such as patulin production enzymes, isoepoxydon dehydrogenase and 6-

methylsalicylic acid synthase (Dombrink-Kurtzman and Engberg, 2006; Puel et al.,

2007), and RNA polymerase beta (Peterson, 2008). Thus, genomic aspects of the heat

resistance of Byssochlamys ascospores are still poorly understood. Research on

transcriptomics and proteomics of heat resistance is limited by the lack of genomic

information.

High temperature can induce expression of various groups of stress-related compounds

such as proteins or sugars in fungal and yeast cells (Albrecht et al., 2010; Chen and

Chen, 2004; Goldani et al., 1994; Piper, 1993). The most studied stress-related

protectors are heat shock proteins (HSPs), which are molecular chaperones in microbial

37

cells, protecting intracellular proteins from thermal denaturation (Li and Srivastava,

2004). The protection of HSPs is based on their interactions with proteins, helping to

maintain their native structures or refolding misfolded proteins (Burnie et al., 2005).

Casella et al. (1990) suggested that HSPs might contribute to the heat resistance of

ascospores of B. nivea but further studies are required to examine the protein profiles of

Byssochlamys ascospores and how they might change during heat inactivation.

2.7 Concluding remarks

Byssochlamys species, particularly B. fulva and B. nivea, have caused problems in the

fruit juice and the canned fruit industry for nearly 80 years. From the early studies, a

considerable body of knowledge has been gained about their taxonomy, morphology,

physiology, sources of contamination and significances in food quality and safety. It is

well established that B. fulva and B. nivea can survive in highly acidic (pH 3.0 – 4.5)

and low water activity (0.892 – 0.993) environments with an extremely small

requirement of oxygen. Ascospores of the fungi are widely distributed in soil; hence,

frequent contact with soil in orchards renders fruits most susceptible to Byssochlamys

contamination. Byssochlamys species can produce mycotoxins, most notably patulin and

byssochlamic acid.

The ability of B. fulva and B. nivea to survive thermal processes is attributed mostly to

the production of highly heat resistant ascospores. Typically, thermal destruction times

(D) are 1 – 12 minutes at 90oC for ascospores of B. fulva and 0.6 – 1.2 minutes at 85oC

for those of B. nivea. Therefore, they can easily survive pasteurisation processes used by

the food industry. Moreover, the heat resistance is greatly enhanced by the presence of

common food components such as sugars and salts. Effects of acids on the ascospores

can either be inhibitory or protective depending on types of acid. However, heat

treatments coupled with low pH (less than 3.6) and food preservatives are effective in

inactivating the ascospores. Additionally, the analysis of heat resistance is complicated

by phenomena such as the presence of a shoulder and tailing on the survival curves.

Current knowledge of the physiology, chemical composition and ultrastructure of

Byssochlamys ascospores, unfortunately, is insufficient to explain their remarkable heat

resistance. Most of the studies have examined only effects of extrinsic factors on the

heat resistance, leaving a gap in our knowledge of how ascospores achieve the property

38

intrinsically. There have been theoretical links between the heat resistance and

ascospore age, ultrastructure, chemical composition, physical arrangements of

intracellular materials and heat-stress proteins. They represent areas for future research

in order to elucidate the ultimate mechanisms of heat resistance of Byssochlamys

ascospores.

Apart from the need for scientific knowledge, research on the bases of heat resistance is

also fuelled by demands of the food industry and consumers. Consumers are urging less

processed, more natural products with minimal preservatives. That in turns drives food

manufacturers to develop novel, milder processing techniques (Sloan, 2010; Zink,

1997). Thus, a better understanding of the heat resistance properties of Byssochlamys

ascospores would be beneficial for the food industry, assisting it to develop effective

processes that could meet consumers’ demands and expectations.

39

Chapter 3 – Thermal inactivation of ascospores of Byssochlamys fulva and Byssochlamys nivea during aging

3.1 Introduction

Heat resistant microorganisms are defined as those that can survive thermal processes

such as pasteurisation which is commonly used by the food industry (Houbraken and

Samson, 2006). They represent a small yet significant group of major concern to the

food manufacturers, especially those whose products rely on thermal processing as the

major hurdle for microbiological stability. In most cases, they acquire the thermo-

tolerance by producing structures such as endospores, ascospores, thick-walled

chlamydospores, cleistothecia and sclerotia which are dormant and resistant to adverse

conditions including heat (Atrih and Foster, 2002; Beuchat and Pitt, 2001; Tournas,

1994). Members of this group include spore-forming bacteria such as Bacillus spp. and

Clostridium spp. (Atrih and Foster, 2002), and some filamentous fungi such as

Byssochlamys spp., Talaromyces spp. and Neosartorya spp.) (Mouchacca, 2007).

Byssochlamys fulva and B. nivea are two of the most heat resistant fungal species of

relevance to the food industry (Samson, 1989; Samson et al., 1995). Their ascospores

are able to survive thermal treatments at 90oC, rendering pasteurisation ineffective

(Bayne and Michener, 1979). Outbreaks of food spoilage linked to these two species

have been regularly reported in many regions including Australia (Hocking and Pitt,

1984; Richardson, 1965), North America (Denny and Brown, 1969; Yates, 1974),

Europe (Kotzekidou, 1997), Asia (Ayesha and Viswanath, 2006) and Africa (Ugwuanyi

and Obeta, 1991). Soil often serves as a reservoir for Byssochlamys ascospores, so food

materials that may come into contact with soil are more likely to be contaminated by

these fungi (Jesenská et al., 1993). B. fulva and B. nivea often spoil canned fruits and

fruit juices because of their tolerance to acidity, low oxygen conditions and the

production of pectinolytic enzymes (Beuchat, 1998; Tribst et al., 2009; Ugwuanyi et al.,

1999). They can also produce the mycotoxin patulin, which may be a significant risk to

public health (Becci et al., 1981; Rice et al., 1977b, 1980; Taniwaki et al., 2010).

Hence, it is important to understand factors that underpin the heat resistance of

40

Byssochlamys ascospores so that they can be effectively controlled during food

processing.

Several studies have reported on the heat resistance of Byssochlamys ascospores.

Research on heat resistance and physiology of Byssochlamys species prior to 1976 has

been reviewed by Beuchat and Rice (1979) and more recent studies have been discussed

by many authors (Beuchat, 1998; Pitt and Hocking, 2009; Tournas, 1994). Thermal

reduction times (D) and thermal resistant coefficients (z) of B. fulva ascospores have

been reported as 1 – 12 minutes at 90oC and 6 – 7oC, respectively, while those of B.

nivea ascospores are 0.6 – 12 minutes at 85oC and 4 – 6.8oC, respectively (Casella et al.,

1990; Michener and King, 1974). The range of D values reflects the effects of several

factors on heat resistance of B. fulva and B. nivea ascospores. While there are

differences in D values between the two species, strains within these species also show

some variations in this property (Bayne and Michener, 1979; Engel and Teuber, 1991).

Food components such as sugar, salt, some types of acids and food solids have been

reported to increase the D values of the ascospores (Beuchat, 1981; Beuchat, 1998;

Beuchat and Toledo, 1977; Splittstoesser and Splittstoesser 1977; Splittstoesser et al.,

1974b). However, the heat resistance of the ascospores decreased in the presence of

food preservatives (e.g. SO2, sorbate and benzoate) or in acidic environments with pH

values below 3.5 (Roland et al., 1984a, b; Splittstoesser et al., 1974b).

The age of the cultures from which ascospores were harvested (ascospore age) was also

noted to affect their heat resistance in such a way that the heat resistance generally

increased with ascospore age (Beuchat, 1988; Conner et al., 1987; Dantigny and

Nanguy, 2009; Engel and Teuber, 1991). For Byssochlamys, only a few studies have

reported the effect of age on ascospore heat resistance. Casella et al. (1990) showed that

B. nivea ascospores became more heat resistant when the ascospore age increased from

4 to 16 weeks. Slongo et al. (2008) reported similar increased thermo-tolerance in fruit

juices for 1 and 2 months old ascospores of B. nivea. However, these studies were

limited to only one strain of B. nivea species and a maximum age of 16 weeks. Age can

undermine the efficacy and design of heat processes because products can harbour

ascospores for significant periods of time prior to processing. Consequently, more

research is needed to better understand the thermal inactivation kinetics of ascospores as

a function of their age.

41

This Chapter reports the thermal inactivation of ascospores obtained from B. fulva and

B. nivea after culture for 4, 6, 8, 12 and 24 weeks. Two strains of each species were

examined as three different inactivation temperatures.

3.2 Materials and methods

3.2.1 Fungal cultures, growth conditions and ascospore harvest

Four fungal strains, B. fulva FRR 2299, B. fulva FRR 2785, B. nivea FRR 4421 and B.

nivea FRR 6002, from the fungal culture collection of the Division of Food and

Nutritional Sciences, CSIRO, North Ryde, Australia were used in this study. The B.

fulva strains were originally isolated from Australian fruit juice samples, B. nivea FRR

4421 was isolated from Brazilian strawberries and B. nivea FRR 6002 was from a New

Zealand dairy product. These strains were screened from others in the collection to

ensure that they were efficient producers of ascospores and had different levels of heat

resistance. During the screening process, ascospores of B. fulva FRR 2785 were more

heat resistant than those of B. fulva FRR 2299, and those of B. nivea FRR 6002 were

more resistant than those of B. nivea FRR 4421 (Appendix 3.1).

The fungi were grown on Malt Extract Agar (MEA) at 30oC for 4, 6, 8, 12 and 24

weeks. At the end of the incubation, plates were flooded with 0.05% (w/v) aqueous

Tween 80 (Merck, VIC, Australia) and the fungal biomass was scraped from the surface

using sterile bent glass rods. Ascospores were freed from asci and mycelium using a

modified version of a previously described method (Splittstoesser et al., 1974b).

Approximately 1.4 mL of harvested biomass was added into 2 mL centrifuge tubes

(Biocorp, VIC, Australia) which contained about 0.3 g of glass beads with a diameter of

maximum 106 µm (Sigma-Aldrich, NSW, Australia). The tubes were then shaken in a

FastPrep® FP120A machine (MP Biomedicals, NSW, Australia) at a speed setting of 5.5

for 180 seconds for B. fulva strains and 90 seconds for B. nivea strains. The

homogenates were filtered through sterile glass wool to remove glass beads and

mycelial fragments. The filtrates were washed three times by centrifugation at 4000g for

20 minutes at 4oC in sterile distilled water (SDW). The pellet was resuspended in SDW,

distributed into 1.5mL aliquots and stored at 4oC for further usage. The numbers of free

ascospores and remaining asci were counted after glass bead treatment by phase contrast

microscopic observation using a haemocytometer (Marienfeld, Germany).

42

3.2.2 Heat resistance of ascospores at different ages

The heat challenges were conducted at 85oC, 87.5oC and 90oC for up to 90 minutes for

B. fulva strains and at 82.5oC, 85oC and 87.5oC for up to 60 minutes for B. nivea strains.

The heating menstruum was citrate phosphate buffer (CPB) pH 4.0 which comprised

citric acid and di-sodium hydrogen orthophosphate at the final concentrations of about

0.0626M and 0.0748M , respectively (Ajax Finechem, Australia).

Ascospore concentrations were adjusted to 107 cells/mL with SDW prior to each

experiment. The suspension was pre-heated at 65oC for 10 minutes to inactivate any

remaining vegetative cells and heat sensitive conidia (Houbraken et al., 2006). One

hundred microlitres of the pre-treated suspension was then added to closed glass tubes

containing 9.9 mL CPB that had been brought up to the pre-determined temperature in a

thermally controlled water bath (Thermoline Scientific). Temperatures of CPB in the

tubes were monitored throughout the experiment via a digital thermocouple placed in an

identical tube containing the similar amount of CPB immersed in the same water bath.

The tubes were heated for 1, 2, 4, 8, 12, 16, 20, 30, 40 and 60 minutes for B. nivea

strains or all the time points up to 90 minutes for B. fulva strains. The sample for time

“0” contained 9.9 mL CPB and 100 µL of pre-treated ascospore suspension and

received no further heat treatment. At the end of the heating time, tubes were cooled

down in an ice slurry for 20 seconds, serially diluted in 0.1% (w/v) aqueous

bacteriological peptone (Amyl Media, Australia) and viable ascospore populations

determined by spread-plating on MEA in duplicate. MEA plates were incubated at 30oC

for up to 5 days and counted for colony forming units (cfu). Each heat treatment was

done in triplicate.

Decimal reduction times (D) were defined as the time to reduce the microbial

population by 90% were calculated from the portion of the (semi-log) survival curves

where surviving counts were linearly reduced (Casella et al., 1990; Hatcher et al., 1979).

Analysis of variances (ANOVA) and t-tests were performed using the statistical

package in Microsoft® Excel and MiniTab 15 (MiniTab Inc., PA, USA). The same

significance level (α = 0.05) was used for all statistical comparisons.

43

3.2.3 Heat resistance of sequential generations of ascospores surviving heat

treatments

To assess whether survivors at the end of the heat treatments were intrinsically more

heat resistant, serial subculturing and heating were carried out with the surviving

populations for four generations. For this experiment, ascospores prepared from 4 week

old B. nivea FRR 6002 and B. fulva FRR 2299 cultures were used. Ascospore

suspensions were heat treated at 85oC and 87.5oC for up to 60 and 40 minutes,

respectively, and spread-plated on MEA in duplicate. From colonies that developed on

MEA plates from surviving ascospores, two were randomly selected. These colonies

were independently subcultured and incubated on MEA at 30oC for 4 weeks to generate

new ascospore crops. The heat resistance of these ascospores was determined using the

same procedure as already described and D values determined. One representative of

the surviving colonies on MEA of the longest heating time was isolated and the process

of culture for 4 week ascospore harvest and heating was repeated. In all this process was

repeated for three generations.

3.3 Results

3.3.1 Effects of age on thermal inactivation of ascospores

Ascospores harvested from 4, 6, 8, 12 and 24 week old cultures were heated at 85, 87.5

or 90oC for B. fulva strains or 82.5, 85, 87.5oC for B. nivea strains. Figures 3.1 – 3.4

show the survival curves for B. fulva FRR 2299, B. fulva FRR 2785, B. nivea FRR 4421

and B. nivea FRR 6002, respectively. The initial viable counts of the ascospores before

heating were 102 – 104 cfu/mL. None of the heat treatments gave complete inactivation

of the ascospores even after heating for 90 minutes, and viable populations of 102 – 103

cfu/mL were detected at end of the treatments.

The survival curves of Byssochlamys ascospores comprised four phases: activation,

shouldering, linear reduction and tailing. The activation phase happened in the first

minute of heating when ascospore viable counts abruptly increased to a maximum

population, which varied with strains, ages and temperatures. For most conditions, there

was a short period of time when the population remained at this maximum level (within

0.3 log10 for at least three consecutive time points) before undergoing a rapid linear

44

inactivation. This short period of time is referred as the shoulder phase. After this

shoulder, progressive inactivation of the ascospores occurred. The rate of inactivation

was approximately linear across several times points, but then the rate decreased giving

an extended period, the tailing phase, where the population did not decrease by more

than 0.5 log10.

Log1

0 (c

fu/m

L)

Heating time (min)



85oC (■), 87.5oC (♦) and 90oC (▲); each point is an average of three experiments; the

approximately linear reduction part of a curve is between the highest activation point

and time point indicated by arrows of the same colour

A B

C D

E

45

Log1

0 (c

fu/m

L)

Heating time (min)



85oC (■), 87.5oC (♦) and 90oC (▲); each point is an average of three experiments; the



A B

C D

E

46

Log1

0 (c

fu/m

L)

Heating time (min)



82.5oC (○), 85oC (□) and 87.5oC (◊); each point is an average of three experiments; the



A B

C D

E

47

Log1

0 (c

fu/m

L)

Heating time (minutes)



82.5oC (○), 85oC (□) and 87.5oC (◊); each point is an average of three experiments; the



A B

C D

E

48

During the activation stage, the viable counts of B. fulva increased from initial levels of

2 – 3 log10 (cfu/mL) to about 5 – 5.5 log10 (cfu/mL) maximum, representing an

approximate 1000 fold increase. For B. nivea, their counts increased slightly less than

the B. fulva, going from 2 – 3 log10 (cfu/mL) up to about 4 – 5 log10 (cfu/mL)

maximum. The maximum viable counts due to activation at 1 minute were much closer

to the initial concentration of ascospores, which was about 5 – 6 log10 (cells/mL),

added at the beginning of the heat treatment, but 100% activation was not achieved.

However, the degree of activation varied between strains, ascospore age and, to a lesser

extent, heating conditions (Figure 3.5). To elaborate the data presented in Figure 3.5,

the detailed comparisons of the thermal activation across heating conditions and

ascospore age are presented in Appendices 3.2 and 3.3.

Age of ascospores affected their activation in the first minute of the heat treatment and

was strain dependent. For most strains, the highest level of activation (2.5 – 3 log10)

predominantly happened with 6 – 12 week ascospores. However, the 24 week samples

followed by the 4 week samples exhibited significantly lower activation (1 – 1.5 log10).

Exceptionally, ascospores of B. fulva FRR 2299 demonstrated a different dynamic of

activation, which significantly increased from about 1.8 log10 at 4 weeks to about 2.7

log10 at 6 weeks but did not change between 6 and 24 weeks.

For all tested strains, the level of activation was not significantly different when

ascospores of the same age were treated at different inactivation temperatures.

Exceptions occurred with the 4 and 24 week B. nivea FRR 4421, and the 24 week B.

nivea FRR 6002. In those cases, higher temperature (e.g. 87.5oC) significantly reduced

the levels of activation compared with less severe treatments at 82.5oC and 85oC.

49

A)

0

0.5

1

1.5

2

2.5

3

3.5

4 6 8 12 24Ascospore age (weeks)

Log

10 (c

fu/m

L)

85oC

87.5oC

90oC

B)

0

0.5

1

1.5

2

2.5

3

3.5


Log

10 (c

fu/m

L)

85oC

87.5oC

90oC

C)

-0.5

0

0.5

1

1.5

2

2.5

3

4 6 8 12 24

Ascospore age (weeks)

Log

10 (c

fu/m

L)

82.5oC

85oC

87.5oC

D)

0

0.5

1

1.5

2

2.5

3

3.5


Log

10 (c

fu/m

L)

82.5oC

85oC

87.5oC

Figure 3.5. Average activation levels of B. fulva FRR 2299 (A), B. fulva FRR 2785 (B), B. nivea FRR 4421 (C) and B. nivea FRR 6002

(D); each experiment started with a population of 5 – 6 log10 (cfu/mL); similar letters indicate statistically different activations (p < 0.05)

between ages at least at two out of three tested temperatures; each column is an average of triplicate experiment

a

a a

ab

cd

ac ac bd

a

abc

abc b

c

abc bc

a

b c

50

The shoulder and tailing phases were more prevalent with B. fulva than B. nivea strains.

The shoulder phase was observed more frequently at low inactivation temperatures in

ascospores from cultures older than 6 weeks (e.g. 85oC for B. fulva and 82.5oC for B.

nivea). The shoulder phase was also longer in older cultures (12 and 24 weeks; Figure

3.1E-D, Figure 3.2E-D). Higher inactivation temperatures completely removed this

phenomenon, although the 12 and 24 week old B. fulva samples showed it more

persistently.

Tailing was found in most survival curves and was dependent on culture age and

inactivation temperatures. Tailing appeared to be delayed in older cultures but increased

temperatures induced it to happen earlier. The tailing phase displayed by B. nivea

ascospores was more inconsistent than for B. fulva, especially with higher ages and

temperatures.

The survival population was reduced by up to 3 log10 (cfu/mL) during the linear

decimal reduction stage. In older cultures, fewer ascospores were killed, demonstrated

by a longer and less steep linear section. However, increased temperatures resulted in a

higher inactivation and a steeper linear phase. For B. fulva, the duration of this section

could be up to 30 – 40 minutes with ascospores from 4 and 6 week old cultures, and 60

– 90 minutes with older ages (Figure 3.1A-E). It was more difficult to compare the

linear phase for ascospores of the more heat sensitive B. nivea since this section was

often indistinct from the shoulder and tail. The estimated duration of this phase was 20

minutes for 4 – 8 week ascospores but it could extend until the end of the heat

treatments for the older samples (Figure 3.3A-E).

3.3.2 Thermal reduction time (D) of Byssochlamys ascospores

Thermal reduction time (D) for the inactivation of the ascospores was derived from the

linear portion of the curves (Figures 3.1 – 3.4). These D values of Byssochlamys

ascospores are given in Table 3.1 and their changes in response to culture age are

illustrated in Figure 3.6.

As expected from the duration of the linear reduction phase, the D values were larger

for the lower heating temperatures and smaller for the higher temperatures for all

strains. This observation was consistent for ascospores of most ages. An exception was

51

that D87.5 values (14.12 ± 0.31 minutes) of 8 week ascospores of B. fulva FRR 2785

were higher than their D85 (10.75 ± 0.49 minutes). Another out-of-trend value was the

D85 times (34.41 ± 6.58 minutes) for 24 week ascospores of B. nivea FRR 4421, being

higher than their D82.5 (16.40 ± 7.14 minutes), respectively (Table 3.1).

Table 3.1. Thermal reduction times (D) in minutes ± standard deviation (SD) of

Byssochlamys ascospores at different ages and inactivation temperatures; each value is

an average of three experiments

Age (weeks)

B. fulva FRR 2299 B. fulva FRR 2785

D85 D87.5 D90 D85 D87.5 D90

4 12.81± 0.86 4.25 ± 0.09 1.66 ± 0.43 6.54 ± 0.85 2.55 ± 0.18 1.42 ± 0.26

6 14.73 ± 0.07 7.01 ± 0.38 2.58 ± 0.36 24.65 ± 1.48 7.09 ± 0.08 2.68 ± 0.31

8 14.12 ± 5.89 10.37 ± 0.92 5.78 ± 0.54 10.75 ± 0.49 14.12 ± 0.31 4.26 ± 0.25

12 27.65 ± 3.75 10.04 ± 0.29 6.48 ± 0.14 26.43 ± 2.02 8.61 ± 0.40 4.66 ± 0.14

24 47.92 ± 7.23 17.10 ± 1.52 6.29 ± 0.61 34.52 ± 1.42 18.52 ± 1.56 5.32 ± 0.69

B. nivea FRR 4421 B. nivea FRR 6002

D82.5 D85 D87.5 D82.5 D85 D87.5

4 5.65 ± 0.71 2.66 ± 1.46 3.01 ± 1.40 12.20 ± 2.90 3.82 ± 1.42 4.41 ± 1.21

6 7.48 ± 0.67 2.96 ± 0.55 2.21 ± 0.78 18.99 ± 2.46 3.15 ± 0.21 1.53 ± 0.28

8 9.37 ± 0.47 3.95 ± 0.11 1.40 ± 0.63 24.76 ± 2.85 4.31 ± 1.27 1.80 ± 0.36

12 12.53 ± 1.14 10.83 ± 2.69 5.20 ± 2.32 11.27 ± 1.75 8.81 ± 1.01 3.06 ± 0.53

24 16.40 ± 7.14 34.41 ± 6.58 3.43 ± 1.17 35.65 ± 7.11 6.28 ± 1.11 4.53 ± 0.75

In most cases, D values significantly increased with increasing ascospore age. They

increased by as much as 4-fold for B. fulva FRR 2299 and 7-fold for B. fulva FRR 2785

at all temperatures for ascospores from 4 to 24 week cultures. The D values of B. nivea

strains also increased up to 4-fold when ascospores of 4 and 24 week cultures were

compared. However, D values of B. nivea ascospores at intermediate ages (6, 8 and 12

weeks) occasionally did not follow the expected increasing trend as consistently as

those of B. fulva.

The trend of increasing D values with ascospore age was most conclusive at the lower

heating temperatures (Figure 3.6). At the higher temperatures (87.5oC for B. nivea and

52

90oC for B. fulva) where the D values were smaller and analytical variation was greater,

this progressive increase with age was not consistently observed. In addition, the higher

temperatures often resulted in maximum D values at 12 weeks with little change at 24

weeks (Figure 3.6).

The heat resistance was also different at species and strain levels. B. nivea ascospores

were more heat sensitive than B. fulva by having smaller D values at all ages. Within

species, B. fulva FRR 2299 ascospores could survive heating significantly better than B.

fulva FRR 2785, with the exceptions of 6 and 8 weeks at 85oC, 8 weeks at 87.5oC and

24 weeks at 90oC. On the other hand, B. nivea FRR 6002 ascospores were significantly

more heat resistant than B. nivea FRR 4421 at 4, 6 and 8 weeks of age, and at 82.5oC.

The variations between B. nivea strains were insignificant when ascospores from

cultures older than 8 weeks were examined or temperatures were 85oC or higher.

53

A) B)

C) D)

Figure 3.6. D values of ascospores of B. fulva FRR 2299 (A) and FRR 2785 (B), B. nivea FRR 4421 (C) and FRR 6002 (D) at different culture

ages and inactivation temperatures; Temperature: 82.5oC (●), 85oC (♦), 87.5oC (■) and 90oC (▲)

54

3.3.3 Heat resistance of sequential generations of ascospores surviving heat

treatments

The tailing of the inactivation curves in Figures 3.1 – 3.4 suggested a possible selection

for ascospores with increased resistance to the heat treatment. If these surviving

ascospores were intrinsically more heat resistant, then this property would be reflected

in increased heat resistance with repeated heat treatments and selection of survivors. To

test this possibility, colonies of the surviving ascospores were isolated and cultured to

produce a new crop of ascospores over 4 generations and their D values compared.

Two colonies each of the 4 week B. fulva FRR 2299 and B. nivea FRR 6002, which

respectively survived a 60-minute treatment at 87.5oC and 40-minute treatment at 85oC

used in the previous experiment (Figures 3.1A and 3.4A), were subcultured sequentially

for three generations and the heat resistance of these ascospore crops was examined.

Figures 3.7 and 3.8 show the inactivation curves of the parents and their progeny

ascospore crops for the B. fulva and B. nivea strains, respectively. Measurements of

ascospore viability were commenced at one minute, after heat activation had occurred.

The inactivation curves of the progeny followed similar patterns as their parents at 4

weeks which mainly comprised the linear reduction and tailing phases. No shoulder

phase was present, being consistent with previous results for the 4 week ascospores

(Figure 3.1A and 3.4A). The tailing of the curves meant incomplete inactivation of the

initial ascospore loads at the temperatures used, even after three generations of the

surviving colonies.

D values for each ascospore crop were determined from the linear portion of the

respective inactivation curves. D87.5 values of ascospores of B. fulva were similar for the

parent (first), second, third and fourth generations (Figures 3.9A). A similar conclusion

can be drawn for B. nivea although D85 values for ascospores of the third generations

showed a statistically significant decrease in heat resistance (Figure 3.9B).

It may be concluded from these experiments that the surviving members in an ascospore

population were more heat resistant but this trait was not transferred as a progressive

increase in resistance from parent generation to progeny.

55

A)

B)

Figure 3.7. B. fulva FRR 2299: representative survivor curves of subsequent

generations of 4 week ascospore cultures derived from the ‘tailing’ phase of the parent

and three sequential generations; A – Colony 1, B – Colony 2; all inactivation

treatments were done at 87.5oC

56

A)

B)

Figure 3.8. B. nivea FRR 6002: representative survivor curves of subsequent

generations of 4 week old ascospore cultures derived from the ‘tailing’ phase of the

parent and three sequential generations; A – Colony 1, B – Colony 2; all inactivation

treatments were done at 85oC

57

A)

B)

Figure 3.9. Comparing D values of 4 week ascospores derived from the parent and

three sequential “tailing” generations of B. fulva FRR 2299 (A) and B. nivea FRR 6002

(B); Asterisk (*) denotes D value pair significantly different from other pairs

*

58

3.4 Discussion

3.4.1 Inactivation kinetics of Byssochlamys ascospores

The inactivation curves of ascospores of B. fulva and B. nivea did not follow the

traditional logarithmic pattern for inactivation of microorganisms (Bigelow, 1921).

Their inactivation kinetics were characterised by multi-phasic survival curves, which

comprised activation, shoulder, exponential reduction and tailing phases. This non-

linear survival pattern of Byssochlamys ascospores has been reported by other workers

(Bayne and Michener, 1979; Casella et al., 1990; Sant’Ana et al., 2009). The non-linear

inactivation curves may be caused by environmental factors other than temperature,

such as pH, water activity or recovery media, and variability of heat resistant properties

(Mafart et al., 2010; Peleg and Cole, 1998).

3.4.1.1 Heat activation phase

The initial activation phase of the survival curves is expected because heating has been

known to break spore dormancy and induce germination (Finley and Fields, 1962;

Kikoku, 2003; Yates, 1973). Treatments at 70 – 75oC could activate less than 20% of

the ascospore population (Hebert and Larson, 1972; Yates et al., 1968) whereas

temperatures above 80oC increased the activation rates at the same time, inactivation of

a proportion of the ascospore population may be initiated (Beuchat, 1986; Splittstoesser

et al., 1971, 1972). The mechanism of heat activation is possibly related to changed

permeability of ascospore cell wall, allowing influx of water and activation/germination

factors, or inducing germinant receptors on the ascospore surface (Cronin and

Wilkinson, 2007; Foster and Johnstone, 1990; Yates et al., 1968). However, such

modifications could be lethal to the ascospores due to loss of essential cellular materials

(Coleman et al., 2007).

The dual activation/inactivation effects could be responsible for the incomplete

activation of all the ascospores initially added in the heat treatments for all strains

tested. This dynamic could also explain the lower inactivation levels occasionally at

high temperatures which caused more inactivation (Turnbull et al., 2007). For B. fulva,

the plate count technique could underestimate the number of activated ascospores

59

because the method treated single ascospores and intact asci similarly (Splittstoesser et

al., 1969).

The heat activation of ascospores was dependent on ascospore age, with rates increasing

for ascospores at 4 – 12 weeks (Figure 3.5). Older and more heat resistant ascospores

were less susceptible to the initial inactivation and therefore, the activation was more

prominent. This result agreed with findings of the study of Hebert and Larson (1972)

where asci older than 2 weeks had higher activation percentages. However, the 24

weeks ascospores could become more constitutionally dormant, reducing the heat

activation efficiency compared with the younger ascospores. A similar decrease in

activation was reported for ascospores of N. fischeri at 114 days which were heated in

apple juices (Conner and Beuchat, 1987). Contrarily, Slongo and Aragão (2006) found

that activation of N. fischeri ascospores in fruit nectars was not significantly affected by

ages between 1 and 3 months. However, the age of the ascospore crops used in the

study of Slongo and Aragão (2006) were younger than those in Conner and Beuchat

(1987) and the current study.

3.4.1.2 Shoulder and tailing phases

The shoulder and tailing phases are characteristics of the heat resistance of

Byssochlamys ascospores. These phenomena have been reported for ascospores of B.

fulva (Bayne and Michener, 1979; King et al., 1979) and B. nivea (Casella et al., 1990)

prior to this study. They are also associated with dormant structures of other heat

resistant moulds, such as T. flavus ascospores (Beuchat, 1988; King and Halbrook,

1987), N. fischeri ascospores (Beuchat, 1986; Salomão et al., 2007) and Aspergillus

niger conidia (Fujikawa and Itoh, 1996). Similar behaviour can be seen with endospores

of bacteria, such as Bacillus spp. (Furukawa et al., 2005) and Alicyclobacillus

acidoterrestris (Ceviz et al., 2009).

In this study, the shoulder phase could be ascribed to the dynamics of simultaneous heat

activation and inactivation of the ascospores. This relationship is in agreement with the

results of Casella et al. (1990). The fact that this phase always followed the initial

activation supported this conclusion because it took time for the ascospores to activate

and decrease the heat resistance. Moreover, increased inactivation temperatures, i.e.

inactivation energy, could shorten or remove the shoulder.

60

A common cause of the shoulder and tailing phases is the existence of ascospores in

groups. For B. fulva, the ascospore suspensions prepared for heat treatments still

contained intact asci at concentration of about 105 cells/mL (Appendix 3.4). Physically

bonded ascospores in the forms of asci and clumps could prevent uniform heat

penetration into individual cells (Cerf, 1977; Furukawa et al., 2005). Activation of these

structures would thus take more time and energy than required for single ascospores

(Splittstoesser et al., 1969). Additionally, all members of an asci or ascospore clumps

have to be killed in order to completely prevent the groups from forming visible

colonies on agar plates (Aiba and Toda, 1966). Thus, the mortality pattern of asci could

be skewed towards the beginning time of the heat treatments, which coincided with the

formation of the shoulder (Peleg and Cole, 1998). Similar delay during thermal

inactivation due to the increased mass has been observed with endospores of Bacillus

species and arthroconidia of Chrysosporium inops (Furukawa et al., 2005; Kinderlerer,

1996).

However, the ‘group’ theory could not account for the tailing phase of B. nivea samples

since their suspensions were almost free from groups of ascospores (Appendix 3.4).

Moreover, as shown in this study (section 3.3.3), the tailing could not be related to the

presence of a genetically heat resistant population because there was no inheritance of

this property across sequential generations of surviving colonies.

A possible explanation of the tailing phase was the presence of a small group of

ascospores that were intrinsically more heat resistant than the rest of the population.

This population could be a result of a normal distribution of the heat resistance

properties among ascospores or a heat adaptation (Casella et al., 1990; Han et al., 1976;

Peleg and Cole, 1998). For the distribution theory, the heat resistant group could have

remained dormant and insusceptible to heat activation and inactivation throughout the

heat treatments, a phenomenon described as super-dormancy in bacterial endospores

(Rodriguez-Palacios and LeJeune, 2011). Meanwhile, factors such as dehydration of the

ascospore core and heat shock proteins could have contributed to the heat adaptation

process (Casella et al., 1990; Mah et al., 2008).

61

3.4.2 Heat resistance and age of Byssochlamys ascospores

The heat resistance of Byssochlamys ascospores harvested from cultures at different

ages and heated at different temperatures was compared. The ascospore crops of these

strains were prepared from cultures that were grown for 4, 6, 8, 12 and 24 weeks.

Thermal treatments of the ascospores were carried out at 82.5 – 90oC for up 90 minutes

in a relatively simple medium (CPB pH 4.0). Their heat resistances were compared

according to patterns of their survival curves and D values. Since the heating medium

did not contain compounds, such as sugars, salts, preservatives, other organic acids,

which can impact on the viability of ascospores (Beuchat, 1981; Rajashekhara et al.,

1996; Splittstoesser et al., 1986), the observed changes in the heat resistance are solely

related to the culture age and inactivation temperatures.

The most significant result was that ascospores of B. fulva and B. nivea became more

heat resistant with increasing culture age (4 – 24 weeks), while more severe thermal

treatments resulted in higher inactivation of the ascospores. The increase of the heat

resistance based on D values was 4 – 7-fold for B. fulva strains and 2 – 4-fold for B.

nivea strains. Some D values at high heating temperatures, 90oC for B. fulva and above

85oC for B. nivea, were slightly out of trend due to increased heat sensitivity of the

ascospores in such severe conditions (King et al., 1979). In a few cases, the standard

deviations of D values were large, for instance 8 week old B. fulva FRR 2299 (D85 =

14.12 ± 5.89 min), which could be attributed to variations within the ascospore crops

and low recovery rate on MEA. However, those outliers did not affect the general trend

of increased heat resistance with age. Although the killing effect of various inactivation

temperatures on Byssochlamys ascospores has been abundantly reported (King et al.,

1969; Kotzekidou, 1997; Sant’Ana et al., 2009), the age-induced resistance has been

studied less intensively, mostly with B. nivea ascospores (Casella et al., 1990; Slongo et

al., 2008). The increased heat resistance observed here agrees with results of the

previous studies.

Casella et al. (1990) showed that D80 values of B. nivea ascospores increased from 15 to

76 minutes between 4 and 16 weeks, representing up to a 5-fold increase in heat

resistance. Meanwhile, the D85 values increased 2.5-fold, from 1.7 to 4.1 minutes. A

similar result between this study and Casella et al. (1990) was that the increase of D

values with age was more prominent at temperatures 85oC or below. At 87oC or above,

62

the change was inconsistent since the D values were mostly less than 1 minute. Thus,

both studies indicate the greater dependence of D values on inactivation temperatures

than on ascospore age.

However, the D85 – 87 values reported by Casella et al. (1990) were less than those found

in this study. The tailing of the survival curves was detected at 88oC or above

irrespective of ascospore age. This temperature was higher than 82.5oC where tailing

was observed in this study. These discrepancies were probably due to different strains

and heating protocols used in the two studies.

Casella et al. (1990) also reported higher viable counts at time 0 than our respective

counts, 5 – 6 log10 and 2 – 3 log10, respectively, which sometimes rendered the

shoulder and exponential reduction phases indistinguishable. The high initial counts

could be attributed to two ingredients, namely 0.1% peptone and 0.85% sodium chloride

(NaCl), of the heating medium. The 0.1% peptone has been found to favour the survival

of yeasts in suspension so it may have worked synergistically with heat to activate the

ascospores (Mian et al., 1997). On the other hand, NaCl was a heat resistant factor

which helped to minimise the inactivating effect during the first stage of the heat

treatments (Beuchat, 1981). Moreover, Casella et al. (1990) did not statistically compare

D values at different ages. That comparison was important at high temperatures where

D values decreased and became apparently similar between ages.

Slongo et al. (2008) examined the heat resistance of 1 and 2 month old ascospores of B.

nivea. The authors observed non-logarithmic survival curves with shoulders. Instead of

calculating D values, they linearised the survival curves by the method of Alderton and

Snell (1970) and determined 1/k values (k is a death rate constant), which were

analogous to D values. With this method, Slongo et al. (2008) showed that 1/k values of

B. nivea ascospores increased from 90.9 to 125 minutes, representing a 1.4-fold increase

in heat resistance. It is impractical to compare numerical results of Slongo et al. (2008)

and this study due to different approaches of determining heat resistance.

The aging effect on heat resistance has also been observed with other heat resistant

moulds. N. fischeri ascospores at 16 weeks old survived thermal treatment better than

those at 3 weeks (Conner and Beuchat, 1987; Slongo et al., 2008). Heat resistance of T.

flavus ascospores quickly increased with age up to 40 days but showed little change

with further aging up to 58 days (Beuchat, 1988; Dijksterhuis and Teunissen, 2004).

63

Because heat resistant fungi are also pressure resistant (Hocking et al., 2006), the

observed results mirror the findings which show age enhances pressure resistance of

various heat resistant moulds (Chapman et al., 2007a).

3.4.3 Proposed mechanisms of age-induced heat resistance in ascospores

Because age is an intrinsic property of the ascospores, increased heat resistance with age

suggests that critical changes occur to the physiology, ultrastructure and chemical

composition of Byssochlamys ascospores. Subsequent chapters of this thesis are devoted

to examining the relationship between age and the heat resistance in a greater depth.

Older ascospores may have time to undergo ultrastructural changes in order to obtain an

optimally strong conformation, without interfering with their sensitivity to germinating

signals (Chapman et al., 2007a). The ultrastructural changes could involve dehydration

of the central region of the ascospores and localisation of important proteins inside the

cells (Conner et al., 1987). That compact configuration can facilitate dehydration of the

ascospores, offering protection and repair of key cellular materials via a glassy state or

heat shock proteins (Dijksterhuis, 2007; Tereshina et al., 2005).

Structural modifications are known to play an important part in the thermo-tolerance of

bacterial spores (Orsburn et al., 2008). Dehydration of the spore protoplast helps to

stabilise molecules and genetic materials necessary for viability (Keynan, 1978; Novak

et al., 2003). Spores achieve a low moisture state by contracting the cortex and

maintaining pressure on the protoplast (Keynan, 1978; Sussman and Douthit, 1973).

Importantly, storage time is a limiting factor for bacterial endospores to modulate their

moisture content so that they achieve more dormant and resistant properties (Rodriguez-

Palacios and LeJeune, 2011).

3.4.4 Calculating D values from non-logarithmic survival curves

In this study, the D values of Byssochlamys ascospores were determined from the

exponential reduction portion of the survival curves. This method did not take into

account shoulder and tailing phases and potentially underestimated D values of the

ascospores. Moreover, determination of the reduction phase was relatively subjective,

particularly with B. nivea, which further reduced the certainty of the calculated D

64

values. Some anomalies were noted in the result section where D values did not always

increase with ascospore age (Figure 3.6). Using the same method to determine heat

resistance, Casella et al. (1990) showed similar inconsistencies in their age-related D

values. Therefore, the results from this experiment reinforce a need for a different

model to study thermal inactivation of Byssochlamys ascospores.

Some researchers have attempted to linearise the non-logarithmic curves and calculated

D-like and z-like values (Bayne and Michener, 1979; King et al., 1979; Slongo et al.,

2008). However, the model mostly dealt with survival curves with a shoulder phase and

would not be applicable to curves with strong tailing effect such as those in this

experiment. The Weibull frequency distribution model is another resource which is

firstly used for bacterial spores to solve the non-linear issue of the survival curves

(Mafart et al., 2002). This model has been recently applied to describe inactivation

kinetics of B. fulva ascospores in clarified apple juice by continuous pasteurisation

(Sant’Ana et al., 2009). Modifying these models so that they can be applied to multi-

phase survival curves of this experiment is out of the scope of the current project but it

deserves more research effort in the future.


This Chapter studied the thermal inactivation of B. fulva and B. nivea ascospores aged

4, 6, 8, 12 and 24 weeks. Two strains of each species were examined. The thermal

inactivation kinetics of the four tested strains strongly deviated from the conventional

linear logarithmic model as they comprised activation, shoulder, exponential reduction

and tailing phases. Thus, the D values were derived from the linear portion of the

inactivation curves

The heat resistance of the ascospores increased with ascospore age but decreased with

inactivation temperatures. Particularly, older ascospores had higher D values and their

survival curves exhibited longer shoulder and less steep reduction. The effects of age

were more prominent for B. fulva strains at inactivation temperatures below 87.5oC, and

for B. nivea strains at temperatures below 85oC. The D values increased up to 12 weeks

and changed little with further aging to 24 weeks.

65

The shoulder and tailing phases are characteristic for heat resistant structures like fungal

ascospores. The shoulder was attributed to the heat activation and aggregation of the

ascospores. The tailing resulted from the aggregation of ascospores and the presence of

an intrinsically more heat resistant population of ascospores. The complex inactivation

kinetics of Byssochlamys ascospores suggest more effort needs to be invested into

developing new models to better assess the heat resistance of these fungi.

Heat resistance (D values) of ascospores derived from ‘tailing’ populations were

generally similar after four successive generations. That means an ascospore population

was intrinsically more heat resistant than the rest of the population but the property was

not genetically transferred to progeny of the surviving colonies.

The age-induced heat resistance of Byssochlamys ascospores strongly suggests

physiological, chemical or structural developments in the cells during the aging process.

Some of these changes possibly involve developing ascospore layers and dehydration of

the central region inside the cells. Later chapters have built on this knowledge to link

heat resistant properties with other intrinsic factors of the ascospores.

66

Chapter 4 – Electron microscopy investigation of ultrastructure of Byssochlamys ascospores at two different ages and after different heating treatments

4.1 Introduction

In Chapter 3, the response of Byssochlamys ascospores to various heat processes was

reported. For some treatments, dormant ascospores were activated to a viable, culturable

state before inactivation to a non-culturable state was observed. In addition, ascospores

harvested from older cultures were more resistant to heat inactivation than those

harvested from younger cultures. Information on the ultrastructure of the ascospores

may assist in understanding the physiology and molecular bases of these changes.

Several studies have investigated the ultrastructure of ascospores of heat resistant

moulds using electron microscopy (Conner et al., 1987; Dijksterhuis et al., 2007).

Conner et al. (1987) used both scanning electron microscopy (SEM) and transmission

scanning microscopy (TEM) to examine differences in the ultrastructure of ascospores

of Neosartorya fischeri var. glaber from 11 and 25 day cultures, of which the older

ascospores were more heat resistant. The authors reported some differences in the

internal structures between the two types of ascospores. However, their findings were

limited by the relatively young age of the cultures examined. Using low temperature

SEM, Dijksterhuis et al. (2007) revealed the ultrastructure and highly viscous state of

ascospores of Talaromyces macrosporus. However, they did not examine development

of the structure during aging of the ascospores.

Only a few studies have used electron microscopy to examine the ultrastructure of

Byssochlamys ascospores. In particular, Partsch et al. (1969) used TEM to identify three

distinctive compartments in the ultrastructure of B. fulva ascospores, which included a

cell-wall, an extremely thick intermediate space and a highly dense cytoplasm in the

centre. The cell-wall of the ascospores was thicker than that of the more heat sensitive

conidia whereas the intermediate space and concentrated cytoplasm were quite unique

to the ascospores. Thus, the cell-wall and the intermediate space together possibly

protect the cytoplasm from thermal destruction. Using SEM, Hatcher et al., (1979)

showed increased thickness of the ascus wall of B. fulva asci when cultures aged

67

between 30 and 90 days were examined. The thick wall of the asci was another potential

heat resistance factor. The studies on the ultrastructure of Byssochlamys ascospores so

far are limited in that they concern only one species, a relatively young culture age (up

to 90 days) and have not examined the ascospores after exposure to different levels of

inactivation by heat treatments.

In this Chapter, SEM and TEM were applied to compare the ultrastructure of ascospores

of B. fulva and B. nivea after heat activation and inactivation, and dormant ascospores

harvested from young, 4 week cultures and older, 24 week cultures.


4.2.1 Fungal samples and heat treatment of ascospores

Ascospores were prepared from 4 and 24 week old cultures of B. fulva FRR 2299 and B.

nivea FRR 4421 as described Chapter 3. Ascospore suspensions of approximately 106 –

107 ascospores/mL were kept in sterile distilled water (SDW) at 4oC until they were

processed for electron microscopy.

Ascospores at three different physiological states were examined by electron

microscopy. These were dormant, heat activated and heat inactivated states. Dormant

ascospores did not receive any heat treatment after harvest and were prepared as

described above. Activated samples suspended in SDW were heated in a water bath at

75oC for 30 minutes while inactivated ascospores were obtained by heating in SDW in a

water bath at 95oC for 30 minutes. The heat activation treatment in SDW was estimated

from results of a previous study which showed maximal viable counts (Splittstoesser et

al., 1972). After the treatments, heated and dormant samples were cooled in ice and

sample preparation for EM was carried out within 2 hours of cooling. Only ascospores

prepared from 24 week old cultures were used for activation and inactivation analyses.

All sample preparation steps and image viewing were carried out at the Electron

Microscopy Unit, Macquarie University (North Ryde, NSW, Australia).

68

4.2.2 Reagents used for sample preparation for EM

Glutaraldehyde prepared from 50% stock solution (EM grade, distillation purified)

came from Electron Microscopy Sciences (Hatfield, PA, USA). Osmium tetroxide,

(OsO4), uranyl acetate and LR white resin (medium grade, C023) were supplied by

ProSciTech (QLD, Australia). Polyethylenimine was obtained from Sigma-Aldrich

(MO, USA). Pure ethanol (Univar) came from Ajax FineChem (NSW, Australia).

Ultrapure low melt agarose was from J.T. Baker Inc. (Phillipsburg, NJ, USA).

Pioloform resin was from Structure Probe® (West Chester, PA, USA).

4.2.3 Sample preparation

One millilitre of ascospore suspensions (approximately 107 cells/mL for B. fulva

FRR2299 and 108 cells/mL for B. nivea FRR4421) in 1.5 mL microcentrifuge tubes

were centrifuged at 4000g for 5 minutes and the supernatant was removed without

disturbing the pellet of sedimented ascospores. Ascospores were then resuspended in 1

mL of 6% glutaraldehyde for an overnight fixation at 4oC. Fixed samples were then

washed three times in phosphate buffer (pH 7.2) before they were processed for either

SEM or TEM.

4.2.3.1 Scanning electron microscopy (SEM)

Washed samples were post-fixed in freshly made 2% OsO4 for 2 hours at room

temperature (ca. 20oC). After sedimentation and washing in Milli Q water, samples

were dehydrated for 15 minute in each of a 50 – 70 – 80 – 90 – 95% graded ethanol

series and twice for 15 minutes in 100% ethanol. Dehydrated ascospores were quickly

dried in CO2 in an EMITECH K850 critical point dryer (EMITECH, Ashford, Kent,

UK), then sputter coated with gold in an EMITECH K550 gold sputter coater unit

(EMITECH) (Waugh et al., 2001). Prepared samples were viewed under a JEOL JSM-

6480 LA Analytical scanning electron microscope (JEOL, Sydney, NSW, Australia).

69

4.2.3.2 Transmission electron microscopy (TEM)

Glutaraldehyde-fixed and washed ascospore pellets were embedded into 1% low melt

agarose. The agar blocks containing the cell pellets were post fixed in 2% OsO4 for 2

hours at room temperature (20oC) and then immersed in 1 mL of 2% uranyl acetate (en

bloc staining) for 30 minutes at room temperature. After a washing step in Milli Q water

for 10 minutes, ascospores were dehydrated in a similar graded ethanol series as SEM

samples. They were then infiltrated with LR white resin by using incremental 3:1, 2:1,

1:1, 1:2, 1:3 pure ethanol:resin mixtures and lastly pure resin. Each infiltration step

involved adding 1 mL of the ethanol-resin mixture and leaving for 1 hour at room

temperature. The pure resin infiltration was carried out overnight at 4oC. Polymerisation

of pure resin to embed the infiltrated ascospores was performed overnight at 60 – 70oC.

Thin sections from resin blocks were prepared using a Reichert ultracut ultramicrotome

(Reichert, NY, USA), collected on copper grids (200 mesh) (ProSciTech) that were

coated with 0.3% Pioloform resin in chloroform. The sections were stained in 7.7%

aqueous uranyl acetate then in lead citrate (Reynolds’ reagent) (Reynolds, 1963), and

observed with a Philips LM 10 transmission electron microscope (Philips, NY, USA)

(McKeown et al., 1996).

4.2.4 Dimensions of the ascospores and image processing

Measurements on electron micrographs were done using the ImageJ software, which

was available at http://www.macbiophotonics.ca/imagej/index.htm. The longest and

shortest diameters were regarded as lengths (L) and widths (W), respectively, of the

ascospores. In order to perform statistical comparisons, the lengths and widths of at

least 40 ascospores on SEM images or 20 ascospores on TEM images were measured.

In particular, only TEM images that clearly showed all compartments of ascospores

were used. For each ascospore, the length and width each were measured five times and

average values were determined. The cross section areas (A) of ascospores were

calculated by the equation: A = ¼ πLW assuming ascospores as ellipsoid (Dijksterhuis

et al., 2002).

For TEM images, L, W and A were also determined for the ascospore section without

the cell-wall and for the central cytoplasm. Because the TEM sections cut through

different positions of the ascospores, the absolute measurements on TEM images could

http://www.macbiophotonics.ca/imagej/index.htm

70

not be directly compared between samples and compartments of the ascospores. Instead,

ratios of L, W and A of each part of the ascospores to those of the whole ascospores

were used for size comparisons. Statistical analyses were performed at the significance

level (α) of 0.05 and using the statistic package in Microsoft Excel.

4.3 Results

4.3.1 SEM study of Byssochlamys ascospores

Scanning electron micrographs of 4 and 24 week old dormant ascospores are shown in

Figures 4.1 and 4.2 for B. fulva FRR 2299 and B. nivea FRR 4421, respectively.

A) B)

C) D)

Figure 4.1. Scanning electron micrographs of B. fulva FRR 2299 dormant ascospores

and ascus at 4 (A, C) and 24 weeks (B, D). The coating layer is observed outside single

ascospores (thin arrow) and asci (thick arrow)

71

The B. fulva samples contained single ascospores, intact asci and some debris whereas

the B. nivea samples comprised mainly free ascospores. All ascospores were ellipsoidal

without surface ornamentation. Dormant ascospores of both strains did not show any

visually significant differences on the surfaces between 4 and 24 weeks. However, B.

fulva ascospores possessed a layer of extracellular material that created roughness on

the outside of the ascospore wall (Figures 4.1 A, B). A similar coating was also

observed to form the ascus wall (Figures 4.1 C, D) and acted to hold the ascospores

together inside the asci. This surface layer was absent in B. nivea single ascospores but

was occasionally observed in the intact asci if they were present (Figure 4.2).

A) B)

C) D)

Figure 4.2. Scanning electron micrographs of B. nivea FRR 4421 dormant ascospores at

4 (A, C) and 24 weeks (B, D)

The heat activation treatment (75oC for 30 minutes) did not cause any significant

changes on the surface or to the shape of 24 week old ascospores of the B. nivea strain

(Figure 4.3 B, D). Conversely, activated ascospores of the B. fulva strain exhibited a

72

shattered outer layer. A smoother and darker surface than the coating layer was revealed

underneath places where the coat was removed (Figure 4.3 A – C).

A) B)

C) D)

E)

Figure 4.3. Scanning electron micrographs of 24 week old B. fulva FRR 2299 (A, C, E)

and B. nivea FRR 4421 (B, D) ascospores activated at 75oC for 30 minutes; the coating

layer was removed to reveal a shiny smooth surface (thin arrow) for the B. fulva

ascospores but not those of B. nivea

73

Heat inactivation (95oC for 30 minutes), on the other hand, induced modifications on

the ascospore surface of both B. fulva and B. nivea strains. B. nivea ascospores were

affected more frequently than B. fulva ascospores. The structural changes observed on

the ascospores included depression in the centre of the cells, swellings and openings in

the outer wall (Figures 4.4 A – D). B. fulva ascospores, in particular, displayed a loss of

the coating layer (Figure 4.4 E) that was usually observed on dormant ascospores.

A) B)

C) D)

E)

Figure 4.4. Scanning micrographs of 24 week old B. fulva FRR 2299 (A, C, E) and B.

nivea FRR 4421 (B, D) ascospores inactivated at 95oC for 30 minutes; modifications

occurred on the surface of the ascospores, include depression in the centre (thin arrow),

holes (thick arrow) or removal of the coating layer (arrow head)

74

Size measurements of dormant and activated ascospores are given in Table 4.1. Because

the shapes of inactivated ascospores were severely distorted after the treatment, they

were not included in this measurement and quantitative comparison.

Dormant ascospores of the B. fulva strain were consistently larger than those of the B.

nivea strain with respect to length (L), width (W) and cross sectional area (A). Their

respective sizes (L x W) were 5.31 – 5.48 x 3.3 – 3.5 µm and 4.04 – 4.18 x 3.04 – 3.06

µm. The cross sectional areas of the ascospores were 14.31 – 14.8 µm2 for the B. fulva

strain and 9.7 – 10.01 µm2 for the B. nivea strain.

The 4 week dormant B. fulva ascospores had significantly smaller average width than

the corresponding 24 week samples whereas the 4 week dormant B. nivea ascospores

had significantly smaller average length than the corresponding 24 week samples. For

both strains, the cross sectional areas (A) of the ascospores were similar for the two

ages. Activation did not cause substantial changes in the cross sectional areas of the 24

week old samples. However, the activated ascospores were shorter than the dormant

cells in either diameter, depending on the strains, particularly the length for B. fulva and

the width for B. nivea (Table 4.1).

During the course of measurement, the sizes of both dormant and activated ascospores

were relatively variable over a wide range. Thus, histograms of all the measurements (L,

W and A) were analysed in order to reveal any distribution in sizes of ascospores

(Figure 4.5). Medians of the distributions of those size parameters were determined and

are presented in Table 4.1.

Most of size measurements could be regarded as normal distribution with the medians

similar to the mean values. However, a bimodal distribution was found for 24 week

dormant B. nivea ascospores although the median and mean values were similar (width,

Figure 4.5D). Some distributions are skewed and their medians are relatively different

from the mean values (Table 4.1). Specifically, the area median of 24 week dormant

ascospores of B. fulva was smaller than the mean value (Figure 4.5E). For the B. nivea

strain, the width and area medians of 24 week dormant ascospores were smaller than the

mean values (Figure 4.5 B, F).

75

Table 4.1. Dimensions of Byssochlamys ascospores at different ages and physiological states; measurements are expressed as average

values (mean) or medians of the distribution of the data sets

Strain Age (weeks) State N

Mean ± SD Median

Length (µm) Width (µm) Cross sectional area (µm2)

Length (µm)

Width (µm)

Cross sectional area (µm2)

B. fulva FRR 2299

4 Dormant 49 5.48 ± 0.38a 3.32 ± 0.17 14.31 ± 1.53 5.48 3.33 14.31

24 Dormant 47 5.31 ± 0.53a 3.54 ± 0.31b 14.80 ± 2.04 5.32 3.56 14.44*

24 Activated 66 5.11 ± 0.45 3.53 ± 026b 14.18 ± 1.70 5.13 3.51 14.20

B. nivea FRR 4421

4 Dormant 76 4.04 ± 0.37 3.06 ± 0.14d 9.70 ± 1.01 4.02 3.05 9.74

24 Dormant 100 4.18 ± 0.46c 3.04 ± 0.15d 10.01 ± 1.32 4.31* 3.06 10.20*

24 Activated 100 4.28 ± 0.31c 3.00 ± 0.16 10.09 ± 1.11 4.30 3.03 10.03

For the same strain, similar superscript letters indicate no significant difference (α = 0.05) between figures in either length, width or cross sectional area. Comparison

was not carried out between the two species because ascospores of B. fulva are generally larger than those of B. nivea (Pitt and Hocking, 2009)

N is the number of ascospores measured on scanning micrographs and used in statistical comparison

* Skewed distributions whose means and medians are greatly different

76

B. fulva FRR 2299 B. nivea FRR 4421

A) B)

C) D)

E) F)

Figure 4.5. Distributions of size measurements of dormant and activated ascospores of

B. fulva FRR 2299 and B. nivea FRR 4421. Size: length (A – B), width (C – D) and

cross sectional area (E – F). Physiological state: dormant, 4 weeks ( ); dormant, 24

weeks ( ); activated, 24 weeks ( )

77

4.3.2 TEM study of Byssochlamys ascospores

Transmission electron micrographs of ultrastructures of B. fulva and B. nivea ascospores

are shown in Figures 4.6 and 4.7, respectively. In the experiment, it was noted that the

fixing and embedding reagents did not enter dormant and activated ascospores

effectively resulting in loss of many cut sections.

Cross section images revealed three major layers in the ultrastructure of Byssochlamys

ascospores: a cell-wall (CW), a cytoplasm (CY), and an intermediate space (IMS)

sandwiched between the other two layers (Figures 4.6 A, B and 4.7A, B). The cell-wall

was a thick and unornamented mono-layer with medium to high electron density,

manifested as grey to dark coloured. The wall was relatively homogenous in structure

without any significant changes of density. There was occasionally a thin outer

membrane on the surface of the wall. Contrarily, the cytoplasm was highly electron

dense, extremely compact and occasionally exhibited a granular structure. It was located

approximately in the centre of the ascospores and had an ellipsoidal to irregular shape.

The rest of the ascospores was occupied by an exceptionally thick intermediate space.

The IMS was heterogeneous in the electron density but it mostly maintained a

translucent appearance.

The major difference between young and old dormant ascospores was observed inside

the IMS. The 24 week ascospores developed extra concentric layers around the

cytoplasm (Figures 4.6 A, B and 4.7 A, B). These layers could be clearly distinguished

by their optical densities. They were found in ascospores of both B. fulva and B. nivea.

However, the frequency of this feature was low since it was only observed in occasional

cut sections.

Activated ascospores had three layers similar to the dormant ascospores at 24 weeks,

including the sub-layers in the IMS. Importantly, there were sometimes discernable

organelles in the cytoplasm of the activated cells. The clear internal content was

observed in ascospores of both B. fulva and B. nivea strains (Figures 4.6D, 4.7D).

Damage to ascospores clearly occurred after heat inactivation since the cells were

significantly distorted and lost their original shapes (Figures 4.6E, 4.7E). The changes

to ascospore structures were relatively random on the cell-wall. The cytoplasm of

inactivated ascospores became less dense, which allowed the differentiation of internal

materials, such as mitochondria or ribosomes (Figure 4.6F, 4.7F).

78

A) B)

C) D)

E) F)

Figure 4.6. Ultrastructures of ascospores of B. fulva FRR 2299. Sample: 4 week,


(E – F); Ultrastructure components: cell-wall (CW), intermediate space (IMS),

cytoplasm (CY); Special features: the thin outer membrane (thin arrow), sub-layers in

the IMS of 24 week ascospores (arrow head), cytoplasmic organelles such as

mitochondria (thick arrow). Scale bar: 1 µm (A – C and E), 0.5 µm (D and F)

CW

IMS

CY

79

A) B)

C) D)

E) F)

Figure 4.7. Ultrastructures of ascospores of B. nivea FRR 4421. Sample: 4 week,


(E – F); Ultrastructure components: cell-wall (CW), intermediate space (IMS),

cytoplasm (CY); Special features: the thin outer membrane (thin arrow), sub-layers in

the IMS (arrow head), cytoplasmic organelles such as mitochondria (thick arrow)

CW

IMS

CY

80

The size measurements of each layer of the ascospores were variable depending on

angles and depths of the sections cut through the cells. Hence, the area-to-area ratios of

each compartment to the whole ascospore were used for the quantitative analysis instead

of the absolute measurements. For B. fulva ascospores, the IMS occupied approximately

70% of the cross sectional area, hence of the total volume, of the cells. The cytoplasm

was about 21 – 24% of the volume and the cell-wall accounted for the least proportion,

10 – 13% (Figure 4.8). Ascospores of B. nivea had a slightly smaller volume of the IMS

(61 – 64%) while the cytoplasm (23 – 25%) and the cell-wall (13 – 15%) both occupied

greater volume percentages than those of B. fulva (Figure 4.9).

The relative proportions for the cell-wall, IMS and cytoplasm components were similar

in dormant ascospores from either 4 or 24 week cultures for both B. fulva and B. nivea

(Figures 4.8A and 4.9A). However, activated ascospores of the B. fulva strain had

significantly larger cell-wall/whole cell ratios and smaller IMS/whole cell ratios than

both 4 and 24 week dormant ascospores. Meanwhile, the cytoplasm/whole cell ratios of

activated ascospores were only significantly larger than those of 24 week dormant

samples (Figure 4.8A). For the B. nivea strain, activated ascospores only had

significantly larger cell-wall/whole cell ratios than the 24 week dormant cells. Other

ratios were similar across ages and physiological states (Figure 4.9A).

An additional comparison was performed on the largest 25% of the length and width

measurements. These larger figures were more likely to be from sections cut through the

centre of the ascospores and therefore, they were closer to the actual ascospore

dimension. Using these numbers for comparison potentially reduced the complications

of variable cutting sections. The second comparison shows that the size ratios of the B.

nivea strain were similar irrespective of age or physiological states (Figure 4.9B).

Dormant ascospores of B. fulva also had similar size ratios irrespective of ages.

Activated ascospores of the B. fulva strain had significantly larger cell-wall/whole cell

ratios at both ages and smaller IMS/whole cell ratios at 24 weeks then the dormant

samples. The cytoplasm/whole cell ratios of B. fulva ascospores were not statistically

different between ages or across heat treatments (Figure 4.8B).

81

A)

B)

Figure 4.8. Area-to-area ratios of different compartments of B. fulva FRR 2299

ascospores using the whole data set (A) or the top 25% based on length and width (B).

Legend: 4 weeks, dormant (A: , B: ); 24 weeks, dormant (A: , B: ); 24 weeks,

activated (A: , B: ); Similar letters indicate no significant differences among three

samples in each category

b b

aa

aa

ccd

d

dd

d

bbc

c

82

A)

B)

Figure 4.9. Area ratios of different compartments of B. nivea FRR 4421 using the

whole data set (A) or the top 25% based on length and width (B). Legend: dormant, 4

weeks (A: , B: ); dormant, 24 week (A: , B: ); activated, 24 week (A: , B: ).

Similar letters indicate no significant difference among three samples in each category

c c c

aa a

b b b

cc c

dd d

abb a

83

4.4 Discussion

Dormant ascospores of B. fulva and B. nivea were examined by electron microscopy

(EM) for differences in their ultrastructure between 4 and 24 weeks. The ultrastructure

of 24 week ascospores was also inspected after heat activation and heat inactivation.

This study expands the previous findings of Partsch et al. (1969) by examining

qualitatively and quantitatively the ascospores at conditions other than the dormant state

at a single age. Both SEM and TEM methods revealed interesting results that could be

attributed to species, ascospore age and effects of the heat treatments used.

4.4.1 Multilayered ultrastructure and heat resistance of Byssochlamys

ascospores

The scanning electron micrographs displayed familiar ellipsoidal and unornamented

ascospores whose sizes were within commonly reported ranges for both species

examined (Pitt and Hocking, 2009). A notable observation was of the coating layer

found only on ascospores of the B. fulva strain. This layer was similar to, and therefore

possibly was a part of the ascus wall of B. fulva and B. nivea asci. For B. fulva, the

coating was particularly resistant since it was only removed after mechanical shaking

with glass beads (section 4.2.1) and thermal treatments. This outer layer may be similar

to that of the ascospore wall of Daldinia concentrica, which has been reported to be

removed by autoclaving in 4.5% potassium hydroxide (Beckett, 1976).

The chemical composition and functions of the outer coating of Byssochlamys

ascospores have not been researched thoroughly. This coating layer is likely to contain

chitin, chitosan and glucan as found in ascospore walls of many fungal species

(Bartnicki-Garcia, 1968; Ruiz-Herrera and Ortiz-Castellanos, 2010). It can function as

an interspore bridge to keep the ascus intact and to hold individual spores together

(Coluccio and Neiman, 2004). Considering its ability to resist mechanical and thermal

stresses, one can conclude that the coating provides the ascospores with a degree of

protection against environmental insults including heat (Coluccio et al., 2008; Hatcher

et al., 1979). The coating layer could be a heat resistance factor that renders B. fulva

ascospores more heat resistant than B. nivea as demonstrated in Chapter 3 (Section 3.3.1

and 3.3.2)

84

TEM revealed the ultrastructure of Byssochlamys ascospores that is characterised by

three major layers, namely cell-wall, intermediate space and cytoplasm. This

compartmentalised arrangement is consistent with previous results for B. fulva

ascospores (Partsch et al., 1969; Put and Kruiswijk, 1964). The names used in the

present study for these layers were kept consistent with the original terminology by

Partsch et al. (1969) to enable comparisons with the literature.

The cell-wall and the IMS were relatively resistant, hindering the penetration of

fixatives into the ascospores. Thus, they are possibly also protective factors against heat

damage. In fact, the spore wall is generally regarded as the physical barrier which

maintains the dormancy and provides resistance against physical and chemical

challenges (Coluccio et al., 2008, Sussman and Douhit, 1973). The coating layer of

Neurospora tetrasperma ascospores was also impermeable to EM fixing reagents

(Lowry and Sussman, 1968). The cell-wall of Saccharomyces cerevisiae ascospores

exhibited low permeability to movement of proteins (Suda et al., 2009). Insufficient

penetration of fixatives through the spore coats of Clostridium endospores also limited

structural details that could be visualised in the mature cells (Hoeniger and Headley,

1968; Stevenson et al., 1972).

The cytoplasm, in particular, was highly compact and electron dense. These properties

are consistent with a viscous and low moisture environment (Dijksterhuis et al., 2007).

These properties could manifest as a special structure called a glassy state inside the

ascospores (Stecchini et al., 2006). The presence of low moisture, glassy state is a

potential mechanism for elevated thermotolerance whereby it protects intracellular

organelles and essential proteins from denaturation (Sunde et al., 2009). A study

presenting evidence for a glassy state in Byssochlamys ascospores will be detailed in

Chapter 5.

The multilayered ultrastructure is relatively common in ascospores of other heat

resistant moulds. However, other species have been shown to have more structural

layers than Byssochlamys ascospores, depending on the microscopic techniques used.

With TEM and barium permanganate stain, the wall of dormant ascospores of N.

fischeri showed four distinct layers (Conner et al., 1987), of which the third one (W3)

was visually equivalent to the IMS of Byssochlamys ascospores. The plasma membrane

outside the cytoplasm of N. fischeri ascospores was also clearly distinguished. With low

85

temperature TEM, dormant ascospores of T. macrosporus showed the same three basic

compartments as seen in those of Byssochlamys. Upon germination, they exhibited two

additional fine layers that were next to the exterior side of the cytoplasm (Dijksterhuis

et al., 2007). Therefore, it may be possible to reveal finer details of the ultrastructure of

Byssochlamys ascospores if different microscopic or staining protocols are used in the

future.

4.4.2 Effects of heat treatments on the ultrastructure of Byssochlamys

ascospores

Ultrastructure of the ascospores was significantly modified by heat and changes

depended on the severity of applied treatments. Heat activation only removed the

coating layer of the ascospores. More detrimental damages to the ascospore structure

occurred with heat inactivation. The corroboration between scanning and transmission

images of inactivated ascospores suggests that heating probably affects the cell-wall and

the IMS first. This is in accordance with the design of ascospores where these two

layers are located outside and serve to protect the cytoplasm.

At the same time the heat treatments altered conditions of the cytoplasm so that its

content became discernable. This change correlated with a loss of viscosity inside

ascospores when they moved from activated to germinating or inactivated states

(Dijksterhuis et al., 2007). Interestingly, many of the visible organelles had a globular

shape and cristae that were similar to mitochondria found in Neurospora tetrasperma

ascospores (Lowry and Sussman, 1968). The presence of mitochondria in ascospores

prior to and following germination is common among ascomycete members, possibly as

a preparation strategy for outgrowth (Murray and Hynes, 2010; Niyo et al., 1986).

Activated ascospores had larger cell-wall area ratios but smaller IMS area ratios than

the dormant cells. This was probably due to the rehydration of activated ascospores that

changed the relative size proportion of each compartments. However, this theory is not

in agreement with the results showing that the cross sectional areas and cytoplasm area

ratios of the activated ascospores were similar to those of dormant ascospores. This may

be a limitation of using relative ratios instead of absolute data in quantitative

comparison.

86

4.4.3 Effects of age on the ultrastructure of Byssochlamys ascospores

The major difference between the 4 and 24 week ascospores was that the older samples

occasionally had sub-layers in the IMS. These additional layers had variable electron

densities and, therefore, different structural arrangements. The development of these

layers could result from post-sporulation modifications occurring inside the IMS when

ascospores matured to achieve a stable dormant state (Bomar, 1962). They may

comprise cross-linked carbohydrates and proteins (Banner et al., 1987), forming a

structure analogous to the peptidoglycan network making up the cortex of bacterial

spores (Atrih and Foster, 2002). The dehydration of the IMS and cytoplasm could then

create pressure inside the ascospores and compress similar networks into layers (Warth,

1985). As time can be a limiting factor, aging enables ascospores to undergo this

internal maturation and structural modification (Conner and Beuchat, 1987). Therefore,

the presence of several layers in the IMS of the matured ascospores was a possible

indicator of a higher thermo-tolerance.

The increasing complexity of ultrastructure of ascospores during aging has been

reported earlier in ascospores of N. fischeri. More defined layers in the cell-wall and

plasma membrane were observed in 25 day old, heat resistant ascospores, but not in 11

day old samples (Conner et al., 1987).

Older ascospores of Byssochlamys species were significantly larger than the younger

ones only in either one of the two diameters. The increase sizes could assist the

ultrastructural development of the older ascospores mentioned above. However, this

change was contradictory to the inverse relationship of size and heat resistance in

endospores of Clostridium species (Beaman et al., 1982; Mah et al., 2008; Novak et al.,

2003; Orsburn et al., 2008).

Additionally, Byssochlamys ascospores had similar area-to-area ratios at both tested

ages. This result was opposed to a trend in bacterial spores where spore coat and cortex

became thicker with heat resistance (Mah et al., 2008). The use of ratios may contribute

to the insignificance in the statistical comparisons. Additionally, Byssochlamys cultures

were grown for a relatively long time (4 and 24 weeks) compared with bacterial

samples. Thus, even the youngest ascospores at 4 weeks may have been already well-

developed and changed their sizes minimally when further aging.

87

4.4.4 Recommendations for future research using electron microscopy

The en bloc staining technique could inadvertently influence the preparation of TEM

samples. It is acknowledged that the method offered a convenient handling of

concentrated ascospores without losing materials after several washing steps. However,

because ascospores were pelletised and embedded into an agar block, this treatment

could hinder a uniform penetration of fixatives and resins into individual ascospores

(Dykstra and Reuss, 2002). It could constitute an extra complication, considering that

the ascospore wall is already highly impermeable.

Results of the present study open up avenues for future research on the relationship

between ultrastructure and heat resistance. Firstly, using different fixing protocols and a

number of stains will enhance quality of transmission images and reveal more details of

the ultrastructure (Conner et al., 1987). Secondly, instead of performing the fixing and

resin infiltration steps in a static condition as done in this experiment, a rotary shaker

can be used to facilitate the penetration of reagents into the ascospores (Kushida, 1969;

Glauert, 1974). Finally, although ultrastructural changes are not always visible, there

may be differences in chemical composition of the ascospores with heat treatments and

age. Chemical composition of the coats, cortex and protoplasm of Bacillus and

Clostridium endospores have been shown to affect their thermo-tolerance (Atrih and

Foster, 2002; Orsburn et al., 2008). Thus, chemical analysis of Byssochlamys

ascospores and each compartment may reveal subtle changes.


SEM and TEM were used in this experiment to study ultrastructure of Byssochlamys

ascospores, and changes brought about by age (i.e. heat resistance) and heat treatment.

There are interesting findings that link all the aspects being examined.

The ultrastructure of Byssochlamys ascospores consists of three distinct layers, namely

cell-wall, intermediate space and cytoplasm. There is also a coating layer outside the

ascospores which helps to hold them together in the asci. The cell-wall is relatively

impermeable to penetration of fixatives. The intermediate space is remarkably thick and

along with the cell-wall, is proposed to maintain the resistance of ascospores against

environmental insults. Sub-layers are occasionally developed in this space, which is the

88

major distinction between young, heat sensitive and old, heat resistant ascospores. The

mechanism of development of those sub-layers is unknown but may be a result of

dehydration of the internal content of the ascospores.

The cytoplasm has a high electron density during dormancy but after heat treatment

becomes less dark with discernable organelles. This phenomenon has been observed in

germinating ascospores of other fungi and it is a result of a viscosity drop and

disappearance of a glass structure. A group of organelles which has internal crossings

similar to the cristae of mitochondria frequently appears in the cytoplasm of heat treated

ascospores.

Heating also causes significant damage to ascospore ultrastructure. The coating layer is

partially removed during heat activation and dramatically lost on inactivated ascospores.

Moreover, inactivated ascospores have severely distorted shapes and modifications on

the cell-wall. These results suggest that the cell-wall and IMS may provide protection

for the cytoplasm against thermal stress.

Old dormant ascospores are larger than the younger ones in one of the two diameters

but their area ratios are similar. The larger size may accommodate the structural

development of the ascospores during aging. However, it contrasts the age-related

decreasing trend of size of bacterial spores, which probably helps the dehydration of

intracellular content and enhances their heat tolerance. Activated ascospores are also

smaller than dormant ones in one of the two diameters. Moreover, they have

significantly higher cell-wall and lower IMS area ratios. Activated ascospores are likely

to be rehydrated and may change in size but, based on the results of the EM

investigation, these findings are inconclusive.

This is the first time Byssochlamys ascospores have been subjects of significant

investigation using electron microscopy. The knowledge gained improves the current

understanding of ultrastructure of ascospores, and the connection to heat resistance

properties and mechanisms of thermal activation or inactivation. The study also points

out possible pitfalls of applying electron microscopy to the study of heat resistant

ascospores and potential directions for future research.

89

Chapter 5 – Application of Differential Scanning Calorimetry (DSC) to examine the arrangement of material in Byssochlamys ascospores

5.1 Introduction

In Chapter 4, ascospores of B. fulva and B. nivea were examined by electron

microscopy, which showed a dense and viscous cytoplasm in the centre of the

ascospores. Cellular organelles in the cytoplasm became discernable, possibly as a

result of rehydration of this region following heat treatments. The results suggested that

the intracellular environment of the ascospores may be protective for the internal

content. Thus, understanding the state of materials inside the ascospores may elucidate a

heat resistance factor of Byssochlamys species.

The cytoplasm of Byssochlamys ascospores is likely to be a dehydrated environment,

which has been observed in ascospores of Talaromyces macrospores (Dijksterhuis et

al., 2007). Byssochlamys ascospores may also contain a high concentration of trehalose,

a non-reducing sugar found abundantly in fungal spores (Thevelein, 1984). The low

moisture and high trehalose contents are often related to the anhydrobiosis phenomenon

and a special state of material called a glassy state (Aguilera and Karel, 1997). In that

condition, molecules are arranged in a quasi solid structure but maintain certain degree

of flexibility of the fluid state (Leopold et al., 1994). The dual property of the glassy

state enables the stabilisation of molecular structures and prevents denaturation.

Therefore, the glassy state has been considered to be a tentative mechanism of thermo-

tolerance of heat resistant moulds (Crowe et al., 1998; Dijksterhuis, 2007).

Differential scanning calorimetry (DSC) is often the chosen method to study glassy

states and other temperature-dependent structural changes in materials (Roos and Karel,

1990). Originally developed for polymers (Watson and O’Neill, 1966) and later

biomaterials (Kalichevsky et al., 1992; Spink, 2008; Sturtevant, 1987), DSC has been

used in the study of Bacillus endospores and has produced evidence to link a glassy

state and their heat resistance property (Katayama et al., 2008; Leuschner and Lillford,

2003; Sapru and Labuza, 1993a, b). In those works, the glassy state was characterised

by an endothermic peak at variable temperatures depending on samples and

90

experimental protocols. To date, the application of DSC in fungal research is very

sparse and mostly unrelated to the heat resistant property (Van Cauwelaert and Verbeke,

1979; Verbeke and Van Laere, 1982).

No studies so far have investigated the arrangement of materials, particularly structure

similar to the glassy state, in Byssochlamys ascospores. Thus, it is relevant to study

ultrastructure of the ascospores from the glassy state point of view. Findings will not

only complement the electron microscopic results but may also identify an essential

heat resistance factor of Byssochlamys ascospores.

This Chapter applies DSC to investigate changes in the structural configurations of

materials in the ascospores of B. fulva and B. nivea. Two strains of each species were

examined after heat activation and heat inactivation, and dormant ascospores harvested

from young, 6 week cultures and older, 24 week cultures. This study also aims to find

evidence of a glassy state in Byssochlamys ascospores.


5.2.1 Fungal strains, ascospore harvest and heat treatment

Four Byssochlamys strains were used in this study, comprising B. fulva FRR 2299, B.

fulva FRR 2785, B. nivea FRR 4421 and B. nivea FRR 6002. The fungal cultures were

grown on MEA for 6 and 24 weeks at 30oC. Ascospores were harvested according to

the protocol described in Chapter 3. The harvesting procedure was briefly described as

follows: mould biomass was gently scraped off the agar surface, free ascospores were

released by vigorously shaking with glass beads of diameter less than 106 µm (Sigma-

Aldrich), samples were then filtered through sterile glass wool to remove mycelium and

cell debris, and the filtrate that mostly contained free ascospores and asci was washed

three time with SDW by repeated centrifugation. Harvested ascospores were stored in

SDW at 4oC until analysis.

Dormant ascospores did not receive any heat treatments prior to DSC analysis.

Activated ascospores were prepared by heating dormant ascospore solutions in SDW at

75oC for 30 minutes (Splittstoesser et al., 1972). Inactivated ascospores were prepared

in a similar way but heat treatment was done at 95oC for 30 minutes. All heated samples

91

were cooled down to room temperature (ca. 22oC) before being prepared for DSC

analysis.

5.2.2 Preparation of ascospore samples for DSC

The ‘dry’ method of preparing samples for DSC analysis was modified from a

previously described procedure (Stecchini et al., 2006). Approximately 1.5 mL of

dormant or heat treated ascospore solutions was centrifuged at 10,000 rpm for 5

minutes. Supernatant was decanted but a small amount of liquid was left to make a

dense suspension. About 30 µL of the dense ascospore solution was pipetted into 40 µL

DSC aluminium crucibles (ME – 26763, Mettler Toledo, VIC, Australia) which had

been weighed previously with their lids. All DSC crucibles were then dried over

granulated silica gel overnight in a desiccator at room temperature until constant

weights were achieved. At the end of the drying process, the pans were hermetically

sealed and weighed to determine dry sample weight. Post-DSC weights of closed pans

with samples were also measured to determine any loss of material. All the weights

were recorded up to two decimal places in milligrams.

5.2.3 Differential scanning calorimetry

All DSC analyses were performed in a Mettler Toledo DSC 822e platform (Mettler

Toledo) equipped with a sample robot TSO 801RO (Mettler Toledo). Nitrogen gas was

used as furnace and purge gases and liquid nitrogen was used for cooling. Heat flow

was calibrated against indium (ME-119442, Mettler Toledo) at the beginning of each

experiment day. An empty 40 µL aluminium crucible was used for reference.

5.2.3.1 Selection of scanning rates

Screening for an optimal scanning rate was conducted on the 6 week dormant

ascospores of B. fulva FRR 2299. Samples used for the screening test were prepared by

the ‘dry’ method. Four heating rates, 2.5 Kelvin per minute (Kmin-1), 5 Kmin-1, 7.5

Kmin-1 and 10 Kmin-1, were tested for potential to reveal reproducible and meaningful

events. The analysing program included alternating two heating and two cooling

segments between 10oC and 120oC (Table 5.1). All scans were performed in duplicate.

92

5.2.3.2 DSC analysis of ascospore samples

Ascospore suspensions prepared under different conditions were submitted for DSC

analysis. Scanning program comprised alternating two heating and two cooling

segments between 5 and 125oC (Table 5.1). The two heating steps (segments I and III)

would be referred to as ‘initial scan’ and ‘immediate rescan’ in the rest of the Chapter.

Scanned samples were then kept in desiccators at room temperature for a week and re-

analysed using the same protocol. All measurements were technically repeated twice

using biological duplicated ascospore crops. Viewing data and analysis of thermograms

were performed using Mettler STARe software (Mettler Toledo).

Table 5.1. DSC scanning programs for analysis of Byssochlamys ascospores

Experiment Scanning program

Segment Scanning range Heating or cooling rate*

Optimal scanning

rate test

I 10 – 120oC 2.5 Kmin-1; 5 Kmin-1; 7.5

Kmin-1; 10 Kmin-1

II 120 – 10oC – 10 Kmin-1

III 10 – 120oC 2.5 Kmin-1; 5 Kmin-1; 7.5

Kmin-1; 10 Kmin-1

IV 120 – 10oC – 10 Kmin-1

Analysis of

Byssochlamys

ascospores

I 5 – 125oC 10 Kmin-1

II 125 – 5oC – 10 Kmin-1

III 5 – 125oC 10 Kmin-1

IV 125 – 25oC – 10 Kmin-1

* Positive rates denote heating segments, negative rates denote cooling segments

5.2.3.3 Other comparative experiments

Two other protocols for preparing ascospore samples, referred to as the ‘wet’ method

(Belliveau et al., 1992) and the ‘open lid’ method (Stecchini et al., 2006) in this

Chapter, were compared against the ‘dry’ method for their effects on DSC results.

These two methods were different from the ‘dry’ method in that no drying step was

involved before sealing DSC pans. For the “wet” preparation, approximately 10 – 15 µL

93

of dense aqueous ascospore solution were pipetted into pre-weighed 40 µL DSC

aluminium crucibles which were then hermetically sealed and immediately analysed.

For the “open lid” method, orifices were made in the lid before crucibles were sealed

and submitted to the DSC instrument.

A vacuum drying method was also compared for any effects on the DSC results.

Dormant ascospores from 6 week cultures were dried in a desiccator under vacuum

instead of at normal atmosphere. Crucibles were then hermetically sealed and analysed

in the same manner as samples of the ‘dry’ method.

5.3 Results

DSC results of Byssochlamys ascospores were recorded on a Cartesian coordinate plane,

called a thermogram, which depicted heat flow through a sample with time. Thermal

transitions were recorded as first-order transitions, or peaks, and second-order

transitions, or step changes. A peak had a similar baseline on both sides whereas the

baseline of a step change was different prior to and after the event. Peaks and step

changes were characterised by peak temperatures (Tp) and mid-point temperatures

(Tm), respectively.

5.3.1 Screening for the optimal scanning rate

In this experiment, the optimal scanning rate which would reveal clearly thermal

transitions in both initial scans and immediate rescans was determined. The DSC

analyses of dormant ascospores of B. fulva at the four scanning rates are presented in

Figure 5.1. Events being recorded had relatively small total enthalpy and the integrated

energy was often less than 5 mW in most experiments. In addition, the four scanning

rates showed a similar pattern of events, although the transitions happened at slightly

different temperatures.

The general event pattern contained a major endothermic peak at 45 – 60oC in the initial

scans that was reversible at relatively similar temperatures in the immediate rescans.

The peak temperatures were variable with the scanning rates (Table 5.2). The scanning

rate of 10 Kmin-1 exhibited more consistent peaks than other rates in both rounds of

scanning (Figure 5.1D).

94

Figure 5.1. DSC thermograms of ascospores of B. fulva FRR 2299 scanned at four

heating rates: 2.5 Kmin-1 (A), 5 Kmin-1 (B), 7.5 Kmin-1 (C) and 10 Kmin-1 (D); Samples

were scanned twice (unbroken line – initial scan; dashed line – immediate rescan);

Exothermic heat flow is upwards and measured in a relative scale (2 mW)

The peak height, or peak strength, that was indicated by the peak enthalpy, increased

with scanning rates (Table 5.2). The fastest rate (10 Kmin-1) also gave the most distinct

peak. The lower rates, such as 2.5 Kmin-1 and 5 Kmin-1, showed relatively weak

transitions. These weak transitions would be more sensitive to variations and could be

easily masked by more intense events or by the baseline. It can be concluded from these

results that the scanning rate of 10 Kmin-1 revealed more and consistent and stronger

thermal transitions than other tested rates and therefore, it was the optimal scanning rate

for subsequent DSC analyses of Byssochlamys ascospores.

A

B

C

D

A

B

C

D

95

Table 5.2. Peak temperatures and enthalpy at different scanning rates

Scanning rate (Kmin-1)

Peak temperature (oC) Peak enthalpy (Wg-1 of samples)^

Initial scan Immediate

rescan Initial scan

Immediate rescan

2.5 56.7 48.9 -0.01331 -0.01441

5 46.3 47.0 -0.01976 -0.03750

7.5 46.3 45.3 -0.14 -0.16

10 53.1 54.2 -0.22 -0.24

^ Negative energy indicates endothermic peaks

5.3.2 DSC analysis of Byssochlamys ascospores

Representative thermograms of ascospores of four tested strains are presented in Figures

5.2 – 5.3 (B. fulva strains) and Figures 5.4 – 5.5 (B. nivea strains). These thermograms

include DSC results of 6 and 24 week dormant ascospores, and ascospores after heat

activation (75oC for 30 minutes) and heat inactivation (95oC for 30 minutes). For clear

presentation, only thermograms of the ascospores after the first round of drying were

shown. The rescans of the same samples after one week conditioning are presented in

Appendices 5.1 – 5.4.

In general, for each strain, the transition patterns and temperatures (Tp and Tm) were

variable depending on ages and heat treatments applied to the ascospores. The DSC

results also varied significantly between B. fulva and B. nivea species but slightly within

each species. DSC features of Byssochlamys ascospores with respect to strains, species,

ages and applied heat treatments will be examined in detail in the sections following

Figures 5.2 – 5.5.

96

6 w

eeks

24 w

eeks



for 30 minutes; Scan: unbroken line – initial scan, dashed line – immediate rescan.

Transition: endothermic peak (broken arrow) and step change (arrow head); Exothermic

heat flow is upwards and measured in relative scales (5 mW)

A

B

C

A

B

C

A

A

B

B

C

C

97

6 w

eeks

24 w

eeks



for 30 minutes; Scan: unbroken line – initial scan, dash line – immediate rescan;

Transitions: endothermic peak (arrow) and step change (arrow head); Exothermic heat

flow is upwards and measured in relative scales (5 mW)

A

A

B

B

C

C

A

A

B

B

C

C

98

6 w

eeks

24 w

eeks



for 30 minutes; Scan: unbroken line – initial scan, dashed line – immediate rescan;

Transition: uneven baseline (arrow), step change (arrow head); Exothermic heat flow is

upwards and measured in relative scales (5 mW)

A

A

B

B

C

C

A

A

B

B

C

C

99

6 w

eeks

24 w

eeks



for 30 minutes; Scan: unbroken line – initial scan, dashed line – immediate rescan;

Transition: uneven baseline (arrow), step change (arrow head). Exothermic heat flow is


A

A

B

B

C

C

A

A

B

B

C

C

100

5.3.2.1 Comparison of dormant ascospores between ages, species and strains

Temperatures (Tp and Tm) of the major transitions exhibited by dormant ascospores at

6 and 24 weeks are tabulated in Table 5.3. They are complementary to the graphical

presentation of DSC data of dormant samples in Figures 5.2 – 5.5.

For B. fulva, the dormant ascospores showed a major endothermic peak at 56 – 59oC at

6 weeks and at slightly higher temperatures, 56 – 60oC, at 24 weeks. This peak was

consistently reversible at similar temperatures (Figures 5.2A and 5.3A). The Tp was

slightly higher for B. fulva FRR 2299 than B. fulva FRR 2785, which were 56 – 58.6oC

and 55 – 57.9oC, respectively (Table 5.3).

In addition, an endothermic step change preceded the major peak but its appearance was

variable between strains and ages. Dormant ascospores of B. fulva FRR 2299 exhibited

the step change at lower Tm than those of B. fulva FRR 2785, which were 28.7 – 48.2oC

and 42.4 – 50.8oC, respectively, regardless of ages and rounds of scanning. However,

the transition displayed by B. fulva FRR 2785 was recovered less consistently than B.

fulva FRR 2299 at both ages. Moreover, the step change displayed by 24 week

ascospores was generally more slowly recovered at higher Tm than the 6 week samples.

In contrast to B. fulva, B. nivea strains had completely different thermograms which

comprised only an endothermic peak with a lower returning baseline. The peak uneven

baseline could result from an overlapping of a peak and a step change, and was

characterised by a Tp in this experiment. At 6 weeks, this peak was at 44 – 49oC and

replaced only by a step change in the rescans (Figures 5.4A and 5.5A, 6 weeks).

However, it was slowly reversible at higher Tp = 43 – 51.8oC after one week for 24

week ascospores (Figures 5.4A and 5.5A, 24 weeks). At both ages, B. nivea FRR 6002

had higher peak temperature Tp than B. nivea FRR 4421, being 43.4 – 51.8oC and 42.4

– 45.6oC, respectively (Table 5.3).

The step changes of B. nivea ascospores consistently appeared in the immediate rescans.

They were more prominent and at higher temperatures at 24 weeks (Tm = 44 – 48.4oC)

than 6 weeks (Tm = 28 – 33.6oC) (Table 5.3). One week conditioning retained the

reversibility of the step changes but increased the Tm in most cases. Ascospores of B.

nivea FRR 6002 (Tm = 29 – 49oC) again showed slightly higher Tm than those of B.

nivea FRR 4421 (Tm = 28 – 47.4oC).

101

Table 5.3. Temperatures of major transitions (peaks and step changes) from the DSC analysis of dormant ascospores of Byssochlamys species

Strain Age (weeks)

Transition temperatures (oC) Peak temperature (Tp) Mid-point temperature (Tm)

Fresh sample One week sample Fresh sample One week sample Initial scan Immediate

rescan First scan Rescan Initial scan Immediate rescan First scan Rescan

B. fulva FRR 2299

6 56.0 – 57.5 56.0 – 58.4 56 – 57.9 56 – 58.6 40 – 47.2 34.2 – 35.7 44 – 46.5 28.7 – 32.9

24 56.0 – 59.5 57.0 – 60.0 56.0 – 59.0 57.6 – 59.9 35.6 – 39.6 46.4 – 48.3 - 44.6 – 48.2 31.6 – 34.5

B. fulva FRR 2785

6 56.2 – 57.9 56.3 – 57.7 55.0 – 58.8 56.5 – 57.6 46.2 – 47.6 33.7 46.0 36.2 – 38.0

24 57.4 – 58.7 58.4 – 59.0 56.1 – 58.4 58.4 – 59.2 42.4 – 50.8 - - -

B. nivea FRR 4421

6 45.4 - 45.6 - - - - 28.0 – 29.9 46.5 – 47.8 47.0 – 47.4

24 42.4 – 43.5 60.3 – 62.1 - 43.0 – 45.0

54.0 – 59.0 - - 45.8 – 46.4 - 40.3 – 52.1

B. nivea FRR 6002

6 44.0 – 49.0 - - - - 29 – 33.6 47 – 47.5 48.4 – 49.0

24 43.3 – 45.7 51.8 – 57.9 - 44.5 – 51.8 - - 44.0 – 48.4 - 44.2 – 47.5

- denotes no transition being observed

Italic denotes inconsistent transition being absent in one of the duplicates

102

Thermograms of the cooling segments were also different between species (Figure 5.6).

For B. fulva strains, an exothermal event consistently occurred at peak temperatures of

about 52 – 54oC (Figure 5.6A, B). This event could correspond with the endothermic

peak observed on the heating curves of this species. However, no exothermic peak was

recorded for any samples of B. nivea strains after the initial heating. This coincided with

the loss of the uneven peak on their heating thermograms (Figure 5.6C, D).

Figure 5.6. The cooling segments during DSC analysis of ascospores of B. fulva FRR

2299 (A), B. fulva FRR 2785 (B), B. nivea FRR 4421 (C) and B. nivea FRR 6002 (D);

Transition: the exothermic peak at 52 – 54oC for B. fulva but not for B. nivea (arrow);

Exothermic heat flow is upwards and measured in a relative scale (5 mW)

5.3.2.2 DSC comparisons of ascospores after heat treatments

Transition temperatures (Tp and Tm) of heat activated and heat inactivated ascospores

are given in Tables 5.4 and 5.5, respectively. For B. fulva, heat treatments did not affect

the reversibility of the major endothermic peak but delayed the recovery of the step

change by a week (Figures 5.2B, C and 5.3B, C). Compared with dormant samples

mentioned earlier, the heat activation did not change Tp and Tm ranges (Table 5.4)

whereas the heat inactivation reduced slightly those temperatures, Tp = 54 – 59oC and

A

B

C

D

103

Tm = 40 – 46oC (Table 5.5). The 24 week ascospores generally had higher Tp and Tm

than the 6 week samples irrespective of heat treatments.

The heat treatments significantly altered the responses of B. nivea ascospores in DSC

analysis. The effects of the heating were either attenuating or removing transitions on

the thermograms depending on the severity of the treatment (Figure 5.4B, C and 5.5B,

C). The activation treatment generally reduced the reversibility of the uneven peak and

replaced it with a step change in the rescans. Effects of the inactivation treatment were

more severe, including removal of the peak and infrequent recovery of the step change.

The 24 week ascospores were more resistant to heat treatments, showing more

reversible transitions than the 6 week samples.

For B. nivea, the peak and step change transitions were relatively variable between sets

of the duplicates for heat treated samples, especially the heat inactivated ones. These

inconsistent results suggested complex effects of heat treatments on B. nivea

ascospores, causing various degrees of changes to the cellular structure (Tables 5.4 and

5.5). Similar variations were observed for the transition temperatures (Tp and Tm),

which were varied in the 40 – 50oC range without following specific trends as observed

with B. fulva ascospores.

104

Table 5.4. Peak and step change temperatures from the DSC analysis of Byssochlamys ascospores activated at 75oC for 30 minutes

Strain Age (weeks)




B. fulva FRR 2299

6 56.9 – 57.3 57.6 – 58.3 56.6 – 57.1 57.2 – 58.1 43.1 – 44.8 - 44.0 – 45.3 -

24 56.7 – 58.2 57.4 – 58.6 57.4 – 59.0 57.5 – 59.8 43.1 – 44.4 - 46.0 – 47.8 -

B. fulva FRR 2785

6 55.8 – 56.1 56.8 – 57.0 56.3 – 56.4 56.3 – 56.8 43.8 – 43.9 - 45.1 – 45.2 -

24 57.0 – 58.6 57.7 – 59.3 57.2 – 59.0 57.9 – 59.9 43.1 – 45 - 45.3 – 46.2 -

B. nivea FRR 4421

6 44.3 – 48 - 48.5 – 48.6 - - 40.8 – 41.0 - 41.8 – 42.6

24 44.5 – 46.5 or 52.2 – 54.7 - 43.9 – 49.5 - - 39.4 – 43.1 or

47.5 – 48.4 - 43 – 46.5

B. nivea FRR 6002

6 - - - - 37.5 or 41.1 – 48.5 48.1 – 49.5 44.9 – 46.9 46.7 – 47.4

24 44.9 – 45.0 or 52.6 - 45.1 or 59.5 - - 39.4 – 43.9 or

46.4 – 46.9 44.8 – 45.5 45.6 – 46.1

- No transition being observed

Italic – Inconsistent transition being absent in one of the duplicates

105

Table 5.5. Peak and step change temperatures from the DSC analysis of Byssochlamys ascospores inactivated at 95oC for 30 minutes

Strain Age (weeks)




B. fulva FRR 2299

6 56.3 – 56.6 56.9 – 57.3 56.5 – 56.8 57.0 – 57.3 39.5 – 42.1 - 43.7 – 45.4 -

24 56.4 – 58.0 57.3 – 58.7 56.8 – 58.1 57.3 – 58.9 40.4 – 43.6 - 44.7 – 45.8 -

B. fulva FRR 2785

6 54.4 – 55.6 56.3 – 55.7 54.4 – 55.4 55.4 – 56.1 40.8 – 43.3 - 43.8 – 45.7 -

24 56.4 – 57.7 57.4 – 58.7 57.3 – 58.2 57.9 – 58.7 42.4 – 43.0 - 44.3 – 46.6 -

B. nivea FRR 4421

6 51.0 – 52.8 - 47.2 – 47.6 - 47.7 – 48.0 42.5 – 45.2 46.7 – 46.9 47.3 – 48.2

24 48.6 – 49.0 - - - 32.5 – 34.3 46.6 – 47.3

36.0 46.0 44.0 – 44.4 43.4 – 43.5

B. nivea FRR 6002

6 - - 53.1 - 47.5 – 49.7 47.5 – 49.3 47.4 – 48.7 47.4 – 48.7

24 47.5 – 48.0 - - - 33.3 – 33.6 47.0 43.8 – 44.5 44.1 – 44.9 43.8 – 46.8

- No transition being observed

Italic – Inconsistent transition being absent in one of the duplicates

106

5.3.3 Effects of methods for sample preparations on DSC profiles of

Byssochlamys ascospores

Since DSC technique is not traditionally used to analyse microbial spores, methods for

preparing this type of sample are not standardised and different approaches can affect

the DSC results. The ‘wet’ and ‘open’ methods and vacuum drying were used to prepare

samples of Byssochlamys ascospores in this particular experiment. The thermograms of

samples made by these methods were compared against those of the ‘dry’ method for

their effects on DSC profiles of the ascospores.

Ascospore prepared by the ‘open’ method produced completely different thermograms

from the ‘dry’ method, comprising only a broad endothermic peak at 100oC with tails

spanning 50 – 120oC. This peak was possibly related to evaporation of water and may

have masked much smaller transitions of the ascospores (Figure 5.7). No transitions

were detected in the rescans or the cooling segments.

Figure 5.7. Thermograms of Byssochlamys dormant ascospores prepared in crucibles

with punctured lids; Species: A – B. fulva, B – B. nivea; Exothermic heat flow is

upwards and measured in relative scale (50 mW)

A

B

107

The lack of meaningful transitions was also seen with samples prepared by the ‘wet’

method. Although using wet samples in hermetically sealed crucibles avoided the broad

water peak, there was only a sharp increase of energy flow after 130oC (Figure 5.8). No

transitions occurred in the rescans. More importantly, crucibles prepared by the “wet”

method usually exploded after the initial scan with consequent loss of contents.

Figure 5.8. Thermograms of Byssochlamys dormant ascospores prepared in aqueous

forms without drying; Species: A – B. fulva, B – B. nivea. Scan: unbroken line – initial

scan, dashed line – rescan. Exothermic heat flow is upwards and measured in relative

scale (100 mW)

The effects of vacuum drying on DSC results were dependent on the species. Vacuum

drying did not change greatly the DSC profiles of B. fulva ascospores compared with

drying under normal atmosphere. The characteristic reversible endothermic peak and

step change were present at slightly lower temperatures, Tp = 56 – 57oC and Tm = 30 –

34.1oC, respectively (Figure 5.9A). However, B. nivea ascospores dried under vacuum

condition lost both the uneven peak and the step change. No transitions were recorded

in the rescans of B. nivea samples (Figure 5.9B).

A

A

B

B

108

Figure 5.9. Thermograms of Byssochlamys dormant ascospores prepared by vacuum

drying; Species: A – B. fulva, B – B. nivea. Scan: unbroken line – first scan, dashed line

– rescan. Exothermic heat flow is upwards and measured in relative scale (2 mW)

5.4 Discussion

5.4.1 Characteristics of DSC transitions of Byssochlamys ascospores

This study was the first application of DSC to investigate the states of intracellular

materials of Byssochlamys ascospores after heat activation, heat inactivation and for

dormant states at 6 and 24 weeks. Age, heat treatments along with strain variation could

significantly affect DSC profiles of the ascospores, which were in turn related to

different arrangements of materials inside the cells (Farkas et al., 2002). The major

transitions observed for Byssochlamys ascospores were endothermic. These events often

result from enthalpy relaxations, such as denaturation and melting, of polymer structure.

In the context of biological samples, those transitions could be related to conformational

changes of macromolecules in the ascospores which led to physiological responses such

as heat activation and denaturation (Leuschner et al., 1998). Although the enthalpy of

those transitions was relatively small, their significance was highly comparable to those

of bacterial spores (Leuschner and Lillford, 2003; Nguyen Thi Minh et al., 2010).

A

A

B

B

109

The major endothermic peak at 56 – 60oC of B. fulva ascospores, in particular, was

close to the optimal activation temperatures for this species (Splittstoesser et al., 1970,

1972). The lower peak temperature of B. nivea ascospores was possibly due to the

higher heat sensitivity of this species, causing earlier activation of the cells compared

with B. fulva (Chapter 3). The heat activation transition in the 56 – 60oC range has been

observed in spores of other microorganisms analysed by DSC. Endospores of Bacillus

cereus and Bacillus megaterium exhibited activation peaks at 56 – 57oC although their

recovery took several weeks (Belliveau et al. 1992; Maeda et al., 1975; Miles et al.,

1986). Activation peaks at 58 – 70oC were reported for Bacillus subtilis endospores and

it was recovered after 5 days (Ablett et al., 1999, Leuschner and Lillford, 2003; Nguyen

Thi Minh et al., 2010).

The baseline displacement observed with B. nivea samples was characteristic for an

increase of specific heat of material making up the ascospores (van Dooren and Müller,

1981b). This transition could indicate a change from a less to a more ordered structure

inside the ascospores. A change in sample mass, which could also cause uneven

baseline and step change (van Dooren and Müller, 1981b), was prevented in this

experiment by using dried samples, hermetical sealing and checking sample weights

prior to and after DSC analysis.

In this experiment, it was not possible to assign the detected DSC transitions to specific

compartments of the ascospores. The ascospore cytoplasm, where cellular materials

were concentrated, was likely to account for some of the changes. Contributions of the

intermediate space and the ascospore wall could not be underestimated because they

occupied a significant volume of the ascospores and are the first to be affected by heat

(Chapter 4). The outer proteinaceous coat and the cortex of the B. subtilis endospores

were both credited for the major activation peak (Leuschner and Lillford, 2003; Nguyen

Thi Minh et al., 2010). In addition, since the composition of Byssochlamys ascospores is

relatively complex (Banner et al., 1979), their DSC responses probably came from

different macromolecules, such as carbohydrate, lipid and proteins, and their polymeric

arrangements in the cells. Relaxation of many polysaccharides hydrated with 5 – 25%

(w/w) water occurred at 50 – 60oC similar to those of Byssochlamys ascospores

(Appelqvist et al., 1993). In bacterial spores, proteins and ribosomes were also

associated with endotherms at 62 – 67oC and 68 – 69oC, respectively (Miles et al., 1986;

Verbeke et al., 1981).

110

5.4.2 Arrangement of intracellular materials and heat resistance of


5.4.2.1 Comparisons between species and ages

The heat resistance property of Byssochlamys species is related to the ultrastructure and

composition of the ascospores (Chapter 4). The DSC results supported a link between

the thermo-tolerance and the arrangement of intracellular materials of Byssochlamys

ascospores. This relationship was elaborated by comparing DSC profiles of the more

heat resistant B. fulva and the more heat sensitive B. nivea ascospores. B. fulva showed

generally more conserved and reversible thermal transitions at higher temperatures than

B. nivea. These features may indicate that B. fulva ascospores have a strong and stable

cellular structure which is less affected by the applied heat treatments. In contrast, the

internal structure of B. nivea ascospores is less ordered and robust, rendering it more

susceptible to heat damage.

The aging process can enhance the heat resistance and complexity of the ascospore

ultrastructure (Chapter 3, 4). The present study with DSC supports a more robust and

resistant arrangement of internal material in the older ascospores i.e. 24 weeks. The

higher temperatures of transitions detected in the older samples possibly meant that the

effects of heating were delayed by the stronger structure of the ascospores. The older

ascospores of B. nivea, in particular, had more reversible transitions, which is another

piece of evidence for the more resistant structure. Even the matured ascospores affected

by heat treatments demonstrated a superior ability to resume nearly the original state,

probably due to the more resilient structure.

5.4.2.2 Comparisons between physiological states induced by heat

DSC profiles of heat treated ascospores exhibited signs of structural modifications in

accordance with the results of electron microscopic study (Chapter 4). The heat

activation caused mild yet sufficient changes to the structures to trigger the rehydration

inside the ascospores. These transformations resulted in a relaxation in the arrangement

of cellular materials, decreasing the recovery of the DSC transitions. Meanwhile, the

heat inactivation was more detrimental to the ascospores, possibly damaging the

structures beyond the point of their own self-recovery, which manifested as frequent

111

loss of DSC transitions. The older ascospores had a more robust structure but the

reversibility still required more time than the dormant cells.

The effects of heat treatments on DSC profiles emphasised the different thermo-

tolerance between B. fulva and B. nivea. Ascospores of B. fulva were less affected by

heat and exhibited less prominent changes in their structures than those of B. nivea.

However, the minimal changes in DSC profiles of B. fulva ascospores could be due to

the insensitivity of the measurement. The frequent presence of asci/aggregations in B.

fulva ascospore crops could defect the heat flow and modify DSC results. Thus, more

research is needed to determine if other variables could contribute to the responses of

ascospores during DSC analyses.

5.4.3 Possible existence of a glassy state in Byssochlamys ascospores

A glassy state in Byssochlamys ascospores has been a favourite theory for heat

resistance because it can help maintain the internal dehydration and protect proteins and

nucleic acids from denaturation (Buitink and Leprince, 2004; Crowe et al., 1996;

Dijksterhuis, 2007). The fact that trehalose is found abundantly in fungal spores also

favours the theory since this di-saccharide readily forms a glassy state under low

moisture conditions (van Laere, 1989). A glassy state is characterised by a glass

transition temperature (Tg) where material melts and loses viscosity (Gee, 1970;

Leopold et al., 1994). On DSC thermograms, Tg is often detected as a reversible

second-order transition, or step change (Bell and Touma, 1996).

In the current study, there are lines of evidence for an orderly arrangment similar to a

glassy state in Byssochlamys ascospores at least under dried conditions as used in this

experiment. The uneven peaks of B. nivea strains contained a step change component

that satisfied the criterion of a glassy state. Similarly, the occasional second-order

changes of B. fulva samples were suggestive for a glassy state.

Biological samples do not always show proper traits of Tg although they form a glassy

state (Appelqvist et al., 1993). In such cases, the endothermic peaks could be associated

indirectly with the glassy state. These peaks often appear near and overlap the glassy

state transition; hence, the detection of endotherms could signal a glass transition in the

absence of its usual indications, especially with biological samples (Ablett et al., 2001;

112

Appelqvist et al., 1993; Tunick et al., 2006). A similar indicator has been used to study

the glassy state in Bacillus spores (Ablett et al., 1999; Sapru and Labuza, 1993a;

Stecchini et al., 2006). The native dormant endospores of B. megarterium and B. cereus

had a slowly reversible, endothermic peak at 56 – 70oC, which was tentative evidence

for materials in the glassy state (Belliveau et al., 1992; Maeda et al., 1974, 1975).

It is noted that evidence of the glassy state does not guarantee a low moisture content

distributed homogenously within the ascospores. Instead, the cells could maintain some

low moisture regions such as cell wall or the cytoplasm where the glassy state was

likely to form locally (Leuschner and Lillford, 2003; Sunde et al., 2006). However,

moisture distribution in Byssochlamys ascospores is poorly understood and needs more

research.

5.4.4 Experimental designs and effects on DSC profiles

Because fungal ascospores are not conventional samples for DSC, prior knowledge

about standard methodology and expected results are relatively limited. There is a wide

range of variables that need to be optimised in order to obtain good DSC thermograms

(van Dooren and Müller, 1981a, b, c). In the present study, two major factors, sample

preparation methods and scanning rates, were considered.

5.4.4.1 Effects of sample preparation methods

Moisture content of samples is an essential determinant of the glass transition (Chen et

al., 2000). The plasticising effect of water on material structure is well-known for

altering its thermal profiles and therefore excessive water is undesirable in DSC

samples (Appelqvist et al., 1993; Frank, 2007). As shown in this experiment, the

moisture content can be influenced by preparation methods. The ‘dry’ method removed

most of the water in samples, resulting in satisfactory DSC profiles. However, the ‘wet’

method left much water in the final samples (Appendix 5.5) and the evaporation of

water masked all of the other transitions. Vapour also burst the sealed crucibles at high

temperatures, causing loss of samples and cross contamination.

Using the ‘dry’ method was supported by Stecchini et al. (2006) but was at odds with

other workers who analysed bacterial spores and vegetative cells apparently without

113

drying (Ablett et al., 1999; Belliveau et al., 1992; Leuschner and Lillford, 2003; Miles

et al., 1986). If moisture contents of samples are strictly monitored, it will become a

known variable and its influences can be effectively controlled (Maede et al., 1974;

Nguyen Thi Minh et al., 2010; van Cauwelaert and Verbeke, 1979; Verbekey et al.,

1981). Alternatively, the water issue could be mitigated by using special crucibles to

prevent loss of volatile components (Maeda et al., 1975).

Vacuum drying did not give as good results as the air drying method, especially for B.

nivea samples. Vacuum drying was supposed to draw more moisture out of samples.

However, it may disrupt structure due to pore formation and puff effect (Hawlader et

al., 2006). The ruptured ultrastructure can explain the loss of the major transition in

thermograms of B. nivea ascospores.

5.4.4.2 Effects of scanning (heating) rates

The heating rate of 10 Kmin-1 selected in this study has been successfully used to probe

thermal changes and the glassy state in bacterial spores (Ablett et al., 1999; Belliveau et

al., 1992; Leuschner and Lillford, 2003; Miles et al., 1986). Slower rates tended to

decrease both transition temperatures and enthalpy (Nguyen Thi Minh et al., 2010).

Samples also take a longer time to undergo a transition and may cause irregularities

(van Dooren and Müller, 1981b). However, it is not uncommon to find slower scans

(0.6 – 2.5 Kmin-1) being used in spore studies (Maeda et al., 1974, 1975; van

Cauwelaert et al., 1979; Verbeke et al., 1981). In those cases, slow scanning rates

allowed sufficient time for individual transition to occur and avoid potential

overlapping.

Meanwhile, rates above 10 Kmin-1 accelerate the scans but reduce significantly the

resolution power because samples have less time to rearrange their structures. Thermal

transitions scanned at higher rates are often shifted to higher temperature ranges and

neighbour events may overlap with one another (Tan and Che Man, 2002). However,

extremely fast scanning rates (100 and 200 Kmin-1) are able to differentiate a glassy

state in spores (Katayama et al., 2008). The argument for the rapid heating is to force

conventionally long transitions to occur in a shorter period of time, which enhances the

clarity and detection.

114

The contradictory uses of different scanning rates in literatures indicate that there are no

fixed rules in selecting this parameter. Each DSC experiment needs to be optimised in

order to determine a heating rate most suitable for the type of samples, purposes of the

analyses and machinery available.


DSC was applied to study the arrangement of intracellular materials and their

relatedness to heat resistance of Byssochlamys ascospores. Because of a lack of a

standard DSC methodology, experimental conditions including sample preparation and

heating rates were optimised. The chosen protocol included drying dense ascospore

solution over silica gel in a desiccator before hermetically sealing DSC crucibles (40

µL, aluminium). The optimal scanning rate was 10 Kmin-1 and repeated twice

alternatively with two cooling rounds at a rate of 10 Kmin-1.

DSC thermograms of the ascospores were significantly different between B. fulva and

B. nivea but less variable within each species. Thermograms of B. fulva ascospores were

characterised by an endothermic reversible peak at 56 – 60oC and occasionally

reversible step change at 40 – 47oC. Thermograms of B. nivea ascospores were much

simpler, comprising an endothermic peak with uneven baselines that was recovered as a

step change in the rescans. The reversibility of the transitions was dependent on strains,

ages and heat treatments applied to the ascospores.

The aging process increased the transition temperatures (Tp and Tm) for both B. fulva

and B. nivea. Older B. nivea ascospores also had more reversible transitions. On the

other hand, heat treatments removed the transitions or slowed down their recovery

process. The detrimental effects of heating were more prominent on thermograms of B.

nivea ascospores than those of B. fulva.

From the cellular structure point of view, the DSC results suggested that B. fulva

ascospores may possess a more robust and resistant structure than those of B. nivea.

Aging increased the resilience of the structure by improving its self-recovery. Heating

caused relaxations of structural materials, leading to a loss of integrity and resistance.

The extent of the effects depended on the severity of the heat treatments.

115

Existence of a glassy state inside Byssochlamys ascospores was considered based on

evidence presented by DSC thermograms. Despite the lack of usual indicators of a

glassy state, the endothermic peak and the step change suggested the presence of an

ordered configuration similar to the glassy state in the ascospores.

This is the first time DSC has been used in research of ascospores of heat resistant

fungi. The findings revealed some aspects of the material arrangements inside

Byssochlamys ascospores, complementing the electron microscopy results (Chapter 4).

The evidence indicating the glassy state theory may shed light on an important

mechanism of heat resistance. Future research can expand this knowledge to

unequivocally prove the presence of the glassy state. For example, it is suggested to

determine and control the moisture content, e.g. aw or percentage of water, of ascospore

preparations. DSC examination of ascospores under controlled moisture conditions, for

instance in buffer at ambient temperatures, can be conducted. It may also be possible to

apply extremely high scanning rates to look for the reversible second-order transition

that is characteristic for a glassy state.

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Chapter 6 – Heterogeneity in ascospore populations of Byssochlamys and its relationship with heat resistance

6.1 Introduction

In Chapter 3, the thermal inactivation of Byssochlamys ascospores was found to be

more complex than that of vegetative cells since it did not follow the conventional log-

linear pattern. The deviation from linearity, which is characterised by the shoulder and

tailing phases, of the heated ascospores can be ascribed to different groups of

ascospores in the treated populations. Ascospores can be different in a number of

aspects including morphology, size, shape, points of sporulation (age), chemical

composition and nucleic acid content (Dantigny and Nanguy, 2009; Gefen and Balaban,

2009). Such heterogeneity in the ascospore population can cause them to respond

differently to thermal treatment (Cronin and Wilkinson, 2008a; Rodriguez-Palacios and

LeJeune, 2011). There have been no systematic studies on the heterogeneity of

Byssochlamys ascospores and the relationship with heat resistance properties. Casella et

al. (1990) suggested that a small group of intrinsically more heat resistant ascospores of

B. nivea might explain tailing in the survival curves. However, their experiment was not

designed to screen different types of cells in the ascospore suspensions. Thus, more

research about the heterogeneity of Byssochlamys ascospores is needed for a better

understanding of their survival strategies.

Flow cytometry (FCM) is a culture-independent, high throughput, multi-parameter and

single-cell analytical method (Boye and Løbner-Olesen, 1990). In a flow cytometer,

individual cells are differentiated by the ability to scatter light in forward angles (FSC,

10 – 15o deflection from the detecting light), which is related to morphology and size, or

in a side angle (SSC, 90o deflection), which is related to internal complexity of the cell

(Davey, 2002; Salzman, 2001). Flow cytometry can also distinguish cells by their

ability to fluoresce after staining with fluorescent probes that differentially react with

and label cells, depending on their physiological states (Sträuber and Müller, 2010).

One such stain is SYTO 9, which is a dye that can pass through the cytoplasmic

membrane of vegetative cells to react with their nucleic acid. Propidium iodide (PI) is

another type of stain that reacts with nucleic acid materials, but can only enter cells

117

when the function of the cell membrane has been impaired. Therefore, SYTO 9 and PI

can be used to indicate the integrity of the cell membrane, which is strongly related to

cell viability (Armstrong and He, 2001; Breeuwer and Abee, 2000). In addition, these

stains may also be used to observe cells with microscopic techniques to elaborate data

from FCM (Ghosh et al., 2009; Rydjord et al., 2007).

FCM has been increasingly applied in food microbiology, especially for monitoring,

detection and quantification of pathogenic bacteria, where the method may produce

more sensitive and faster results than plate culturing techniques (Budde and Rasch,

2001; Dumain et al., 1990; Gunasekera et al., 2000; Laplace-Builhé et al., 1993; Lavilla

et al., 2009). Viability and germination of microbial spores can also be assessed by

FCM (Kong et al., 2010; Laflamme et al., 2005a; Cronin and Wilkinson, 2010). Several

studies using FCM coupled with fluorescent dyes have shown that stresses from heating

and long time storage can result in heterogeneous cell populations of Rhizopus

oligosporus sporangiospores (Thanh et al., 2007) and bacterial spores (Cronin and

Wilkinson, 2008a, b).

This Chapter investigates the use of FCM to characterise the heterogeneity within

ascospore preparations of Byssochlamys, with respect to sizes and cell wall integrity

(i.e. viability) at their dormant state and after heat treatments. Flow cytometry was used

alone and with staining using SYTO 9 and PI to determine sub-populations of

ascospores. Two strains of each species, B. fulva and B. nivea, were examined at 4, 8

and 12 weeks of culturing. Since commercially available fluorogenic dyes have not

been used previously for ascospore staining, a preliminary study based on an Optical

Microscope eXperimental (OMX) platform was performed to probe the penetration of

fluorescent dyes into the ascospores. Another preliminary evaluation of the two stains,

SYTO 9 and PI, in differentiating sub-populations of the ascospores was also

conducted.

6.2 Methodology

6.2.1 Fungal strains and ascospore preparation

Four Byssochlamys strains, B. fulva FRR 2299, B. fulva FRR 2785, B. nivea FRR 4421

and B. nivea FRR 6002, were used in this study. Fungal cultures were grown on MEA

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and incubated at 30oC for 4, 8 and 12 weeks. At the end of the incubation, ascospores

were harvested from the MEA plates using the protocol described in Chapter 3. Some

modifications were included in the process in order to obtain ascospore suspensions that

were more suitable for FCM analysis. In particular, MEA plates were flooded with

0.05% (w/v) aqueous Tween 80 (Merck, VIC, Australia) solution that had been filtered

through 0.20µm syringe filters (Sartorius Stedim, VIC, Australia) and autoclaved.

Biomass was scraped from the surface of the plates using sterile bent glass rods. Free

ascospores were prepared from asci and mycelium using the glass bead shaking method

(Splittstoesser et al., 1974). About 1.4 mL of fungal biomass and 0.3 g of glass beads

(Chapter 3) were shaken in a tissue lyser (Retsch, PA, USA) at 30 Hertz for 15 minutes

for B. fulva strains and 7.5 minutes for B. nivea strains. The homogenates were filtered

through glass wool, and the ascospores in the filtrates were sedimented and washed by

centrifugation (4000g for 20 minutes at 4oC), and resuspended in filtered sterile distilled

water (SDW). The final suspension was layered on top of a 60% (w/w) sucrose (food

grade, Woolworths, NSW, Australia) solution, and purified by density centrifugation

(4000 rpm, 30 minutes, room temperature) in a Jouan CR3i Multifunction swinging

bucket centrifuge (Thermo Scientific, MA, USA). The sucrose solution was made with

filtered SDW to minimise particles interfering with flow cytometry. After removing the

supernatant, the pellets were washed twice in filtered SDW, resuspended in 0.05%

filtered aqueous Tween 80, and distributed as 1 mL aliquots that were kept at 4oC until

analysis. Ascospore concentrations in the aliquot were 108 – 109 cells/mL as determined

by microscopic observation using a haemocytometer (Marienfeld, Germany).

6.2.2 Thermal treatments of Byssochlamys ascospores for flow cytometry

Aliquots (1 mL) of the harvested Byssochlamys ascospores were diluted 20-fold in

filtered SDW. They were then further diluted 10-fold into capped glass tubes containing

filtered citrate phosphate buffer (CPB) pH 4.0 that had been pre-heated in a water-bath

set at 87.5oC for B. nivea samples and 90oC for B. fulva samples. The final volumes of

10 mL were heated for 1, 4, 12 and 30 minutes, then quickly cooled down in an ice

slurry and transferred to 28 mL sterile plastic tubes for easy pipetting. Mixtures of

dormant ascospore and buffer that were not heat treated were regarded as time “0”

samples. Experiments were performed in triplicate.

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6.2.3 Staining of ascospores with fluorescent dyes

Ascospore preparations were stained immediately after heat treatment. Two nucleic acid

binding fluorescent dyes, SYTO 9 or propidium iodide (PI) (Molecular Probes,

Invitrogen Australia Pty Ltd, VIC, Australia), were used. The two stains were obtained

from the LIVE/DEAD® BacLightTM Bacterial Viability Kit applicator sets (L13152,

Molecular Probes). Each stain, which was supplied in dehydrated form, was thoroughly

mixed with 0.5 mL of Baxter water (Product code AHF7113, Baxter, NSW, Australia)

that had been filtered through 0.20 µm syringe filters. The stain solutions were kept

frozen at -18oC and used within 4 weeks of preparation.

Ascospores were stained by mixing 495 µL of diluted ascospore suspension with 5 µL

SYTO 9 or 490 µL of diluted ascospore suspension with 10 µL PI. The final dye

concentrations were similar to the manufacturer’s recommendations for this particular

BacLight Viability Kit, which was 1.2 µM for SYTO 9 and 12 µM for PI

(http://tools.invitro-gen.com/content/sfs/manuals/mp07007.pdf). The staining was

carried out in the dark for 10 minutes at room temperature (ca. 22oC) and samples were

analysed immediately after staining.

Negative controls were prepared from dormant ascospores (time “0” samples) without

staining. The positive controls were prepared from ascospores treated at 96oC for 60

minutes and stained as above. The ascospores treated at 96oC without staining were

used as the positive controls for unstained samples.

6.2.4 Microscopic examination of SYTO 9-stained ascospores

Microscopy was used to determine if the nucleic acid binding stain SYTO 9 was able to

penetrate into the ascospores. Ascospores from 8 week cultures of B. fulva FRR 2299

and B. nivea FRR 6002 were used in this experiment. Dormant ascospores and heat

treated ascospores were tested. The heat treatment was conducted in capped glass tubes

containing SDW for 5, 15 and 30 minutes at 87.5oC for B. nivea FRR 6002 and 90oC for

B. fulva FRR 2299. The ascospore suspensions were concentrated by centrifugation and

stained with SYTO 9 at a final concentration of 1.2 µM for at least 10 minutes. Wet

mounts were prepared from the stained samples on glass microscope slides and covered

with cover slips. The slides were then examined under a DeltaVision Optical

http://tools.invitro-gen.com/content/sfs/manuals/mp07007.pdf

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Microscope eXperimental (OMX) 3D-Sim Super-Resolution microscope (Applied

Precision, WA, USA) at The University of Technology, Sydney (Sydney, NSW,

Australia).

6.2.5 Flow cytometric analysis

Approximately 0.5 mL of stained ascospores was transferred to a 5 mL Falcon round-

bottom tube (BD Biosciences, CA, USA) for use in the flow cytometer. Flow cytometry

was performed in a FACSCaliburTM instrument (BD Biosciences) equipped with a 15

mW air-cooled 488 nm argon-ion laser. The flow cytometer was calibrated weekly by

BD CellQuest Pro software. Calibration was performed with CaliBRITE beads (BD

Biosciences) for three colour set-up using the FACSComp software. IsoflowTM sheath

fluid (Beckman Coulter, NSW, Australia) was used. Green fluorescence (FL1) was

collected through a 550 nm long-pass filter and red fluorescence (FL3) through a 650

nm long-pass filter. The voltage settings were manually adjusted to keep the forward-

angle light scatter (FSC), side scatter (SSC) and fluorescence signals on log-scale. The

typical setting was: FSC E00, SSC 200 mV, FL1 380 mV, FL2 378 mV and FL3 470

mV. Event rates were controlled to be less than 1000 events per second in all

experiments. An event in flow cytometry can be a single ascospore or an aggregation

which is hydro-dynamically focused and intercepts the light detection system.

6.2.6 Data collection and analysis

When samples were analysed by the flow cytometer, the number of events collected for

each sample was fixed at 10,000 events that occurred in a specified area of the forward

scattering (FSC) versus side scattering dot plots (Region R1, Figure 6.1). This region

was drawn in such a way that it excluded most of the noise and contained the key sub-

population that was always recognised by the flow cytometer. It was adjusted according

to species, strains and condition of ascospores.

Flow data were handled by FlowJo 7.6 software (Tree Star Inc., OR, USA). Statistical

analysis was performed using Minitab® 15 software (Minitab, PA, USA) and the

statistics package in Microsoft Excel. The one-way analysis of variance (ANOVA) and

121

Tukey’s test were used for multiple pair comparisons. The significance level of all

statistic comparisons was 95% (α = 0.05).

A) B)

C) D)

Figure 6.1. Data collecting area on forward scattering (FSC) versus side scattering

(SSC) dot plots for B. fulva FRR 2299 (A), B. fulva FRR 2785 (B), B. nivea FRR 4421

(C) and B. nivea FRR 6002 (D); the number of collected events was fixed at 10,000 in

the R1 region; noise (thin-lined circle) was removed during the data analysis

6.2.7 Procedure of differentiating groups of ascospores

Data of ascospore samples recorded by the flow cytometer were firstly displayed on the

FSC versus SSC dot plots. Events associated with ascospores were mostly in the upper

right quadrant (Q2) of the dot plot, clearly separated from noise (Figure 6.2A). The

subset of events in Q2 area were further analysed on different fluorescent intensity dot

plots depending on the stains being used. For unstained samples, the selected events in

Q2 were viewed on autofluorescence (FL1) versus size (FSC) dot plots. Data of SYTO

122

9-stained samples were viewed on green fluorescence (FL1) versus FSC dot plots while

those of PI-stained samples were on red fluorescence (FL3) versus FSC dot plots.

Groups of ascospores, or sub-populations, were distinguished on the basis of ascospore

size (FSC intensity) and green/red fluorescence (FL1/FL3 intensity) of the cells. Dot

plots of the time points that showed most clear differentiation of sub-populations were

used to set up the sub-population templates (Figure 6.2B). Sub-populations were

identified on dot plots as distinct groups of events, usually shaped as an ellipsoid. A

region, namely a gate, was drawn around each population borders. Gates of each sample

were adjusted to capture collectively 99% or more of the events selected from Q2. The

gate information (position, shape) was transferred without modifications across the

triplicates and the positive controls for each strain at one age. The event abundance (in

percentage) of each sub-population was automatically calculated out of the total events

in Q2. Gates were adjusted to accommodate variations between strains, ages and

staining options.

A) B)

Figure 6.2. Procedure to differentiate sub-populations of Byssochlamys ascospores: A –

Events associated with ascospores were collected in Q2 separated from noise; B –

Events in Q2 were replotted on FL1 versus FSC dot plots and distinct sub-populations

were gated; percentages of sub-population were calculated out of total events in Q2;

gate information was maintained across triplicates of one strain at one age. Axis: FSC –

forward scattering; SSC – side scattering; FL1 – (auto-) fluorescence. Dot plots are

representative for illustration only and will be different with each sample

Q2

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6.3 Results

6.3.1 Penetration of SYTO 9 into ascospores

SYTO 9 penetrated poorly into the dormant ascospores of B. fulva FRR 2299 and B.

nivea FRR 6002. The stain only accumulated on the cell periphery, forming a distinct

fluorescent ring around the ascospores (Figure 6.3A, E). Intracellular configurations of

the dormant ascospores were not clearly shown. On the other hand, SYTO 9 was able to

penetrate heat treated ascospores and highlighted some details of the internal content as

well as the cell wall. Apart from the peripheral staining, the central cytoplasm of heated

ascospores fluoresced brightly and was clearly differentiated from the rest of the cells.

There was a void between the outer ring and the central area where no fluorescence was

detected (Figure 6.3D, H).

The intensity of staining by SYTO 9 increased with the length of the heat treatment,

indicating a higher permeability of ascospores to the dye. The central cytoplasm became

clearer and more distinctive in more severely heated ascospores (Figure 6.3B – D, F –

H). However, modifications on the surface of the ascospore wall also became more

prominent as heat progressed, especially after 30 minutes (Figure 6.3D, H).

124

Dor

man

t

A) E)

5 m

inut

es

B) F)

15 m

inut

es

C) G)

30 m

inut

es

D) H)

Figure 6.3. Penetration of SYTO 9 into ascospores in response to heat treatment at

90oC for B. fulva FRR 2299 (left) or 87.5oC B. nivea FRR 6002 (right). Note the stained

periphery (block arrow), the central cytoplasm (arrow) and the surface modifications

(arrow heads). Scale bar = 1 µm

125

6.3.2 Evaluation of fluorescent dyes for differentiating sub-populations of


Four week ascospores were subjected to three staining options: unstained, SYTO-

stained and PI-stained. The staining results for B. fulva FRR 2299 and B. nivea FRR

4421 are presented in Figures 6.4 and 6.5, respectively. For clear presentation, only data

at time 0 (dormant) and 30 minutes are shown. Dot plots of treatments at 1, 4 and 12

minutes showed intermediate states between 0 and 30 minutes. Similar staining patterns

were observed for 8 and 12 week ascospores (Appendices 6.1 – 6.4).

Unstained ascospores, as expected, had relatively low autofluorescence intensity,

irrespective of strain and age. Progressive heat treatment (1 – 30 minutes) resulted in a

concomitant increase in the autofluorescence but the change was relatively small even

after the longest heating time. Sub-populations could be observed in dormant and heated

samples. For B. fulva strains, sub-populations were separated better by autofluorescence

(FL1) than by size (FSC). Even then, the sub-populations were closely stacked on top of

each other (Figure 6.4 A, B, E, F). For B. nivea strains, sub-populations were well

separated by size and auto-fluorescence (FL1) (Figure 6.5 A, B, E, F). For both species,

the whole populations were partially clustered on the FSC axis, making it difficult to

distinguish sub-populations.

SYTO 9-stained ascospores exhibited about 10 times as much fluorescence (FL1) as the

unstained samples irrespective of strain and age, therefore elevating all events into the

centre of the dot plots. The fluorescence intensity increased slightly after 30 minute heat

treatment compared with the dormant samples. SYTO 9 staining could also separate

sub-populations of B. nivea strains better than the unstained ascospores (Figure 6.5 C,

D). However, the differentiation of sub-populations was not improved as much for B.

fulva ascospores (Figure 6.4 C, D).

PI-stained ascospores displayed slightly increased fluorescence on FL3 by about 3-fold

compared with unstained samples. This change occurred with both dormant and heat

treated samples irrespective of strain and age. For B. fulva strains, PI staining did not

enhance the separation of sub-populations better than SYTO 9 staining or no stain

(Figure 6.4 G, H). For B. nivea strains, PI-stained ascospores were differentiated

marginally better than unstained samples, but not as effectively as those stained with

126

SYTO 9. Additionally, a part of the PI-stained populations was still shadowed by the

FSC axis (Figure 6.5 G, H).

In conclusion, unstained ascospores can be separated by size and, to a lesser extent, by

autofluorescence. For staining options, SYTO 9-stained ascospores showed higher

fluorescence intensity and clearer separation of sub-populations, particularly for B.

nivea, than PI-stained samples. Thus, SYTO 9 was a better fluorogenic dye than PI in

differentiating sub-populations of Byssochlamys ascospores after aging and heat

treatments.

127

Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)

FSC intensity – Size

Figure 6.4. Comparative staining of 4 week ascospores of B. fulva FRR 2299 with

different nucleic acid stains. Staining option: A, B, E, F – unstained, C and D – SYTO 9

stain, G and H – PI stain; ascospores were heat treated at 90oC for up o 30 minutes

128

Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


Figure 6.5. Comparative staining of 4 week ascospores of B. nivea FRR 4421 with

different nucleic acid stains. Staining option: A, B, E, F – unstained, C and D – SYTO 9

stain, G and H – PI stain; ascospores were heat treated at 87.5oC for up to 30 minutes

129

6.3.3 Differentiating sub-populations of unstained ascospores

Sub-populations of unstained ascospores could be separated based on size (FSC) and

autofluorescence (FL1) as shown in Section 6.3.2. Autofluorescence can indicate the

viability state of the ascospores, whereby dead cells exhibit more autofluorescence than

live cells (Wu and Warren, 1984). The patterns of sub-populations of unstained

ascospores were clearly different between B. fulva and B. nivea but quite similar within

each species. In this section, the sub-populations of unstained ascospores are described

in more detail for each species and strain.

6.3.3.1 Sub-populations of unstained ascospores of B. fulva

Unstained samples of B. fulva FRR 2299 and B. fulva FRR 2785 at 4, 8 and 12 weeks

are shown in Figures 6.6 and 6.7, respectively. Only dot plots of dormant (0 minute) and

30 minutes ascospores are presented. The other heating time points showed transitional

states between 0 and 30 minutes, and are available in Appendices 6.5 and 6.6.

Figures 6.6 and 6.7 indicate the presence of four major sub-populations in dormant

samples with different sizes and autofluorescence properties: Abf – medium size and low

autofluorescence, Bbf – small size and low autofluorescence, Cbf – large size and

medium autofluorescence, and Dbf – large size and high autofluorescence. These sub-

populations were relatively close, almost on top of each other. The comparison

‘low/small – medium – high/large’ was only relative among the four sub-populations

and did not indicate actual values.

Heat treatments, such as 90oC for up to 30 minutes and 96oC for 60 minute, increased

slightly the autofluorescence intensities of all sub-populations by less than two fold.

Sub-population Bbf became gradually less abundant and apparently moved into Abf as

the heating progressed. The sub-population patterns of dormant and heated samples

were similar between the two B. fulva strains across three experimented ages.

130

4 weeks 8 weeks 12 weeks

90o C

, 0 m

in

FL1

– A

utof

luor

esce

nce

inte

nsity

90

o C, 3

0 m

in

96o C

, 60

min



ages (4,8 and 12 weeks) and after a heat treatment at 90oC for up to 30 minutes; sub-

populations are Abf, Bbf, Cbf and Dbf; treatment at 0 minute is dormant ascospores;

ascospores treated at 96oC for 60 minutes is the positive control

131

4 weeks 8 weeks 12 weeks 90

o C, 0

min

FL

1 –

Aut

oflu

ores

cenc

e in

tens

ity

90o C

, 30

min

96

o C, 6

0 m

in



ages (4, 8 and 12 weeks) and after a heat treatment at 90oC for up to 30 minutes; sub-

populations are Abf, Bbf, Cbf and Dbf; treatment at 0 min is dormant ascospores;

ascospores treated at 96oC for 60 minutes is the positive control

Because of the much similar patterns of sub-populations before and after heat

treatments, identities of the sub-populations could not be fully elucidated. Prediction of

identities is attempted for some sub-populations based on size and autofluorescence

characteristics. Abf is predicted to be aggregated ascospores, which are partially coming

from Bbf sub-population of single ascospores after heat treatments. Abf may also

comprise heat affected ascospores. Cbf and Dbf are likely to be aggregated, severely

heat-damaged ascospores, including heat inactivated, due to their high levels of

132

autofluorescence. However, it is impossible to assign any specific viability state for

three sub-populations Abf, Cbf and Dbf. Properties and the predicted identities of sub-

populations for B. fulva ascospores are tabulated in Table 6.1.

Table 6.1. Predicted identities of sub-populations of unstained B. fulva ascospores

Sub-population

Relative cell size

Relative auto-fluorescence intensity

Predicted identity

Abf Medium Low Aggregated or heat inflicted

ascospores

Bbf Small Low Single, dormant ascospores

Cbf Large Medium Aggregated or heat damaged or

inactivated ascospores

Dbf Large High Aggregated or severely

damaged/inactivated ascospores

6.3.3.2 Sub-populations of unstained ascospores of B. nivea

Unstained ascospores of B. nivea FRR 4421 and B. nivea FRR 6002 at 4, 8 and 12

weeks are shown in Figures 6.8 and 6.9, respectively. As for B. fulva, only dormant and

30 minutes samples are shown here and data of the intermediate time points are

presented in Appendices 6.7 and 6.8.

Unstained, dormant ascospores displayed three major sub-populations: Abn – low

autofluorescence and small sizes, Bbn – low autofluorescence as Abn and large sizes, and

Cbn – high autofluorescence and medium sizes. These sub-populations were separated

better than those of B. fulva. The comparison of size and autofluorescence among the

sub-populations is only relative. Heating at 87.5oC for up to 30 minutes increased the

autofluorescence of all sub-populations by less than two fold. Sub-populations Bbn and

Cbn also became more abundant after the heat treatment. Moreover, as a result of

heating, a new sub-population Dbn appeared as an extension from Bbn, which had similar

size but higher autofluorescence than Bbn. Ascospores that were heated severely at 96oC

for 60 minutes had only two sub-populations Bbn and Dbn.

The developments of sub-populations for dormant and heated ascospores are similar

between two B. nivea strains. However, the abundance of some sub-populations was

133

different with ascospore age. Sub-population Bbn was more prominent at 4 weeks than 8

and 12 weeks both before and after the heat treatment. Sub-population Dbn was also

more pronounced at 4 weeks than the other ages. Contrarily, sub-population Cbn was

more abundant at 8 and 12 weeks than 4 weeks.


87.5

o C, 0

min

FL

1 –

Aut

oflu

ores

cenc

e in

tens

ity

87.5

o C, 3

0 m

in

96o C

, 60

min

utes



ages (4, 8 and 12 weeks) and after a heat treatment at 87.5oC for up to 30 minutes; sub-

populations are Abn, Bbn, Cbn and Dbn; ascospores treated at 96oC for 60 minute is the

positive control

134


.5o C

, 0 m

in

FL1

– A

utof

luor

esce

nce

inte

nsity

87

.5o C

, 30

min

96

o C, 6

0 m

inut

es



ages (4, 8 and 12 weeks) and after a heat treatment at 87.5oC for up to 30 minutes; sub-

populations are Abn, Bbn, Cbn and Dbn; ascospores treated at 96oC for 60 minute is the

positive control

The predicted states of B. nivea sub-populations are summarised in Table 6.2. The clear

difference between sub-population patterns of dormant ascospores and those treated at

96oC indicates that Abn may comprise single dormant ascospores whereas Bbn and Dbn

are aggregated, heat inactivated cells. Bbn possibly contain damaged cells with low

internal content, causing low level of autofluorescence. Sub-population Cbn could be

heat activated ascospores or smaller aggregations than Bbn.

135

Table 6.2. Predicted identities of sub-populations of unstained B. nivea ascospores

Sub-population

Relative cell size

Relative auto-fluorescence intensity

Predicted identities

Abn Small Low Single, dormant ascospores

Bbn Large Low Aggregated, dormant or heat


Cbn Medium High Smaller aggregation than Bbn or

heat activated ascospores

Dbn Large High Aggregated, dormant or heat


6.3.4 Differentiating sub-populations of SYTO 9-stained ascospores

SYTO 9 was used to differentiate sub-populations because of its higher fluorescence

intensity and better separation of ascospore groups than PI (Section 6.3.2). The

separation principle of SYTO 9 is based on the integrity of the ascospore wall whereby

increased fluorescence (FL1) corresponds with a compromised cell-wall, allowing

SYTO 9 to penetrate and stain nucleic acid material in the central cytoplasm of the

ascospores.

SYTO 9-stained ascospores displayed different sub-populations from the unstained

sample. The sub-populations are dissimilar between B. fulva and B. nivea but follow the

same patterns within each species. The identification of sub-populations for each

species and strains are described in detail below.

6.3.4.1 Sub-populations of SYTO 9-stained ascospores of B. fulva

Fluorescence (FL1) versus size (FSC) dot plots of SYTO 9-stained ascospores of B.

fulva FRR 2299 and B. fulva FRR 2785 for 4, 8 and 12 week preparations are shown in

Figures 6.10 and 6.11, respectively. Only dormant and 30 minutes heated samples are

presented. Other intermediate time points, which exhibit transitional states between 1

and 12 minutes, are shown in Appendices 6.9 and 6.10.

The SYTO 9-stained, dormant ascospores had four major sub-populations with different

relative size and relative fluorescence property: A*bf – medium size and low

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fluorescence, B*bf – small size and low fluorescence, C*bf – large size and medium

fluorescence and D*bf – large size and high fluorescence. Compared with unstained

ascospores, sub-populations of SYTO 9-stained samples were still closely arranged to

each other. Sub-populations A*bf and B*bf were often crossed over and were the most

prominent groups in the dormant samples.


90o C

, 0 m

in

FL1

– SY

TO 9

fluo

resc

ence

inte

nsity

90

o C, 3

0 m

in

96o C

, 60

min



different ages (4, 8 and 12 weeks) and after a heat treatment at 90oC for up to 30

minutes; sub-populations are A*bf, B*bf, C*bf and D*bf; time 0 is dormant ascospores;

ascospores treated at 96oC for 60 minute is the positive control

137

The heat treatment at 90oC for up to 30 minutes resulted in a movements of sub-

population B*bf into A*bf while the other two sub-populations were relatively

unchanged. Ascospores that received the treatment at 96oC (positive control) had only

two major sub-populations C*bf and D*bf.


90o C

, 0 m

in

FL1

– SY

TO 9

fluo

resc

ence

inte

nsity

90

o C, 3

0 m

in

96o C

, 60

min


different ages 4, 8 and 12 weeks) and after a heat treatment at 90oC for up to 30

minutes; sub-populations are A*bf, B*bf, C*bf and D*bf; time 0 is dormant ascospores;

ascospores treated at 96oC for 60 minute is the positive

The spatial arrangements of sub-populations before and after heat treatments are

relatively similar across the two strains and three ages. The difference in sub-population

arrangements between dormant and positive control suggests that A*bf and B*bf contain

138

mostly dormant ascospores whereas C*bf and D*bf are predominantly damaged and heat

inactivated ascospores. B*bf possibly comprises single ascospores and A*bf contains

both single and aggregated forms. A*bf may also have sub-lethally injured or heat

activated ascospores due to its increased abundance after heat treatments. Ascospores in

D*bf potentially suffer more damage than C*bf, causing a higher fluorescence intensity.

The characteristics and predicted identities of these sub-populations are summarised in

Table 6.3. The identification with SYTO 9 complemented the previous prediction using

no stains (Table 6.1). Thus, the equivalent identification without stain is also included in

Table 6.3.

Table 6.3. Predicted identities of sub-populations of SYTO 9-stained B. fulva

ascospores

Sub-population (without stain)

Relative cell size

Relative fluorescence intensity


A*bf (Abf) Medium Low Larger or aggregated,

dormant or sub-lethally

injured or heat activated

ascospores

B*bf (Bbf) Small Low as A*bf Single, dormant ascospores

C*bf (Cbf) Large Medium Aggregated, damaged or

heat inactivated ascospores

D*bf (Dbf) Large

(> C*bf)

High Larger aggregation than

C*bf or heat inactivated

ascospores

The movement of B*bf into A*bf was evidenced by their changed percentages during

heat treatment. The percentages of the predicted large dormant ascospores (A*bf)

increased significantly. This change mirrored the decreased percentages of the smaller

dormant sub-populations (B*bf). The simultaneous changes of these two sub-

populations quickly reached equilibrium after 4 minutes of heating for B. fulva FRR

2785 and 12 minutes for B. fulva FRR 2299. The percentages of the other sub-

populations of aggregated, heat inactivated ascospores (C*bf and D*bf) often showed

little change or even a slight decrease after 30 minutes of heat treatment. At 30 minutes,

139

the smaller and larger dormant sub-population accounted for 10 – 20% and 40%,

respectively of the total population. The corresponding figures of heat inactivated sub-

populations were 10 – 30% each. Figure 6.12 illustrates the changed abundance of sub-

populations with heating time for 4 week ascospores. Results are similar at 8 and 12

weeks, and are provided in Appendices 6.11 and 6.12.

A)

B)

Figure 6.12. Changed abundances of sub-populations of B. fulva ascospores stained by

SYTO 9 after a heat treatment at 90oC; ascospores are 4 weeks old. Strain: A – B. fulva

FRR 2299, B – B. fulva FRR 2785. Sub-population: A*bf (♦), B*bf (■), C*bf (▲) and

D*bf (●); each point is an average of three experiments

140

6.3.4.2 Sub-populations of SYTO 9-stained ascospores of B. nivea

The sub-populations of SYTO 9-stained ascospores of B. nivea FRR 4421 and B. nivea

FRR 6002 are shown in Figures 6.13 and 6.14, respectively. Only dormant and 30

minute heated samples for 4, 8 and 12 week ascospore preparations are presented here.

Results of other time points are shown in Appendices 6.13 and 6.14.

Staining SYTO 9 revealed five major sub-populations of B. nivea ascospores with

different relative size and fluorescence intensity: A*bn – small size and low

fluorescence; B*bn – large size and low fluorescence; C*bn – small size and medium

fluorescence occurring as an extension of A*bn; D*bn – large size and medium

fluorescence; and E*bn – large size and high fluorescence.

There were differential distributions of these sub-populations with heat treatments and

ages. Dormant ascospores comprised predominantly sub-population A*bn followed by

D*bn. Sub-populations B*bn and D*bn were more abundant in 4 week than 8 and 12

week samples. After the heat treatment at 87.5oC for up to 30 minutes, the abundance of

A*bn decreased while those of C*bn, D*bn and E*bn increased significantly. Particularly,

sub-population B*bn also became more pronounced at 4 weeks, but not at 8 and 12

weeks. Sub-population E*bn, however, was more prominent at 8 and 12 weeks than 4

weeks after the heat treatment.

The treatment at 96oC completely removed two sub-populations A*bn and C*bn. The 4

week ascospores only had D*bn as the predominant sub-population. Older ascospores, 8

and 12 weeks, were distributed mostly in B*bn, D*bn and, to a lesser extent, E*bn.

141


.5o C

, 0 m

in

FL1

– SY

TO 9

fluo

resc

ence

inte

nsity

87

.5o C

, 30

min

96

o C, 6

0 m

in



different (4, 8 and 12 weeks) ages and after a heat treatment at 87.5oC for up to 30

minutes; sub-populations are A*bn, B*bn, C*bn, D*bn and E*bn; treatment at time 0 is

dormant ascospores; ascospores treated at 96oC for 60 minute is the positive control

142


.5o C

, 0 m

in

FL1

– SY

TO 9

fluo

resc

ence

inte

nsity

87

.5o C

, 30

min

96

o C, 6

0 m

in



different ages and after a heat treatment at 87.5oC for up to 30 minutes; sub-populations

are A*bn, B*bn, C*bn, D*bn and E*bn; treatment at time 0 is dormant ascospores;

ascospores treated at 96oC for 60 minute is the positive control

The identities of each sub-population can be predicted from the changes following heat

treatments described above. Sub-population A*bn is most likely to contain single

dormant ascospores due to its absence in the positive control. B*bn is possibly

aggregated, dormant or heat inactivated ascospores. Occurring only after a heat

treatment and absent in the positive controls, C*bn can be associated with heat activated

or (mildly) sub-lethally injured ascospores. D*bn and E*bn are potentially heat

143

inactivated ascospores since they became more pronounced after heating. In addition,

B*bn and D*bn may also contain mechanically damaged ascospores which are observed

frequently during harvesting ascospores for this experiment. The prediction with SYTO

9-stained ascospores improves the results of unstained samples (Table 6.2). Thus, the

predicted identities of the sub-populations with and without SYTO 9 stain are

summarised together in Table 6.4.

Table 6.4. Predicted identities of sub-populations of SYTO 9-stained B. nivea

ascospores

Sub-population

(without stain)

Relative cell size

Relative fluorescence intensity


A*bn (Abn) Small Low Single, dormant ascospores

B*bn (Bbn) Large Low Larger or aggregated, dormant

or mechanically damaged or heat


C*bn (Cbn) Small Medium Heat activated or (mildly) sub-

lethally injured ascospores

D*bn (Dbn) Large Medium Mechanically damaged or heat


E*bn (Dbn) Large High Heat inactivated ascospores

The percentages of sub-populations reflected the concurrent, opposite changes of the

single dormant ascospores (A*bn) and the inactivated population (D*bn) during the heat

treatments. As the heating progressed, the abundance of dormant sub-population

decreased significantly whereas that of the heat inactivated sub-populations increased.

The percentages of other sub-populations increased slightly but significantly at 1 minute

and showed little changed until the end of the treatment. After 30 minute treatment, the

single dormant ascospores accounted for about 20% of the total population, aggregated

ascospores (B*bn) 20 – 30%, activated sub-population (C*bn) less than 10%, and

inactivated ascospores (D*bn and E*bn) 40% or more. These changes were similar at the

three experimented ages. Results of the 4 week ascospores are presented in Figure 6.15

and those of other ages are in Appendices 6.15 and 6.16.

144

A)

B)

Figure 6.15. Changed percentages of sub-populations of B. nivea ascospores stained by

SYTO 9 after heat treatment at 87.5oC. Ascospores are 4 weeks old; Strain: A – B. nivea

FRR 4421, B – B. nivea FRR 6002; Sub-population: A*bn (♦), B*bn (■), C*bn (▲), D*bn

(●) and E*bn (◊); each point is an average of three experiments

145

6.4 Discussion

For the first time flow cytometry combined with fluorescent probes was used to

examine the heterogeneity of Byssochlamys ascospores of different ages before and

after heat treatment. Flow cytometry was chosen because of its capability of analysing

single cells in a high throughput and multi-parameter manner (Davey and Kell, 1996).

The culture-independence of the method also allowed the quick analysis of samples

immediately after heat treatments. These technical advantages enabled information to be

obtained about the sub-populations of Byssochlamys ascospores.

6.4.1 Penetration of SYTO 9 into Byssochlamys ascospores and the

protective roles of the cell wall of the ascospores

SYTO 9 showed poor penetration into dormant ascospores but its entrance occurred

after heating the ascospores preparation. The heat-dependent penetration of SYTO 9

indicates increased permeability of the ascospore wall following heat treatment. Heating

has been known to compromise the integrity of ascospore walls, thereby facilitating the

penetration of dyes into the cells (Cronin and Wilkinson, 2008a, b; Kelly and Gay,

1969). This result highlights the protective roles of the outer wall of Byssochlamys

ascospore. The external wall or coat is an essential structural factor of heat resistance of

microbial spores because it forms the first line of defence against thermal and chemical

insults (Atrih and Foster, 2002; Driks, 1999).

The outer wall of dormant ascospores is possibly a micro-sieve that mediates the

penetration of materials into the ascospores. The ingress of SYTO 9 molecules into the

ascospores to stain the central cytoplasm (OMX experiment, Section 6.3.1) was

observed only when the outer wall was apparently disrupted by heat treatments. The

result using OMX agrees with the data obtained using electron microscopy (Chapter 4).

Better penetration of fixatives and resins was observed with heat treated than with

dormant ascospores. In addition, this phenomenon is analogous to the modifications that

heat treatments caused to the wall of Neurospora tetrasperma ascospores, and the coat

and cortex of Bacillus endospores, which, after treatment, allowed transportation of

chemical molecules in and out of these cells (Kong et al., 2010; Laflamme et al., 2005a;

Sussman, 1954; Tabit and Buys, 2010).

146

6.4.2 Evaluation of SYTO 9 and PI in differentiating sub-populations of


In flow cytometry, the use of appropriate fluorescent markers can assist differentiation

of cell sub-populations because different fluorogenic dyes can indicate different aspects

of cellular viability (Sträuber and Müller, 2010). The selection of fluorescent probes for

use in this study was limited by the lack of dyes developed for fungal applications.

Within this constraint, SYTO 9 and PI were potential candidates because they have

been previously applied in assessing the heterogeneity of fungal sporangiospores as well

as bacterial endospores, in response to environmental stresses (Cronin and Wilkinson,

2008a; Thanh et al., 2007; van Melis et al., 2011).

The SYTO 9 and PI dyes used in this study are two components of the LIVE/DEAD

BacLight kit which is intended to differentiate viable and dead bacterial cells,

respectively (Berney et al., 2007). SYTO 9 is permeable to all cells whereas PI is only

permeable to those with compromised plasma membranes (Berney et al., 2007).

However, because Byssochlamys ascospores are quite different from bacterial cells with

respect to cellular structure, the application of these two stains in the present study was

more experimental. They were used as an indicator of the integrity of the ascospore

ultrastructure, particularly the cell-wall (Armstrong and He, 2001; Breeuwer and Abee,

2000). Increased fluorescence intensity corresponded with a higher permeability of the

wall to SYTO 9 and, therefore, more damage to ascospores from heat treatments.

The results showed that SYTO 9 had a superior performance over PI in differentiating

many sub-populations of Byssochlamys ascospores, especially those of B. nivea. This

could be attributed to the more versatile permeability of SYTO 9 into cells than PI as

mentioned already. Moreover, SYTO 9 has much higher emission intensity than PI, so it

can separate different types of cells more effectively. In fact, upon binding to DNA and

RNA, the fluorescence intensity of SYTO9 and PI increases by 300 and 8 times,

respectively (Stocks, 2004). The better permeability and fluorescence of SYTO 9

justified its use as the major probe to investigate sub-populations of Byssochlamys

ascospores.

147

6.4.3 Fluorescence mechanisms of SYTO 9 in Byssochlamys ascospores

In dormant, non-heated ascospores, the outer wall prevented the penetration of SYTO 9

and allowed only peripheral staining, resulting in relatively low fluorescent emission

from the stained ascospores. Since nucleic acid materials, which are the main targets for

binding and fluorescence of SYTO 9, are unlikely to be on the ascospore wall (Setlow,

2007), the fluorescence from dormant, non-heated ascospores may involve non-specific

binding with non-nucleic acid molecules on the cell-wall. A possible target for stain

binding is proteins since they can be present on the cell-wall of the ascospores (Neiman,

2005) and bind to dyes to fluoresce (Ferencko and Rotman, 2010; Magge et al., 2009).

The peripheral fluorescence of SYTO 9 on the cell wall is not unusual since it has been

reported for other dormant structures, including the ascospores of Neurospora crassa

stained by immuno-fluorescent labels (Hecker and Sussman, 1973) or endospores of

Bacillus subtilis stained with nucleic acid markers (Ferencko and Rotman, 2010; Melly

et al., 2002; Setlow et al., 2002).

For heated ascospores, SYTO 9 was shown to penetrate into the cytoplasm of the

ascospores and probably stain the nucleic acid materials here, causing the fluorescence

intensity of SYTO 9-stained ascospores to increase tenfold. However, this change is

much lower than the theoretical 1000-fold increase when SYTO 9 binds to nucleic acid

molecules (Stocks, 2004). This weak fluorescence could be attributed to a number of

reasons. Firstly, DNAs or RNAs that were present in the dormant ascospores may have

been broken down by heat treatments (Kozyavkin and Lyubchenko, 1984), causing a

loss of substrates for SYTO 9 binding. Secondly, the low moisture condition inside the

ascospores could induce conformational changes in the DNA and RNA (Osborne and

Boubriak, 1994). These structural modifications might affect the ability of nucleic acid

materials to interact with SYTO 9 molecules, which in turns reduces the fluorescent

intensity.

Conformational changes in DNA and RNA could also result from their binding with

other molecules in the cytoplasm, such as proteins, which might interfere with the

intercalation of the fluorogenic molecules. In bacterial endospores, a group of α/β-type

small acid soluble proteins has been known to bind DNA in the core to stabilise the

structure and reactivity of DNA (Setlow, 1992, 1994, 1995, 2006). Lastly, as mentioned

already, the weak fluorescence after heat treatments might be ascribed to the non-

148

specific binding between SYTO 9 molecules and proteins that could be found

abundantly in the ascospore cytoplasm (Setlow, 2007).

6.4.4 Differentiating sub-populations of Byssochlamys ascospores

6.4.4.1 Sub-populations of dormant ascospores

Flow cytometric analysis showed that dormant ascospores of both B. fulva and B. nivea

comprised a mixture of single dormant, aggregated dormant and mechanically damaged

ascospores. This result was unexpected because the harvest and purification process,

which involved glass wool and density centrifugation in 60% sucrose solution, were

supposed to produce relatively homogeneous ascospore crops.

The heterogeneous composition of the ascospore preparations could result from several

factors. Byssochlamys species produces ascospores which remain aggregating in asci,

and which need to be disintegrated mechanically. The mechanical treatment with glass

beads used in this experiment did not completely break up the asci into individual

ascospores. Thus, there were still intact asci and ascospore groups of variable sizes in

the ascospore crops. Meanwhile, the glass bead treatments were possibly vigorous

enough to cause damage to some ascospores. The preparations of B. nivea contained

more mechanically damaged ascospores than those of B. fulva. This difference may be

due to the lack of the external coating layer on the asci and ascospores of B. nivea,

making them more susceptible to mechanical treatments than those of B. fulva (Chapter

4). The use of density centrifugation and filtration did not effectively separate single

ascospores from ascospore groups, asci and damaged ascospores. These structures were

all variable in size, physiology and, therefore, ability to absorb stain. Thus, they were

characterised as different sub-populations with flow cytometry (Allman, 1992).

Variations in the ascospore crops can also occur even if the ascospores are apparently

similar in size. The heterogeneity may arise from different sporulation time point, or

ascospore age, which could affect their ultrastructure (Chapters 4 and 5) and heat

resistant properties (Chapter 3). Additionally, basic mutational variability, in both

genotype and phenotype, can be manifested as different external or internal structure

and heat resistance, which, therefore, can be another cause of heterogeneity within the

ascospore preparations (Davey and Kell, 1996; Eijlander et al., 2011; Kong et al., 2010).

149

6.4.4.2 Sub-populations of heat activated ascospores

At the first minute of the heat treatments, FCM analysis showed significant changes in

the abundances of the ascospore subpopulations compared with the dormant samples. In

particular, the subpopulation of predicted single, dormant ascospores diminished

whereas those of predicted aggregated and heat inactivated ascospores became more

prominent. This meant that the applied heat treatments affected the cell-wall integrity of

the ascospores, causing ingress of SYTO 9 stain and increased size, within a relatively

short time after exposure to the wet-heat conditions.

The results obtained by flow cytometry supported a previous observation that the first

minute was the heat activation stage in the survival curves of Byssochlamys ascospores

(Chapter 3). Moreover, the effects of heating on the cell-wall as described above may

elaborate the activation mechanism of Byssochlamys ascospores. The activation of

fungal ascospores generally requires rehydration of the cytoplasm in order to activate

key enzymes for the germination (Dijksterhuis et al., 2007; Setlow, 2003). Since

dormant ascospores are relatively resistant to penetration of moisture and chemicals,

activating factors such as heating can break down the impermeable cell-wall, facilitating

the influx of water into the ascospores and the rehydration of the central cytoplasm.

However, the results of flow cytometry could not explain specifically the causes of the

increase in cell size during the heat activation stage. This change could result from

either the aggregation or the uptake of water into the heated ascospores. Clumping of

ascospores during heating may occur as the surface proteins of the cell-wall are

denatured, increasing the hydrophobicity of the ascospore wall surface (Furukawa et al.,

2005). For the water uptake theory, water upon entering the ascospores may expand the

cytoplasm (Setlow, 2007), resulting in an enlargement of the whole structure. The

increase of spore dimensions following hydration and expansion of the spore core

during activation has been observed in endospores of Bacillus subtilis (Katz, 2005a, b;

Leuschner and Lillford, 2000). However, Dijksterhuis et al. (2007) found no major

changes in size of the ascospores of Talaromyces macrosporus during activation.

6.4.4.3 Sub-populations of heat-inactivated ascospores

Heat treatment increased the heterogeneity of the ascospore populations, as indicated by

the appearance of new sub-populations that were absent in the dormant samples. The

150

heat-induced sub-populations included not only inactivated ascospores but also in-

between cells, which were stressed at different levels but could not be categorised

clearly as live or dead cells (Davey, 2011). These sub-populations may comprise heat

activated and several classes of sub-lethally injured ascospores (Cronin and Wilkinson,

2008a). This spectrum of responses to heat treatments from various ascospore groups

could be manifested as a non-linear semi-logarithmic survival curves (Peleg and Cole,

1998), which agrees with an observation of the thermal inactivation experiment

(Chapter 3).

Moreover, the presence of asci and clumps in the ascospore preparations can contribute

to their heterogeneous responses to heat. These aggregated structures are more heat

resistant than free, single ascospores since the heat penetration into them is not uniform,

causing different effects on individual members (Furukawa et al., 2005; Hatcher et al.,

1979; Splittstoesser et al., 1969).

At the end of the heat treatments, the sub-populations of dormant ascospores only

accounted for less than 20% of the total population whereas the aggregated and heat

inactivated ascospore sub-populations were 30 – 40% each. The remainder of the

dormant sub-populations somewhat agreed with a previous finding where the tailing

phase of the survival curves consistently occurred from 20 minutes until the end of the

heat treatment (Chapter 3). Thus, studies using thermal inactivation kinetics and flow

cytometry both indicate persisting sub-populations of heat resistant ascospores that are

present throughout the heating process.

Ascospores showed a general trend of increased size and autofluorescence or SYTO 9

fluorescence at the end of the heat treatments. The larger size could be attributed to

clumping of heated ascospores due to elevated hydrophobicity of the cell-wall as

mentioned for heat activated samples. The increased autofluorescence with longer

heating time may indicate damage inflicted on the ascospores, particularly the cell-wall.

Autofluorescence is common in fungal spores and often related to phenolic compounds

in the cytoplasm (Hecker and Sussman, 1973; Wu and Warren, 1984). This

phenomenon has been considered as a viability marker for bacterial spores whereby the

higher intrinsic fluorescence is associated with reviving spores and more permeable

cell-wall (Laflamme et al., 2005b, 2006). The results of autofluorescence agrees with

those of SYTO 9 fluorescence, which also suggest that further heating increased the

151

permeability of the ascospore wall, allowing the penetration of more stain molecules

into the cytoplasm.

However, differentiating the sub-populations using flow cytometry exhibited some

limitations. The sub-population maps of the ascospores before and after heat treatments

were not always clearly different, especially those of B. fulva strains. Thus, the

predicted identities of ascospore sub-populations in Table 6.1 – 6.4 were empirical and

would require more research for confirmation. Additionally, the use of nucleic acid

stains such as SYTO 9 can affect colony-forming ability of the ascospores and,

therefore, limit further viability tests using plating techniques.


This study examined the heterogeneity of ascospores of B. fulva and B. nivea following

heat treatments and at different ages by using flow cytometry and two fluorescent

probes, SYTO 9 and PI. To the author’s knowledge, this is the first study to apply single

cell analysing techniques to investigate the survival mechanisms of ascospores of a heat

resistant mould. This study was also exploratory in determining the use of fluorescent

dyes to evaluate viability states of the ascospores.

SYTO 9 was better than PI with respect to fluorescent intensity and separation of

various sub-populations. The penetration of SYTO 9 into ascospores was dependent on

the integrity of the ascospore wall. Dormant ascospores were stained peripherally

whereas heated ascospores with compromised cell-wall integrity allowed more SYTO 9

to enter and stain the cytoplasm.

Ascospore sub-populations of B. fulva and B. nivea could be differentiated with respect

to cell sizes and autofluorescence or SYTO 9 fluorescence. The identities of all sub-

populations could be predicted from the size and fluorescence characteristics, which

included dormant (single, asci, aggregated ascospores), heat activated, sub-lethally

injured and inactivated ascospores. Sub-populations of B. nivea samples were

differentiated and identified better than those of B. fulva with or without staining.

The heat treated ascospores moved towards the higher fluorescence and larger size areas

on the dot plots. This movement indicates that heat treatments caused cell aggregation,

152

cell enlargement and damage to the ascospore wall which allowed more (auto)-

fluorescence to be detected.

The results of this study were complicated by the heterogeneous composition of

ascospore crops and the heterogeneous responses of ascospores to heat treatments.

Future research can focus on developing sample preparation methods which produce

more homogeneous ascospore crops. Research also needs to elucidate the mechanisms

by which various fluorescent probes can be used in flow cytometric research on

ascospores.

153

Chapter 7 – Proteomic profiles of ascospores of Byssochlamys species detected by two dimensional gel electrophoresis during aging and heat treatment

7.1 Introduction

Resistance to environmental stresses such as high temperatures is an intrinsic property

of microorganisms and, therefore, may be tracked through molecular activities, such as

genomics or transcriptomics, of cells (Brul et al., 2011; Setlow and Setlow, 1996). For

vegetative cells of bacterial and fungal species, the acquired thermo-tolerance often

involves a global change in the physiology and metabolism of the cells, for instance via

the production of various groups of stress-related compounds such as proteins or sugars

(Goldani et al., 1994; Piper, 1993; Raju et al., 2007). For spores and ascospores, the

synthesis of new components may not be applicable but it is possible that stress-related

materials are already present intracellularly so that they protect cells during the course

of dormancy and activation (Brambl, 2009; Ruijter et al., 2003).

The most studied group of stressed-cell protectors are heat shock proteins (HSPs). They

are molecular chaperones found in different cell compartments and play a pivotal role in

protecting essential proteins from denaturation (Li and Srivastava, 2004). HSPs are

prevalent in fungal cells, and along with another abundant stress protector, namely

trehalose, represent a strategy to acquire thermo-tolerance (Elliot et al., 1996; Piper,

1993; Raggam et al., 2011; Singer and Lindquist, 1998; Tereshina, 2005).

Byssochlamys ascospores contain about 9% (w/w) of proteins which was similar

between strains of different heat resistance. The contents of individual amino acids were

also the same regardless the heat resistance level (Banner et al., 1979). Those proteins

may comprise stress proteins or components of protein synthesis pathways that enable

fungal cells to manufacture HSPs prior to dormancy (Brambl, 2009). However, no

research has been conducted to identify proteins and their functions in Byssochlamys

ascospores, especially whether HSPs are present and contribute to thermo-tolerance.

Proteomics provide researchers with an ability to examine global changes of the whole

protein content (proteome) in cells. It combines the resolving power of protein

154

electrophoresis with identification by mass spectrometry (MS). In particular, two-

dimensional gel electrophoresis (2D GE) is able to separate proteins extracted from cells

based on their molecular weight and charges (Lilley et al., 2001). If cells differentially

express proteins in varying conditions, 2D GE will facilitate a clear comparison of

proteomic profiles down to single proteins (Lee et al., 2008). Proteins of interest can

then be excised from gels and identified by different MS methods such as the matrix

assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI TOF-

MS) (Bonk and Humeny, 2001).

Proteomic approaches using 2D GE and MS have been applied to study responses of

fungal cells to environmental stresses such as antibiotic stress (Melin et al., 2002),

osmotic stress (Kim et al., 2007), oxidative stress (Lessing et al., 2007) and high

temperatures (Raggam et al., 2011). Research on proteomics of Bacillus endospores has

also generated novel information about the molecular aspects of heat resistance (Brul et

al., 2011; Lai et al., 2003).

Proteomics have not been used to study heat resistant ascospores of Byssochlamys

species. Compared with genomics and transcriptomics, proteomics is less dependent on

knowledge of the genome of Byssochlamys, the sequence of which is unavailable at the

moment. Genomic sequences and associated information are available for other fungal

species, such as some species of Aspergillus, Neurospora, Penicillium and Talaromyces

(Fedorova et al., 2007; Galagan et al., 2003; Pel et al., 2007; van den Berg et al., 2008)

and can be used as a reference point with respect to Byssochlamys.

Using 2D GE, this Chapter examines the proteomic profiles of ascospores of B. fulva

and B. nivea as affected by heat treatments and the aging process. Ascospores of 4 week

old cultures of each species were examined after heating at either 87.5 or 90oC for 1, 4

and 30 minutes. The proteomic profiles of dormant ascospores from 4 week and 12

week cultures were also compared.

155


7.2.1 Isolation and preparation of ascospore samples

Two Byssochlamys strains, B. fulva FRR 2299 and B. nivea FRR 6002, were selected

for proteomic analysis. Their cultures were grown on Malt Extract Agar for 4 and 12

weeks after which ascospores were harvested and prepared according to the procedures

described in Chapter 6. Dormant ascospores were stored in a suspension of filtered

(0.20 µm syringe filter, Sartorius Stedim, VIC, Australia) distilled water at 4oC and used

within seven days after harvest.

7.2.2 Heat treatment of ascospores

Proteomes of ascospores were analysed according to the protocol given in Figure 7.1.

This allows a comparison of the proteomic profiles with respect to (i) ascospore age and

(ii) heat treatments as follows.

i. Dormant ascospores harvested from 4 and 12 week cultures

ii. Four week ascospores heat treated at 90oC for 1, 4 and 30 minutes for B. fulva

FRR 2299 and 87.5oC for 1, 4 and 30 minutes for B. nivea FRR 6002. These

times and temperatures were selected to compare the proteomic profiles at the

points of activation, linear inactivation and the tailing phase as determined from

the thermal inactivation studies found in Chapter 3

Suspensions of dormant ascospores were adjusted to a concentration of 108 ascospores

per mL. One millimetre of the ascospore suspension was then added into glass test tubes

containing filtered (0.20 µm syringe filter, Sartorius Stedim) citrate phosphate buffer

(pH 4.0) that had been pre-heated in a water bath to either 87.5oC (B. nivea) or 90oC (B.

fulva). Heating at these temperatures was carried out for either 1, 4 or 30 minutes before

the tubes were removed, and quickly cooled in an ice slurry. The contents were then

instantly frozen in dry ice. Samples of dormant ascospores without any heat treatment (t

= 0) were also frozen in dry ice at approximately the same time as the heated samples.

Both dormant and heat treated samples were then stored at -80oC until the time of

analysis. The experiment was done in triplicate, using three different ascospore crops

from each fungal strain (Figure 7.1).

156

Figure 7.1. Treatment of Byssochlamys ascospores used for proteome analysis

7.2.3 Proteomic analysis of ascospores

Proteomic analysis including protein extraction, 2D GE, gel image analysis and protein

spot identification were carried out at the Australian Proteome Analysis Facility (APAF,

Macquarie University, North Ryde, NSW, Australia). The analysis process is

summarised in Figure 7.2.

B. fulva FRR 2299

4 week old Dormant

4 week old Heated at 90oC for

1, 4 and 30 minutes

12 week old Dormant

1 sample

3 samples

1 sample

1 sample

1 sample

3 samples

5 samples per strain

5 samples per strain

Biological triplicates (x 3) Total number of samples = 30

B. nivea FRR 6002

4 week old Dormant

4 week old Heated at 87.5oC

for 1, 4 and 30 minutes

12 week old Dormant

157

Figure 7.2. Proteome analysis of Byssochlamys ascospores; boxes with broken borders

and broken arrows could be omitted if not required

Beating with glass beads (x 3 times)

Reduction and alkylation (1 hr at ~22oC)

Centrifugation and collecting supernatant

Conductivity checking

Buffer exchange or concentration

Quantifying protein concentration (Bradford assay)

1D gel electrophoresis (SDS-PAGE)

Product checking

SDS-PAGE (test run)

Isoelectric focusing (pH 3 – 10, 20 hrs, 100 kVh)

SDS-PAGE (8 – 18%, 40mA per gel)

Image analysis and spot selection

Spot sequencing by MALDI – TOF/TOF

Coomassie staining (fix, stain and destain)

Search against database (NCBInr)

Ascospores in 2D buffer + protease inhibitor cocktail

2D gel electrophoresis

158

7.2.3.1 Reagents

The reagents used in the proteomic analysis and their compositions are presented in

Table 7.1. Most reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, MO,

USA). Coomassie Brilliant Blue G-250 dye and acrylamide were obtained from Bio-

Rad (Bio-Rad, CA, USA).

Table 7.1. Reagents used in the proteomic analysis of Byssochlamys ascospores

Solution description Compositions in final concentration

2D buffer 7M urea, 2M thiourea, 4% CHAPS (3-[(3-

cholamidopropyl)- dim- ethylammonio]-l-

propanesulfonate), 40mM Tris, 10mM acrylamide,

5mM TBP (Tri-n-butylphosphine), MilliQ water

Protein inhibitor cocktail (Catalogue # P8215 from Sigma-Aldrich)

Equilibration buffer 6M Urea, 2% SDS (sodium docecyl sulphate), 20%

glycerol, 5x Tris-HCl (pH 8.8), MilliQ water

Fixing solution 24.8% methanol, 2.48% phosphoric acid

Coomassie staining

solution

0.15% w/v Coomassie Brilliant Blue G-250, 20% v/v

methanol, 2.32% v/v orthophosphoric acid, 10% w/v

Ammonium sulphate

Gel plug wash solution 50% v/v acetonitrile, 50mM ammonium bicarbonate

Zip Tip activation solution 90% v/v acetonitrile, 0.1% trifluoroacetic acid

Zip Tip equilibration and

wash solution

0.1% trifluoroacetic acid

Matrix solution for

MALDI-TOF mass

spectrometry

4 mg/mL α-cyano-4-hydroxycynnamic acid, 70%

acetonitrile, 0.06% trifluoroacetic acid, 1mM

ammonium citrate

7.2.3.2 Protein extraction and quantification

Frozen ascospore suspensions were quickly defrosted and ascospores were collected by

centrifugation at 2000g for 10 minutes at 4oC and subsequent removal of the

supernatant. Protein was extracted by dissolving the ascospore pellet in 1 mL of 2D

buffer (Table 7.1) and 100 µL of a protein inhibitor cocktail. The mixtures were then

transferred into 2 mL screw capped tubes containing 0.5 g of acid washed glass beads

159

(diameter 425 – 600 µm) (Sigma-Aldrich). Breaking ascospores was achieved by

shaking the solutions with glass beads three times in a FastPrep® FP120 bead beater

(BIO 101/Savant, MP Biomedicals, OH, USA) for 45 seconds at a speed of 6.5. After

each beating round, the tubes were cooled down in ice for 10 minutes.

Reduction and alkylation of proteins were carried out by adding 25 µL of 200 mM TBP

(Tri-n-butylphosphine) (Sigma-Aldrich) and 15 µL of 1M acrylamide (Bio-Rad) in

every 1 mL of beaten samples. The mixtures were then mixed by one round of shaking

in FastPrep as described above. They were cooled in down in ice for 10 minutes and

incubated at room temperature (ca. 22oC) for 1 hour. The reduction and alkylation

process was terminated by another round of bead beating (45 seconds) and ice cooling

(10 minutes). Extracted proteins from ascospores were collected in the supernatant layer

after a centrifugation step at 20,000g for 15 minutes at RT. The extract was distributed

into smaller aliquots and stored at -80oC until used.

Conductivity of the protein product was checked by a handheld Horiba B-173 Twin

Cond conductivity meter (Horiba, CA, USA) calibrated against a conductivity

calibration solution (Hanna Instruments, RI, USA). The conductivity of protein samples

was adjusted by buffer exchange or concentration so that a value of 0.3 milli Siemens

per centimetre (mS/cm) was achieved. Buffer exchange and concentration were done

with Vivaspin 5 kD centrifugal filter units (Sartorius Stedim) at 10,000g, 10oC for about

30 minutes. Proteins were retained in the filtered unit and were eluted into new 1.5 mL

centrifuge tubes.

Protein concentrations of the extracts were determined by the Bradford assay so that

optimal amounts of proteins were loaded onto gels. The extracts were diluted five-fold

with 2D buffer and 10 µL duplicates were pipetted into a 96 well polystyrene multititer

plate (Greiner Bio-one, Frickenhousen, Germany). Two hundred microlitres of Bradford

reagent (Sigma-Aldrich) was then added into each well and reactions were allowed to

take place for 5 minutes at RT under shaking conditions. Absorbance of solutions was

measured at 595 nm in a Fluostar Optima microplate reader (BMG Labtech, Offenberg,

Germany). Protein concentrations were determined from a standard curve that was

constructed from bovine serum albumin (BSA) (Sigma-Aldrich) solutions. Standard

BSA solutions were prepared at concentrations of 0.05, 0.1, 0.15, 0.25, 0.5, 0.75 and 1

160

mg/mL. Blank solution contained 2D buffer only. All standards and the blank were

measured in triplicate.

7.2.3.3 Two-dimensional (2D) gel electrophoresis

The protein extracts from ascospores was separated by 2D gel electrophoresis using

protocols previously described (Herbert et al., 2001; Lam et al., 2010) with some

modifications.

Isoelectric focusing (IEF) of proteins (first dimensional separation)

Approximately 100 µg of protein (determined from the Bradford assay) was made up to

300 µL with 2D buffer. IPG (Immobilised pH gradient) buffer pH 3 – 10 (GE

Healthcare BioSciences AB, Uppsala, Sweden) and bromophenol blue were added to

1% of the solution volume. Protein solutions were then absorbed onto 17 cm IPG strips

(GE Healthcare BioSciences AB) with linear pH gradient 3 -10 in rehydration trays (GE

Healthcare Bio-Sciences AB) for 6 hours at room temperature. Proteins in rehydrated

strips were focused on an Ettan IPGphor II (Amersham Biosciences, Uppsala, Sweden)

in Ondina medicinal-grade paraffin oil (Shell, Sydney, Australia) (Sewell and Bishop,

2009). The focusing program comprised 4 hours at 300V, 8 hours at linearly increased

voltage to 8000V, and at least 8 hours at 8000V. The total voltage product was

approximately 100 kVh. An average current of 40 mA per strip was applied during the

focusing. Focused IPG strips were equilibrated in equilibration buffer (Table 7.1) twice

for 15 minutes each, directly before the second dimensional electrophoresis step.

SDS-PAGE (second dimensional separation)

The second separation was a size-dependent SDS polyacrylamide gel electrophoresis

(SDS-PAGE). The equilibrated IPG strips were loaded onto SDS-PAGE gels and

separation was performed at 5 mA per gel until the bromophenol blue front just ran off

the bottom of the gels. Gel dimension and polyacrylamide gradient were 21 cm x 22 cm

and 8 – 18%, respectively. Gels were prepared in the APAF laboratory using Tris-

acetate buffer pH 7. Gels were transferred to a fixing solution (Table 7.1) for 2 hours,

then stained with Coomassie Brilliant Blue G-250 solution (Table 7.1). Background

stain was removed by immersing gels in 1% acetic acid for 30 minutes. Destained gels

were then stored in new 1% acetic acid solution on a gel rocker, prior to the protein

161

visualisation step. Before scanning, destained gels were transferred into MilliQ water

for 30 minutes.

7.2.3.4 Protein visualisation and gel image analysis

Gels were scanned by an Odyssey infrared scanner (Li-cor Biosciences, NE, USA) set

at 700 nm wavelength and medium resolution. Gel images were analysed and compared

across treatments and between ages using Progenesis SameSpots 4.0 coupled with

Progenesis PG240 Image Analysis Software (Nonlinear Dynamics, NC, USA). Protein

spots were first identified by the software and individually reviewed by a trained

operator to confirm their authenticity.

The relative abundance of proteins was measured by normalised volumes of spots on

gels. They were then compared within the triplicates, across treatment times and ages.

Normalisation was necessary to remove technical noise on 2D gels due to preparation

and electrophoretic process (Levänen and Wheelock, 2009). Spot volumes were

normalised by expressing the volume of each spot in a gel relative to the total spot

volume of the entire gel (Keeping and Collins, 2011). The normalisation algorithm was

built in the Progenesis Samespots 4.0 software and automatically performed.

Changes of normalised spot volumes indicated differential expressions of proteins in

various tested conditions. In this experiment, meaningful changes were based on

statistical significance by ANOVA one-way test (α = 0.05) and at least a two-fold

change. The protein spots also had to appear consistently in all triplicate gel images.

A subset of the significantly changed spots was selected for identification. Apart from

the criteria mentioned above, the chosen spots needed to be clearly visible on the

stained gels so that the excision could be carried out without aided scanning methods.

7.2.3.5 Sample preparation for mass spectrometry

Selected spots on Coomassie stained gels were automatically excised from 2D gels by

ExQuest Spot Cutter (Bio-Rad) and collected in a 96-well microtitre plate. Three

abundant spots on a gel were also processed as internal controls for this step and the

subsequent identification. The gel plugs were destained in 250 µL of wash solution

(Table 7.1) at 37oC for 5 minutes with shaking. The washing and destaining step was

162

repeated three times. The gel pieces were washed in 20 µL of acetonitrile for 10 minutes

at room temperature and then removed followed by drying in an oven at 37oC for 30

minutes. Trypsin (Sigma-Aldrich) at a final concentration of 4 ng/µL (10 µL) in 25 mM

ammonium bicarbonate (20 µL) was added to each sample and the in-gel digestion

occurred overnight (ca. 16 hours) in an oven set at 37oC.

After digestion, the resultant solution was acidified with 10 µL of 0.1% trifluoroacetic

acid and peptides were extracted from gel pieces in a sonic water bath for 20 minutes.

The extracted peptides were desalted and concentrated by the zip tipping method as

follows. For each peptide extract solution, a ZipTipsTM C18 micro-column tip (Millipore,

MA, USA) was used to withdraw and dispense repeatedly 10 µL Zip Tip activation

solution (Table 7.1) 3 times, then 10 µL of Zip Tip equilibration solution (Table 7.1) 3

times, then 10 µL of the peptide extract 15 times, then 10 µL of Zip Tip washing

solution (Table 7.1) 3 times and 1µL of matrix solution (Table 7.1) 10 times. The last

matrix-peptide solution (1 µL) was deposited directly onto a 192-well stainless steel

target plate (Applied Biosystems, CA, USA) and allowed to air dry.

7.2.3.6 Peptide mass fingerprinting by mass spectrometry

Peptides were identified by Matrix-assisted Laser Desorption Ionization tandem mass

spectrometry (MALDI-TOF MS/MS) using a 4800 Plus MALDI TOF/TOFTM Analyser

(Applied Biosystems). A Nd:YAG (neodymium-doped yttrium aluminium garnet) laser

(355 nm) was used to irradiate samples. The spectra were acquired in reflectron mode in

the mass range 700 – 4000 Da and were externally calibrated using known peptide

standards including bradykinin, neurotensin, angiotensin and ACTH

(adrenocorticotropic hormone). The instrument was then switched to MS/MS mode

where the eight strongest peptides from the first MS scan were isolated and fragmented

by collision-induced dissociation using filtered laboratory air, then re-accelerated to

measure their masses and intensities. A near point calibration was applied and would

give a typical mass accuracy better than 50 ppm.

163

7.2.3.7 Database search and protein identification

The peptide peak lists were submitted to the database search program, Mascot (Matrix

Science Ltd., London, UK). Because the genome of Byssochlamys was unknown, all

samples were searched against all Fungi data in the non-redundant protein database,

NCBInr (9.0 x 106 entries in May 2011) (National Center for Biotechnology

Information, MD, USA). The selected search parameters were set as in Table 7.2.

Table 7.2. Search parameters set in the database search program Mascot

Search parameters Set values

Type of search PMF (Peptide mass fingerprinting) and MSMS

Enzyme used Trypsin

Variable modifications Methyl de-esterification, methionine oxidation and

cysteine modification of propionamide

Mass values Monoisotopic

Protein mass Unrestricted

Peptide mass tolerance ± 50ppm

Fragment mass tolerance ± 0.8 Da

Peptide charge state 1+

Max missed cleavages 1

When at least four peptide masses were matched to protein sequences in the database, a

number of parameters were considered to confirm meaningful identifications. The

MOWSE (Molecular Weight Search) score of the matching result given by the search

engine had to be at least 70 which increased with higher confidence (Perkins et al.,

1999). Positions of the matched peptides in the matched protein sequences had to be

close to each other. The molecular weight (MW) and pI values of the identified proteins

needed to match reasonably with experimental data of the 2D gel electrophoresis.

Putative functions of matched proteins were partially obtained from databases including

The Universal Protein (UniProt) Resource (The UniProt Consortium, 2011), The

Hierarchical Catalog of Eukaryotic Orthologs (OrthoDB) (Waterhouse et al., 2011) and

164

the World Health Organisation/International Union of Immunological Societies

(WHO/IUIS) Allergen Nomenclature database (Chapman et al., 2007b).

7.3 Results

7.3.1 General features of proteome of Byssochlamys ascospores

Protein concentrations and conductivity measurements of all protein extracts are

summarised in Appendix 7.1. They were used to determine the amounts of proteins

loaded to the 2D gels.

On typical 2D gels, a total of 509 and 502 protein spots were resolved for B. fulva FRR

2299 and B. nivea FRR 6002, respectively. The overall separation and clarity of the

spots were comparable to other 2D gels of Aspergillus nidulans conidia (Oh et al.,

2010) and Bacillus subtilis cells (Movahedi and Waites, 2000) and better than those

reported for Rhizopus oligosporus sporangiospores (Thanh, 2004). As also observed in

the cases of A. nidulans and B. subtilis, the protein contents were relatively high within

the pH range 4 – 7, which caused smearing and apparently overlapping regions.

Identified spots of the B. fulva strain had MWs varying from 10 to 170 KDa and pI

values from 4 to 10. About 70% of the spots had MWs of 10 – 59 KDa and about 80%

had pI of 4 – 7.9 (Appendix 7.2). The MW of identified spots of the B. nivea strain

varied over a smaller range, 10 – 100 KDa but their pI spanned a slightly wider range,

3.5 – 10. Approximately 70% of the spots had MW of 10 – 49 KDa while nearly 80%

had pI of 3.5 – 7.4 (Appendix 7.2).

Less than 4% of the 500 plus protein spots significantly changed their normalised

volumes (p < 0.05, minimum two-fold change) with either heating or aging. The

differential proteomic profiles of ascospores in response to each condition are described

in more detail in the following sections.

7.3.2 Proteomic profiles of ascospores following heat treatments

Protein spots that significantly changed (p < 0.05, minimum two-fold change) with heat

treatments are summarised in Table 7.3 for both tested B. fulva and B. nivea strains. The

MW and pI of each spot were estimated from its migrating position on the 2D gels.

165

Table 7.3. Significantly changed protein spots (in terms of normalised spot volumes) of

Byssochlamys ascospores following a heat treatment

Strain Spot no. ANOVA p-value

Fold change#

Estimated MW (KDa)

Estimated pI

B. fulva FRR 2299

1106 0.004 -4.3 40 4.75

1132 0.035 -2.7 45 7.79

1145 0.029 -3.4 38 9.05

1270 0.048 -6.1 39 4.65

1220* 0.056 -5.7 46 8.47

1137* 0.059 -3.1 51 7.27

Total number of spots = 507

Number of spots significantly increasing = 0

Number of spots significantly decreasing = 4

B. nivea FRR 6002

628 0.02 2.5 87 6.56

1197 0.0005 2.7 48 6.09

1202 0.027 2.6 51 6.1

1193 0.038 -2.1 31 4.31

Total number of spots = 501


Number of spots significantly decreasing = 1 * Spots that did not change significantly at α = 0.05 but significantly at α = 0.1 # Negative values denote decreased normalised volume; positive values denote increased normalised

volume

For B. fulva FRR 2299, no protein spots significantly changed after 1 and 4 minute

treatments. However, 4 out of 509 spots (spot number 1106, 1132, 1145 and 1270) had

significantly lower abundance levels after 30 minutes of treatment (Table 7.3). Two

additional spots (1120 and 1137) decreased more than two fold but their changes were

only significant at α = 0.1. However, the statistical tests of these two spots were close to

the confidence level of 0.05. Therefore, they were also included for the identification

step later. The estimated MWs of reported spots were mostly below 51 KDa while their

estimated pI values were variable between 4.65 and 9.05. A representative 2D gel

illustrating positions of the described spots of B. fulva ascospores is presented in Figure

7.3A. The changed normalised volumes of those spots are shown in Figure 7.3B.

166

Figure 7.3. (A) Positions of significantly changed protein spots of B. fulva FRR 2299 in

response to heat treatment at 90oC (broken arrows – decreased); (B) normalised

volumes of protein spots changing with heating time; each data point is an average of

three measurements on triplicate gels

Estimated MW (KDa)

Estimated pH

1106 1145

1132

1270

1220*

1137*

5 7 9

75

100

25

50

20

10

A

1145

1220

1332

1137

1106

1270

B

167

For B. nivea FRR 6002, there were no significant changes at 1 minute but 3 out of 502

identified spots (628, 1197 and 1202) significantly increased their normalised volumes

at 4 minutes. After 30 minutes, four spots (628, 1193, 1197 and 1202) showed

significant changes (Table 7.3). Three of them (628, 1197, 1202) had increased

normalised volumes and only one (1193) decreased its relative abundance at the end of

the heating. Three increased spots had estimated pI values in the neutral region (6.09 –

6.56) while the last one was more in the acidic environment (pI = 4.31). Spot 628 had a

MW of 87 KDa, but the MWs of the three other proteins were 31, 48 and 51 KDa. The

positions and changes of the four mentioned spots of B. nivea ascospores on 2D gels are

shown in Figure 7.4.

For a clearer presentation, the gel images of individual spot showing its differential

expressions after various heating times are presented in Appendices 7.3 (B. fulva FRR

2299) and 7.4 (B. nivea FRR 6002).

168

Figure 7.4. (A) Positions of significantly changed protein spots of B. nivea FRR 6002

in response to heat treatment at 87.5oC (unbroken arrows – increased, broken arrows –

decreased); (B) normalised volumes of protein spots changing with heating time; each

data point is an average of three measurements on triplicate gels

628

1197

1202

1193

A

Estimated pH9 54 7

Estimated MW (KDa)

100

75

50

25

20

10

1202

1193

1197

628

B

169

7.3.3 Proteomic profiles of ascospores at two ages

More protein spots significantly changed their normalised volumes when ascospore age

increased from 4 to 12 weeks compared with the heat treatment. Ascospores of B. fulva

FRR 2299 had 6 out of 509 detectable spots satisfying the significant change criteria

(Table 7.4). Two of those proteins were more abundant and four were less in 12 week

ascospores than the 4 week ones. The two proteins showing an increase had higher

estimated MWs and pI values than those that decreased. In particular, the MWs and pI

of the proteins that increased were above 55 KDa and 6.5, respectively whereas those of

the proteins that decreased were below 33 KDa and 5.8, respectively. Positions of the

six investigated spots of B. fulva FRR 2299 on 2D gels and their changes with ages are

shown in Figure 7.5.

B. nivea FRR 6002 had the most number of spots (17) that significantly changed their

expressions in response to aging (Table 7.4). Out of those 17 protein spots, 9 spots

increased their expression levels and the remaining 8 spots decreased during the aging

of ascospores. The MW ranges of both groups were overlapping between 18 and 52

KDa. The pI range of the proteins that decreased was 5.3 – 8.69, being slightly more

basic than those that increased, which was 4.47 – 7.77. The separation of the 17 protein

spots on 2D gels and the changes of their expression levels are shown in Figure 7.6.

The differential expression of each of the protein spots that significantly changed

between 4 and 12 weeks is presented in Appendices 7.5 (B. fulva FRR 2299) and 7.6 (B.

nivea FRR 6002).

170

Table 7.4. Significantly changed protein spots (in terms of normalised spot volumes)

between 4 and 12 week Byssochlamys ascospores

Strain Spot no. ANOVA p-value

Fold change#

Estimated MW (KDa) Estimated pI

B. fulva FRR 2299

83 0.018 2.4 81 7.52

1225 0.019 2 56 6.54

1222 0.009 -3.2 23 5.35

1224 0.023 -2 24 5.35

1228 0.029 -2 30 5.71

1236 0.024 -2.2 33 5.44


Number of spots significantly decreasing = 4

B. nivea FRR 6002

343 0.041 2.4 28 4.57

1061 0.014 2 27 6.69

1197 0.013 3.2 48 6.09

1241 0.016 3.7 27 7.77

1278 0.003 3.1 49 5.29

1302 0.015 2.4 30 6.45

1310 0.025 3.3 25 4.47

1327 0.036 2.9 31 7.16

1331 0.013 3.9 18 5.34

1275 0.008 -2.3 48 7.48

1281 0.036 -2.3 52 5.3

1299 0.001 -3.4 28 6.77

1304 0.004 -2.4 35 6.68

1335 0.034 -3.2 18 5.7

1340 0.034 -2.4 31 8.69

1343 0.036 -2 35 5.58

1347 0.023 -5.2 19 6.01


Number of spots significantly decreasing = 8 # Negative values denote decreased normalised volume; positive values denote increased normalised

volume

171

Figure 7.5. Changed proteomic profiles of B. fulva FRR 2299 ascospores during aging.

(A) Significantly different proteins spots between 4 and 12 weeks (unbroken arrows –

increased at 12 weeks, dashed arrows – decreased at 12 weeks; dotted arrows –

reference spots for identification); (B) Changed abundance of selected protein spots at 4

and 12 weeks

1222

83 1225

1224

1236 1228

A

Estimated pH5 7 9

Estimated MW (KDa)

75

100

25

50

20

10

1052

1020

821

831225

12241228

1222

1236

B

172

Figure 7.6. Changed proteomic profiles of B. nivea FRR 6002 ascospores during aging. (A) Significantly different spots between 4 and 12

weeks (unbroken arrows – increased at 12 weeks, broken arrows – decreased at 12 weeks); (B) Increased abundance of selected protein spots at

4 and 12 weeks; (C) Decreased abundance of selected protein spots at 4 and 12 weeks

1331

12781281

1299

A

1304

12751197

1061

1302

1241

1347

1310

1335

1340

13271343

343

Estimated pH

Estimated MW (KDa) 100

75

50

25

20

10

B1310

1331 1061

1302

12413431197

13271278

12991335 1340

1343 1304

13471281

C

1275

95 4 7

173

7.3.4 Protein identification

A total of 20 protein spots, including 8 spots from B. fulva FRR 2299 and 12 from B.

nivea FRR 6002, were selected from those shown in Tables 7.3 and 7.4 for protein

identification. Three highly intense spots (821, 1020 and1052) from B. fulva FRR 2299

were chosen as internal controls for the identification process (Figure 7.5A). The

identifications of the selected spots and their putative functions are shown in Tables 7.5

(B. fulva FRR 2299) and 7.6 (B. nivea FRR 6002). For each protein, the top two results

based on MOWSE scores are included only if they score above 70. The closest matches

of spots with scores below 70 are also given in Tables 7.5 and 7.6. The MWs estimated

from 2D gels of all spots are repeated in those tables to assist the comparison with those

of matched proteins. For further reference, amino acid sequences of the matched

proteins and peptide coverage are given in Appendix 7.7.

All reference spots were identified with high MOWSE scores, confirming that the

preparation and identification processes were working properly (Table 7.5). Of

particular interest, spot 1052 was matched with heat shock protein (HSP) 70 from

Talaromyces and Aspergillus species. The other two reference spots, 821 and 1020,

were similar to members of the oxidoreductase and mannose-6-phosphate isomerase

families, respectively. Their estimated MWs and pI values were relatively close to those

of the matched proteins. Exceptionally, spot 1052 showed large differences between the

estimated MW (75 KDa) and that of the matched protein (69 KDa).

7.3.4.1 Identification of proteins from B. fulva ascospores

Of the 8 spots from B. fulva FRR 2299, only four were identified with scores above 70

and they all had decreased expressions with heat treatment. They had good sequence

coverage and peptide matches with the theoretical proteins (Appendix 7.7). However,

comparing MWs and pI values did not show strong agreements between the estimated

results from 2D gels and those of the matched proteins. The identified proteins were

predominantly enzymes involving in the citric acid cycle (TCA cycle) for metabolism

and energy generation, such as oxidoreductase, citrate synthase and its mitochondrial

precursor. The hypothetical protein An07g00820 (1132) possibly had oxidoreductase

activity specific to flavin-containing compounds. Another protein (1270) was identified

as the pepsin precursor, pepsinogen, or aspartic protease pepE (Table 7.5).

174

For spots scoring below 70, the best matches included a variety of protein types

including enzymes (aspartic endopeptidase enzyme), nucleic acid binding and mRNA

splicing proteins. One protein (1224) was similar to a predicted protein from Botryotinia

fuckeliana B05.10 with currently uncharacterised functions. The estimated MWs and pI

values of the proteins scoring below 70 were weakly correlated with those of the

matched proteins (Table 7.5).

7.3.4.2 Identification of proteins from B. nivea ascospores

For B. nivea FRR 6002, 2 out of 4 selected heat-related spots and 6 out of 8 age-related

spots had matches with scores above 70. Both heat-related proteins were identified as

putative erythromycin esterase. Among the 6 age-related spots, two proteins, 1281 and

1299, were matched with HSP 60 and general stress protein (GSP) 39, respectively.

Two other proteins (1331 and 1335) were similar to allergen Asp F3 usually found in

Aspergillus clavatus or N. fischeri. This allergen protein also functions as an

oxidoreductase to maintain the intracellular reduction-oxidation environment. The last

two spots, 1061 and 1241, were the TCA cycle enzyme, malate dehydrogenase, and the

pentose-phosphate pathway enzyme, transaldolase, respectively. In the case of MWs

and pI values, 3 spots (1202, 1331 and 1335) had close results from 2D gels and the

matched proteins whereas other protein spots were not matched.

Three out of 4 spots that scored below 70 were best matched with various types of

hypothetical proteins and conserved hypothetical proteins with uncharacterised

functions. Only one spot (1193) was closer to a precursor for protease A in vacuoles.

The estimated MWs and pI values of those proteins showed large discrepancies with

those of the matched results.

175

Table 7.5. Identification of selected proteins extracted from B. fulva FRR 2299 ascospores after heat treatments and at different age

Spot NCBI code Protein (putative function) Microorganisms MOWSE

score Matched peptides

Coverage %

MW KDa#

pI#

Proteins changing significantly with heating times

1132 145237054 Hypothetical protein An07g00820 (NADH-dependent flavin oxidoreductase putative)

Aspergillus niger CBS 513.88 101 14 42% 45.5 (45)

6.1 (7.8)

218723676 NADH-dependent flavin oxidoreductase, putative

Talaromyces stipitatus ATCC 10500

101 14 36% 46.2 (45)

6.4 (7.8)

1145 238935537 KLTH0E15532p (Oxidoreductase) Lachancea thermotolerans 73 10 36% 34.5 (38)

5.8 (9.1)

1220* 18141566 Citrate synthase Emericella nidulans 241 18 35% 52.1 (46)

8.8 (8.5)

67902576 CISY_EMENI Citrate synthase, mitochondrial precursor

Aspergillus nidulans FGSC A4 241 19 35% 52.2 (46)

8.7 (8.5)

1270 530795 Pepsinogen Aspergillus niger 136 5 16% 43.4 (39)

4.9 (4.7)

145232965 Aspartic protease pepE Aspergillus niger CBS 513.88 136 5 16% 43.4 (39)

4.9 (4.7)

Spots with MOWSE scores below 70

1106 218720012 Aspartic endopeptidase Pep2 Talaromyces stipitatus ATCC 10500

40 2 6% 42.8 (40)

4.9 (4.8)

1137* 85105528 Hypothetical protein NCU07735 (Nucleic acid binding, putative)

Neurospora crassa OR74A 38 9 27% 37.8 (51)

7.8 (7.3)

1224 154298888 Predicted protein (Uncharacterised function)

Botryotinia fuckeliana B05.10 45 4 56% 9.4 (24)

4.9 (5.4)

176

1225 211591546 Pc22g04920 (mRNA splicing, putative) Penicillium chrysogenum Wisconsin 54-1255

53 28 44% 81.5 (56)

6.6 (6.5)

Reference proteins

821 212528726 Oxidoreductase, short-chain dehydrogenase/reductase family

Penicillium marneffei ATCC 18224

174 9 35% 32.2 (33)

8.1 (7.5)

238508858 Aspergillus flavus NRRL3357 138 6 31% 24.1 (33)

5.7 (7.5)

1020 154290333 Hypothetical protein BC1G_15797 (Putative mannose-6-phosphate isomerase and zinc ion binding)

Botryotinia fuckeliana B05.10 113 11 29% 44.7 (45)

5.5 (6.3)

119500846 Mannose-6-phosphate isomerase, class I Neosartorya fischeri NRRL 181 105 11 39% 44.8 (45)

6.1 (6.3)

1052 218715619 Molecular chaperone HSP70 (Protein folding, cytoprotection)

Talaromyces stipitatus ATCC 10500

738 31 51% 69.5 (75)

5.1 (5.4)

67537918 HS70_TRIRU Heat shock 70 kDa protein (Protein folding, cytoprotection)


5.0 (5.4)

# Estimated MW and pI of the matched proteins; asterisks (*) are spots with 0.05 < p < 0.1

# Numbers in brackets are MW and pI estimated previously from 2-D gels

177

Table 7.6. Identification of selected proteins extracted from B. nivea FRR 6002 ascospores after heat treatments and at different ages

Spot NCBI code Protein name and functions Microorganisms MOWSE

score Matched peptides

Coverage %

Mass KDa# pI#

Proteins changing significantly with heating time

1197 121702629 Erythromycin esterase, putative Aspergillus clavatus NRRL 1 115 12 32% 51.7 (48)

5.9 (6.1)

145254965 Hypothetical protein An18g03600 (Erythromycin esterase, putative)

Aspergillus niger CBS 513.88 115 14 31% 54.1 (48)

6.3 (6.1)

1202 121702629 Erythromycin esterase, putative Aspergillus clavatus NRRL 1 77 15 36% 51.7 (51)

5.9 (6.1)

Proteins changing significantly with age

1061 226283156

Malate dehydrogenase, NAD-dependent

Paracoccidioides brasiliensis Pb01

133 15 37% 34.6 (27)

6.4 (6.7)

121703522 Aspergillus clavatus NRRL 1 128 14 42% 35.8 (27)

6.9 (6.7)

1241 115400267

Transaldolase

Aspergillus terreus NIH 2624 221 14 41% 35.0 (27)

6.2 (7.8)

39970315 Magnaporthe oryzae 70-15 214 13 45% 35.6 (27)

5.4 (7.8)

1281 115443330HSP 60, mitochondrial precursor (Protein refolding, prevent aggregation of denatured proteins, proapoptosis)

Aspergillus terreus NIH2624 261 22 46% 62.2 (52)

5.5 (5.3)

238844339 Arthroderma otae CBS 113480

258 26 53% 62.3 (52)

5.4 (5.3)

1299 119501350 Oxidoreductase, short-chain dehydrogenase/reductase family

Neosartorya fischeri NRRL 181

238 6 19% 33.6 (28)

6.0 (6.8)

115401132 General stress protein 39 (Oxidoreductase, short-chain dehydrogenase/reductase family)

Aspergillus terreus NIH 2624 187 5 26% 29.9 (28)

5.4 (6.8)

178

1331 121704078Allergen Asp F3 (Oxidoreductase, cell redox homeostasis)

Aspergillus clavatus NRRL 1 190 7 44% 18.6 (18)

5.1 (5.3)

119467218 Neosartorya fischeri NRRL 181

186 6 36% 18.5 (18)

5.4 (5.3)

1335 121704078Allergen Asp F3 (Oxidoreductase, cell redox homeostasis)


5.1 (5.7)

119467218 Neosartorya fischeri NRRL 181

192 6 29% 18.5 (18)

5.4 (5.7)

Spots with MOWSE scores below 70

628 67522969 Hypothetical protein AN1941.2 (Putative uncharacterised protein)


4.6 (6.6)

1193 189211129 Vacuolar protease A precursor Pyrenophora tritici-repentis Pt-1C-BFP

69 4 16% 43.5 (31)

5.1 (4.3)

1310 116194057 Hypothetical protein CHGG_06746 (Putative uncharacterised protein)

Chaetomium globosum CBS 148.51

46 7 56% 20.6 (25)

9.7 (4.5)

1347 121698176 Conserved hypothetical protein (Putative uncharacterised protein)


9.9 (6.0)

# Estimated MW and pI of the matched peptides

# Numbers in brackets are MW estimated on 2-D gels

179

7.4 Discussion

This Chapter reports the first application of proteomics in studying heat resistant

ascospores of Byssochlamys species. The proteomic profiles of 4 week ascospores of

one strain of B. fulva and one strain of B. nivea were compared at various time points

during heat treatments (dormant, 1, 4 and 30 minutes) at 90oC and 87.5oC, respectively.

These sampling times corresponded with some distinct responses exhibited by

ascospores when subjected to heat inactivation at these temperatures (Chapter 3). In

particular, they show a clear activation stage during the first minute, followed by a

linear inactivation (usually 2 – 20 minutes) and then a tailing stage (after 20 minutes)

where surviving ascospores show little further inactivation. In addition, proteomic

analysis was conducted on dormant, non-heated ascospores from 4 and 12 week cultures

to examine the roles of proteins in age-related heat resistance, since it was found in

Chapter 3 that ascospores from older cultures were more heat resistant than those from

younger cultures.

7.4.1 Proteomes of Byssochlamys ascospores after heat treatments and aging

Proteins occur in dormant B. fulva ascospores and account for 8 – 12% of the total

ascospore weight (Banner et al., 1979). Proteomic analysis has now shown that heating

and aging caused small but significant changes to ascospore proteins. The selection of

significantly changed protein spots based on stringent criteria, namely ANOVA p <

0.05, minimum two fold change, clear spots and triplicate analyses, minimised possible

experimental and technical errors, ensuring validity of the results.

Heat treatments caused fewer variations in the proteomes than that of the aging,

suggesting time was an important factor. This was supported by the increasing number

of protein spots that were observed to change when the time of heating progressed to 30

minutes. No significant changes were observed at 1 minute (B. nivea FRR 6002) or 4

minutes (B. fulva FRR 2299) of heating. The delayed response may be ascribed to the

special ultrastructure of Byssochlamys ascospores, especially the cell wall and the thick

intermediate space (Chapter 4), which are likely to slow down the rate of thermal

disruption. Moreover, since the results of Chapter 3 show that the first minute is related

to the activation stage of the ascospores, this initial response could be predominantly

caused by modifications of the physical structure and less involved chemical (protein-

180

linked) process. Ascospores of B. nivea reacted more quickly and displayed earlier

proteomic changes than those of B. fulva and this observation was consistent with the

fact that they were more abundant as single forms (not clumps) and more prone to heat

inactivation.

7.4.1.1 Changed proteomic profiles of ascospores after heat treatments

The abundance of proteins in ascospores may either increase or decrease as a result of

heat treatments. The attenuation of particular proteins could be due to several causes.

Firstly, the heating process could disrupt the physical structure and integrity of the

ascospores, allowing extracellular loss of protein concentration or decrease in

concentration to levels that are no longer detectable on gels. It has been reported that

wet heat could moisten the structure of endospores of Bacillus sporothermodurans and

cause proteins to leak out of the cells (Tabit and Buys, 2010). Secondly, heating may

completely denature proteins due to unfolding of their tertiary and secondary structures,

leading to protein degradation and loss of materials detectable on gels (Dynlacht et al.,

2000). Temperatures above 40oC are sufficient to induce denaturation of many cellular

proteins, with the severity progressively increasing at higher temperatures (Belliveau et

al., 1992; Lepock, 2005; Nguyen et al., 1989). Intracellular proteins of wet heat-killed

endospores of Bacillus cereus and Bacillus megaterium underwent critical structural

modifications towards denatured states (Coleman et al., 2010). Thirdly, heating may

activate protein degradation by proteolytic enzymes. The environment of unheated

ascospores may not be conducive to such reactions because of high viscosity and

compartmental separation of substrates from enzymes (Setlow, 2003). Heating breaks

down these barriers such as rehydration of the cytoplasm thereby allowing enzymatic

proteolysis to occur (Cowan et al., 2003).

Factors that could explain an increase in the amount of a protein after heating of the

ascospores are protein synthesis, fragmentation of proteins into peptides and

aggregation of denatured proteins. The first cause, protein synthesis, is common

outcome of up-regulated genes during spore germination and outgrowth (Armstrong et

al., 1984; Oh et al., 2010). However, gene up-regulation and protein synthesis were

unlikely to occur in heated ascospores in the present study. The moist heat environment

was considerably hostile to any anabolic pathways and newly formed proteins (Zhang et

181

al., 2009). Additionally, the maximum heating time, 30 minutes, may be insufficient for

proper protein syntheses in high temperature conditions.

Fragmentation of proteins into peptides may occur as a result of thermal denaturation.

Instead of losing the peptide products as mentioned earlier, they could be retained by

the ascospores and subsequently detected by electrophoresis. Long heating time could

increase the quantity of those peptides, with their concentrations reflected by elevated

spot volumes on 2D gels (Havea et al., 1998).

Aggregation of proteins could be induced by heat treatments (Havea et al., 1998). In

fact, denatured proteins due to heat shock could act as nuclei for the aggregation of

more proteins, both native and denatured ones, thereby protecting them from further

denaturation (Lepock, 2005). Therefore, this could improve the concentration of

proteins detected on gels.

7.4.1.2 Changed proteomic profiles of ascospores during aging

The increased abundance of a protein during aging could be attributed to a latent protein

synthesis. Dormant ascospores of could maintain a relatively low respiration rate for

respiration purpose, or endogenous respiration, based on the oxidation of lipid

(Mandels, 1963; Mandels and Maguire, 1972). This low but important metabolism may

indicate protein turnover, which could be the main reason why older Byssochlamys

ascospores had increased expression of proteins. The latent protein synthesis was found

in dormant ascospores of Neosartorya fischeri during aging between 11 and 25 days,

manifesting an accumulation of proteins around the germ-tube area (Conner et al.,

1987). As also suggested for conidia of Aspergillus oryzae (Sinohara, 1970), some

enzymes of the dormant ascospores could be significantly induced and carried out their

specific reactions without signs of germination and swelling of the cells.

A decrease in expression of particular proteins during aging could be ascribed to

denaturation. As discussed in Chapter 4, aging could promote dormancy and heat

resistance by enhancing dehydration inside the ascospores. This reduced moisture

environment may denature protein structures and subsequently affect the proteomic

profiles of the matured ascospores. However, the results of proteomics and protein

identification in the present study were not sufficient to substantiate these theories.

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7.4.2 Protein identification and functions in Byssochlamys ascospores

7.4.2.1 Stress related proteins

Stress related proteins have been suggested to be the molecular mechanism for spores to

acquire thermotolerance (Movahedi and Waites, 2000; Setlow, 2006). In the present

study, three stress related proteins, namely HSP 60, HSP 70 and the smaller GSP 39,

were identified in Byssochlamys ascospores for the first time. However, these proteins

were reported in ascospores of either B. fulva FRR 2299 (HSP 70) or B. nivea FRR

6002 (HSP 60, GSP 39) but not in ascospores of both species. This is probably because

only a subset of proteins spots of each species was selected for identification. More

research will be needed to determine whether they are common stress proteins of

Byssochlamys species.

The stress proteins reported in this study are found at high concentrations in vegetative

cells, and act as a natural defence mechanism against environmental stresses including

heat (Goldani et al., 1994; Kim et al., 2007; Lindquist and Craig, 1988). However, the

synthesis of these proteins requires much energy and their activities are also ATP-

dependent (Kregel, 2002; Plesofsky-vig and Brambl, 1985). Thus, ascospores are not

likely to expend much energy on synthesising these proteins when stress conditions

arise. Instead, as a proactive strategy based on the model of Bacillus species (Eaton and

Ellar, 1974; Movahedi and Waites, 2000) and Neurospora crassa (Rensing et al., 1998),

most of these proteins are probably already synthesised and stored inside the cytoplasm

during ascospore formation.

Both HSP 60 and HSP 70 are abundant in different types of cells and function as

‘molecular chaperones’, which protects native proteins via correct folding and re-

folding (Bukau and Horwich, 1998; Panaretou and Zhai, 2008). In Byssochlamys

ascospores, they are likely to localise in the central cytoplasm where macromolecules

that require chaperone protection are located. Their functions could be exploited by the

ascospores during heat treatment so that proteins essential for germination and

metabolism are safeguarded.

Members of HSP 60 family are found and function strictly in the mitochondrial matrix

(Panaretou and Zhai, 2008). The expressions of their encoding genes are constitutively

expressed in many fungal species, such as Aspergillus spp., Penicillium spp., Candida

spp. and Saccharomyces spp., under various stresses including elevated temperatures

183

(Raggam et al., 2010). Their major operation is to stabilise intracellular proteins under

stress conditions, which is achieved by binding proteins to prevent them from

inactivation, blocking the aggregation of denatured proteins, and mediating the

refolding of mis-folded proteins (Burnie et al., 2005; Martin et al., 1992). The protective

activities of HSP 60 are often complemented by a group of smaller proteins, HSP 10,

together forming a chaperone machine inside mitochondria (Gomes and de Cássia

Garcia Simão, 2009). The HSP 60 family are also proapoptotic proteins that assist the

programmed breakdown of cellular components (Kregel, 2002).

Members of HSP 70 family are located in various compartments of the cells, including

the cytoplasm, mitochondria and endoplasmic reticulum (Gomes and de Cássia Garcia

Simão, 2009). The protections mechanisms offered by the HSP 70 family are relatively

diverse, with major functions involving assisting correct folding of newly formed or

denatured proteins, and maintaining native structures of proteins, preventing

aggregation of denatured proteins (Bukau and Horwich, 1998; Mayer and Bukau, 2005).

The HSP 70 also helps translocation of proteins across membranes into different

compartments (Panaretou and Zhai, 2008).

The general stress protein GSP 39 was first identified in stressed Bacillus subtilis cells

(Antelmann et al., 1997) but information about its functions is very sparse, probably

because of its specific expression in a limited numbers of species. The GSP 39 belongs

to the dehydrogenase/reductase family which plays a role in maintaining the redox

homeostasis in cells (Kavanagh et al., 2008). A similar function may be carried out by

the GSP 39 in Byssochlamys ascospores so that the intracellular environment is suitable

for protein conservation.

The activity of the GSP 39 is quite different from those of other stress proteins with

similar MW ranges. The HSP 40 (MW ~ 40 KDa), for instance, is co-chaperones for

HSP 70 (Qiu et al., 2006). Similarly, a class of small HSPs with MW 15 – 30 kDa

possess chaperone functions which include preserving native protein structures and

preventing protein aggregation. Interestingly, the activities of those small HSPs are ATP

independent so they may be compatible with ascospores which are low in energy

expenditure (Jakob et al., 1993; Mchaourab et al., 2009).

184

7.4.2.2 Other identified proteins and their potential functions in ascospores

Apart from the stress related proteins, some of the other identified proteins belonged to

various groups of enzymes which participate in key metabolic pathways in vegetative

cells. Citrate synthase and malate dehydrogenase are a part of the TCA cycle which is

central for the respiration and metabolism in vegetative cells (Xie et al., 2004).

Transaldolase involves in the pentose phosphate pathway which synthesises the

essential coenzyme NADPH (Xie et al., 2004). The pepsin precursor, pepsinogen, and

the pepsin-like aspartic protease pepE are necessary for catabolism of proteins (Fruton,

2002).

The finding of metabolic enzymes in Byssochlamys ascospores was comparable to that

of other species. Malate dehydrogenase and alcohol dehydrogenase were found in

dormant ascospores of Neurospora tetrasperma (Lingappa et al., 1970). Several

enzymes for metabolism and germination have also been detected in forespores and

endospores of Bacillus spp. and Clostridium difficile (DelVecchio et al., 2006; Kuwana

et al., 2002; Lawley et al., 2009). Conidia of A. oryzae contained inducible forms of

proteases such as α-amylase and invertase (Sinohara, 1970). Therefore, one can

conclude that enzymes required for metabolism have been pre-synthesised and stored in

dormant ascospores. This ensures a quick revival of the ascospores when environmental

conditions are hospitable for germination. The availability of metabolic enzymes also

avoids need for protein synthesis and energy requirements at the beginning of the

activation and germination processes.

The rest of the identified proteins possess unique functions and their involvement in

ascospores may present some competitive advantages in surviving in an unfavourable

environment. The hypothetical protein An07g00820 is a putative NADH-dependent

oxidoreductase for flavin-containing compounds, which are required in catabolism of

nitrogen-organic compounds (Morokutti et al., 2005). Many nitrogen-containing

compounds such as drugs and antibiotics are probably lethal to the mould, thus the

enzyme would allow the ascospores to survive and germinate in a special niche such as

the soil in an orchard. Similarly, the erythromycin esterase may offer a resistance

against the antibiotic erythromycin (Arthur et al., 1986). The identification of allergen

Asp F3 may be a possible invasive factor which could also provide an advantage in

mixed soil populations. The larger groups of allergen Asp F are often associated with

185

Aspergillus fumigatus species which are well-known for invasive and allergic

pulmonary aspergillosis in humans and birds. When conidia of A. fumigatus enter the

body and germinate, Asp F3 acts as an antigen triggering the immune responses of

various cells such as CD cells, T helper cells and interferon lambda (Chaudhary et al.,

2010). However, there is no available evidence to support any possible link between the

Asp F3 found in ascospores of Byssochlamys with the aspergillosis caused by A.

fumigatus.

7.4.3 Low percentage of significant matches and poor agreement between

estimated MW and pI values

A third of the proteins (8 out of 23 spots) chosen for MS/MS were not satisfactorily

matched (MOWSE score < 70) with proteins in the NCBInr fungal protein database.

Attempts to expand the search to include all species in the NCBInr protein database

(prokaryotes and human) did not improve the current results. Moreover, there were poor

agreements between MW and pI of matched proteins with the estimated values from 2D

gels. The identification process being employed in this experiment involved matching

the eight most abundant peptides of the test proteins with peptides generated by

theoretically enzymatic digestion of proteins in the database. The advantage of this

approach is rapid identification but it requires knowledge of the species’ genomes so

that protein sequences can be derived. The lack of information about Byssochlamys

genomes could explain the low rate of good identification and the low agreement of

MW and pI. Most of the successfully matched proteins came from a few well-known

fungi such as Aspergillus, Penicillium and Neosartorya whose genome sequences are

available. However, their proteins may not be homologous to those of Byssochlamys

species. The present study has highlighted a knowledge gap in the genomics of

Byssochlamys species which needs to be filled so that advances in understanding the

molecular aspects of heat resistance in the ascospores can be realised.

186

7.4.4 General assessment of the proteome of Byssochlamys ascospores

Ascospores of B. fulva FRR 2299 and B. nivea FRR 6002 strains exhibited more than

500 detectable spots on 2D gels irrespective of heating times and ages. This figure is

comparable to those of spores of other fungi. Proteomic profiles of conidia of A.

fumigatus generated on 2D gels contained 485 spots (Teutschbein et al., 2010). Using a

directly comparative technique called isobaric tagging for relative and absolute

quantitation (iTRAQ), Cagas et al. (2011) could identify 461 proteins in A. fumigatus

conidia. However, the proteome of Penicillium marneffei conidia showed only 270

detectable spots (Chandler et al., 2008).

However, the number of proteins may be less than that because one protein could be

presented by multiple spots as observed in the result. This phenomenon is usually due to

post-translational modifications (PTMs), which are common activities in protein

synthesis in eukaryotic cells (Mann and Jensen, 2003; Vödisch et al., 2009). In the

current experiment, the identification of various allergen Asp F3 proteins represented by

more than one spot supported the occurrence of PTMs in ascospore proteins. Series of

spots with similar MW but incrementally increasing pH are other lines of evidence of

PTMs in this experiment (Jacob and Turck, 2008; this study, Figures 7.3 and 7.4).

The wide MW and pI ranges of protein spots of Byssochlamys ascospores were not

uncommon considering similar variations in proteomic profiles of other fungal species.

Different strains, species and ascospores at various physiological states had dissimilar

proteomes, as could be expected, but there were no definite relationships between these

factors. Germinating and actively growing cells have highly active metabolism so they

could be expected to have more proteins expressed than dormant structures like

ascospores (Armstrong et al., 1984) although some proteins could be down-regulated or

disappear during germination and growth (Thanh, 2004). The number of proteins

detected in A. fumigatus conidia varied between 310 and 461 depending on the methods

used (Cagas et al., 2011; Teutschbein et al., 2010). Proteomic profiles of Trichophyton

rubrum conidia showed about 1026 proteins (Leng et al., 2008). Only 381 spots on 2D

gels representing 334 proteins were identified for the mycelium of A. fumigatus. The

figures for mitochondria of A. fumigatus were even less, with 234 spots and 147

proteins (Vödisch et al., 2009).

187

The spot resolution on 2D gels could vary with the chosen separation ranges. The wide

ranges of MW (10 – 100+ KDa) and pI (3 – 10) used in the current study greatly

contributed to a large number of detectable spots. Shorter ranges could resolve fewer

spots (Crespo-Sempere et al., 2011) but may separate groups of close proteins better,

giving more spots on gels (Khan et al., 2008).

The sensitivity of the dyes and viewing techniques also affected spot visualisation.

Coomassie Blue is less sensitive for viewing protein spots than other dyes such as

SYPRO Ruby or silver staining, so it could underestimate the number of spots (Harris et

al., 2007). In fact, the difference could be as many as 500 spots when Coomassie and

silver stains were applied to 2D gels of similar germinating conidia of Emericella

nidulans (Oh et al., 2010). The sensitivity of Coomassie Blue was significantly

enhanced in the present study by using an infrared fluorescence imaging system (Harris

et al., 2007).

7.4.5 Recommendations for future research

Because 2D GE has not been used to study Byssochlamys ascospores before, the results

present here are also exploratory and can be improved in some aspects. First, gels of

narrower MW and pH ranges than those used in the present study can be used to focus

on subsets of proteins so that the resolution and separation power of 2D gels are

improved. In particular, several groups of overlapping proteins are found in the pI range

4 – 7 and these proteins can be differentiated more clearly on gels with the similar pH

range (Khan et al., 2008). Second, apart from Coomassie blue, more sensitive protein

stains such as SYPRO Ruby can be used to enable better visualisation and quantitative

comparison of protein spots (Albrecht et al., 2010; Harris et al., 2007). Third, more

spots should be chosen for identification to reveal more information about functions of

proteins in ascospores. Another approach is to compare proteomic profiles of mycelium

and dormant ascospores of Byssochlamys, which may reveal proteins specific at

different developing stages of the fungi.

188


This study examined the proteome of Byssochlamys ascospores using 2D gel

electrophoresis and MALDI-TOF/TOF mass spectrometry. The analysis was conducted

on 4 week old ascospores of one B. fulva and one B. nivea strain when they were

subjected to 30-minute heat treatment at 90oC and 87.5oC, respectively. Comparison

was also carried out with proteomic profiles of the dormant ascospores at 4 and 12

weeks. More than 500 protein spots were detectable on 2D gels for both strains but only

a small percentage of these proteins (< 4%) significantly changed their relative

abundance with heating or aging.

During heat treatments, significant changes in proteomic profiles of the ascospores

occurred at least after one minute. This delay could be due to the resistant structure of

the ascospores. The initial heat activation is more likely to involve changes of physical

structure than chemical states of the ascospores. The decreased expression may be

associated with thermal denaturation proteins, extracellular loss of proteins due to

structural disruption or enzymatic proteolysis. The increased abundance was unlikely to

involve up-regulation of genes because the heat conditions were hostile to protein

synthesis. Instead the higher protein content may due to fragmentation of proteins into

peptides or protective aggregations of proteins.

More proteins were observed to change significantly with age than heating, and this

may be because time was a limiting factor for the alterations. Latent protein synthesis

could occur during the aging process, contributing to the increased expression of several

spots. Proteins could be denatured with time, thus reducing their abundance in aged

ascospores.

Twenty three protein spots were selected for identification. Protein matching was

significant for 12 spots whereas the results for the rest were less reliable due to low

MOWSE scores. There were three types of stress-related proteins among the identified

proteins, being HSP 60, HSP 70 and GSP 39. These proteins are well-known for their

chaperones’ functions which protects other proteins from denaturation. This is also the

first time that HSPs have been identified in Byssochlamys ascospores, supporting a long

held theory of the roles of HSPs in thermotolerance of this species.

189

Other identified proteins belong to groups of enzymes either participating in key

metabolic pathways or supporting competitive survival of ascospores in natural

environments such as soil.

The proteomic results suggest a survival strategy in Byssochlamys ascospores whereby

proteins essential for ascospore survival and germination are pre-synthesised and

packed inside the central cytoplasm, where they are protected by heat shock chaperones.

That arrangement minimises the need for protein synthesis and energy generation at the

beginning of the germination process.

The findings of this experiment were limited by the lack of genomic information on

Byssochlamys species. This knowledge gap needs to be addressed before the molecular

basis of heat resistance can be further elaborated. Future research can identify proteins

with a greater confidence, and relate them back to genes and transcriptomes during

aging or exposure to stresses such as heating.

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Chapter 8 – General discussion and conclusions

8.1 Introduction

The studies reported in this thesis aimed to elucidate the contributions of some intrinsic

factors to the heat resistance of fungi in the genus Byssochlamys. Like other heat

resistant moulds, Byssochlamys species acquire thermo-tolerance through the sexual

production of ascospores, dormant structures which are much more tolerant to

environmental stresses than vegetative cells (Pitt and Hocking, 2009). Several previous

investigations have explored the heat resistance of Byssochlamys ascospores mostly in

complex media or other conditions, thereby introducing potential complications from a

number of external factors (Bayne and Michener, 1979; Beuchat and Toledo, 1977;

Splittstoesser et al., 1986). The studies reported here predominantly focused on

determinants of the heat resistance that are intrinsic to the ascospores, and would

therefore be expected to provide explanations of mechanisms more closely linked to the

properties of the cells. The factors investigated in this study included: thermal

inactivation of the ascospores from cultures at different ages, ascospore ultrastructure,

proteomic profiles and, population heterogeneity at the community level.

Each of these aspects and their relationship to the heat resistant properties were

separately investigated and discussed in previous chapters. However, it has been shown

on several occasions that these properties are not independent of each other, for instance

the relationship between aging and ultrastructure of the ascospores (Chapter 4). The

various components are likely to operate at different cellular levels and together

contribute to a greater heat resistance of the ascospores. This multifaceted heat

resistance is analogous to similar systems that have been elucidated in endospores of

bacterial species such as Bacillus spp. and Clostridium spp. (Orsburn et al., 2008;

Setlow, 2006).

This Chapter builds on the findings and discussions of other studies reported earlier to

elaborate further the interactions among these intrinsic factors, which partially define

the thermo-tolerance of Byssochlamys ascospores. The factors, except inactivation

temperatures, and their inter-dependent relationships with respect to the heat resistance

191

of the ascospores that have been proposed in the present studies, are illustrated in Figure

8.1.

Figure 8.1. Relationships between the heat resistance and intrinsic properties of

Byssochlamys ascospores investigated in this thesis. Some relationships are described

and discussed based on the experimental results presented in previous chapters

(unbroken arrows). Other relationships are not clearly shown by the conducted

experiment (broken arrows). Arrows indicate the influencing direction of one factor

onto another

8.2 Age, dormancy and heat resistance of ascospores

Age is an important fundamental property and heat resistance factor of Byssochlamys

ascospores. Therefore, it is an underpinning variable in all studies reported in this thesis.

The increase in heat resistance of the ascospores with age could be related to changes in

Ascospore age

Multilayered ultrastructure

Glassy state Proteins

Ascospore population

heterogeneity

Heat resistance

Individual ascospores

More layers and lower moisture

More robust and resistant structure

Structure stabilisation

Physical protection

Low moisture

Increase individual viability

Increase group viability

Individual variations

192

the physiology, ultrastructure, chemical composition and population distribution of the

cells.

Dormancy is a unique physiological feature of microbial spores. It is generally

associated with elevated resistance to environmental stresses including heating

(Newsome, 2003; Sussman and Douthit, 1973). For Byssochlamys species, aging may

enhance the level of dormancy and consequently increase the heat resistance of the

ascospores. In Chapter 3, older ascospores exhibited lower percentage of activation than

the younger ones. If the decreased activation had been due to dead ascospores, one

would have expected a sudden drop in the survival counts following heat treatments.

Contrarily, the thermal inactivation kinetics of the matured ascospores showed a long

shoulder stage, a gradually linear inactivation and a tailing at a similar count as that

observed for younger cells. Thus, aged ascospores may become more heat resistant as a

result of an enhanced dormancy.

The relationship of three aspects, including age, dormancy and heat resistance, is not

limited to ascospores of B. fulva and B. nivea. Conner and Beuchat (1987) proposed a

similar interaction in ascospores of Neosartorya fischeri after the fungal cultures were

grown for up to 114 days. At the end of the longest incubation, ascospores became more

dormant and required more energy to germinate and inactivate. Thus, the age-induced

dormancy could be postulated to be a driving force behind the higher heat resistance of


8.3 Ultrastructure and heat resistance of ascospores

The unique ultrastructure of microbial spores is essential for their dormancy and,

therefore, plays a crucial role in their stress resistance (Driks, 2003; Sussman and

Douthit, 1973). Protection of the dormancy involves physically preventing the entering

of water and harmful chemicals into the spores as well as the loss of DNA and

important macromolecules from the cells. For the ascospores of B. fulva and B. nivea,

the cell-wall and the thick intermediate space (IMS) could be regarded as the protective

shells safe-guarding materials in the central cytoplasm from physical insults.

Additionally, in dormant, non-heated ascospores, these two layers may also be

impermeable to water and chemicals, which would be crucial in maintaining a

dehydrated intracellular core.

193

The function of the cell wall and the IMS as physical barriers was highlighted by their

ability to hinder SYTO9 or fixatives for electron microscopy from entering freely the

ascospores (Chapters 4 and 6). This obstruction was partially removed when the wall

and structure was compromised by heating. Modifications inflicted by heat treatments

to the ascospores could be opening pores or disrupted integrity of the cell-wall and the

IMS. Consequently, following thermal treatments, the cytoplasm of the ascospores was

transformed, being either stained brightly with SYTO 9 (Chapter 6), or less dense and

displaying many discernable organelles, as observed in the EM studies (Chapter 4).

Similar results have been reported for ascospores of Talaromyces macrosporus

(Dijksterhuis et al., 2007) and endospores of Bacillus stearothermophilus (Beaman et

al., 1988). In these cases, the heated spores allowed higher permeation of water and

chemical compounds, changing the structural appearance of the central region.

It has been noted in Chapter 4 that ascospores of B. fulva appeared to have extra

protection from remnants of the ascus wall which was mostly absent in those of B.

nivea. This layer could also help by bonding individual ascospores in the asci, which

hindered uniform heat penetration into these structures to inactivate them. However,

there have not been any reports on the differences between the outer coats of ascospores

of B. fulva and B. nivea. The formation and chemical composition of this layer may

contribute to this discrepancy and, therefore, can be a topic for further research.

While the central cytoplasm is the target of the protection offered by the two outer

layers, this compartment also contributes to the general stress resistance of the

ascospores. Its high density and special configuration are protective factors for the

enclosed constituents. Chapter 5 presents evidence of a glass state and a low moisture

environment existing in Byssochlamys ascospores. These observations support a long

held theory about the glassy state and its protective mechanisms underpinning the heat

resistance of spore structures such as the ascospores (Dijksterhuis, 2007; Leuschner and

Lillford, 2003).

Once formed, the ultrastructure of the ascospores may not be static but may develop

with time, or ascospore age, as shown in Chapters 4 and 5. The appearance of additional

sub-layers in the IMS, the better recovery of DSC transitions and the higher transition

temperatures all indicated a more robust and resilient ultrastructure of the more matured

194

ascospores. These developments possibly enable an enhanced level of dormancy and a

greater heat resistance of the ascospores.

Despite the potential roles of each ultrastructural compartment in the heat resistance of

Byssochlamys ascospores, the chemical composition of these layers is poorly

understood and deserves more research effort. It would be useful to understand the

construction of the cell-wall and the IMS from the molecular point of view i.e. genes

and encoded enzymes that take part in synthesising the layers. In yeast ascospore walls,

networks of polysaccharides, such as chitin, chitosan or glucan, and amino acids, such

as dityrosine, may partially contribute to the structures of those compartments because

of their prevalence and stress resistance functions (Adams, 2004; Coluccio et al., 2008;

Suda et al., 2009). Moreover, information about the composition of the cytoplasm

would also be useful to explain the arrangement of the intracellular materials, which

were reported in Chapter 5. Trehalose, in particular, is commonly found in fungal

spores and has been associated with the glassy state (Dijksterhuis et al., 2002;

Thevelein, 1984). Detection of trehalose in Byssochlamys ascospores could not be done

using DSC and this will require a thorough chemical analysis of the ascospores in future

studies. Additionally, the states and spatial distribution of water in the ascospores would

be a pertinent research topic for a better understanding of the glassy state, dormancy and

heat resistant properties.

8.4 Proteins and heat resistance of ascospores

Proteomic profiles of Byssochlamys ascospores exhibited many proteins, several of

which were enzymes necessary for key metabolic pathways such as the TCA cycle,

pentose-phosphate pathway and glycolysis (Chapter 7). Another group of proteins

consisted of molecular chaperones, such as HSP 60, HSP 70 and GSP 39, which help to

protect the active structures of other proteins (Chapter 7). The existence of these

proteins is in accordance with the dormancy theory, which together may indicate the

storage of pre-synthesised materials essential for cellular viability inside the ascospores,

possibly in the cytoplasm compartment, for post-activation utilisation. This strategy has

been reported for bacterial endospores where newly synthesised proteins accumulate

during the forespore state and are then packaged in matured spores (Eaton and Ellar,

1974). With this approach, proteins of the ascospores could be preserved by a low

195

moisture environment and a glassy state, protecting them from environmental insults

and extracellular loss. Concurrently, the stress-related proteins act synergistically with

the intracellular environment to protect other enzymes, which further increases the

survival ability of the ascospores in harsh conditions.

Aging affected the proteomic profiles of Byssochlamys ascospores by increasing or

decreasing the abundances of particular proteins (Chapter 7). This result further

supports the theory that culture age could be an important factor in changing intrinsic

properties of the ascospores. More importantly, the age-associated changes in proteomes

indicate that the intracellular content of dormant ascospores may not be static but

transform incrementally towards an optimal state of dormancy and resistance. This may

corroborate the findings of ultrastructure studies with EM and DSC (Chapters 4 and 5)

to explain why ascospores of Byssochlamys species become more heat resistant when

aging.

In Chapter 7, the selection of identified proteins mainly focused on the ‘significant

change’ parameters. Thus, only a small percentage of the total proteins observed was

chosen for identification, leaving a wealth of uncharacterised ascospore proteins. The

unidentified proteins could harbour valuable knowledge about cell-wall assembly,

intermediate space composition, ascospore formation and other stress-related proteins. It

is also worth reiterating the knowledge gap in the genomics of Byssochlamys spp. which

needs to be addressed if molecular mechanisms of heat resistance are to be better

elucidated.

8.5 Population heterogeneity and heat resistance of ascospores

Sections 8.2 – 8.5 are predominantly concerned with individual ascospores that acquire

thermo-tolerance by maintaining a high level of dormancy via the ultrastructure and

intracellular contents. However, the heat resistant factors of one ascospore are not

necessarily similar to those of their neighbouring cells even though they may be derived

from genetically similar cultures. The differences between individual ascospores may

result from variations in time of sporulation, access to nutrients, rates of ultrastructural

development and chemical composition (Allman, 1992; Dantigny and Nanguy, 2009).

Thus, the heat resistant properties within a population may be expected to vary

196

significantly among individual ascospores (Casella et al., 1990; Junior and Massaguer,

2007).

The heterogeneity of populations of Byssochlamys ascospores was partially resolved by

flow cytometry, based on cell sizes and the cell integrity. The heterogeneity of dormant

ascospores was maintained when they were heat treated, indicating that inactivation of

ascospores was not as simple a process as the transition from dormancy to death. A

proportion of the dormant sub-populations changed minimally their flow cytometric

properties during the course of heat treatment. These persistent groups of dormant

ascospores could result from the tailing stage on the survival curves of Byssochlamys

ascospores (Chapter 3). The similar heat resistance of the tailing ascospore populations

after several generations excludes the possibility of genetically more heat resistant

ascospores (Chapter 3). These ascospores may result from the natural selection of the

heat resistant properties or phenotypic variations, such as more compact and dehydrated

ultrastructure (Kort et al., 2005; Peleg and Cole, 1998). The persistent sub-populations

and the tailing phenomenon may also coincide with the ‘super-dormancy’ theory in

bacterial spores (Ghosh et al., 2010; Rodriguez-Palacios and LeJeune, 2011) although

the mechanisms of their existence in populations of Byssochlamys ascospores are a

question for further research.

The heterogeneous responses of ascospores to heat treatments was reflected as a

spectrum of thermally affected ascospores, such as those predicted as activated, sub-

lethally injured, inactivated and the persistently dormant ascospores (Chapter 6). These

diverse populations could increase the survival chance of particular sub-populations

during heat treatments. Moreover, heat inflicted ascospores often exhibited higher levels

of fluorescence with SYTO 9, indicating the presence of compromised cell-wall

(Chapter 6). These results of flow cytometry reiterate the important roles of the

ultrastructure, particularly the cell wall, in protecting the ascospores from chemical and

physical stresses.

Effects of aging on the heterogeneity of Byssochlamys ascospores could not be clearly

demonstrated in the flow cytometry analysis (Chapter 6). There was much similarity

between ages in regards to the sub-population maps of ascospores in their dormant

stages and after heat treatments. For similar reasons, the identification of the exact

physiological states of all sub-populations was empirical and needs confirmation from

197

more studies. If future research is to investigate the heterogeneity of Byssochlamys

ascospores, there is a need for a harvesting process to generate homogeneous ascospore

crops comprising one type of dormant ascospores so that their changes with heating or

aging can be more clearly characterised. Efforts would also be needed in selecting more

compatible fluorescent probes to resolve sub-populations and track them during heat

treatment.

8.6 Recommendations for future research

Several suggestions for future research have been made in previous chapters when the

limitations of each experiment were discussed. In summary, more studies are required

in the following areas in order to gain a great understanding of the heat resistance of


Firstly, it is necessary to know the chemical composition of the ascospores. The focus

should be on the cell wall and the intermediate space which form the first line of

defence for the ascospores against thermal insults. Moreover, knowledge about moisture

content and viscosity of the internal environment would be useful in understanding the

existence of a glassy state inside the ascospores. It is then also important to determine

the distribution of water inside the ascospores since this result would reveal if the glassy

state is uniform or compartmentalised in the ascospores.

Secondly, the ultrastructure of Byssochlamys ascospores needs to be further investigated

using more effective electron microscopic protocols. For TEM, particularly, it is

important to ensure that ascospore samples are properly fixed and infiltrated by fixatives

and resins. Other microscopic techniques such as fluorescence, confocal or OMX

microscopy might be employed to observe ultrastructural changes in a timely manner

with less invasive preparation procedures.

Thirdly, in order to study the heterogeneity of Byssochlamys ascospores especially by

flow cytometry, it is essential to understand the properties (size, cell-wall integrity) of

more homogeneous populations. That will require more effective methods for

harvesting and purifying ascospores. It is equally important to focus on selecting

appropriate detection staining probes to track the transformation of heated ascospores.

198

The next consideration is to sort subpopulations and characterise them unequivocally in

terms of morphology, ultrastructure, viability and heat resistance.

Fourthly, the proteomic findings presented in this thesis can be improved by using more

sensitive techniques to visualise and compare 2D gels. Some direct and quantitative

comparison methods such as Isotope-coded Affinity tags, isobaric peptide tags for

relative and absolute quantification or 2D fluorescence difference gel electrophoresis

can be used for that purpose (Albrecht et al., 210; Gygi et al., 1999; Ross et al., 2004).

Moreover, the sequences of genomes of Byssochlamys species will be absolutely

necessary to make further progresses in understanding the molecular mechanisms of

heat resistance of their ascospores.

8.7 Conclusions

In this project, the heat resistance of Byssochlamys ascospores was studied from the

perspective of their intrinsic properties, which included ascospore age, the

ultrastructure, presence of the glassy state, population heterogeneity and their

proteomics. Integrated results of all the experiments have shown that the heat resistance

mechanism is multifactorial, involving contributions from all the factors at different

levels of the ascospores.

At the cellular level, Byssochlamys ascospores acquire their thermo-tolerance by the

specific multilayered ultrastructure. The two compartments, cell-wall and intermediate

space, are impermeable to water and many chemicals and, therefore, they represent the

protective layers of the ascospores. Their major function is to maintain the dehydrated

environment inside the ascospores, which is equivalent to promoting dormancy of the

cells. They are also the first line of defence against environmental stresses, such as

heating, and extracellular loss of internal content.

Ascospores also maintain a glassy state at least in the central cytoplasm where materials

essential for germination and surviving are stored. The glassy state can effectively

preserve macromolecules by hindering harmful chemical reactions and denaturation.

Additionally, heat shock proteins are pre-synthesised and present in the cytoplasm,

providing proteins with further protection. The packaging of stress proteins and

metabolic enzymes inside the ascospore is likely to take place prior to or during

199

sporulation, which is also a strategy for reproduction and surviving in adverse

conditions.

Aging can increase heat resistance by inducing structural and biochemical changes

inside the ascospores. A noticeable change is the development of sub-layers in the

intermediate space, probably for better dehydration and dormancy. Additionally, the

protein content of dormant ascospores may also change significantly towards an optimal

composition for heat resistance.

At the population level, ascospores are not homogenous with respect to morphology,

cell integrity and possibly age. This heterogeneity can result in different responses of

individual ascospore to heat treatments. Some sub-populations are less affected by heat

than others and, therefore, survive better.

Although definitive answers to all the questions about heat resistance may not have

been answered, this study has advanced knowledge about heat resistant fungi, bringing

researchers incrementally closer to understanding the mechanisms behind this important

characteristic of fungal ascospores. Future research can build on the findings presented

here to advance understanding in this field. An ultimate goal is to provide better

information to enable food manufacturers to more effectively control food and beverage

spoilage caused by heat resistant fungi.

200

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Appendices

Appendix 3.1. Screened heat resistance of the four Byssochlamys strains selected for

usage in this thesis. Comparative survival curves at 85oC of ascospores isolated from 6

weeks old cultures of B. fulva FRR 2299 (●), B. fulva FRR 2785 (▲), B. nivea FRR

4421 (♦) and B. nivea FRR 6002 (■)

Estimated D85 values: B. fulva FRR 2299: 14.7 min, B. fulva FRR 2785: 31.25 min, B.

nivea FRR 4421: 4.47 min. B. nivea FRR 6002: 5.91 min

241

Appendix 3.2. Proportions (in %) of the activated population at 1 minute out of the initially added ascospore concentration (5 – 6 log10); A –

B. fulva FRR 2299, B – B. fulva FRR 2785; C – B. nivea FRR 4421 and D – B. nivea FRR 6002; each column is an average of triplicates

A)

0

20

40

60

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100


Act

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)

85oC

87.5oC

90oC

B)

0

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80

100


Act

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leve

l (%

)

85oC

87.5oC

90oC

C)

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leve

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)

82.5oC

85oC

87.5oC

D)

0

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87.5oC

242

Appendix 3.3. Average activation levels of the four B. fulva and B. nivea strains being tested in this experiment; each experiment started with a

population of 105 – 106 ascospores/mL

Strain Temp.

(oC)

Average activation ± SD (Log10 (cfu/mL))*

4 weeks 6 weeks 8 weeks 12 weeks 24 weeks

B. fulva FRR 2299

85 1.79 ± 0.67AB 2.80 ± 0.04A 2.69 ± 0.04aB 2.06 ± 0.01 2.62 ± 0.35

87.5 1.58 ± 0.47ABC 2.54 ± 0.40A 2.60 ± 0.16bB 2.32 ± 0.40 2.82 ± 0.12aC

90 1.88 ± 0.27 2.51 ± 0.31 1.93 ± 0.08ab 1.96 ± 0.33 2.09 ± 0.10a

B. fulva FRR 2785

85 1.17 ± 0.46ABC 2.77 ± 0.07AD 3.05 ± 0.04BE 2.83 ± 0.18CF 0.87 ± 0.20DEF

87.5 0.91 ± 0.01ABC 2.79 ± 0.05AD 2.71 ± 0.31BE 2.75 ± 0.16CF 0.73 ± 0.18DEF

90 1.71 ± 0.93 2.66 ± 0.08A 2.90 ± 0.11B 2.47 ± 0.62 0.84 ± 0.22AB

B. nivea FRR 4421

82.5 2.35 ± 0.07abA 2.67 ± 0.15B 2.00 ± 0.41C 1.86 ± 0.59D 0.86 ± 0.03abABCD

85 1.40 ± 0.05aABC 2.46 ± 0.25A 2.22 ± 0.24B 1.86 ± 0.10C 0.32 ± 0.32aABC

87.5 1.34 ± 0.17bA 2.16 ± 0.18B 1.60 ± 0.15C 1.76 ± 0.42D -0.14 ± 0.01bABCD

B. nivea FRR 6002

82.5 2.67 ± 0.60AB 2.45 ± 0.10C 2.25 ± 0.25 1.50 ± 0.46A 1.40 ± 0.04aBC

85 2.23 ± 0.52A 2.43 ± 0.22BC 2.41 ± 0.21DE 1.63 ± 0.05BD 1.39 ± 0.13bACE

87.5 1.44 ± 0.54A 2.26 ± 0.12BC 2.34 ± 0.36ADE 1.25 ± 0.02BD 0.93 ± 0.18abCE

* For each strain, similar normal letters (e.g. abc) indicate significance difference (p < 0.05) across temperatures at each age (column); similar capital letters (e.g. ABC)

indicate significant difference (p < 0.05) across ages at each temperature (row); each value is an average of triplicate

243

Appendix 3.4. Representative ascospores suspensions after glass bead treatment of B.

fulva FRR 2299 (A) and B. nivea FRR 4421 (B); note the presence of asci (arrow) in B.

fulva but not B. nivea sample. Examined by light a light microscope

A)

B)

244


conditioning; I – 6 week old, II –12 week old. Ascospores were either dormant (A),

activated at 75oC for 30 minutes (B) or inactivated at 95oC for 30 minutes (C). Scan:

Unbroken line – initial scan, dashed line – immediate rescan. Exothermic heat flow is


I)

II)

A

A

A

A

B

B

B

B

C

C

C

C

245




unbroken line – initial scan, dashed line – immediate rescan. Exothermic heat flow is


I)

II)

A

A

B

B

C

C

A

A

B

B

C

C

246






I)

II)

A

A

B

B

C

C

A

A

B

B

C

C

247






I)

II)

A

A

A

A

B

B

B

B

C

C

C

C

248

Appendix 5.5. Excess water in dense ascospore solutions being removed by drying over

granulated silica gel in a desiccator

Drying time (hours)

Sample weight (mg) B. fulva FRR 2299(Dense solution)

B. nivea FRR 6002(Dense solution)

B. nivea FRR 6002 (Lyophilised)

0 10.91 9.96 2.02

1 4.06 4.77 2

2 3.18 4.37 -

3 - - 1.96

4 3.16 4.18 -

6 - - 1.96

7 3.13 4.19 -

20 - - 1.95

21 3.12 4.18 - Final sample weight (mg) 3.12 4.18 1.95

Water loss (mg)^ 7.79 5.78 0.07

Water loss (% w/w)* 71 58 3

^ Water loss weight (mg) = Weight at time 0 – Final sample weight

* % water loss = (Water loss weight/Weight at time 0) x 100

249


SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


250


2299 with SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


251


SYTO 9 and PI; ascospores are treated at 90oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


252



0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


253



0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


254


SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


255


4421 with SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


256


SYTO 9 and PI; ascospores are treated at 87.5oC for up to 30 minutes Unstained Stained

0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


257



0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


258



0 m

in

FL1

– G

reen

fluo

resc

ence

inte

nsity

(SYT

O 9

)

A) C)

30 m

in

B) D)

0 m

in

FL3

– R

ed fl

uore

scen

ce in

tens

ity (P

I)

E) G)

30 m

in

F) H)


259

Appendix 6.5. Subpopulations of unstained 4 week ascospores of B. fulva FRR 2299

following a heat treatment at 90oC for up to 30 minutes; the positive control is

ascospores heated at 96oC for 60 minutes

FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


260

Appendix 6.5. (cont’d) Subpopulations of 8 week unstained ascospores of B. fulva FRR

2299 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


261


FRR 2299 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


262

Appendix 6.6. Subpopulations of 4 week unstained ascospores of B. fulva FRR 2785

following a heat treatment at 90oC for up to 30 minutes; the positive control is

ascospores heated at 96oC for 60 minutes

FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


263

Appendix 6.6. (cont’d) Subpopulations of 8 week unstained ascospores of B. fulva FRR

2785 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


264


FRR 2785 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


265


following a heat treatment at 87.5oC for up to 30 minutes; the positive control is

ascospores heated at 96oC for 60 minutes FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


266


FRR 4421 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


267


FRR 4421 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


268


following a heat treatment at 87.5oC for up to 30 minutes; the positive control is

ascospores heated at 96oC for 60 minutes FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


269


FRR 6002 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


270


FRR 6002 FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


271

Appendix 6.9. Subpopulations of SYTO 9-stained ascospores of B. fulva FRR 2299 at 4

weeks old following a heat treatment at 90oC for up to 30 minutes; subpopulations are

A*bf, B*bf, C*bf and D*bf; time 0 is dormant ascospores; positive control is ascospores

treated at 96oC for 60 minutes

FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


272


2299 at 8 weeks old FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


273



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


274

Appendix 6.10. Subpopulations of SYTO 9-stained ascospores of B. fulva FRR 2785 at

4 weeks old following a heat treatment at 90oC for up to 30 minutes; subpopulations

are A*bf, B*bf, C*bf and D*bf; time 0 is dormant ascospores; positive control is

ascospores treated at 96oC for 60 minutes

FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


275



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


276



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


277


2299 after a heat treatment at 90oC; Age: A – 8 weeks, B – 12 weeks. Subpopulation:

A*bf (♦), B*bf (■), C*bf (▲) and D*bf (●); each point is an average of three experiments

A)

B)

50

l 40 l

-.- I + = -

•

0 10 15 20 25 30

70

~-I

60 .I __.... ~ '-"

l I

~l) so I r i 40 .vr ______________________________________ ___ <.I ... ~ c. = 30 Q

:c ~ = =Q

~

0 5 10 15 20 25 30 Heating time (min)

278


2785 after a heat treatment at 90oC; Age: A – 8 weeks, B – 12 weeks. Subpopulation:

A*bf (♦), B*bf (■), C*bf (▲) and D*bf (●); each point is an average of three experiments

A)

B)

70

60 _,-.....

~ '-' ~ 50 I:)J)

~

~ 40

I ...........,.

CJ .. ~

~ 30 j~~========~==================~T ~ I ~ . I = 20 .

2 t. • ! !

~: r=:s;---. ------~ 0 5 10 15 20

Heatin g time (min) 25 30

80

70 _,-.....

-:!f.. ~ '-' 60 ~ I:)J)

~ 50 = ~ CJ ..

40 ~ :=.

T I I _I I I T

= ¢ 30 i: ~ -= 20 :=. ¢ ~

10

= ~ ,.. - -

J: - .... - I - "" . ..-- .. ~ -0

0 5 10 15 20 25 30 H eating time (min)

279


4 weeks old following a heat treatment at 87.5oC for up to 30 minutes; subpopulations

are A*bn, B*bn, C*bn, D*bn and E*bn; time 0 is dormant ascospores; positive control is


FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


280


FRR 4421 at 8 weeks old FL

1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


281



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


282


4 weeks old following a heat treatment at 87.5oC for up to 30 minutes; subpopulations

are A*bn, B*bn, C*bn, D*bn and E*bn; time 0 is dormant ascospores; positive control is


FL1

inte

nsity

– G

reen

fluo

resc

ence

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


283



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


284



1 in

tens

ity –

Gre

en fl

uore

scen

ce

0 min (dormant)

1 min

4 min

12 min

30 min

Positive control


285


4421 after a heat treatment at 87.5oC; Age: A – 8 weeks, B – 12 weeks. Subpopulation:

A*bn (♦), B*bn (■), C*bn (▲), D*bn (●) and E*bn (◊); each point is an average of three

experiments

A)

B)

70

60 -. ~ e ..._., ~ 50 I:)J)

,a = 40 ~ ~ .. ~ - 30 = Q

-.c ~ - 20 = c ~

10

\ ~

T

"! .I_

r- - - ---- ... ~r

~

i i 0

0 5 10 15 20 25 30 H eatin g time (m in)

80

70 -. ~ e 60 ..._., ~ I:)J)

.a 50 = ~ ~

t 40 Q.

= Q 30 -.c

\

~ _L ~ r-------r

1 .... -"' .... "' ~ -= 20 Q.

Q

~

r I

4

10

0 0 5 10 15 20 25 30

Hea ting time (min)

286


6002 after a heat treatment at 87.5oC; Age: A – 8 weeks, B – 12 weeks. Subpopulation:

A*bn (♦), B*bn (■), C*bn (▲), D*bn (●) and E*bn (◊); each point is an average of three

experiments

A)

B)

287

Appendix 7.1. Standard curve of the Bradford assay (A); protein concentrations and

conductivity measurements of triplicate protein extracts (S1 – S3) from Byssochlamys

ascospores (B); shaded samples were subjected to buffer exchange and concentration

A)

y = 0.4777x + 0.6919R2 = 0.9923

0.6

0.7

0.8

0.9

1.0

1.1

1.2

0 0.2 0.4 0.6 0.8 1 1.2

Concentration (mg/mL)

Abso

rban

ce (a

bita

ry u

nit)

B)

Sample description Concentration (mg/mL) Conductivity (mS/cm) S1 S2 S3 S1 S2 S3

B. nivea FRR 6002, dormant, 4 weeks 2.90 4.81 3.10 0.64 0.65 0.63

B. nivea FRR 6002, dormant, 12 weeks 3.59 4.67 4.14 0.76 0.88 0.85

B. nivea FRR 6002, 4 weeks, 87.5oC, 1 min 4.28 4.33 4.20 0.79 0.64 0.82



B. fulva FRR 2299, dormant, 4 weeks 4.27 6.10 3.60 0.86 1.37 0.69

B. fulva FRR 2299, dormant, 12 weeks 3.23 3.10 2.25 0.62 0.7 0.65

B. fulva FRR 2299, 4 weeks, 90oC, 1 min 4.82 3.68 2.10 1.47 1.43 0.94



288

Appendix 7.2. Molecular weight (A, C) and isoelectric point (B, D) distribution of protein resolved on 2D-gels of B. fulva FRR 2299 (top)

and B. nivea FRR 4421 (bottom) ascospores

A)

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140 160 More

MW (Kda)

Freq

uenc

y

0%

20%

40%

60%

80%

100%

Cum

ulat

ive

%

Frequency Cumulative % B)

0

20

40

60

80

100

3 4 5 6 7 8 9 10pI

Freq

uenc

y

0%

20%

40%

60%

80%

100%

Cum

ulat

ive

%

Frequency Cumulative %

C)

0

20

40

60

80

100

120

140

160

0 10 20 30 40 50 60 70 80 90 100 More

MW (KDa)

Freq

uenc

y

0%

20%

40%

60%

80%

100%

Cum

ulat

ive

%

Frequency Cumulative % D)

0

20

40

60

80

100

3 4 5 6 7 8 9 10pI

Freq

uenc

y0%

20%

40%

60%

80%

100%

Cum

ulat

ive

%

Frequency Cumulative %

289

Appendix 7.3. Differentially expressed proteins of B. fulva FRR 2299 ascospores

during a heat treatment at 90oC

Dormant (0 min) 1 min 4 min 30 min

1106

1145

1132

1270

1220

1137

290

Appendix 7.4. Differentially expressed proteins of B. nivea FRR 6002 ascospores

during a heat treatment at 87.5oC

Dormant (0 min) 1 min 4 min 30 min

1197

628

1202

1193

291

Appendix 7.5. Differentially expressed proteins of B. fulva FRR 2299 ascospores at 4

and 12 weeks

4 weeks 12 weeks 4 weeks 12 weeks

1222

1224

83

1236

1225

1228

292

Appendix 7.6. Protein spots of B. nivea FRR 6002 on 2D gels that significantly

changed with ascospore ages

4 weeks 12 weeks 4 weeks 12 weeks

1299

1347

1278

1310

1304

1335

1275

1340

1331

1327

1197

1343

1061

1281

1302

343

1241

293

Appendix 7.7. Amino acid sequences of the matched proteins of Byssochlamys

ascospores; matched peptides between the searched proteins and the theoretical proteins

are in red

Spot NCBI code Peptide sequence

628 67522969

1 MHLISSLSLFLAFALSALAM TITSPKSTNQ KVDFSKPFTI RWTTVPSDPK

51 QFTITLVNMD GHNVDQDLAV DVDASEEEYT IDKIEDIPIA NNYQINFRST

101 EKNNMGILAQ SPRFNVTKVA EDEETAEPTA NATRTQSNMA PTETDANGAG

151 RAMGVFSGSV AMAGVMALAV FAL

1061 226283156

1 MVKAVVLGAS GGIGQPLSLL LKASPLVDEL ALYDVVNTPG VAADLSHIST

51 VATIKGYLPD NDGLKNALTG ADIIVIPAGI PRKPGMTRDD LFKVNAGIVQ

101 TLVKGIAEFS PKAFILVISN PVNSTVPIAA EVLKTAGVFD PKRLFGVTTL 151 DVVRAETFTQ EFTGQKDPSK ASIPVIGGHS GETIVPLFSQ AKPPVTIPAD

201 RYDSLVNRVQ FGGDEVVKAK DGAGSATLSM AYAGFRFAES VIKASKGEKG 251 IVEPTYIYLS GVDGGEAIKR EVGLDFFSIP VELGTSGAEK AHNILGGITE

301 QEKKLLEACT KGLKGNIEKG IEFAKNPPPK L

121703522

1 MVKAGKKRTD AYWIAVLGAS GGIGQPLSLL LKACPLVDEL ALYDVVNTPG

51 VAADLSHISS VAKITGYLPK DDGLKHALTG TDIVVIPAGI PRKPGMTRDD 101 LFKVNAGIVR DLVKGIAQYC PKAFVLIISN PVNSTVPIAA EILKNEGVFD

151 PKRLFGVTTL DVVRAETFTQ EYSGQKDPST VQIPVVGGHS GETIVPLFSK

201 ASPALNIPAD KYDALVNRVQ FGGDEVVKAK DGAGSATLSM AYAGFRFAES 251 VIKAAKGQSG IVEPTFVYLP GITGGDEIAK EAGADFFSTL VELGPNGAEK

301 AINILQGVTE QEKKLLEACI KGLKGNIEKG IDFIKNPPPK

1106 218720012

1 MKTASVLAAA ALVGAASAKV HKLKLDKVPL SEQFDKRSMN DHMRSLGQKY

51 MGVVPEGVYE DTSIRPEGGH DVLVDNFLNA QYFSEITIGT PPQNFKVVLD

101 TGSSNLWVPS ASCNSIACYL HNKYDSSSSS TYKKNGSEFA IQYGSGSLEG

151 FVSRDVVTIG DITIKDQDFA EATNEPGLAF AFGRFDGILG LGFDTISVNK

201 IVPPFYNMLN QKTLDEPVFA FYLGDSNKEG DNSEATFGGI DKSHYTGELV 251 KIPLRRKAYW EVDFDAVAFG DNVAELENTG VILDTGTSLI ALPSTLAELL

301 NKEIGASKSW NGQYTVDCTK RDSLPDLTVT LSGHNFSITA HDYVLEVQGS

1132 145237054

1 MTVTDLVNHP AQGISYFTPA QNIPAGTAAN PQSSGKPVPK LFHPLTVRGV

51 TFQNRLGLAP LCQYSAQDGH MTPYHIAHLG GIAQRGPGLM MIEATAVQAE 101 GRITPQDVGL WKDSQIGPMR QVVEFAHSQN QKIGVQLAHA GRKASTVAPW

151 ISATAMATEE LGGWPDRVIG PSDIPFADTF PKPKAMTKDD IVQFKNDWAA

201 AVKRALEVGV DFIEIHGAHG YLISSFLSPA ANNRTDEYGG SFENRIRLPL 251 EIAQITRDIV GPNVPVFLRV SASDWLEETL PEQSWGTEDT VKFAKALADQ 301 GAIDLIDIST GGVHAAQKVK SGPLFQAPFA VAVKQAVGDK LLVSAVGAIT

351 NGRQANDLLE KEGLDVALVG RGFQKDPGLT WTFAQHLDTE ISMASQIRWG

401 FTRRGGREYI DPSVYKPSIF DA

294

218723676

1 MARRDNPPDL QNTPAKGISY FSPEQSPPAG TAANPQTDGT VPPKLFQPLT

51 IRGVTFHNRI GLSPLCQYSA KDGHMNDWHI AHLGGIAQRG PGFIMVEATS

101 VLPEGRITPE DLGLWQDSQI EPLRRTVEFV HSQNQIIGVQ IAHAGRKAST

151 VAPWLSFGDV ALEKNGGWPN NVKGPSDIPF SDSFPLPKAM TKQDIEDLKK 201 AWVDAVKRAV KAGADFVEIH GAHGYLLCSF MSPQSNNRTD EYGGSFENRI 251 RLVLEIAQLT RETVGPNFPV FLRISATEWL EESKAGEPSW TLEDSVKLAK 301 ILADQGAIDL LDVSSGGNNA AQKIKGGPAF QAPFAVEIKK AVGDKLLVSS

351 VGSITSGKLA NKLLEEDGLD FTFVGRGFQK NTSLAWTWAE ELDLEVSAAN

401 QIRWAFSHRG AGSKFLQKPS TRSHH

1137 85105528

1 MSAVVEKITD AAAAAVNDVT NALANTSLTG NKSADEKPAA NDAVLASAAE

51 GRRLYIGNLA YATTEGELKE FFKGYLVESV SIPKNPRTDR PVGYAFVDLS

101 TPSEAERAIA ELSGKEILER KVSVQLARKP ESNEKAEGAN GEGHEGTRRR

151 QSTRGRGRAG RGRGGRARGD RGEKKEEGAE GSTSPATEAL KDITNETHNS

201 DNKAGKPHNR SPRERRERGP PADGIPSKTK VMVANLPYDL TEEKLKELFA 251 AYQPSSAKIA LRPIPRFMIK KLQARGEPRK GRGFGFVTLA SEELQQKAVA 301 EMNGKEIEGR EIAVKVAIDS PDKTDEEANE PEGEAQAPTN GTTEQAGAAV

351 TASA

1145 238935537

1 MSGPTNSSAV GKLRTGATIP LLGFGTWRST EEDGYNAVLT ALKVGYRHID 51 TAAVYGNEAA VGRAIRDSGV PRDQLFVTTK LWNTQHRDPR LALDQSLQRL

101 GLDYVDLYLI HWPLPLRTQR ITDGNLFTVP TKEDGKPDVD EQWSYVKTWE

151 MMQDLPDTSK TRAVGVSNFS VTQLQNLLAA SGTKVIPAVN QVETHPLLPQ 201 DKLYKFCSEN NILLEAYSPL GSQGSPLVEE PAVKEIASKH NADPAQVLIS

251 WGIKRGYVIL PKSVTPSRIE SNFKIVDLTD DDFAKINNIH KEKGEKRIND

301 PDWFHFDEN

1193 189211129

1 MKTAMLLAAA GSATAAVYKT PLKKVSLSEQ LEFANIETQM AGLKQKYSQQ

51 HMAGFKPESH MQAAFKAPYI ADGTHPVPVT NFLNAQYFSE ISLGTPPQTF

101 KVILDTGSSN LWVPSSSCNS IACYLHTKYD SSSSSTYKKN GTEFEIRYGS

151 GSLSGFVSND VFQIGDLKVK NQDFAEATSE PGLAFAFGRF DGIMGLGYDT

201 ISVKGIVPPF YNMLEQGLLD EPVFAFYLGD TNQQQESEAT FGGIDESKYT

251 GKMIKLPLRR KAYWEVELDA LTFGKETAEM DNTGIILDTG TSLIALPSTI

301 AELLNKEIGA KKSFNGQYTV ECDKRDSLPD LTFTLTGHNF TISAYDYILE

351 VQGSCISALM GMDFPEPVGP LAILGDAFLR KWYSVYDLGN SAVGLAKAK

1197 121702629

1 MSQLQELFNQ AARPLPPIAD PNFASHFDSF GNYSVVLLGD GSHGTSEFYT

51 ARAEITKRFI ERHGYTMVAV EADWPDAEAI DRYVRQRPGP NASIGGKARD 101 QESFNRFPTW MWRNREMQDL IEWMRDRNSK VPDEQKAGFY GLDLYSMGAS 151 IRAVIEYLDR VDPGAGKEAR KRYGCLQPWV DDPTSYGLAS LRGMADCESQ 201 VLQMLRDLLE KRLQYSKHDV HDGEEFHSSE QNAFLVRDAE RYYKAMYYSS

251 ASSWTLRDTH MFDTLRRLFR HKPANAKAVV WAHNSHCGDA RYTSMGTRRN

301 EVNIGQLIRE NYGREKVAIL GCGTHTGTVA AAHEWDEDME AMKVRPSRDD

351 SWEMIAHDTG IPSFVIDLRP DRIDPDLRRA MAAENNRLER FIGVIYRPDT 401 ERISHYSQAY LHNQFDAYIW FDQTTAVNPL EKVQPKTPLG LDETYPFGL

145254965

1 FDRKSSVIVC FDRYFLSKRK KKIMSLQNLL STAARPLPPI HDPNFASHFD

51 SFANYRVVLL GDGSHGTSEF YAARAEITKR LIEHHGFTTV AVEADWPDAE

101 AIDRYVRQRP GPQAHLTNDK HEPFQRFPTW MWRNREMQDL VEWMRDHNAN

151 LPDHQKAGFY GLDLYSMGAS IRAVVDYLDR VDPEAGKLAR RRYGCLQPWV

295

201 DDPTAYGLAS LRGLADCEKG VVDMLRDLLR KRLQYAEEDP HDGEESHSSQ

251 QNAYLVRDAE RYYKAMYYSS ASSWTLRDTH MVDTLRRILR HKPPGSKAVV 301 WAHNSHCGDA RYTGMGIRRN EVNIGQLCRE QFGRDKVALI GCGTHTGTVA

351 AAHEWDEDME IMKVKPSRPD SWEWLAHQTG IESFLLDLRP TKMSPEMRDL

401 MAAEDQRLER FIGVIYRPDT ERMSHYSQAD LQNQFDAYVW FDSTEAVKPL

451 ETVQPRTAMG TEETYPFGL

1202 121702629

1 MSQLQELFNQ AARPLPPIAD PNFASHFDSF GNYSVVLLGD GSHGTSEFYT

51 ARAEITKRFI ERHGYTMVAV EADWPDAEAI DRYVRQRPGP NASIGGKARD

101 QESFNRFPTW MWRNREMQDL IEWMRDRNSK VPDEQKAGFY GLDLYSMGAS 151 IRAVIEYLDR VDPGAGKEAR KRYGCLQPWV DDPTSYGLAS LRGMADCESQ 201 VLQMLRDLLE KRLQYSKHDV HDGEEFHSSE QNAFLVRDAE RYYKAMYYSS

251 ASSWTLRDTH MFDTLRRLFR HKPANAKAVV WAHNSHCGDA RYTSMGTRRN

301 EVNIGQLIRE NYGREKVAIL GCGTHTGTVA AAHEWDEDME AMKVRPSRDD 351 SWEMIAHDTG IPSFVIDLRP DRIDPDLRRA MAAENNRLER FIGVIYRPDT 401 ERISHYSQAY LHNQFDAYIW FDQTTAVNPL EKVQPKTPLG LDETYPFGL

1220 18141566

1 MASTLRLSTS ALRSSTLAGK PVVQSVAFNG LRCYSTGKTK SLKETFADKL

51 PGELEKVKKL RKEHGNKVIG ELTLDQAYGG ARGVKCLVWE GSVLDSEEGI 101 RFRGLTIPEC QKLLPKAPGG EEPLPEGLFW LLLTGEVPSE QQVRDLSAEW 151 AARSDLPKFI EELIDRVPST LHPMAQFSLA VTALEHESAF AKATAKGINK

201 KEYWHYTFED SMDLIAKLPT IAAKIYRNVF KDGKVAPIQK DKDYSYNLAN

251 QLGFADNKDF VELMRLYLTI HSDHEGGNVS AHTTHLVGSA LSSPMLSLAA

301 GLNGLAGPLH GLANQEVLNW LTEMKKVVGN DLSDQSIKDY LWSTLNAGRV 351 VPGYGHAVLR KTDPRYTSQR EFALRKLPDD PMFKLVSQVY KIAPGVLTEH 401 GKTKNPYPNV DAHSGVLLQY YGLTEANYYT VLFGVSRALG VLPQLIIDRA

451 FGAPIERPKS FSTEAYAKLV GAKL

67902576

1 MASTLRLSTS ALRSSTLAGK PVVQSVAFNG LRCYSTGKTK SLKETFADKL

51 PGELEKVKKL RKEHGNKVIG ELTLDQAYGG ARGVKCLVWE GSVLDSEEGI 101 RFRGLTIPEC QKLLPKAPGG EEPLPEGLFW LLLTGEVPSE QQVRDLSAEW 151 AARSDLPKFI EELIDRCPST LHPMAQFSLA VTALEHESAF AKAYAKGINK

201 KEYWHYTFED SMDLIAKLPT IAAKIYRNVF KDGKVAPIQK DKDYSYNLAN

251 QLGFADNKDF VELMRLYLTI HSDHEGGNVS AHTTHLVGSA LSSPMLSLAA

301 GLNGLAGPLH GLANQEVLNW LTEMKKVVGN DLSDQSIKDY LWSTLNAGRV 351 VPGYGHAVLR KTDPRYTSQR EFALRKLPDD PMFKLVSQVY KIAPGVLTEH 401 GKTKNPYPNV DAHSGVLLQY YGLTEANYYT VLFGVSRALG VLPQLIIDRA

451 FGAPIERPKS FSTEAYAKLV GAKL

1224 154298888 1 MASCQDGIIT DCAEKVSIVT YNGAAIASDK SPRRVCTPSR VCTTMEVSTF 51 FGFSKADYSA YTCFNGREAL NTRDLISEYD HSLDSV

1225 211591546

1 MTLEDFEKSL AEGREQRQEK SEGRHHRDRN RDRDRSKERS RQHRHRSSHH

51 HRRQSSRSRD RDSERVRESR HRDDDGHRHK RSRRSDDQGD ERGHKRHHRN

101 SKDEGEGVPL VVVQEEPKNI KRDAWMEAPS ALDVDYVHRP DATRQEEPKP 151 IMLSADYELK IHDKELNDHL RDLKDGRTVD EIEDEPAQHE VDYTIGDSGS

201 QWRMTKLKGV YREAEESGRA IDDIAIERFG DLRSFDDARE EETELDRRNT

251 YGKAYVGKDK PSGELFQERK LQSDIRRDPR EHLRDPEQEL NAEGQGKKID

301 TAPPPQTSRH LDMTALNRLK AQMMKAKLRK APDAANLEEQ YNTAAAAMSN

351 RKESDVVVLD VMHNPMLAGA RNEVKAIDTK RGRERGQVEA NEDMTIEDMV

296

401 REERKTRGQP GGEGRRLADQ IGRDAKFENR LEYMDDNASK LAKRVHRSEI 451 DLKNSTINDF HKMNRILDSC PLCHNEDKGT PPLAPVVSLA TRVFLTLPTE

501 PEVSEGGATI VPTQHRTNLM ECDDDEWEEI RNFMKSLTRM YHDQGRDVIF

551 YENAAQPERK RHASMEVVPL PYSLGETSPA FFKEAILSAE SEWSQHRKLI 601 DTLAKSKQGL GRSAFRRTLV KEMPYFHVWF ELDGGLGHIV EDSHRWPRGD 651 LFAREIIGGM LDIAPDVIKR QGRWNRGDRR VEGFRKRWKK FDWTRILVEG

1241 115400267

1 MSSSLEQLKA TGTVVVCDSA IGKYKPQDAT TNPSLILAAS KKPEYAALID

51 AAVAYGKQHG KTVDEQVDAT LDRLLVEFGK EILKIIPGKV STEVDARFSF 101 DTQASIDKAL HIIKLYEEIG IPKDRILIKI ASTWEGIKAA QVLQSQHGIN

151 CNLTLMFSTV QAIAAAEAGA YLISPFVGRI LDWYKAAHKR DYTAQEDPGV

201 KSVQAIFNYY KKYGYKTIVM GASFRNTGEI TELAGCDYLT ISPNLLEDLY

251 NSTAAVPKKL DAAAAASQDI PKRSYINDEA LFRFDFNEEA MAVEKLREGI 301 SKFAADAVTL KDLLKQKIQA

39970315

1 MSNSLDQLKS SGTTVVCDSG DFATIGKYKP QDATTNPSLI LAASKKAEYA

51 KLIDDAIAYA KKQGGSVDDQ VDASLDRLLV EFGKEILKII PGKVSTEVDA 101 RFSFDTKASV DKALHIIKLY EAEGISKDRV LIKIASTWEG IKAAEILQRD

151 HGINCNLTLM FSLVQAIAAA EADAFLISPF VGRILDWYKA AHKKEYKKEE

201 DPGVESVKNI FNYYKKFGYK TIVMGASFRN TGEITELAGC DYLTISPNLL

251 EELLNSSEAV PKKLDAQGAA ALDIQKKTYI SDEPLFRFDF NEDQMAVEKL 301 REGISKFAAD ADTLKGILKE KIQA

1270 530795

1 MKSASLLTAS VLLGCASAEV HKLKLNKVPL EEQLYTHNID AHVRALGQKY

51 MGIRPSIHKE LVEENPINDM SRHDVLVDNF LNAQYFSEIE LGTPPQKFKV

101 VLDTGSSNLW VPSSECSSIA CYLHNKYDSS ASSTYHKNGS EFAIKYGSGS

151 LSGFVSQDTL KIGDLKVKGQ DFAEATNEPG LAFAFGRFDG ILGLGYDTIS 201 VNKIVPPFYN MLDQGLLDEP VFAFYLGDTN KEGDESVATF GGVDKDHYTG

251 ELIKIPLRRK AYWEVELDAI ALGDDVAEME NTGVILDTGT SLIALPADLA

301 EMINAQIGAK KGWTGQYTVD CDKRSSLPDV TFTLAGHNFT ISSYDYTLEV

351 QGSCVSAFMG MDFPEPVGPL AILGDAFLRK WYSVYDLGNS AVGLAKAK

145232965

1 MKSASLLTAS VLLGCASAEV HKLKLNKVPL EEQLYTHNID AHVRALGQKY

51 MGIRPSIHKE LVEENPINDM SRHDVLVDNF LNAQYFSEIE LGTPPQKFKV

101 VLDTGSSNLW VPSSECSSIA CYLHNKYDSS ASSTYHKNGS EFAIKYGSGS

151 LSGFISQDTL KIGDLKVKGQ DFAEATNEPG LAFAFGRFDG ILGLGYDTIS 201 VNKIVPPFYN MLDQGLLDEP VFAFYLGDTN KEGDESVATF GGVDKDHYTG

251 ELIKIPLRRK AYWEVELDAI ALGDDVAEME NTGVILDTGT SLIALPADLA

301 EMINAQIGAK KGWTGQYTVD CDKRSSLPDV TFTLAGHNFT ISSYDYTLEV

351 QGSCVSAFMG MDFPEPVGPL AILGDAFLRK WYSVYDLGNS AVGLAKAK

1281 115443330

1 MQRALSSRTS VLSAASKRAP FYRSTGLNLQ QQRFAHKELK FGVEARAQLL

51 KGVDTLAKAV TSTLGPKGRN VLIESPYGSP KITKDGVTVA KAIQLQDKFE

101 NLGARLLQDV ASKTNELAGD GTTTATVLAR AIFSETVKNV AAGCNPMDLR 151 RGIQAAVDAV VDYLQQNKRD ITTGEEIAQV ATISANGDTH VGKLISTAME

201 RVGKEGVITV KEGKTLEDEL EVTEGMRFDR GYTSPYFITD PKAQKVEFEK 251 PLILLSEKKI SAVQDIIPAL EASTTLRRPL VIIAEDIEGE ALAVCILNKL

301 RGQLQVAAVK APGFGDNRKS ILGDLAVLTN GTVFTDELDI KLEKLTPDML 351 GSTGAITITK EDTIILNGEG SKDSIAQRCE QIRGVMADPT TSEYEKEKLQ

401 ERLAKLSGGV AVIKVGGASE VEVGEKKDRV VDALNATRAA VEEGILPGGG

297

451 TALLKASANG LSDVKSANFD QQLGVSIIKN AITRPARTIV ENAGLEGSVI 501 VGKLTDEFSK DFNRGYDSSK SEYVDMIATG IVDPLKVVRT ALVDASGVAS 551 LLGTTEVAIV EAPEEKGPAA PAGGMGGMGG MGGMGGGMF

238844339

1 MQRALSTSSR ASVLASAAAT RSQVSQFRPA LAAGVSLQQQ RFAHKEIKFG

51 VEGRASLLKG IDTLAKAVTA TLGPKGRNVL IESPYGSPKI TKDGVTVAKA 101 VSLEDKFENL GARLLQDVAS KTNEVAGDGT TTATVLARAI FSETVKNVAA 151 GCNPMDLRRG IQAAVQSVVE YLQANKRDIT TTEEIAQVAT ISANGDLLVG 201 KLISNAMEKV GKEGVITVKD GKTIEDELEV TEGMRFDRGY TSPYFITDPK 251 TQKVEFEKPL ILLSEKKISA VQDILPALEA STTLRRPLVI IAEDIDGEAL

301 AVCILNKLRG QLQVAAVKAP GFGDNRKSIL GDIGILTNAT VFTDELDMKL

351 DKATPDMLGS TGSITITKED TIILNGEGSK DAIAQRCEQI RGVIADPATS 401 DYEKEKLQER LAKLSGGVAV IKVGGASEVE VGEKKDRVVD ALNATRAAVE 451 EGILPGGGTA LLKAAANGLA DVKPTNFDQQ LGVSIVKSAI TRPARTIVEN 501 AGLEGSVVVG KLTDEFASDF NRGFDSSKGE YVDMIASGIV DPLKVVRTAL

551 VDASGVASLL GTTEVAIVDA PEPKSAPAPG GMPGMGGMGG MGGMY

1299 119501350

1 MQEYTEKPAS GAESQFQPGH RIPIQHQKKP GLQAELEDPK PASTRIPTDD

51 YGYQTYKAAG KLAGKRAIIT GGDSGIGRAV AILFAMEGAS SLIVYLPEEE

101 VDAQETKRRV QESGKECHCL AVDLRKRENC QKVVDVALQC LGGIDILVNN

151 AAFQNMVQDI SELDEDQWHR TFDTNIHPYF YLSKYSLPHM RSGSTIINCS 201 SVNHYIGRGD LLDYTSTKGA IVAFTRGLSN QQIGKGIRVN CVCPGPIWTP

251 LIPSTMDTSA MEQFSSVPMG RPGQPSEVAT CFVFLASHDS SYISGQSLHP

301 NGGVMVNG

211587330

1 MPVTQSIERE ASGSKSQFAP GHKIPVQHLK KPGLQSDLGD PKPVSTHIPT

51 EDYGYQTYKA AGKLEGKKAI ITGGDSGIGR AIAILFAMEG ASSVIVYLPE

101 EESDAQTTKK RVEEHGQQCH TLAIDLRQKE NCRKVINTAL EKMGGIDILV

151 NNAAFQDMLS DISELEESQW EKTFNTNIHS FFYLSKYVLP HMKEGSTIIN 201 CASVNPYIGR GDLLDYTSTK GAIVAFTRGL SNQQIKKGIR VNCVCPGPIW

251 TPLIPATMQT EAMEQFHAVP IGRPGQPSEV ATCFVFLASQ DSSYISGQCL

301 HPNGGMMVNG

1310 116194057

1 MGLFSRHAEP GQEPAAQRQP AYEERQPVYE DPPRRHGLFG SKHHSPTRAP 51 TNATHSTRSS TTTSSPDRST GGGGIFRRST DASHNPSMNG HQRTGGGGLL

101 HKFGGNREEM DPSIVQARER VMGAEAAEME ADRALIAARE SVREAREHVR

151 MLELEAKEEA RRAKIKEHHA KEFSKRGKLL GRHDY

1331 121704078

1 MSGLKTGDSF PSDVVFSYIP WSEEQGEITS CGIPINYNAS KEWADKKVIL

51 FALPGAFTPV CSARHVPEYI ERLPEIRAKG VDVVAVLAYN DAYVMSAWGK 101 ANQVTGDDIL FLSDPEARFS KSIGWADEEG RTRRYAIVID HGKVTYAALE

151 PAKNHLEFSS AENVIKQL

119467218

1 MSGLKAGDSF PSDVVFSYIP WSEDKGEITA CGIPINYNAS KEWADKKVIL

51 FALPGAFTPV CSARHVPEYL EKLPEIRAKG VDVVAVLAYN DAYVMSAWGK 101 ANQVTGDDIL FLSDPEARFS KSIGWADEEG RTKRYAIVID HGKVTYAALE

151 PSKNHLEFSS AETVLKHL

1335 121704078 1 MSGLKTGDSF PSDVVFSYIP WSEEQGEITS CGIPINYNAS KEWADKKVIL

51 FALPGAFTPV CSARHVPEYI ERLPEIRAKG VDVVAVLAYN DAYVMSAWGK

298

101 ANQVTGDDIL FLSDPEARFS KSIGWADEEG RTRRYAIVID HGKVTYAALE

151 PAKNHLEFSS AENVIKQL

119467218

1 MSGLKAGDSF PSDVVFSYIP WSEDKGEITA CGIPINYNAS KEWADKKVIL

51 FALPGAFTPV CSARHVPEYL EKLPEIRAKG VDVVAVLAYN DAYVMSAWGK

101 ANQVTGDDIL FLSDPEARFS KSIGWADEEG RTKRYAIVID HGKVTYAALE 151 PSKNHLEFSS AETVLKHL

1347 121698176

1 MSSYMYRERE RDRDWDEPRS STVSIKRYVI PSEEERERDL IFRREDSIAG

51 DRELVIRRST EREEPVMVQR YEREIDYDSR PYDYRSERDY YEREYPSCST

101 RPCPVQLILI CEARGPLTVY PHEPDYAVVH RSEVEREPAY YYHRRVREYD

151 DDRRLRRELS PGDSVSQASR RRDDQEEYSS DDSIVHIRKE VREYDDHPHH

201 RRHLAEGALV GLGAAELLRN HRKKEGEEVS SGLGRVGKDL GAGALGAVAV

251 SAASRAREYY RSKSRRRSHS YDDDRSSRHR HQHYHHSHSR SGRSRSRSHS

301 HSRAKTLAEI GLGAAAIAGA VALARKKSKN DRRSRSRHRR ASSSSRPEKD

351 AKSEKGSQSR MRKHMAEAGL AGAAVAGLVE RARSRSRSRK GSRSHSRLRH

401 ALPVVAAGLG TAVATGLYEK HKMKEGEEGK HRERRRARSR SRAPSEIYPD

451 PNRDSAGLIE YGDHPVAGSI PSAHYYGRPA SQQGYYHSDA SDSAARRGRG

501 RSHSRTRSRG GRYSSSPSDS DRDGSRRRSR HRKHRSRSRE IAEAALAATG 551 VGYAAHKLKQ HRDHKKEERE RSKYEDDSFS EPPYPPSPLP PAQQPAETQF

601 YPNTNYFPPP PGPTPVPIAN NMPYNPADYP PPPGAVPPAQ GYGYPPESGP

651 NRYAPRPRRA DENVSAAWNS SPPTAQYPMQ HGVDEPPSPL RTPRADTARR

701 ARAVSQPAHP KSVAFDLIPD AISIPSSSDT HPIDPGYETD DSDSTIESSI

751 ARHHRDRGSH RRRHSSSYPS SSSSTRRSHH KPTPSHPSHH TTKHQESDSD

801 STIDLPARFD AQGRLLPERG HDDLPDPLAD RVERLLRGIN RVFA

Contribution of intrinsic factors to heat resistance ... - UNSWorks

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