STUDIES ON INDOOR FUNGI by James Alexander Scott A thesis submitted in conformity with the ...

472 Pages·2004·3.52 MB·English

by  James Scott

STUDIES ON INDOOR FUNGI

by

James Alexander Scott

A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy in Mycology,
Graduate Department of Botany in the University of Toronto

Copyright by James Alexander Scott, 2001
When I heard the learn’d astronomer,
When the proofs, the figures, were ranged in columns before
me,
When I was shown the charts and diagrams, to add, divide, and
measure them,
When I sitting heard the astronomer where he lectured with
much applause in the lecture-room,
How soon unaccountable I became tired and sick,
Till rising and gliding out I wander’d off by myself,
In the mystical moist night-air, and from time to time,
Look’d up in perfect silence at the stars.

Walt Whitman, “Leaves of Grass”, 1855
STUDIES ON INDOOR FUNGI

James Alexander Scott
Department of Botany, University of Toronto
2001
ABSTRACT

Fungi are among the most common microbiota in the interiors of buildings, including homes.
Indoor fungal contaminants, such as dry-rot, have been known since antiquity and are
important agents of structural decay, particularly in Europe. The principal agents of indoor
fungal contamination in North America today, however, are anamorphic (asexual) fungi mostly
belonging to the phyla Ascomycota and Zygomycota, commonly known as “moulds”.

Broadloom dust taken from 369 houses in Wallaceburg, Ontario during winter, 1994, was
serial dilution plated, yielding approximately 250 fungal taxa, over 90% of which were moulds.
The ten most common taxa were: Alternaria alternata, Aureobasidium pullulans, Eurotium
herbariorum, Aspergillus versicolor, Penicillium chrysogenum, Cladosporium cladosporioides, P. spinulosum,
Cl. sphaerospermum, As. niger and Trichoderma viride. Chi-square association analysis of this
mycoflora revealed several ecological groups including phylloplane-, soil-, and xerophilic food-
spoilage fungi.

Genotypic variation was investigated in two common dust-borne species, Penicillium
brevicompactum and P. chrysogenum. Nine multilocus haplotypes comprising 75 isolates of P.
brevicompactum from 50 houses were detected by heteroduplex mobility assay (HMA) of
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polymorphic regions in beta-tubulin (benA), nuclear ribosomal RNA spanning the internal
transcribed spacer regions (ITS1-2) and histone 4 (his4) genes. Sequence analysis of the benA
and rDNA loci showed two genetically divergent groups. Authentic strains of P. brevicompactum
and P. stoloniferum clustered together in the predominant clade, accounting for 86% of isolates.
The second lineage contained 14% of isolates, and included collections from the rotting fruit
bodies of macrofungi.

Similarly, 5 multilocus haplotypes based on acetyl coenzyme-A synthase (acuA), benA, ITS1-2
and thioredoxin reductase (trxB) genes comprised 198 isolates of P. chrysogenum obtained from
109 houses. A strictly clonal pattern of inheritance was observed, indicating the absence of
recombination. Phylogenetic analyses of allele sequences segregated the population into three
divergent lineages, encompassing 90%, 7% and 3% of the house dust isolates, respectively.
Type isolates of P. chrysogenum and its synonym P. notatum clustered within the secondary
lineage, confirming this synonymy. No isolates of nomenclatural status clustered within the
predominant lineage; however, this clade contained Alexander Fleming’s historically
noteworthy penicillin-producing strain from 1929. Similarly, there was no available name for
the minor lineage.
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ACKNOWLEDGEMENTS

I am profoundly grateful to my advisors, Dave Malloch and Neil Straus, for their thoughtful
mentorship, both intentional and unintentional, on all matters of science and life. They have
given me gifts of their patience and wisdom that I cannot repay. I thank the members of my
supervisory committee, Jim Anderson, David Miller and Richard Summerbell, for their gentle
encouragement and sincere enthusiasm which helped me to stay on-track. Linda Kohn and
Keith Seifert are thanked for their excellent and thoughful feedback on the thesis in
conjunction with the senate oral examination.

I could not have completed this thesis nor the research it presents without the friendship,
dedication and benevolence of Brenda Koster, Bess Wong and Wendy Untereiner.

I have laughed, cried and grown immeasurably from my friendships and discussions, often
over beer, pizza or #49, with my fellow graduate students Jacquie Bede, Cameron Currie,
Laurie Ketch and Simona Margaritescu, along with many others. I am grateful for the
assistance of Len Hutchison, Brett Couch, Wendy Malloch, Colleen McGee, Emily Taylor and
Megan Weibe during the early stages of this work. More recently, Michael Warnock helped
with proofreading and correcting this thesis. Barry Neville and Shanelle Lum kindly kept me
informed of many news items on indoor fungi that otherwise I would certainly have missed.
Carolyn Babcock and Steve Peterson graciously provided cultures. I thank John Pogacar for
insightful conversations on building science and for help in final thesis preparation. Financial
support was provided by NSERC as operating grants and a strategic grant to DM and NS, and
a doctoral scholarship to JS. Jim Gloer generously funded my early mycological training.
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Wieland Meyer has been a superlative collaborator on the aspects of this project involving
DNA fingerprinting of Penicillium chrysogenum. Steve Peterson, John Pitt and Rob Samson are
thanked for many insightful discussions on Penicillium taxonomy.

I owe the greatest debt of gratitude to my parents, Kay and Alex Scott, for nurturing my
ecclectic interests from a very young age, for their compassion and understanding of the many
turns my life has taken, and for the unconditional freedom, support and love that they have
given me as I have pursued my dreams. For all this and much more, I dedicate this thesis to
them.

On a muggy, summer day when I was a very young boy, my cousin, Jim Guillet inspired me to
study biology. As we stood together in my grandmother’s garden, Jim explained that the stems
of the rhubarb plant could be eaten but that the leaves could not, because they were
poisonous. How could it be so? And why? I stared in utter disbelief and hotly challenged this
absurd idea, while my parents, grimacing, looked on. I stopped just short of biting into a leaf
myself to see its effect. In the very many intervening years since, I have reflected on our
exchange countless times. Jim, perhaps unintentionally, taught me four very valuable lessons
that day: 1) Nature is fascinating and intricate, her properties and processes are rarely apparent
or intuitive; 2) Never be afraid to question any notion proffered as fact, no matter how high
the authority; 3) Science embodies a set of methods that can provide insight into the delicate
inner workings of Nature when applied thoughtfully and skillfully; and above all, 4) Don’t eat
rhubarb leaves.

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TABLE OF CONTENTS
PAGE
ABSTRACT ................................................................................................................II
ACKNOWLEDGEMENTS.......................................................................................................IV
TABLE OF CONTENTS..........................................................................................................VI
LIST OF TABLES ................................................................................................................ X
LIST OF FIGURES ...............................................................................................................XI
LIST OF APPENDICES.........................................................................................................XIII
LIST OF ABBREVIATED TERMS...........................................................................................XIV
LIST OF NOMENCLATURAL ABBREVIATIONS.....................................................................XVI
CHAPTER 1. INTRODUCTION.......................................................................................1
The biology of house dust.....................................................................................................................1
Interactions between mites and fungi.............................................................................................3
Fungi in household dust....................................................................................................................3
Indoor sources of dust mycoflora...................................................................................................5
Penicillium in indoor environments...................................................................................................6
Health effects of exposure to indoor fungi........................................................................................9
Allergic rhinitis and sinusitis...........................................................................................................10
Type I allergic syndromes...........................................................................................................10
Dust mites and allergy.................................................................................................................11
Hypersensitivity syndromes............................................................................................................12
Asthma...............................................................................................................................................12
Mycotoxins........................................................................................................................................15
Volatile fungal metabolites.............................................................................................................17
Objectives of the current study..........................................................................................................17
CHAPTER 2. ANALYSIS OF HOUSE DUST MYCOFLORA.................................................19
Abstract..................................................................................................................................................19
Introduction..........................................................................................................................................19
Materials and methods.........................................................................................................................20
Collection of dust samples..............................................................................................................20
Analysis of dust samples.................................................................................................................23
Identification and isolation of cultures.........................................................................................25
Reliability of identifications............................................................................................................26
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Storage of cultures...........................................................................................................................26
Organization and analysis of data..................................................................................................27
Results....................................................................................................................................................29
Species diversity and distribution..................................................................................................29
Efficiency of sampling.....................................................................................................................29
Species abundance...........................................................................................................................35
Association analysis.........................................................................................................................35
Discussion..............................................................................................................................................45
Conclusions...........................................................................................................................................50
CHAPTER 3. A REVIEW OF TECHNIQUES FOR THE ASSESSMENT
OF GENOTYPIC DIVERSITY......................................................................52
Introduction..........................................................................................................................................52
The detection of genetic variation......................................................................................................53
The use of proteins to distinguish variation.................................................................................53
Hybridization-based markers.........................................................................................................54
Low-stringency PCR........................................................................................................................55
Randomly amplified polymorphic DNA (RAPD)..................................................................55
Amplified fragment length polymorphism (AFLP)................................................................57
High-stringency PCR -- site-specific polymorphisms.................................................................59
Detection of low-level sequence variability..................................................................................60
Restriction endonuclease digestion of PCR products............................................................61
Denaturing-gradient gel electrophoresis (DGGE).................................................................62
Single-strand conformation polymorphism (SSCP)...............................................................62
Heteroduplex mobility assay (HMA)........................................................................................64
Summary................................................................................................................................................69
CHAPTER 4. DEVELOPMENT OF METHODS FOR THE ASSESSMENT
OF GENOTYPIC DIVERSITY OF PENICILLIA.............................................70
Abstract..................................................................................................................................................70
Isolate selection................................................................................................................................70
Isolation of DNA from Penicillium conidia...................................................................................71
Materials and methods, DNA isolation....................................................................................71
Results and discussion, DNA isolation....................................................................................73
Heteroduplex mobility assay..........................................................................................................74
Materials and methods, HMA...................................................................................................74
DNA amplification.................................................................................................................74
Cloning and sequencing of PCR products..........................................................................75
Preparation and analysis of DNA heteroduplexes.............................................................76
Electrophoresis and imaging..........................................................................................................77
Resolution of heteroduplexed DNAs on Phast system.........................................................77
Results and discussion, HGE................................................................................................81
Vertical gel electrophoresis (VGE) in HMA...........................................................................84
HMA Screening approaches......................................................................................................85
Overlapped pairs.....................................................................................................................85
Universal heteroduplex generator.........................................................................................86
Results and discussion, VGE.....................................................................................................86
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Identification of polymorphic genetic loci...................................................................................90
Phylogenetic analysis of Penicillium sensu stricto.........................................................................91
Penicillium chrysogenum........................................................................................................................99
Penicillium brevicompactum...................................................................................................................99
CHAPTER 5. ASSESSMENT OF GENETIC VARIATION IN INDOOR ISOLATES
OF PENICILLIUM BREVICOMPACTUM....................................................101
Abstract................................................................................................................................................101
Introduction........................................................................................................................................102
Materials and methods.......................................................................................................................103
Collection and characterization of isolates.................................................................................103
Sequence analysis...........................................................................................................................105
Results..................................................................................................................................................109
Heteroduplex mobility assay........................................................................................................110
DNA sequence analysis.................................................................................................................110
Discussion............................................................................................................................................118
Penicillium stoloniferum......................................................................................................................122
Penicillium paxilli..............................................................................................................................122
Affiliations of P. brevicompactum....................................................................................................123
Conclusions.........................................................................................................................................124
CHAPTER 6. ASSESSMENT OF GENETIC VARIATION IN INDOOR ISOLATES
OF PENICILLIUM CHRYSOGENUM........................................................125
Abstract................................................................................................................................................125
Introduction........................................................................................................................................126
Materials and methods.......................................................................................................................128
Isolation and identification of strains..........................................................................................128
DNA preparation and heteroduplex analysis.............................................................................132
PCR fingerprinting.........................................................................................................................132
Random primer-pair fingerprinting.............................................................................................135
Cluster analysis...............................................................................................................................136
DNA sequencing............................................................................................................................137
Sequence analysis...........................................................................................................................137
Spatial analysis................................................................................................................................138
Dendrogram construction............................................................................................................138
Results..................................................................................................................................................138
Heteroduplex analysis....................................................................................................................138
Individual data sets........................................................................................................................143
Combined sequence data..............................................................................................................148
Spatial analysis................................................................................................................................155
PCR fingerprinting.........................................................................................................................155
Random primer fingerprinting.....................................................................................................163
Discussion............................................................................................................................................169
Morphological and physiological variation.................................................................................171
History of P. chrysogenum................................................................................................................173
Nomenclatural stability of P. chrysogenum....................................................................................174
Synonyms of P. chrysogenum...........................................................................................................177
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Penicillium chrysogenum and the indoor environment...................................................................178
Conclusions.........................................................................................................................................179
CHAPTER 7. CONCLUSIONS AND GENERAL SUMMARY..............................................180
Fungal decay in early structures...................................................................................................181
Ventilation.......................................................................................................................................182
Early housing and urbanization...................................................................................................183
Modern era......................................................................................................................................184
The changing urban landscape.....................................................................................................186
Suburban life...................................................................................................................................187
North American dependence on petroleum..............................................................................187
The energy crisis and the sick building.......................................................................................188
Heating and ventilation.................................................................................................................189
Gypsum-based wallboard products.............................................................................................194
The dawn of “Sick Building Syndrome”....................................................................................195
Ecology of dust-borne fungi........................................................................................................195
Association analysis of dust-borne fungi....................................................................................196
Penicillium brevicompactum.................................................................................................................198
Penicillium chrysogenum......................................................................................................................200
Summary..............................................................................................................................................202
LITERATURE CITED..........................................................................................................205

APPENDIX A Summary of dust mycoflora by house............................................................229
APPENDIX B Chi square statistics for association analysis of dustborne taxa..................290
APPENDIX C Calculations of sampling efficiency.................................................................340
APPENDIX D Alignment of sequences of nuclear ribosomal DNA,
ITS1-5.8S-ITS2 and partial 28S region from 85 Penicillium species............344
APPENDIX E Alignment of sequences of nuclear ribosomal DNA,
ITS1-5.8S-ITS2 region from 46 taxa from Subgenus Penicillium.................393
APPENDIX F Alignments of P. brevicompactum sequences
F-1 Beta-tubulin (benA), partial sequence.............................................................406
F-2 Nuclear ribosomal RNA, ITS1-5.8S-ITS2.....................................................409
APPENDIX G Alignments of P. chrysogenum sequences
G-1 Acetyl coenzyme A synthase (acuA), partial sequence.................................413
G-2 Beta-tubulin (benA), partial sequence.............................................................418
G-3 Nuclear ribosomal RNA, ITS1-5.8S-ITS2.....................................................426
G-4 Thioredoxin reductase (trxB), partial sequence.............................................436

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