Modern Concepts in Penicillium and Aspergillus Classification

Modern Concepts in
Penicillium and Aspergillus

Classification
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Series A: Life Sciences
Modern Concepts in
Penicillium and Aspergillus
Classification
Edited by
Robert A. Samson
Centraalbureau voor Schimmelcultures
Baarn, The Netherlands
and
John I. Pitt
CSIRO Division of Food Processing
North Ryde, New South Wales, Australia
Springer Science+Business Media, LLC
Proceedings of the Second International Pnicillium

and Aspergillus NATO Advanced Research Workshop,
held May 8-12, 1989,
in Baarn, The Netherlands
Library of Congress Catalog1ng-1n-Publ1cat1on Data
I n t e r n a t i o n a l Pnicillium and Aspergillus NATO Advanced Research

Workshop (2nd : 1989 : Baarn, Netherlands)
Modern concepts 1n pencilllun and a s p e r g l l l u s c l a s s i f i c a t i o n /
edited by Robert A. Sanson and John I. P i t t .
p.
en. (NATO ASI s e r i e s . Series A, Life sciences ; v.
185)
"Proceedings of the Second I n t e r n a t i o n a l Pnicillium and
Asperglllus NATO Advanced Research Workshop, held May 8-12, 1989, In
Baarn, The Netherlands"Verso of t.p.
"Published In cooperation with NATO S c i e n t i f i c A f f a i r s Division."
Includes bibliographical r e f e r e n c e s .
ISBN 978-1-4899-3581-6
1. P n i c i l l i u m C l a s s i f i c a t i o n C o n g r e s s e s . 2. Asperglllus-ClasslficationCongresses.
I. Sanson, Robert A. I I . P i t t , John
I. I I I . North A t l a n t i c Treaty Organization. S c i e n t i f i c A f f a i r s
Division. IV. T i t l e . V. S e r i e s .
QK625.M7I57 1989
589.2'3dc20
90-7008
CIP
ISBN 978-1-4899-3581-6
DOI 10.1007/978-1-4899-3579-3
ISBN 978-1-4899-3579-3 (eBook)
1990 Springer Science+Business Media New York

Originally published by Plenum Press, New York in 1990
Softcover reprint of the hardcover 1st edition 1990
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PREFACE
In our view, the First International Penicillium and Aspergillus Workshop held in Baarn
and Amsterdam in May, 1985, was a great success. The assembly in one place of so many
specialists in these two genera produced both interesting viewpoints and lively
discussions. But more particularly, a remarkable cohesion of ideas emerged, borne
primarily of the realisation that taxonomy has passed from the hands of the solitary
morphologist. The future of taxonomy lay in collaborative and multidisciplinary studies
embracing morphology, physiology and newer methodologies.
The Second International Penicillium and Aspergillus Workshop was borne logically
from the first, and was held in Baarn on May 8-12, 1989. It was attended by 38 scientists
from 16 countries. At this Workshop we have attempted to move further into new
methods, especially by bringing together molecular biologists, medical and food
mycologists and biochemists as well as more traditional taxonomists. We feel that the
meeting contributed greatly to dialogue between taxonomists, and also fundamental and
applied mycologists. At the meeting, we became aware that the approach to taxonomy of
these genera is now becoming more pragmatic, with an increasing emphasis on
consensus, and on stability of names. This is a noteworthy development, which we, as
editors, welcome. So many species in Penicillium and Aspergillus are economically
important in biotechnology, foods and medicine, and practical, stable taxonomy is of vital
importance.
These Proceedings comprise 40 papers divided into 9 chapters. Discussions relating to
each paper were taped and after careful editing, have been included following the
relevant paper. Some general discussion is also included. Dr Keith Seifert kindly helped us
with typing the discussions, and we are extremely grateful for his assistance. We also wish
to thank Mr Guido van Reenen, who prepared the layout and camera ready copy of both
the Abstracts and these Proceedings.
The Second International Penicillium and Aspergillus Workshop was sponsored by the
NATO ARW programme. Cosponsors were the Dutch Programmebureau Biotechnology,
the Royal Academy of Sciences and some industrial companies. We wish to sincerely
thank all of these sponsors.
Local arrangements for the Workshop were very efficiently and smoothly organised
with the assistance of Mrs Ans Spaapen-de Veer, Tineke van der Berg and Marjolein van
der Horst; we wish to thank each of them, as well as many other colleagues from the
Centraalbureau voor Schimmelcultures.
The Editors
Baarn, September 1989
vii
CONTENTS
Chapterl
INTRODUCTION
Systematics of Penicillium and Aspergillus - past, present and future - J.I.Pitt and R.A. Samson ..................... 3
Chapter 2
TECHNIQUES AND PRACTICAL ASPECTS FOR IDENTIFICATION OF PENICILLIUM AND
ASPERGILLUS
Standardization in Penicillium identification - O. Constantinescu........................................................................ 17
Identification of Penicillium and Aspergillus species in mixed cultures in petri dishes using secondary
metabolite profile - O. Filtenborg and J.e. Frisvad ........................................................................................... 27
Variation in Penicillium and Aspergillus conidia in relation to preparatory techniques for scanning electron
and light microscopy - P. Staugaard, RA Samson and M.I. van der Horst ................................................. 39
Variants of Penicillium expansum: an analysis of cultural and microscopic characters as taxonomic criteria
- J.F. Berny and G.L. Hennebert ........................................................................................................................... 49
Penicillium and Aspergillus in the food microbiology laboratory - A.P. Williams. ............................................. 67
Chapter 3
NOMENCLATURE: CONSERVATION AND STABILITY OF NAMES OF ECONOMICALLY
IMPORTANT SPECIES
Problems and prospects for improving the stability of names in Aspergillus and Penicillium
- D.L. Hawksworth ................................................................................................................................................ 75
Proposals to conserve important species names in Aspergillus and Penicillium - J.e. Frisvad,
D.L. Hawksworth, Z. Kozakiewicz, J.I. Pitt, RA Samson and Ae. Stolk.. ..................................................... 83
Nomenclature stability in Penicillium and Aspergillus, an alternative view - W. Garns .................................... 91
Chapter 4
TAXONOMIC SCHEMES OF PENICILLIUM
Speciation and synonymy in Penicillium Subgenus Penicillium - towards a definitive taxonomy
- J.I. Pitt and RH. Cruickshank .......................................................................................................................... 103
The systematics of the terverticillate Penicillia - Ae. Stolk, RA Samson, J.e. Frisvad and
O. Filtenborg ............................................................................................................................................................ 121
A reappraisal of the terverticillate Penicillia using biochemical, physiological and morphological features
- P.D. Bridge, D.L. Hawksworth, Z. Kozakiewicz, AH.S. Onions, RR.M. Paterson, M.J. Sackin and
P.H.A. Sneath ................................................................................................................................. 139
Evaluation of the diagnostic features of some species of Penicillium section Divaricatum - O. Fassatiova
and A. Kubatova ..................................................................................................................................................... 149
Revision of Penicillium Subgenus Furcatum based on secondary metabolites and conventional characters
- J.e. Frisvad and O. Filtenborg .......................................................................................................................... 159
The Penicillium funiculosum complex - well ddined species and problematic taxa
- E.S. van Reenen-Hoekstra, J.C Frisvad, RA Samson and A.C Stolk.. ..................................................... 173
Identification of Penicillium species isolated from an agricultural loess soil in Germany
- H.I. Nirenberg and B. Metzler .......................................................................................................................... 193
ChapterS
TAXONOMIC SCHEMES OF ASPERGILLUS
Chemotaxonomy and morphology of Aspergillus fumigatus and related taxa - J.e. Frisvad and
RA. Samson ............................................................................................................................................................. 201
Exocellular polysaccharides from Aspergillus fumigatus and related taxa - J.P. Debeaupuis, J. Sarfati,
A Goris, D. Stynen, M. Diaquin and J.P. Latge .................................................................................................. 209
viii
Biotyping of Aspergillus fumigatus and related taxa by the yeast killer system - L. PoloneIli, S. Conti,
L. Campani and F. Fanti ......................................................................................................................................... 225
Analysis of components of Aspergillus and Neosartorya mycelial preparations by gel electrophoresis
and western blotting procedures - V.M. Hearn, M. Moutaouakil and J.-P. Latge ...................................... 235
Taxonomy of Aspergillus section Restricta - J.I. Pitt and R.A. Samson ................................................................ 249
Isoenzyme patterns in Aspergillus flavus and closely related species - R.H. Cruickshank and J.I. Pitt ......... 259
Chapter 6
COMPUTER-ASSISTED IDENTIFICATION OF PENICILLIA AND ASPERGILLIA
Computer applications in Penicillium and Aspergillus systematics - M.A. Klich ............................................. 269
Penname, a new computer key to common Penicillium species - J.I. Pitt .......................................................... 279
Identification of terverticillate Penicillia from a matrix of percent positive test results - P.D. Bridge .......... 283
Identification of Penicillium and Aspergillus computer-assisted keying - A.P. Williams ................................ 289
Chapter 7
NEW APPROACHES FOR PENICILLIUM AND ASPERGILLUS SYSTEMATICS: MOLECULAR
BIOLOGICAL TECHNIQUES
A review of molecular biological techniques for systematic studies of Aspergillus and Penicillium
- E.J. Mullaney and M.A. Klich ........................................................................................................................... 301
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics - JH. Croft,
V. Bhattacherjee and KE. Chapman .................................................................................................................... 309
Variation in pectinolytic enzymes of the black Aspergilli: a biochemical and genetic approach
-M.A. Kusters - van Someren, H.CM. Kester, R.A. Samson and J. Visser ................................................. 321
A molecular assessment of the position of Stilbothamnium in the genus Aspergillus - J. Dupont,
M. Dutertre, J.-F. Lafay, M.-F. Roquebert and Y. Brygoo .................................................................................. 335
Ribosomal RNA comparisons among taxa of the terverticillate Penicillia - A. Logrieco, S.W. Peterson
and D.T. Wicklow ................................................................................................................................................... 343
Ribosomal DNA restriction studies of Talaromyces species with Paecilomyces and Penicillium
anamorphs - J.W. Taylor, J.I. Pitt and A.D. Hocking ...................................................................................... 357
ChapterS
NEW APPROACHES FOR PENICILLIUM AND ASPERGILLUS SYSTEMATICS: BIOCHEMICAL AND
IMMUNOLOGICAL TECHNIQUES
Secondary metabolites as consistent criteria in Penicillium taxonomy and a synoptic key to Penicillium
subgenus Penicillium - J.C Frisvad and O. Filtenbor& .................................................................................... 373
Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillus taxa: (1) Aspergillus
sects. Ornati and Cremei - J. Sugiyama and K Yamatoya. ............................................................................. 385
Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillus taxa: (2) Aspergillus
sect. Flam - K Yamatoya, J. Sugiyama and H. Kuraishi... .............................................................................. 395
The ubiquinone system as a taxonomic aid in Aspergillus and its teleomorphs - H. Kuraishi, M. Itoh,
N. Tsuzaki, Y. Katayama, T. Yokoyama and J. Sugiyama ................................................................................ 407
Immunological differentiation between Penicillium and Aspergillus taxa - B. Fuhrmann, M.F. Roquebert,
V. Lebreton and M. van Hoegaerden ................................................................................................................... 423
The significance of yeast extract composition on metabolite production in Penicillium - O. Filtenborg,
J.C Frisvad and U. Thrane .................................................................................................................................... 433
Chapter 9
TAXONOMIC STUDIES ON THE TELEOMORPHS OF PENICILLIUM AND ASPERGILLUS
Chemotaxonomy of Eupenicillium javanicum and related species - J.C Frisvad, R.A. Samson and
A.C Stolk ..................................................................................................................................... 445
The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
- R.A. Samson, P.V. Nielsen and J.CFrisvad ................................................................................................... 455
ix
Participants
Species Index
......................................................................................................................................................469
...............................................................................................................................................473
1
INTRODUCTION
SYSTEMATICS OF PENICILLIUM AND ASPERGILLUS

- PAST, PRESENT AND FUTURE
J.I. Pittl and R.A. Samson2

lCSIRO
Division of Food Processing

North Ryde, NSW 2113, Australia
'lCentraalbureau voor Schimmelcultures

3740 AG Baarn, The Netherlands
SUMMARY
The great pioneers in the systematics of Penicillium and Aspergillus were Charles Thorn and Kenneth B.
Raper, who together developed the first workable and widely accepted taxonomies of these genera.
Both were instrumental also in the development of culture collections for these industrially important
fungi. Raper made the invaluable contribution of developing freeze drying techniques suitable for
fungi, and this has proved to be of great practical importance. However, they also left a legacy of
nomenclatural and taxonomic problems which have been addressed only recently. Moreover, inherent
difficulties in achieving consensus on species concepts and on identification of isolates have provided
a challenge for modem students of these genera.
This paper describes the new approaches which have led to clarification of the systematics of these
genera. On the one hand, increased awareness of the principles of priority, typification and type
specimens has helped to bring order to nomenclature. On the other, the use of gross physiological
characters, secondary metabolites and isoenzyme patterns has greatly assisted in clarifying taxonomy.
Collaborative studies on difficult aspects of both genera have been initiated both by individuals and
under the control of an international working group, the Subcommission on Penicillium and
Aspergillus Systematics.
Great progress has been made. In particular, the taxonomy of the important and notoriously
difficult subgenus Penicillium has now become firmly established, and criteria for differentiating
aflatoxin producing and nontoxigenic Aspergillus species have been clarified.
The immediate end of all this work lies in a better understanding of relationships within these
genera, and improved identifications. The wider benefits lie in a greater knowledge of the role of
particular species in food spoilage, toxicology, biodeterioration, biotechnology and ecology.
INTRODUCTION
Penicillium and Aspergillus are two of the most economically important genera of fungi.
Much of their economic impact is deleterious, with food spoilage, mycotoxin production
and biodeterioration heading the list, but in fact their potential for economic utility is
equally great. Penicillin has been the great success story in the utilisation of Penicillium,
but the physiological and biochemical diversity of both genera is such that their potential
for benefiting mankind has only just begun to be tapped. The future includes cheaper
production of chemicals such as citric and other organic acids, biochemical syntheses and
conversions of intricate molecules not amenable to chemical techniques, and a whole
range of enzymes, proteins and other organic molecules of value in commercial
applications. Genetic engineering will playa major role.
It is imperative, then, that the systematics of these genera be clarified and stabilised,
as a matter of urgency. Ideally, identification should be unequivocal, accurate, simple and
J.I. Pitt & R.A. Samson
immutable. In some parts of Penicillium we are approaching this goal, a situation which
even a few years ago would have been regarded as unlikely to ever be reached.
The majority of the significant recent work has dealt with Penicillium, the larger and
more complex of these two genera, so it will be primarily discussed in this paper.
Aspergillus will be considered also where appropriate.
THE PAST: A SHORT HISTORY
During the 19th century, the taxonomy of anamorphic genera such as Penicillium and
Aspergillus was strictly botanical. Descriptions were based on observations of the fungi on
natural substrates and were based mainly on microscopical observations. Because fruiting
structures are ephemeral, especially in Penicillium, descriptions were usually rather
meagre. Many such species subsequently went unrecognised.
With the advent of pure culture techniques around the turn of the century, colony and
fruiting structure development began to be observed. As a result of improvements in
microscopes and microscopic techniques, great advances occurred in descriptions.
Recognition of different types of fruiting structure in both genera led to the splitting out of
genera such as Citromyces and Sterigmatocystis, but most taxonomists retained Penicillium
and Aspergillus as monolithic and broadly based, a situation still existing today. As they
were less ephemeral than the anamorphs, teleomorphs of these genera were described
early. The connection between Eurotium Link 1809 and Aspergillus, established by de Bary
in 1854. Ludwig described Eupenicillium in 1892, though this teleomorph genus was not
accepted until the 1970s.
The great pioneer in the study of Penicillium and Aspergillus was Charles Thorn (18721956). Thorn (1906) produced the first readily recognisable descriptions of Penicillium
species and Thorn (1910) the first taxonomy. He was author or coauthor of two further
monumental works on Penicillium. Thorn (1930) was a compendium of the 300 species
described to that time, with keys and some indications of synonymy. Raper and Thorn
(1949) published "A Manual of the Penicillia", which was the authoritative taxonomy in
almost exclusive use until 1980.
Thorn and his colleagues also studied Aspergillus taxonomy. Thorn and Church (1926)
produced the first complete taxonomy of the genus, which was expanded by Thorn and
Raper (1945), and enlarged and refined again by Raper and Fennell (1965).
Other taxonomists.
Other taxonomists have also made significant contributions to the taxonomy of
Penicillium, especially in Europe. Westling (1911) and Sopp (1912) both described a
number of Penicillium species from Scandinavian soils. In France, Bainier and Sartory (e.g.
Bainier, 1907; Bainier and Sartory, 1912) published a series of short papers on new species.
Biourge (1923) published an extensive taxonomy, and revived and characterised some
names of Dierckx (1901) which had been inadequately described.
In Aspergillus, description of new species appears to have proceeded more or less
piecemeal, with no major taxonomies other than those mentioned above.
Culture collections.
At the same time as the taxonomy of Penicillium and Aspergillus developed, collections of
fungal cultures began to be established. The role of culture collections in taxonomy,
allowing the preservation and distribution of living ex type cultures and other important
Systematics of Penicillium and Aspergillus
isolates, cannot be overemphasized. Their significance in the effective utilisation of fungi

for beneficial purposes will prove equally great in the coming years.
The Centraalbureau voor Schimmelcultures in Baarn, and the Commonwealth
Mycological Institute in Kew, Surrey, have played a major role in Penicillium and
Aspergillus taxonomy by providing a vital continuity in cultures. Many of the important
European taxonomists sent their cultures to CBS, including Westling and Biourge. Both
Thom and Raper visited Baarn, and both obtained cultures which laid the foundation of
their own collection, at the USDA Northern Regional Research Center in Peoria, lllinois, a
collection which has played a pivotal role in Penicillium and Aspergillus taxonomy.
Culture preservation.
It is worth noting here also that KB. Raper pioneered the technique of freeze drying for
the preservation of fungal cultures. This has also been of great importance. Studies by Pitt
(1979) were based on the Raper collection at Peoria, which in many cases provided him
with better quality cultures than were available elsewhere, from collections where cultures
were maintained by less effective preservation techniques, or had been freeze dried later.
Shortcomings of Raper and Thom's work.

Charles Thom and Kenneth Raper were taxonomic giants, whose perceptions of species
and species relationships were without parallel among their contemporaries. Thom, Raper
and their colleagues dominated the taxonomy of these two genera for half a century.
However, as the number of species described in Penicillium and Aspergillus increased, so
nomenclatural and taxonomic difficulties multiplied. Without any disrespect to their
memories, it can be stated in the late 1980s that some of Thom and Raper's concepts have
proved to be less than satisfactory and, equally importantly, that their very conservative
approach to nomenclatural changes ultimately led to serious difficulties in the systematics
of both genera: in Penicillium, especially, to a state little short of chaos.
Nomenclatural problems.
The nomenclatural problems which arose as a consequence of the Raper and Thom
taxonomies can be summarised in three points: (a) failure to observe priority of names; (b)
failure to typify species; and (c) failure to give precedence to teleomorphs in naming
species.
(a) Priority.
In the sense used in the International Code of Botanical Nomenclature (ICBN; Greuter,
1988) "priority" has the specific meaning of "requiring use of the earliest validly
published name". "Validity" is governed by a number of Articles, most of which are not
relevant here. Suffice to say that Thom and Raper sometimes did not accept names which
had priority, on various grounds which are not acceptable now. Ignoring Biourge's
neotypifications, Thom and Raper rejected many names published by Dierckx (1901) on
the grounds of inadequate descriptions, unless it suited them, as in the case of an apt
name like Penicillium brevicompactum. Valid names of other authors were sometimes
rejected for similar reasons. Name changes made much later (Samson et al., 1976; Pitt,
1979), with the inevitable confusion that name changes cause, could have been avoided in
many cases.
(b) Failure to typify.

Among the criteria for valid publication, the ICBN demands that species by "typified", i.e.
that a representative herbarium specimen be preserved from material used to produce the
original description. Raper and Thorn failed to specify dried types, though in their
defense, it must be said that many other taxonomists were equally negligent. More
seriously, however, Raper and Thorn frequently failed to identify even living type
material in their standard descriptions of species, and sometimes ignored existing type
material altogether.
(c) The problem of naming sexual states.

Thorn and Raper, and to a lesser extent, Fennell, maintained that teleomorph species with
anamorphs in Aspergillus or Penicillium, including those with teleomorphs, should be
named in the anamorph genera, making new combinations as necessary (Thorn and
Raper, 1946; Thorn, 1954; Raper, 1957; Fennell, 1973). For example, Eurotium amstelodami
Mangin was renamed Aspergillus amstelodami (Mangin) Thorn & Church (Thorn and
Church, 1926) and maintained this way in their later taxonomies. However the ICBN, and
the weight of nomenclaturalists' opinions, dictate that teleomorph names have priority
over anamorph names.
A further problem concerns species described in an anamorph genus but which
included the teleomorph, for example, Penicillium javanicum van Beyma. Raper and Thorn
(1949) accepted such species as being correctly placed in Penicillium, contrary to the ICBN.
Hawksworth and Sutton (1974) showed that the ICBN then current was ambiguous in the
naming of a species such as Penicillium javanicum. As a consequence, the International
Botanical Congress in Sydney altered articles in the new ICBN (Voss, 1983) to make
naming of such species unequivocal. The price was that some well established names,
especially in Aspergillus and Penicillium, had to be changed. The nomenclatural problem
was overcome, but at the expense of introducing new and unfamiliar names. Pitt (1979)
made the necessary changes to Penicillium. For example, "Penicillium javanicum van
Beyma" is now known correctly as Eupenicillium javanicum (van Beyma) Stolk & Scott
when the teleomorph, or the whole fungus, is under consideration, and as Penicillium
indonesiae Pitt when the anamorph alone is being considered. Samson and Gams (1985)
carried out the necessary alterations in Aspergillus: "Aspergillus amstelodami (Mangin)
Thorn & Church" is correctly called Eurotium amstelodami Mangin when the teleomorph is
present, and Aspergillus hollandicus Samson & Gams for the Aspergillus state alone. For a
more detailed discussion of this point see Pitt (1988).
"Groups".
Thorn and his coworkers arranged clusters of species in Aspergillus and Penicillium into
"Groups", a subgeneric classification without nomenclatural status under the ICBN. This
has caused confusion, because most mycologists believe that if there is to be
nomenclatural stability, there must be a a code of nomenclature (the ICBN), and if there is
an ICBN, it must be accepted in all aspects. In industrially important genera, stability is
essential, and stability at the present time comes only from strict adherence to the ICBN.
Pitt (1979) replaced the "Group" structure in Penicillium by a subgeneric and sectional
structure. Gams et al. (1985) carried out the same changes to Aspergillus.
Taxonomic problems.
The taxonomy of Penicillia has always been difficult. All taxonomists have agreed that the
genus has a lot of species separated by very little. Raper and Thorn (1949) produced the
first definitive taxonomy, in its day a masterpiece, and in almost exclusive use for nearly
30 years. But it had shortcomings. First, the "taxonomic base", i.e. the range of characters
used to distinguish species, was rather narrow, so that species were difficult to distinguish
from each other, even to the expert. Also, Raper and Thorn (1949) placed too much
reliance on colony texture. For example, in the Raper and Thorn classification
terverticillate species were spread across Sect. Asymmetrica subsects. Velutina, Fasciculata,
Funiculosa and Lanata, mainly on the basis of differences in colony texture. Without expert
guidance, the classification was largely unworkable.
Samson et al. (1976) approached this problem by reducing a number of difficult to
distinguish Raper and Thorn species to the status of varieties. Pitt (1979) disagreed both
with the Raper and Thorn reliance on texture, and the varietal approach of Samson et al.
(1976). Instead, he relied heavily on colony diameters and morphology, especially conidial
colour. However, this approach was relatively ineffective in subgenus Penicillium where
the majority of species respond similarly to temperature and reduced water activity (aw),
and differences in colony colours are small and rather subjective.
The competition among three classification systems with quite different approaches,
and the shortcomings of all three, resulted in confusion (Onions et al., 1984).
THE PRESENT: NEW APPROACHES
Clearly, new approaches were needed to improve the taxonomy of Penicillium and
Aspergillus; changes which would broaden the taxonomic base, clarify species concepts
and bring both nomenclature and taxonomy into line with the ICBN. Change started
slowly, then gathered pace, on several fronts.
Nomenclatural changes.
The first developments came with nomenclatural changes relating to the use of
teleomorph names. Benjamin (1955) introduced Talaromyces for Penicillium teleomorphs
with gymnothecia, and took up Carpenteles Langeron for teleomorphs with cleistothecia.
Raper (1957) resisted these changes. Stolk and Scott (1967) revived the earlier name
Eupenicillium for Carpenteles and, for example, correctly renamed Penicillium javanicum van
Beyma as Eupenicillium javanicum (van Beyma) Stolk & Scott. This approach won general
approval over the next decade.
In search of a broader base.
A variety of attempts to improve taxonomy occurred more or less simultaneously. Abe
(1956) published a modified version of Raper and Thorn (1949) in which he made use of
some physiological characters as an aid in Penicillium classification. These included the use
of 37C as a growth temperature and the use of nitrite as a selective agent for growth. Pitt
(1973) developed this idea, making use of both SoC and 3~C as measures of the effect of
temperature on the growth of Penicillium isolates. He also introduced 25% glycerol nitrate
agar (G25N, aw 0.935) as a medium of reduced water activity, which provided some
measure of physiological adaptation to low aw . To make effective use of these concepts,
Pitt (1973) used a carefully standardised set of media and incubation conditions. He
proposed quantifying the influences of temperature and aw by measurements of colony
diameters grown for a standard incubation time of 7 days.
A variety of other approaches followed, all aimed at broadening the base of
Penicillium taxonomy. Pyrolysis gas liquid chromatography (Kulik and Vincent, 1973;
Bums et al., 1976; S"derstrf1J1l1 and Frisvad, 1984) and differences in long chain fatty acids
(Dart et al., 1976) each showed the capacity to distinguish genera or species when
relatively few isolates were examined, but became impossible to interpret when a large
genus like Penicillium was studied. Moreover, the match of long chain fatty acids with
morphological taxonomy was poor (Pitt, 1984). This was the case also with the production
of nigeran, a macromolecular cell wall constituent studied by Bobbitt and Nordin (1978).
Secondary metabolite production.
The quest for taxonomically valuable physiological characters started along another line
when Ciegler et al. (1973) suggested that secondary metabolites, particularly mycotoxins,
might be of taxonomic value. The concept was applied on a limited scale to Penicillium
viridicatum (Ciegler et al., 1973, 1981). The development of simpler techniques followed. Of
particular interest was the concept of directly spotting TLC plates with small samples of
culture cut directly from Petri dishes with a cork borer, followed by chromatography
(Filtenborg and Frisvad, 1980; Filtenborg et al., 1983). Mycotoxin and secondary metabolite
production can be assessed qualitatively much faster by this method than by conventional
extraction, clean up and concentration techniques.
These approaches met with some early opposition because of the complexity of the
concepts (Frisvad and Filtenborg, 1983; Frisvad, 1985, 1986). However, difficulties with
misidentified cultures, and the relationship of mycotoxin profiles to morphological
taxonomy have now been clarified, especially with the introduction of another new
technique, electrophoretic comparison of certain enzymes (see below). Patterns of
secondary metabolites have now become an effective taxonomic tool, especially when
used in conjunction with traditional taxonomy as governed by the ICBN (Pitt, 1984).
Frisvad and Filtenborg (1983), Paterson (1986) and EI-Banna et al. (1987) have
published detailed thin layer chromatographic solvent systems and R values for a wide
variety of Penicillium metabolites, so that metabolite profiles can now be used by the
determinative taxonomist.
Isoenzyme electrophoresis.
Cruickshank (Cruickshank and Wade, 1980; Cruickshank, 1983) developed effective
methods for separation of species of Sclerotinia, Botrytis and other genera by examining
patterns of pectic enzymes after separation by gel electrophoresis. Small samples of
culture fluid were subjected to electrophoresis at low temperatures, then the separated
enzymes allowed to act on methoxy pectin incorporated into the gel, and the sites of
enzyme action visualised by ruthenium red staining.
To enable the differentiation of the many species in subgenus Penicillium,
Cruickshank's technique was broadened by including the examination of amylase and
ribonuclease isoenzymes. For amylase production, soluble starch was incorporated in the
gels as a substrate, and for ribonucleases, ribosomal RNA. Fungi were cultured on wheat
grains for the production of both these sets of enzymes (Cruickshank and Pitt, 1987a).
A study of the isoenzyme patterns (zymograms) for all species accepted by Pitt (1979)
in subgenus Penicillium has now been successfuly completed (Cruickshank and Pitt,
1987b). For the first time, an objective physiological test has been correlated to an
acceptable degree with classical taxonomic methods. All isolates examined from the great
majority of species accepted by Pitt (1979) and now agreed on by Samson and Pitt (985)
showed an absolute correlation with specific zymogram patterns, greatly reinforcing our
confidence in current species concepts in this subgenus. In certain species, notably P.
aurantiogriseum and P. viridicatum, correspondence was less clear; however, a lack of
clarity in classical taxonomic concepts was already evident.
By 1989, the taxonomy of subgen. Penicillium has been greatly clarified. Close agreement
on taxonomy by classical techniques, secondary metabolites and isoenzyme patterns has
resulted in well defined species (Pitt and Cruickshank, 1990i Stolk et al., 1990i Frisvad and
Filtenborg, 1989).
Examination of herbarium specimens.
As was stated earlier in this paper, the name of any fungus is linked to a herbarium
specimen. The fruiting structures in some genera, including Penicillium and Aspergillus,
are of a relatively emphemeral nature, and taxonomists have frequently ignored
herbarium specimens in reaching taxonomic conclusions. Recently Seifert and Samson
(1985) undertook a study of herbarium specimens in an obsolete genus, Coremium, and
were able to recognise some old, valid species which needed to be transferred to
Penicillium. As a result, the names of a few species have been changed. These include P.
claviforme Bainier to P. vulpinum (Cooke & Massee) Seifert & Samson, P. granulatum Bainier
to P. glandicola (Oudem.) Seifert & Samson, and P. concentricum Samson et al. to P.
coprophilum (Berk. & Curt.) Seifert & Samson.
Collaborative examination of cultures.
To assist in clarification of the taxonomy of afiatoxigenic fungi, Klich and Pitt (1985, 1988)
carried out a collaborative study involving the detailed morphological and gross
physiological examination of more than 200 isolates of Aspergillus flavus and related
species. Cultures were examined in two different locations, with isolates identified only by
code. Features distinguishing A. flavus from A. parasiticus and from the closely related
food fermentation species, A. flavus and A. sojae, were documented.
A subcommission of the International Commission on Taxonomy of Fungi (IUMS) has
been formed, which will carry out collaborative studies on the taxonomy of Penicillium
and Aspergillus. Known as the Subcommission on Penicillium and Aspergillus Systematics
(SPAS), this group has twelve members from seven countries, and comprises both
morphological taxonomists and those skilled in the newer techniques which have been
described here. A significant impact is expected.
Genetic studies.
Until recently, the genetic tools of DNA hybridisation and analysis of DNA and RNA
sequences have been little used in the systematics of Penicillium and Aspergillus.
On the basis of studies of DNA homology among A. flavus and related species,
Kurtzman et al. (1986) reduced several well known species to subspecies or varietal status.
Specifically A. parasiticus became A. flavus subspecies parasiticus Kurtzman et al.i A. oryzae
became A. flavus subspecies flavus variety oryzae Kurtzman et a1.i and A. sojae became A.
flavus subspecies parasiticus variety sojae Kurtzman et al.
Klich and Mullaney (1987) disagreed with this conclusion, arguing that DNA
restriction enzyme fragment polymorphism showed differences between the DNA of A.
flavus and A. oryzae. Klich and Pitt (1988) also did not accept this change, on the grounds
that morphological differences existed between these two species, and on the more
practical grounds that it was important that species used for food fermentations possess
different species names from those which are known to be mycotoxigenic.
10
J.I. Pitt & RA Samson
THE FUTURE
Finally, a look at the future. Some aspects of prediction are not difficult, because some
techniques of the future are already in use, and are described more fully in the papers
which follow in these Proceedings.
In the future, increased reliance will be placed on secondary metabolites and
isoenzyme profiles as aids in taxonomy -not, perhaps, in routine identifications, but
certainly in more specialist laboratories and those where fundamental taxonomic studies
are carried out.
Care is required in interpreting results from both of these techniques. The fact that
two isolates or sets of isolates have similar profiles by either technique does not prove a
close relationship. However, in our view, similar profiles with both techniques, and close
similarities in morphological characters, provide an excellent guide to species definition.
Second, genetic techniques will be increasingly employed -again, for basic studies, not
as a determinative tool. Genetic studies will range from fundamental work on RNA,
which will enable clarification of phylogeny on a generic scale, and the setting of
evolutionary clocks, to comparisons of nuclear and mitochondrial DNA as an aid in
delimiting species. DNA probes will be of great value, once suitable probes have been
developed. Again it needs to be emphasised that genetic techniques, like other taxonomic
methods, are tools, not the ultimate solution. Similarities or differences in genetic material,
measured by whatever technique, must be assessed in conjunction with other taxonomic
yardsticks.
No doubt other new and innovative genetic methods will appear in the next few
years. Of particular interest is the new technique of "gene amplification", which promises
to make it possible to identify the most limited herbarium material by copying DNA from
even a single spore until there is sufficent to hybridise or sequence. The impact of this
technique on current taxonomy is difficult to assess, but may be profound, unless carefully
managed.
Third, computer based systems will be increasingly applied to Penicillium and
Aspergillus taxonomy. Culture collections, the indispensible libraries of the taxonomist,
will become computerised, in time enabling access for all interested scientists to much
more data than is currently available, and much more rapidly. Networks of information of
this kind will be established, though rather slowly.
At a different level, computers will be used to produce databases relating to
individual genera, related series, or isolates within a species. Keys will be developed from
this information, and will to some degree replace current written taxonomies.
Fourth, more rapid taxonomic methods will be devised. The trend will be towards
systems which can be used directly on isolation plates, and may involve computer
databases, recognition of specific or more general antibodies, or secondary metabolites
(Filtenborg and Frisvad, 1990).
Fifth, a trend towards protection of the names of well recognised species will develop
in the next few years. Penicillium, especially, will be in the forefront of these studies,
because general agreement on species concepts throughout much of the genus is already
close, or will become so, especially through international collaboration under SPAS. Such
protection is becoming imperative (Hawksworth, 1990).
However, regardless of the introduction and use of new techniques, we will not, and
indeed should not, see the replacement of current morphological and gross physiological
identification methods, at least into the next century. The reason is simple, and
compelling: it is most important that taxonomy, at the determinative level, be accessible,
11
as far as possible, to those in Bangkok or Bogota, Berlize or Bhutan, as well as those

working in the favoured environments of Baarn, Berlin or Britain.
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12
GREUTER, W. et al. 1988. International Code of Botanical Nomenclature adopted by the Fourteenth
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and London: Plenum Press.
SAMSON, RA. and pm, T.I. 1985. Check list of common Penicillium species. In Advances in Penicillium and
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13
STOLK, Ae., SAMSON, R.A., FRSIVAD, J.e. and FlLTENBORG, O. 1990. The systematics of the
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DIALOGUE FOLLOWING DR. PITT'S PRESENTATION
It's interesting to see new methods for identification being developed for these
genera. Alternative methods of identification may involve genetic methods or secondary
metabolites. Right now, we're seeing some of those methods used in medical mycology.
Later on, we may see some of them being applied to food.
TAYLOR:
How about identification of Penicillium and Aspergillus by serological

techniques?
SAMSON:
don't mind what methods we use. Food and pharmaceutical people are desperate
for faster methods. Presently, there are no simple methods that anyone can use. I intend
to concentrate on developing some new ideas in this area over the next few years.
PITT: I
2
TECHNIQUES AND PRACTICAL ASPECTS FOR
IDENTIFICATION OF PENICILLIUM AND ASPERGILLUS
17
STANDARDIZATION IN PENICILLIUM IDENTIFICATION
o. Constantinescu
The Herbarium
Uppsala University
751 21 Uppsala, Sweden
SUMMARY
The cultural and morphological characters of 36 species of Penicillium were examined after growth at
25C on Czapek yeast extract agar (CYA), malt extract agar (MEA), and 25% glycerol nitrate agar
(G25N), in glass and in unsealed and sealed plastic Petri dishes. As a result, the following suggestions
for the standardization of Penicillium identification are made: a conidial suspension in sterile water
supplemented with a wetting agent to be used as inoculum; addition of trace elements solution to the
CYA; the possible use of unsealed plastic Petri dishes provided with ribs instead of glass ones for the
cultivation on CYA and MEA, and the same but partially sealed for cultivation on G25N; the
inoculation in one point instead of three as a better alternative for the fast growing species.
INTRODUCTION
The identification of Penicillia is still to a large extent based on the examination of macroand micromorphological characters. Moreover, the results of the new approaches in
Penicillium identification, such as the use of secondary metabolites and biochemical tests,
are always checked against the traditional identification procedures. The interest in
developing standardized media and growth conditions in order to guarantee
reproducibility in Penicillium identification goes back to 1893 when Biourge made the first
attempt to monograph the genus (Hennebert, 1985) and was continued until recently (Pitt,
1985; Samson and Pitt, 1985 b). However, in spite of almost one hundred years of efforts, it
seems that a consensus is far from attained. It is symptomatic that no complete agreement
has been reached even on the formulation of malt extract agar, one of the two most used
media for Penicillium identification. This is rather unfortunate, particularly for the nonspecialist who is confronted with controversial procedures and approaches in his attempt
to name Penicillium isolates.
During routine identification of Penicillia with the aid of recent monographic and
other studies (Pitt, 1979, 1985; Williams and Pitt, 1985), certain difficulties were
encountered. Atypical micromorphology was present in most species when grown on
Czapek yeast extract agar (CYA). The evaluation of the growth was difficult in fast
growing species because interference between the colonies. When, for time-saving
reasons, disposable, without ribs, plastic Petri dishes were used instead of glass ones, the
diameter of the colonies on CYA and malt extract agar (MEA) was reduced by 25 to 40
mm by occasional tightening of the dishes during incubation. Similarly, the colony
diameters on glycerol-nitrate agar (G25N) were consistently smaller than expected in
almost all species. Wrapping the dishes in polyethylene film did not reduce variation in
colony diameters, but increased the variation on both CYA and MEA.
In an attempt to critically evaluate and standardize the procedures for inoculum
preparation, inoculation and growth conditions, a series of experiments were initiated.
o. Constantinescu
18
MATERIAL AND METHODS
Fungi.
Thirty six species of Penicillium preserved at UPSC were tested (Table 1).
Table 1. Variation in colony diameters (in mm) of cultures grown in glass Petri dishes, and
occurrence (+) of some cultural and microscopical features in 36 species of Penicillium
number of species
strain
CYA
P. aurantiogriseum
P. brevicompactum
P. camemberti
P. canescens
P. chrysogenum
P. citreonigrum
P. citrinum
P. corylophilum
P. crustosum
P. decumbens
P. digitatum
P. echinulatum
P. expansum
1297
1267
1718
1536
1503
2161
1831
2495
1590
1733
980
100S
12%
2736
2697
2444
1577
1828
1036
1354
2488
1974
2178
2019
2737
2715
2040
972
2492
1597
1695
1735
1624
2700
830
31-38
21-25
31-34
32-33
29-37
23-24
22-25
27-32
*41-50
20-23
*55-56
33-38
34-40
*45-48
26-36
16-19
P.glabrum
P. hirsutum
P. islandicum
P. italicum
P. janczewskii
P. janthinellum
P.jensenii
P.lividum
P. miczynskii
P. minioluteum
P.olsonii
P. puberulum
P. purpurogenum
P. restrictum
P. roqueforti
P. rugulosum
P. sclerotiorum
P. spinulosum
P. variabile
P. verrucosum
P. viridicatum
P.vulpinum
colony diameter
G25N
MEA
32-39
14-19
45-49
1-35
~48-56
18-22
15-21
~42-45
*33-50
25-28
*62-70
31-38
*43-50
~50-58
24-31
17-19
~40-48
~56~1
27-33
32-43
27-32
26-32
20-27
34-39
26-37
22-25
14-15
27-29
28-35
40-50
13-18
32-42
20-26
*48-48
22-28
23-24
34-38
15-25
*61-70
13-15
*59~
7-10
35-41
~43-50
~42-58
~55-57
7-10
18-28
24-34
26-34
16-20
15-17
~33-40
24-28
14-18
15-16
20-27
19-23
14-20
10-13
10-14
13-17
18-26
12-17
4-5
20-22
19-21
17-19
19-25
4~
14-18
12-16
13-15
13-17
10-16
9-16
2-4
19-21
13-14
0
14-18
19-22
3-5
13-19
15-23
4-7
15-17
16-20
9-14
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Values affected by interference of the colonies

A = Abnormal microscopical structures on eVA; B = Reduction of exudate production in plastic Petri
dishes; e = Additional colonies in plastic Petri dishes; 0 = Interface effect
Standardization in Penicillium identification
19
Media.
MEA and CYA were prepared according to Samson and Pitt (1985b), and G25N according
to Pitt (1979). In addition, CYA supplemented with the trace elements solution according
to Smith (1949) was also used. Yeast extract, malt extract, peptone and agar (all Difco),
sucrose (BDH Analar), and other components (all Merck) were used.
Culture vessels.
Glass Petri dishes 90 x 14 mm, and plastic dishes 87 xIS mm provided with ribs, were
used. Eighteen ml of medium was used for each Petri dish. Inoculum was prepared by
Figure 1. The influence of medium and growing procedure on the micromorphology of some
Penicillia. a-c. Abnormal structures in P. Toqueforti; a. from the interface zone on CYA; b-c.
from the interface zone on MEA. d; P. puberulum, swollen branch on CYA; e-f. P. canescens;
e. regular and smooth-walled penicilli on MEA; f. irregular and rugose penicilli on CYA;
g. P. miczynskii. Abnormal, reduced penicilli on CYA.
20
O. Constantinescu
mixing conidia with sterile distilled water containing 0.1% Tween 20 (KEBO), in a deepwell slide. A lid of a plastic dish, in which three holes 1.5 mm diam and 38 mm from each
other had been drilled, was used to mark the places of inoculation on the back of the Petri
dish. The plates were inoculated upside-down by dipping the inoculation needle into the
conidial suspension and piercing the medium at these previously marked points. The
plates were incubated upside-down, at 25C for 7 days, in darkness, in an incubator
provided with vertical ventilation. For some fast growing species (i.e. with colonies of 30
mm or more after 7 days) a series of cultures with single colonies were also made. One of
the two series of cultures grown in plastic Petri dishes was kept during incubation in
partially sealed polyethylene bags. Two diameters of each colony, one along the radius
and one perpendicular to it, were measured by using a transparent sheet of plastic on
which both these diameters and the inoculation points were traced. Two duplicates were
made for each combination of medium, isolate and type of culture vesseL The experiments
were carried out in duplicate.
Figure 2. Variation in colony diameters of 36 species of Penicillium grown on CYA and MEA
in unsealed (left) and partially sealed (right) plastic Petri dishes, as compared to cultures
grown in glass dishes. Bars indicate the variation in the averages of two series of
measurements.
21
RESULTS AND DISCUSSION
Preparation of inoculum. and inoculation.

The addition of 0.1 % Tween 20 to the sterile water for inoculum preparation prevented the
formation of additional colonies from stray conidia, a frequent phenomenon when dry
conidia or water alone (Raper and Thorn, 1949) is used. This procedure, as well as the one
described by Frisvad (1981), is simpler than the one employing molten agar as dilution
agent and screw cap vials (Raper and Thorn, 1949; Pitt, 1979). Moreover, it proved very
suitable for rapid inoculation of a large number of Petri plates. Inoculating the plates by
piercing the medium assures that a rather large number of conidia are placed on a
minimum of surface.
Micromorphology on CYA.
Eleven out of 36 species tested showed frequent atypical microscopic structures when
grown on CYA (Table 1). These include reduction in number of the metulae and/or
phialides, swollen branches or metulae, presence of rudimentary, non developing metulae
etc. (Fig. 1 d, f-g). By comparison, the micromorphology on MEA was far more regular
(Fig. 1 e). This observation supports the claim that better development of Penicillia occurs
on MEA than on CYA (Samson and Pitt, 1985a, p. 100). By adding trace elements solution
of Smith (1949) to CYA, as recommended by Frisvad and Filtenborg (1983) and Pitt (1988),
the presence of these abnormal structures was almost eliminated.
8
I IIII I,
I
~ 0
-2
II ,I 'I" 'I rI
-4
-6
-8
15101520253035
1".I""'""I!!"I ...
"I",!
SPECIES NO
1
5
10
15
20
25
30
35
1,.,I,,,ol,,.,I,,,.!,,''''II"'II,I.
SPECIES NO
Figure 3. Variation in colony diameters of 36 species of Penicillium grown on G25N in

unsealed (left) and partially sealed (right) plastic Petri dishes, as compared to cultures grown
in glass dishes. Bars indicate the variation of the averages of two series of measurements.
Glass versus plastic Petri dishes.

No significant differences were observed in the colony shape, margin, colour and
production of diffusible pigments when the fungi were grown in glass or plastic dishes.
However, the production of exudate was diminished in 7 species when plastic dishes were
used (Table 1). Although inoculated at three points, frequent additional colonies were
formed in 14 species when grown in plastic dishes (Table 1). In some cases the whole
surface of the plate was covered with colonies which made the measurement of an
22
O.
Constantinescu
individual colony impossible. The presence of additional colonies is extremely rare when
glass dishes are used. It seems that species belonging to subgenus Aspergilloides
particularly have the tendency to form such additional colonie, as five out of the seven
tested showed this problem. The remaining two, P. sclerotiorum and P. restrictum, showed
late, and rather poor sporulation, which may explain why no additional colonies were
formed. The static electricity is, most probably, responsible for this phenomenon.
However, many heavily sporulating species do not form additional colonies. This
different behaviour is certainly connected with characters of conidiogenesis, conidium
size, weight and surface morphology.
The variation in colony diameters of cultures grown in unsealed or partially sealed
plastic Petri dishes, compared with glass dishes are summarized in figures 2-4. Low
variation (ca. 1 mm or less) on CYA occurred in 13 species when grown in unsealed dishes
and in seven species when the dishes were sealed. The corresponding values for MEA are
16 and 10, and for G25N 6 and 8. Higher variation (4 mm or more) between the two series
of measurements was present on CYA in four species when unsealed and in eight species
when sealed. Corresponding values for MEA are 4 and 8, and for G25N 9 and 5. Only a
few species showed higher than 8 mm variation between two measurements: two on each
sealed CYA and MEA, and two on unsealed G25N. These figures indicate that sealing the
dishes results in an increase of variation between two series of measurements on CYA and
MEA, but not on G25N. In most of the species the cultivation in plastic dishes gives higher
or lower values of the colony diam compared to the control (Fig. 4). Sealing the dishes
induced an increase of these differences on both CYA and MEA. On G25N most of the
species responded by a reduction of the diameter when grown in unsealed dishes, but an
increase occurred in all but two species when the dishes were sealed. This is certainly
caused by the rapid drying of this low water activity medium when plastic dishes
provided with ribs are used.
The use of glass Petri dishes is recommended for Penicillium identification. According
to Pitt (1979, p. 19) plastic dishes may be used if they are wrapped in polyethylene film.
Our results show that unsealed plastic dishes provided with ribs, in which the aeration is
relatively constant, provide more uniform results on CYA and MEA. A partial sealing is
beneficial in the case of G25N medium.
Three inoculum points versus one.
When the colony diameter is ca. 30 mm, a mutual influence between adjacent colonies
becomes noticeable. This effect mostly consists of the inhibition of growth and
sporulation, presence of abnormal micromorphological structures (Table 1, Fig. 1 a-c), and
alteration of the colony texture. The inhibitory effect on the growth was measured in eight
fast or relatively fast growing species: P. camemberti, P. chrysogenum, P. crustosum, P.
digitatum, P. expansum, P. italicum, P. roqueforti and P. spinulosum. These were grown in
glass Petri dishes both as one or three colonies. Single colonies had larger diameters, in
average by 9.5 mm in P. roqueforti, 5.8 mm in P. italicum, 4.5 mm in P. spinulosum and 1.7 in
P. crustosum. The remaining species showed no significant differences in this respect. In P.
jensenii a reduction in exudate production was noticed at the interference zone. The
interface effect is clearly evident in P. vulpinum and P. expansum in which no synnemata
are formed at the interference zone. Traditionally, the Penicillia are inoculated in three
points, although at least one of the monographers, Abe (1956), used one colony in his
study. According to Raper and Thorn (1949: 71) "one can study the mature growth and
fruiting habits of the mold best in the area where the colonies approach one another".
However, our experience does not support this view. The only advantage of growing
23
three colonies in each plate may be that three instead of one colony are examined and
occasional variation may be noticed. The practice in cultivating Penicillia shows, however,
that significant variation between colonies obtained in the same plate is extremely rare.
It>
'"
0
It>
'"
0
'"
GN
~
It>
II)
W
II.
IL
II)
MEA
a::
w
GI
It>
:I
:I
Z
..
It>
~
It>
10
0
mm
-2
-4
-6
-8
-10
Figure 4. Histogram showing the differences in colony diameters of 36 species of Penicillium

grown on CYA, MEA and G25N in unsealed (shaded area) and partially sealed (unfilled
area) plastic Petri dishes as compared to cultures grown in glass dishes.
24
O. Constantinescu
CONCLUSIONS
For a better standardization of the procedures for Penicillium identification the following
suggestions are made: Preparation of inoculum in sterile distilled water supplemented
with a wetting agent; addition of trace elements solution to the CYA; inoculation in one
point, at least for fast growing species; if plastic Petri dishes are used, those provided with
ribs should be preferred; cultures on G25N medium should be partially sealed if plastic
dishes are used.
ACKNOWLEDGEMENTS
Thanks are due to Mrs Ulla-Britt Sahlstmm for printing the photographs. This work was
supported by grants from the Swedish Natural Science Research Council and Swedish
Council for Forestry and Agricultural Research.
REFERENCES
ABE, S. 1956. Studies on the classification of the Penicillia. Journal of General and Applied Microbiology 2: 1-344.
FRISVAD, J.C 1981. Physiological criteria and mycotoxin production as aids in identification of common
asymmetric Penicilia. Applied and Environmental Microbiology 41: 568-579.
FRISVAD, J.e. and FILTENBORG, O. 1983. Classification of terverticillate Penicillia based on profiles of
HENNEBERT, G.L. 1985. Dierckx' contribution to the genus Penicillium. In Advances in Penicillium and
Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 9-21. New York and London: Plenum Press.
PITT, J.1. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
- - 1985. A Laboratory Guide to Common Penicillium Species. North Ryde, N.S.W.: CSIRO, Division of
Food Research.
- - 1985. Media and incubation conditions for Penicillium and Aspergillus taxonomy. In Advances in
Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.1. Pitt, pp. 93-103. New York and
- - 1988. A Laboratory Guide to Common Penicillium Species. Second edition. North Ryde, N.S.W.:
CSIRO, Division of Food Processing.
RAPER, K.B. and THOM, e. 1949. A Manual of the Penicillia. Baltimore: Williams and Wilkins.
SAMSON, R.A. and PITT, J.I., eds. 1985a. Advances in Penicillium and Aspergillus Systematics. New York
- - 1985. General recommendations. In Advances in Penicillium and Aspergillus Systematics, eds. R.A.
Samson and J. I. Pitt, pp. 455-460. New York and London: Plenum Press.
SMITH, G. 1949. The effect of adding trace elements to Czapek-Dox medium. Transactions of the British
Mycological Society 32: 280-283.
WILLIAMS, A.P. and PITT, J.1. 1985. A revised key to Penicillium subgenus Penicillium. In Advances in
Penicillium and Aspergillus Systematics, eds. R. A. Samson and J. I. Pitt, pp. 129-134. New York and
DIALOGUE FOLLOWING DR. CONSTANTINESCU'S PAPER
PITT: I'd like to make some comments rather than ask questions about what you've done.
Much of it is already known. I think any mycologist doing morphological work now uses
Petri dishes with ribs. Fungi are sensitive to carbon dioxide, so ribbed Petri dishes are
essential. Ones without ribs are not suitable for mycological work.
25
As for the use of CYA as the standard medium for morphological examination, this is
based on what Dr. Raper did. I followed his lead in using Czapek as the primary
medium. He used malt very little, and his descriptions on malt are often quite
incomplete. I followed him with reasonable justification. If a consensus of people now
feel that malt extract agar is preferable for morphological examination, then go ahead. It
is impractical for me to change my system now. I still prefer CYA. You do get more
morphological variation on CYA so you must examine more than one penicillus and find
typical forms.
As for using three colonies rather than one, this gives you a replication of your
colony sizes. If a culture is old or moribund, you will find an unacceptable variation in
the size of colonies. This gives an indication of problems with the culture. If there is too
much variation, the culture should be reinoculated. Certainly faster growth is possible
from single colonies, but the three colony system is a standard system and is what is
used in the literature. The fact that you can get faster growth with a single colony is
irrelevant.
The differences in morphology between margin and the centre of the colonies is also
unimportant. Normally, one works near the margins of the colony where the growth is
youngest. On the other hand, if you have weakly sporulating colonies, as happens with
some Eupenicillium species, spores may form near the colony centres. The system is
designed to obtain results in one run. Industry wants results now.
One other point. In your presentation and your table 1 on variations of colony
diameter. You have measured colonies both on the radial and tagential diameters and
averaged them. The data in Pitt (1979), however, is based only on the largest diameter i.e.
the tangential diameter, as it is evident that colonies must interfere with each other in the
radial direction when they become large. The methodology you used seems therefore to
be different.
CONSTANTINESCU: I was just reporting my observations, not intending to make a criticism
of your work. In my paper, I state that the only advantage to having three colonies on
one plate instead of only one, is that one can observe the morphological variaton
between the three colonies. Significant variation in three colonies is extremely rare
provided that the colonies are the same distance apart and have the same amount of
inoculum. I would not recommend changing the system.
SAMSON: At CBS, we still use Czapek and 2% malt extract agar. We find that the 2% malt
extract, without any additions, is excellent for morphology of Penicillium and the work
that we will present later in this workshop is based on this medium. We particularly like
2% malt extract for micromorphology of Penicillium and also of Aspergillus.
CONSTANTINESCU: At Uppsala, we want to follow a single system, so we are following
Pitt.
SAMSON: We use CYA only for growth diameters, not micromorphology.
GAMS: We are still in the phase of learning about taxonomic relations from molecular
biology, genetics, biochemistry and so on. If we get clues from these disciplines that help
us distinguish between entities, we will have to make all efforts to correlate these
distinctions with morphology. In these cas(!s, we may have to use not only malt extract
agar, but also oatmeal agar, SNA and other weak media to get optimal expression of
morphology. Only under such circumstances, will we be able to find the crucial
distinguishing features.
27
IDENTIFICATION OF PENICILLIUM AND ASPERGILLUS SPECIES IN

MIXED CULTURES IN PETRI DISHES USING SECONDARY METABOLITE
PROFILE
O. Filtenborg and J.C. Frisvad
Department of Biotechnology
The Technical University of Denmark
2800 Lyngby, Denmark
SUMMARY
Identification of mould species is a time consuming procedure, which usually takes at least 14 days,
including isolation and subculturing. We have investigated the possibility of performing identification
directly on the isolation media, using TLC screening of secondary metabolite profiles. An
identification procedure is proposed using standard isolation media, based on comparison of
secondary metabolite profiles from a selected number of isolates. The procedure has been tested on a
number of food samples, and a high degree of agreement was found with results from normal
identification procedures with pure cultures. An international testing of this identification procedure
is proposed and we are willing to provide test samples and photos of standard secondary metabolite
profiles.
INTRODUCTION
Over the years a lot of effort has been devoted to the development of general purpose
isolation and diagnostic media for mould species important in foods and feeds. Many
isolation media have been devised, of which Dichloran 18% Glycerol nitrate agar (DG18:
Hocking and Pitt, 1980) and Dichloran rose bengal chloramphenicol agar (DRBC: King et
al., 1979) are good examples. These media support the growth of all important species of
Penicillium, Aspergillus and several other genera. Pure cultures for identification can be
readily isolated from them (King et al., 1986). However, this procedure is time consuming,
requiring at least five to seven days for initial growth on the selective medium and seven
days for identification on diagnostic media. An alternative approach is the development
of selective media,. However, only two successfull media exist so far: AFPA for the
screening of Aspergillus flavus and A. parasiticus (Pitt et al., 1983) and PRYES for the
screening of P. verrucosum chemotype n (Frisvad, 1983).
As another alternative to the procedure just described, it may be easier to identify the
moulds directly on the isolation plates. One way may be the determination of secondary
metabolite profiles of the fungi directly on these media, because these profiles are specific
for particular fungal species (Frisvad and Filtenborg, 1983; Frisvad, 1989). In this paper we
report on investigations on the ability of a number of food spoilage and myotoxinogenic
moulds to produce secondary metabolites on the isolation media DRBC and DG18 and
modifications of them. The aim is to determine if such fungi can be identified directly in
mixed culture on isolation media, by using simple TLC screening procedures to detect
their production of secondary metabolites.
o Filtenborg & J.e.
28
Frisvad
Fungi. The investigation of metabolite production in pure culture was performed on

isolates from commercial, not visibly mouldy, food products. They were: Penicillium
roqueforti from artichoke, P. expansum from apple, P. brevicompactum from potatoes, A.
alutaceus from chili, A. versicolor and A. flavus from pepper, P. verrucosum II, P.
chrysogenum, P. hordei and P. aurantiogriseum from barley. Mixed cultures originating from
commercial food products were tested as well.
Table 1. Influence of media on secondary metabolite production of selected Penicillium and
Aspergillus species after incubation for seven days.

species
metabolite
CYA
P. roqueforti
PR-toxin
Roquefortine C
Patulin
Citrinin
Xanthomegnin
Viomellein
PeniciIlic acid
Xanthomegnin
Viomellein
Roquefortine C
Roquefortine C
Citrinin
Ochratoxin A
Brevianarnid A
Sterigmatocystin
Aflatoxin Bl
ic
ec>ic
ec>ic
ic
ic
ec>ic
ic
ic
ic
ic
ec>ic
ec>ic
ic
ic
ec>ic
P. expansum
A. alutaceus
P. aurantiogriseum
P. chrysogenum
P. hordei
P. verrucosum
P. brevicompactum
A. versicolor
A. flavus
(a) ec = extracellular detection, ic = intracellular detection; better than intracellular detection.
YES
medium
DRBC
ectal
ic
ec>ic
ec>ic
ic
ic
ec>ic
ic
ec>ic
ec>ic
ic
ic
ec>ic
DRYES
ec>ic
ec>ic
ic
ic
ec>ic
ic
ec>ic
ec>ic
ec>ic
ic
ec>ic
ec>ic
ec>ic
ic
ec>ic
ec>ic
ec>ic
ec>ic
ic
ec>ic
= no detection, ec>ic = extracellular detection
Media.
The media used were Dichloran rose bengal yeast extract sucrose agar (DRYES; Frisvad,
1983), DG18 and DRBC. DRYES was used instead of the similar medium PRYES, although
it is less restrictive towards Rhizopus, since the pentachloronitrobenzene used in PRYES is
considered a possible cancinogen (King et al., 1979)
Inoculation.
Pure cultures were inoculated in triplicate, either as three point inoculations or spread
plates. Samples of food products were homogenized, diluted and inoculated on the media
by spread plates in triplicate. The TLC agar plug method (Filtenborg and Frisvad, 1980;
Filtenborg et a!., 1983) was used for screening for metabolite production. Plug samples
were taken from all three colonies. Variations in metabolite concentrations between
triplicates were never significant. TLC conditions were adjusted to be optimal for
individual metabolites during the pure culture investigations. When mixed cultures were
used the following protocol was chosen: elution in chloroform/acetone/isopropanol
Identification of Penicillium and Aspergillus species in mixed cultures
29
85:15:20 (CAP) followed by Ce(S04)2-spray for DRBC and elution in toluene/

ethylacetate/90% formic acid 4:4:1 (TEF) followed by anisaldehyde-spray for both DRBC
and DRYES. For details see Filtenborg and Frisvad (1980) and Filtenborg et al. (1983).
Production of secondary metabolites on isolation media by pure cultures.

The ability of the selected Penicillium species to produce secondary metabolites in pure
culture was compared on isolation media, DRYES, DG18 and DRBC and media known to
support production of secondary metabolites: Czapek yeastextract agar (CYA) and yeast
extract sucrose agar (YES)(Frisvad and Filtenborg, 1983) (Fig. 1).
Figure 1. TLC-metabolite profiles of pooled pure cultures of several Penicillium species on

CYA and YES, extracted with chloroform/methanol. The metabolite-extracts have been
concentrated by evaporation. TIC-condition: Eluation in TEF, spraying with anisaldehyde
and 365 nm UV-illumination.
o FiHenborg & J.e. Frisvad
30
Most of the metabolites investigated were produced on DRYES and/ or DRBC within
seven days (Table 1). However, none of the metabolites investigated were detected after
growth on DG18 for seven days. This is probably due to the lower water activity level of
this medium (0.95), compared to 0.995 or more for the other media. Reduced water
activity is well known to inhibit metabolite production dramatically, so an extended
incubation time is needed to detect secondary metabolites on DG18.
On DRYES and DRBC generally the metabolite concentrations were equal to or
slightly lower than those on YES and CYA but a detection was still possible with the
rather insensitive TLC screening method. Detection is significantly improved, if
incubation time is extended to nine days. Extracellular metabolites were normally
produced in greater amounts in DRYES than in DRBC while the reverse was true for
intracellular metabolites.
Table 2. Production of secondary metabolites by Penicillium and Aspergillus colonies in

mixed culture on DRYES and DRBC.
food
medium
Walnut
DRYES
DRBC
Pepper (white) DRYES
identity
metabolites1
P. crustosum [2]3
P. expansum [2]
P. aurantio-griseum
P. echinulatum chemotype II
P. brevicompactum [2]
? [2]
P. aurantiogriseum
P. crustosum [2]
P. citrinum [4]
A. versicolor [2]
Grapes
DRYES
Barley
DRYES
A. candidus [2]
P. brevicompactum [4]
P. glabrum [4]
P. verrucosum [5]
DRBC
P. aurantio-griseum [3]
P. hordei
?
P. aurantio-griseum [2]
secondary
metabolites
Terrestric acid, etc.
Citrinin, etc.
Penicillic acid, etc.
several
several
weak profile
Verrucosidin, etc.
Penitrem A, etc.
Citrinin, etc.
Sterigmatocystin,
etc.
several
Brevianamid A, etc.
several
Citrinin,
Ochratoxin A, etc.
few
several
weak profile
Xanthomegnin,
viomellein, etc.
identity
morphology2
P. crustosum
P.expansum
P. aurantio-griseum
P. echinulatum
chemotype II
P. brevicompactum
P. solitum
P. aurantio-griseum
P. crustosum
P. citrinum
A. versicolor
A. candidus
P. brevicompactum
P.glabrum
P. verrucosum
P. aurantio-griseum
P. hordei
P.solitum
P. aurantio-griseum
lFrisvad and Filtenborg (1983); 2Pitt, (1979), Raper and Fennell (1965), Frisvad (1981);
3Number of isolates tested.
Besides the metabolites listed in Table 1, several other members of the expected
metabolite-profile of each species were detected (Frisvad and Filtenborg, 1983), but this
will not be discussed in detail here. Generally the profiles were almost complete on
DRYES and/or DRBC, but at concentrations slightly lower than in YES and/or CYA.
31
Some metabolites appeared on DG18, especially in the mycelium. In Aspergillus species,

DG18 sometimes supported the highest production of metabolites of all the media tested.
Some metabolites are mainly excreted in the medium, but usually can be detected into
the mycelium too, although in lower concentrations. This is important in this context, as
the screening method is intended to be used on mixed cultures, where extracellular
metabolites from different isolates may mix in the medium, but probably not in the
mycelium.
The results listed in Table 1 are based on inoculations with a great number of conidia.
This is unlike the normal spread plate technique where conidia are present on countable
plates in low numbers, 10-50 per plate. To test if the results with mass inocula could be
repeated using this inoculation technique, the metabolite screening was repeated with
three isolates of P. aurantiogriseum, P. verrucosum and P. chrysogenum. Generally the
secondary metabolite profiles obtained were the same, but a few differences were seen.
Xanthomegnin and viomellein was now detected in DRBC, while penicillic acid and
roquefortine C were no longer observed in this medium.
Figure 2. TLC-metabolite profiles of isolates from a paprika sample in a mixed culture on

DRYES. TLC-conditions: Eluation in TEF, no spraying and 365 nm UV-illumination. Single
metabolites were not identified, but profiles characteristic of Eurotium species (profile 2 and
4 representing different species) are dominating, and also Aspergillus niger (profile 17) is
present.
32
Production of secondary metabolites in isolation media: mixed cultures.

Metabolite production may be influenced by a competition of different species. Reduced
production of secondary metabolites could be the result. Hence, the screening method
with mixed cultures may be expected to produce different results from those obtained in
pure cultures. Therefore, the metabolite screening method was tested on the fungal flora
of several food products, inoculated on DRYES and DRBC (Figs 2-6). The metabolite
profiles of the mixed flora on DRYES and on DRBC were used to identify the fungi
present according to Frisvad and Filteborg (1983) and by comparation with photos of
metabolite profiles of well known isolates, based on culture extracts (Frisvad and Thrane,
1987). Verification of the identification was carried out on pure cultures according to Pitt
(1979), Raper and Fennell (1965), and Frisvad (1981). Some of the results are listed in
Table 2.
Figure 3. TIC-metabolite profiles of isolates from a ginger sample in mixed cultures on

DRBC and CYRBC (DRBC with added 0.5% yeast extract). TIC-conditions: Eluation in TEF,
no spraying and 254 nm UV-illumination. Metabolites are brevianamides and raistrick
phenoles. A metabolite profiles characteristic of Penicillium brevicompactum.
33
Table 2 shows that even with a mixed flora on DRYES and DRBC, several secondary
metabolites were produced detectable with the simple TLC screening method used in this
investigation. Sometimes profiles were weak, for example with P. solitum, making
identification impossible unless several colonies of the same species were present which
had more distinct but comparable profiles. This was often the case. Some metabolites were
not detected for example roquefortine C and patulin, due to unfavourable production or
detection conditions or simply because the fungus did not have the ability to produce
them. Nevertheless, it was often possible to correctly identity the important species in a
sample.
The most significant metabolite profiles were often produced on DRYES (Fig. 2 and 5).
When the intracellular metabolite profiles were tested, no interfering metabolites from
adjacent colonies were observed. However when testing extracellular profiles we
observed a certain degree of metabolite diffusion. However, valuable information was
often obtained by comparing intra- and extracellular metabolites as well as profiles of the
same sample from both DRYES and DRBC.
Figure 4. TLC-metabolite profiles of isolates from a buckwheat sample in mixed cultures on

DRBC and CYRBC (DRBC with added 0.5% yeast extract). TLC-conditions: Eluation in TEF,
spraying with anisaldehyde and 254 nm UV-illumination. A metabolite in profile 1 and
several others is penicillic acid. A metabolite in profile 8 and 12 is verrucosidin. Profiles
characteristic of P. aurantiogriseum, P. roqueforti and P. brevicompactum are present.
34
CONCLUSIONS
From these investigations it can be concluded that the production of several Penicillium
and Aspergillus secondary metabolites can be detected in mixed cultures on the mould
isolation media DRYES and DRBC, using a simple TLC screening method (Filtenborg and
Frisvad, 1980; Filtenborg et al., 1983). Not all of the metabolites in the profile of the
individual species could be detected in the same analysis, as TLC conditions, media and
competitive fungal flora plays an important role in this respect. Roquefortine C,
xanthomegnin and viomellein, for example, were rarely detected under these conditions.
However, as only a few metabolites of the total profile, even two or three, can be
sufficient, to identify the individual colony (Frisvad and Filtenborg, 1989), we believe that
by screening for their secondary metabolite profiles on the media DRYES and / or DRBC
will enable identification of several important moulds directly in the mixed flora. The
most significant metabolite profiles were often observed on DRYES. The choice of medium
and TLC conditions, however, must depend on the species and metabolites which are of
interest in the particular situation.
Figure 5. TLC-metabolite profiles of isolates from a wallnut sample in a mixed culture on

DRYES. TLC-conditions: Eluation in TEF, spraying with anisaldehyde and 365 nm UVillumination. The tale in profile 2 and 7 is citrinin. A metabolite in profile 5 and 10 is
terrestric acid. Several other metabolites can be identified. Profiles characteristic of P.
expansum, P. aurantiogriseum, P. solitum and P. crustosum are present.
35
This method was tested on several food products and the majority of the Penicillium and
Aspergillus species could be identified. For example, the important species A. versicolor,
which grows to a colony diameter of only a few millimeters on these media, produced a
spectacular and very specific metabolite profile, including sterigmatocystin. Using this
method we also often observed that colonies, with very different appearances especially
on DRBC, had the same metabolite profile and thus represented the same species. P.
brevicompactum and P. aurantiogriseum in particular showed this effect. This is time saving
information when trying to separate and identify such species.
The application of this method of course depends on available metabolite standards
and data on metabolite profiles. These standards and methods exists to a certain degree
(Frisvad and Filtenborg, 1983, 1990) and the number can be expected to increase
considerably in the future. We are also using extracts (Frisvad and Thrane, 1987) of well
known isolates as standard profiles, and can recommend this as an interim solution until a
sufficient number of standard metabolites are available.
Figure 6. TLC-metabolite profiles of isolates from a white pepper sample in mixed cultures
on DRBC and CYRBC (DRBC with added 0.5% yeast extract). TLC-conditions: Eluation in
TEF, no spraying and 365 nm UV-illumination. The tale in profile 1 and several others is
citrinin. One of the metabolites in profile 11 and others is sterigmatocystin. Profiles
characteristic of P. citrinum and A. versicolor are present.
36
o Filtenborg & J.e.
Frisvad
The described identification procedure needs further testing to prove its value. This may
lead to the need for modification of isolation media to take account of the possibilities of
enhance metaboliteproduction. The addition of MgS04 to DRYES is highly recommended.
Investigations have proven (Filtenborg et aI., 1990) that the production of secondary
metabolites varies significantly with the brand of yeast extract used in YES on which
DRYES is based, but that the addition of MgS04 compensates for this.
REFERENCES
FILTENBORG, O. and FRISVAD, J.e. 1980. A simple screening-method for toxigenic moulds in pure
cultures. Lebensmittel Wissenschaft und Technologie 13: 128-130.
FILTENBORG, 0., FRISV AD, J.e. and SVENDSEN, J.A. 1983. Simple screening method for molds producing
intracellular mycotoxins in pure cultures. Applied and Environmental Microbiology 45: 581-585.
FILTENBORG, 0., FRISVAD, J.e. and THRANE, U. 1990. The significance of yeast extract composition on
metabolite production in Penicillium. In Modern Concepts in Penicillium and Aspergillus Oassification,
eds. R.A. Samson and J.I. Pitt, pp. 433-440. New York and London: Plenum Press.
FRISVAD, J.e. 1981. Physiological criteria and mycotoxin production as aids in identification of cornmon
asymmetric Penicillia. Applied and Environmental Microbiology 41: 568-579.
FRISVAD, J.e. 1983. A selective and indicative medium for groups of Penicillium viridicatum producing
different mycotoxins in cereals. Journal of Applied Bacteriology 54: 409-416.
FRISVAD, J.e. 1989. The use of high-performance liquid chromatography and diode array detection in
fungal chemotaxonomy based on profiles of secondary metabolites. Botanical Journal of the Linnaean
Society 99: 81-95.
FRISVAD, J.e. and FILTENBORG, 0.1983. Classification of terverticillate Penicillia based on profiles of
FRISVAD, J.e. and FILTENBORG, O. 1990. Secondary metabolites as consistent criteria in Penicillium
taxonomy. In Modern Concepts in Penicillium and Aspergillus Oassification, eds. R.A. Samson and J.!.
FRISVAD, J.e. and THRANE, U. 1987. Standardized high-performance liquid chromatography of 182
mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VIS spectra
(diode array detection). Journal of Chromatography 404: 195-214.
HOCKING, A.D. and PITT, J.!. 1980. Dichloran-glycerol medium for enumeration of xerophilic fungi from
low moisture foods. Applied and Environmental Microbiology 39: 480-492.
KING, A.D., HOCKING, A.D. and PITT, J.I. 1979. Dichloran-rose bengal medium for enumeration and
isolation of molds from foods. Applied and Environmental Microbiology 37: 959-964.
KING, A.D., PITT, J.I., BEUCHAT, L.R. and CORRY, J.E.L., eds. 1986. Methods for the Mycological
Examination of Foods. New York and London: Plenum Press,
PITT, J.I. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press,
PITT, J.I., HOCKING, A.D. and GLENN, D.R. 1983. An improved medium for the detection of Aspergillus
flavus and A. parasiticus. Journal of Applied Bacteriology 54: 109-114.
RAPER, K.B. and FENNELL, DJ. 1965. The genus Aspergillus. Baltimore: Williams and Wilkins.
DIALOGUE FOLLOWING DR. FILTENBORG'S PRESENTATION

SAMSON: I'd be interested to see what kind of results you would get on DG18. This has
become a commonly used general purpose medium.
FILTENBORG: On DG18, the water activity is a little low, and the Penicillia do not produce
secondary metabolites well within a week under these conditions. But Aspergilli, they
are wonderful on DG18. You can separate a lot of Aspergilli very well on DG18.
37
SAMSON: What happens when you have other fungi, like Cladosporium or Ulocladium, on
these plates?
FIL TENBORG: They were there!
GAMS - As you are taking agar plugs from mixed cultures, isn't there a problem of
diffusion of metabolites from one colony to another?
FILTENBORG: There is no problem, because we are taking intracellular metabolites from
the mycelium, not diffusible ones from the agar. This could be a problem with some
metabolites, such as citrinin, which diffuses easily. But in general we don't have many
problems.
PITT: Do you tum the plug over so that the mycelium is in contact with the TLC plate, or
do you use the substrate side?
FILTENBORG: We tum it over and put the mixture of chloroform and methanol on the plug,
then tum it around and press it on the TLC plate.
PATERSON: On your slides, there was some variation in the penicillic acid spot between the
isolates. Is this a particular problem with penicillic acid?
FILTENBORG: It is varying, yes. You see this with citrinin, as well.
PATERSON: Why do you see this variability?
FILTENBORG: It depends on the position of the plate, proximity of other colonies,
competition and so on.
PATERSON: Some of the spots are very close together. Is there coelution of some
compounds?
FILTENBORG: We can usually separate them.
HEARN: Does the brand of TLC plates have any effect on your analysis?
FILTENBORG: I hope not. We haven't tested this.
39
VARIATION IN PENICILLIUM AND ASPERGILLUS CONIDIA IN

RELATION TO PREPARATORY TECHNIQUES FOR SCANNING
ELECTRON AND LIGHT MICROSCOPY
P. Staugaard* , R.A. Samson and M.I. van der Horst
CentrQlllburetlu VOOT Schimmelcultures
3740 AG, BQIlTn, The Netherlands
SUMMARY
Conidia and conidiophores of selected species of Penicillium and Aspergillus were examined by light
microscopy and scanning electron microscopy. SEM micrographs were taken from frozen hydrated
material and of critical point dried material after various chemical fixation procedures. Comparison
between the techniques were made with emphasis on the conidial dimensions and surface detail of
the conidia, conidiogenous cells and conidiophore elements. Frozen hydrated specimens reveal
conidial dimensions closest to the living material and show significant shrinkage in chemical fixed
and critical point dried specimens.
INTRODUCTION
The gross microscopic features of Penicillium and Aspergillus isolates can be readily
observed by light microscopy, but in many species it is difficult to elucidate the shape and
ornamentation of the conidia, which are especially small. Therefore scanning electron
microscopy (SEM) has been increasingly used to investigate the pattern of ornamentation
or to measure conidial dimensions (Ramirez, 1982, Bridge et al., 1985; Kozakiewicz, 1989 a
and b).
Beckett et al. (1984) and Read et al. (1983) have discussed the various preparation
techniques for SEM and found Significant differences in the dimensions of uredospores of
Uromyces viciae-fabae (Pers.) Schroeter and ascospores of Sordaria humana (Fuckel) Winter.
The authors concluded that low temperature SEM of frozen hydrated samples was the
best technique for studying fungal spores under conditions as near to their natural
environment.
This paper reports a comparative study of selected Penicillium and Aspergillus species
using light microscopy and various preparation techniques for SEM.
Fungi.
The following isolates were examined: Penicillium aurantiogriseum ex moulded food, P.
digitatum ex Citrus, P. echinulatum CBS 317.48, P. expansum CBS 489.75, P. griseofulvum CBS
384.48, P. italicum CBS 278.58, P. megasporum CBS 529.64, P. oxalicum CBS 460.67, P.
variabile CBS 385.48, Aspergillus flavus CBS 573.65, A. niger CBS 554.65, A. phoenicis CBS
135.48, A. tamarii CBS 104.13, A. terreus CBS 601.65 .
.. Present address: RIVM, Laboratory for Bacterial Vaccins, P.O. Box 1, 3720 BA Bilthoven,
The Netherlands
40
P.
Staugaard et al.
Cultures of the different species were grown on 2% maltextract agar at 25C. Five to seven
days after inoculation samples were taken for microscopical examination.
Light microscopy.
With a glass needle, a piece of the sporulating culture was suspended in a drop of 80% DL
lactic acid on a microscope slide and covered with a coverslip. Samples were observed
and photographed using an Olympus BHZ photo microscope equipped with Nomarski
interference contrast optics.
Low temperature SEM of frozen hydrated material.
Blocks of approximately 5 x 5 mm were cut from the agar containing a sporulating section
of a colony. The blocks were mounted in a cup specimen holder with cryo-glue, freshly
made up as a 50/50 mixture of colloidal graphite (Ems cope No A1533 Aquadag) and
Tissue-Tek mounting medium (Miles Scientific).
The specimen holder was designed as a cylindrical cup made of copper. By providing
radiation cooling this provided maximum protection to the sensitive hyphal material from
damage during warming up. Samples were frozen in slushed nitrogen inside the slushing
chamber of the Hexland CTlOOOA cryo system and quickly transferred under vacuum to
the cold stage in the working chamber. In the working chamber the samples were
sputtered with gold at low temperature (T= 170C) for three to five minutes (thicknes
approximately 20 nm). After transfer to the cold stage (T= -140C) of the Jeol 840A
scanning electron microscope specimens were observed and photographed at 3 to 7 kV
acceleration voltage.
Chemical fixation & critical point drying (CPO).

Blocks of approximately 3 x 7 mm, cut from a sporulating colony on the plate, wereplaced
in a perforated beem capsule and immersed in fixing fluid. Fixation was carried out in a
5% aqueous solution of glutaraldehyde (GA) overnight (16 hours) at 40 C. Potassium
permanganate (1%) was used for 30 minutes and osmium tetroxide (1% solution) for 30
minutes both at room temperature. Mter fixation, the samples were generally dehydrated
in methoxy ethylene glycol in two steps of 20 min. followed by two changes of absolute
acetone (Samson et al., 1979). A few samples were slowly dehydrated (30 min. each step)
in an ethanol series (15,30,45,60,70,80,90,96,100,100% ethanol in distilled water. After
dehydration was completed, the samples were critical point dried in a Balzers critical
point drying apparatus using liquid carbon dioxide.
The blocks were mounted on specimen stubs with colloidal silver paint (Agar G302)
and sputtered with gold for 150 s in a Polaron E5200 sputter coater. Specimens were
observed and photographedin a Jeol840A SEM at 7 to 10 kV accelerating voltage.
Measurements of conidial diameter.
Micrographs were enlarged and printed to a final magnification of 2000 x or in a few cases
for very small conidia, 4000 x. From each series of photographs 100 to 300 conidia were
measured and counted. The average diameter, measured at right angles to the growth
axis, was taken as a quantitative measurement for shrinkage and swelling comparisons.
Variation in Penicillium and Aspergillus conidia
41
Dimensions: conidial diameter measurements.

When P. aurantiogriseum conidia were relatively rapidly dehydrated in methoxy ethylene
glycol as described by Samson et al. (1979) or by gentle dehydration in an ethanol series no
significant differences in conidial size could be determined (Fig. 1).
diameter (11m)
1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
30
120
avg=2.01
---------
100
80
20
..
GI
til
..
.
GI
til
as
C
GI
as
cG)
..
60
()
CI
0.
40
10
()
GI
0.
E
~
()
20
0
0
diameter (11m)
1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
40
120
------------
100
30
80
G)
..
CI
til
as
..
c
CI
20
60
CI
0.
40
()
CI
0.
E
~
()
10
20
o L -_ _ _ _a..
0
Figure 1. Average diameter of conidia of P. aurantiogriseum of critical point dried specimens
after in (A) gentle dehydration in an ethanol series and (B) rapid dehydration in methoxy
ethylene glycol.
P.
42
Staugaard et al.
As shown in Table I, both Aspergillus and Penicillium conidial diameters measured after
chemical fixation and critical point drying were smaller than in the frozen hydrated state
(Table 1). Shrinkage ranged from 8 to 34%, and in most species an average of 20-30 %
could be measured when comparing lightmicroscopy and SEM.
Table 1. Conidial diameters of various Aspergillus and Penidllium species.
Spedes
Light microscopy
Frozen hydrated
GAfixCPD
A. flavus
6.16
A. tamarii
6.62
P. digitatum
3.71
P.expansum
3.09
P. italicum
2.84
P. megasporum
6.75
P.oxalicum
3.27
P. variabile
2.7
4.91
20%
4.76
28%
3.62
2%
2.92
6%
2.4
15%
4.81
29%
2.99
8%
2.15
20%
4.88
21%
4.37
34%
3.05
18%
2.55
18%
1.95
31%
5.15
24%
2.88
12%
2.01
27%
Average diameters are expressed in

microscope value.
~m.
Shrinkage is expressed as a percentage compared with the light
When conidia of P. aurantogriseum, P. echinulatum and P. griseofulvum were fixed in OS04,

glutaraldehyde or potassium permanganate small differences in conidial size could be
measured but they appear not to be consistent (Table 2). The three species also show a
different degree of shrinkage.
Table 2. Conidial diameters after different SEM preparatoration techniques.
Spedes
Light
microscopy
Frozen
hydrated
GAfix
CPD
OsOdix
CPD
KMn04fix
CPD
P. echinulatum
4.68
P. aurantiogriseum
2.49
P. griseofulvum
2.87
4.43
5%
2.38
4%
2.32
19%
3.93
16%
2.14
14%
2.24
22%
4.57
2%
1.98
20%
2.17
24%
4.38
6%
2.07
17%
2.34
18%
Average diameters are expressed in ~m. Shrinkage is expressed in percentage compared with the light
microscopi cal value.
43
Conidial diameters of all examined Aspergillus and Penicillium isolates, measured in the
light microscope after suspension in 80 % lactic acid were significantly larger than those
measured in the SEM.
Ultrastructural detail.
The conidia of P. echinulatum and P. megasporum appeared more regular and spherical in
light microscopy and frozen hydrated specimens than in chemically fixed samples. Frozen
hydrated samples showed well preserved conidia with regular spines but in fixed and
critical point dried samples the conidial wall was more wrinkled. This indicates that
shrinkage leads to artificial ornamentation in fixed specimens. The conidia of P.
aurantiogriseum and P. digitatum have a very fine surface structure in the frozen hydrated
state. This structure can not been observed in fixed samples and is too small to be resolved
in light microscopy.
Stipes and metulae show significant shrinkage in fixed samples as septa, which have
relatively a more rigid structure, form protruding rings with lower shrinkage than
elsewhere. These rings are rarely observed in frozen hydrated samples. In LM and Cryo
SEM the stipes of P. echinulatum showed a smooth surface with clearly defined warts as
ornamentation. In fixed samples the surface of the stipes is less smooth and the warts are
deformed. Apart from shrinkage and deformation, a lot of debris is to be seen in OS04 and
KMn04 fixed samples, probably material from the exudate which is fixed unto the surface
during immersion in the fixing fluid.
Phialides of P. echinulatum and P. aurantiogriseum are smooth in LM and in frozen
hydrated state; however in fixed and CPD specimens the phialides have a wrinkled
surface, again indicating shrinkage.
DISCUSSION
The preparation of soft biological material to be observed in the dried state inevitably
results in its shrinkage. Conidial size is determined during the final drying process,
although the actual amount of shrinkage varies depending on the actual mode of drying
and the specimen. Read et al. (1983) found that air-dried conidia probably showed most
shrinkage, while frozen hydrated spores of Sordaria humana provided superior
preservation. However, in some cases expansion of the fungal cell may occur due to the
conversion to ice and this may explain the variation observed in the present study. In
addition the structure of the conidial wall of Penicillia and Aspergillia can also vary,
which may influence the degree of shrinkage.
In our SEM investigations conidial ornamentation was not significantly different than
when observed in light microscopy. In some species with very small conidia, e.g.
Aspergillus terreus, striations on the conidial wall were observed, which confirmed
observations by Miegeville (1987) and Kozakiewicz (1989b). This ornamentation could not
be seen in LM. The "microverrucate" and "reticulate" ornamentation reported by
Kozakiewicz (1989a) for conidia of P. corylophilum and P. hirsutum have not been seen in
many isolates representing these taxa (Samson, unpubl. data). However, she examined airdried conidia from three weeks old cultures grown on Czapek agar, which probably had
become dehydrated and collapsed naturally.
The measurements of conidial dimensions are not only dependant on the age and
growth of the fungal material, but to a great extend to the preparatory techniques
involved. Frozen hydrated samples will give dimensions closest to those of living
44
P. Staugaard et al.
Figure 2. Scanning electronmicrographs of conidia and conidiogenous structures of P.

echinulatum CBS 317.48. a. air-dried; b-d. critical point dried; b. fixed with glutaraldehyde, c.
potassium permanganate, d. osmiumtetroxide; e-f. frozen hydrated (magnification alI2000x)
Figure 3. a-b. Scanning electronmicrographs of conidia and conidiogenous structures of P.

hirsutum CBS 201.57, frozen hydrated, a. conidiophore (1500x), b. conidia (25OOx). Note the
finely roughened surface of the conidia; c-e. stipes of the penicillus of P. echinulatum, Co
frozen hydrated, d. CPD with osmiumtetroxide fixation, f. CPD with potassium
permanganate fixation (magnification all 2000x)
45
46
P. Staugaard et al.
Figure 4. Scanning electronmicrographs of Penicillium conidia, a-b. P. digitatum, a. CPO

fixed with glutaraldehyde, b. frozen hydrated; c-d. P. megasporum CBS 529.64, c. CPO fixed
with glutaraldehyde, d. frozen hydrated; e-f. P. expansum CBS 486.75, e. CPO fixed with
glutaraldehyde, f. frozen hydrated (magnification all2000x).
Figure 5. Scanning electromicrographs of Aspergillus conidia, a-b. A. tamani CBS 104.13, a.

frozen hydrated, b. CPD fixed with glutaraldehyde; cod. A. niger CBS 554.65, c. frozen
hydrated, d. CPD fixed with glutaraldehyde. Note that with both methods the conidia show
longitudinal bars; e-f. A. terreus CBS 601.65, e. frozen hydrated, f. CPD fixed with
glutaraldehyde. Note the fine striations(magnification a1l2OOOx).
47
48
P. Staugaard et al.
material. We agree with Read et al. (1983) that, if SEM is used, frozen hydrated material
should be used as a base line, although it should be not considered as the panacea for
morphological studies.
It is tempting to employ cryo SEM for the examination of fungal material, but
particularly for the important and common species of Penicillia and Aspergillia a simple
technique, usable in any well-equiped laboratory should be employed. For taxonomic
studies light microscopical observations are preferable and in our view SEM has only a
limited value in resolving taxonomic problems in Penicillium and Aspergillus anamorphs. It
is especially emphasised that conventional SEM techniques should be used with great
care, as dehydration may cause artefacts in ornamentation.
REFERENCES
BECKETT, A., READ, N.D. and PORTER, R. 1984. Variation in fungal spore dimensions in relation to
preparatory techniques for light microscopy and scanning elctron microscopy. Journal of Microscupy 136:
87-95.
BRIDGE, P.D., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.s., PATERSON, R.R. and
SACKIN, M.J. 1985. An integrated approach to Penidllium systematics. In Advances in Penidllium and
Aspergillus Systematics. eds. R.A. Samson & J.I. Pitt, pp. 281-309. New York and London: Plenum Press.
MIEGEVILLE, M. 1987. Apport de la microscopie electronique a baiayage dans l'observation de divers
Aspergillus. Bulletin Society France Mycologie Medicinal 16: 133-140
KOZAKIEWICZ, Z. 1989a. Ornamentation types of conidia and conidiogenous structures in fasciculate
Penicillium species using scanning electron microscopy. Botanical Journal of the Linnean Sodety 99: 273-293.
--1989b. Aspergillus species on stored products. Mycological Papers 161: 1-188.
RAMIREZ, C. 1982. Manual and Atlas of the Penicillia. Amsterdam: Elsevier Biomedial Press.
READ, N.D., PORTER, R. and BECKETT, A. 1983. A comparison of preparative techniques for the
examination of the external morphology of fungal material with the scanning electron microscope.
Canadian Journal of Botany 61: 2059-2078.
SAMSON, R.A., STALPERS, J.A. and VERKERKE, W. 1979. A simplified technique to prepare fungal
specimens for scanning electron microscopy. Cytobios 24: 7-11
49
VARIANTS OF PENICILLIUM EXPANSUM: AN ANALYSIS OF CULTURAL

AND MICROSCOPIC CHARACTERS AS TAXONOMIC CRITERIA
J.F. Berny and G.L. Hennebert
Llborataire de Mycologie Systematique et Appliquee, MUCL
Catholic University of Louvain
1348 Louvain-la-Neuve, Belgium
SUMMARY
The strain Penicillium expansum MUCL 29412, a monoconidial culture derivated from the wild type
LCP 3384, after being submitted to divers physical factors, has bred ten variants. The variations are
maintained from one generation to an other. They concern mainly the cultural characters and some
microscopic characters. The implications for taxonomic studies and identification on a culture issued
from a monospore are discussed. The value of cultural and microscopic characters, stable or not, as
taxonomic criteria is investigated.
INTRODUCTION
Variants of Penicillium notatum NRRL 1249 Bl differing in cultural characters have been
reported to occur spontaneously in successive single conidium transfers on malt agar
under laboratory conditions (Sans orne, 1947). Two distinctive variants resulted: one dark
coloured and heavily sporulating, the other pale coloured and poorly sporulating.
Morphological variants producing double sized conidia have also been obtained from the
same strain on a mutagenic camphor culture medium (Sans orne, 1949). In single conidium
lines of Penicilium viridicatum P. crustosum and P. aurantiocandidum, two or three types of
variation have been obtained on Czapek agar. These differed in growth rate, sporulation
rate, colony reverse colour, presence and colour of diffusible pigment, presence of white
mycelial outgrowth and conidium ornamentation. Two were reported as stable, the third
one reverted partly into the two stable types (Bridge et al., 1986). Samson et al. (1976), Pitt
(1979) and Onions et al. (1984), have observed an intergradation between the fasciculate
species of Penicillium subg. Penicillium and reported degradation of significant taxonomic
characters in strains after long cultivation.
For taxonomic purposes, it is most important to assess the degree of variability within
particular isolates (an isolate usually being a population), and the degree of variability of
the isolates within the species and between close species. To check the stability of type
strains during subculturing and preservation in culture collections, variations within the
strain depending on conditions needs to be critically examined in order to facilitate the
recognition of deviated elements amongst typical ones within the strain population.
Preservation techniques applied in culture collections and incidental X-ray exposure
in customs have induced nine types of variants in subcultures of the single conidium
strain P. expansum MUCL 29412. These variants differ from the original parent strain on
some of their cultural and microscopic characters.
This study shows the extent of variability of those characters and investigates its
implication in their taxonomy.
50
J.F. Berny & G.L. Hennebert
Strain.
P. expansum MUCL 29412 is a single conidium strain derived from the 1984 wild type
isolate P. expansum LCP 3384.
Origin of variants.
In the course of a CEC research programme (BAP 0028) on the improvement of
preservation techniques of fungal strains, subcultures of P. expansum MUCL 29412 were
submitted to 19 different preservation techniques by freeze-drying and by freeze-storage
at -20 to -196C, 11 strains being exposed incidentally to uncontrolled X-ray exposure
soon after being revived from preservation. Mass conidia (5000 conidia) and 200 single
conidium transfers were prepared from each of the 19 revived cultures after treatment and
from the parent strain MUCL 29412 preserved at 4C. The mass conidia transfers and the
4000 single conidium isolates were grown on 2% malt yeast agar (MYA2: malt extract 2%,
yeast extract 0.1 %, agar 2%, pH 7) and on Czapek agar and incubated for 21 days in the
laboratory under diffuse light. The mass conidia transfers showed numerous sectors on
both culture media, while 9 variant types were recognized amongst the single conidium
isolates on MYA2. One strain of each variant has been isolated. The variant strains and the
parent strain have been preserved on MYA2 slants at 4C.
Growth conditions for strain characterization.
Ten single conidium replicates of the original parent strain and of each variant type were
inoculated on MYA2 in Petri dishes and incubated at 24C in the dark and at 20C in
diffuse light in the laboratory. Observations of cultural and microscopic characters were
recorded after 7 days.
Scanning electron microscopy.
Three day old conidia taken at the half radius of 7 day old colonies were mounted on
double sided cellophane tape and examined by scanning electron microscopy (Jeol JSM35,20 KV, magnification 10000 X).
Phytopathogenicity testing.
Ten day old conidia from a single conidium isolate of each of the 8 sporulating variants
and of the parent strain were inoculated into subepidermic scars made in apples of
cultivar Gloster and incubated in a moist chamber for 4 weeks.
Nuclear staining.
Nuclei were visualized in conidia of the parent strain in 21 day old cultures according to
the HCl-Giemsa method (Robinow, 1981).
Stability testing.
Three successive generations of the 9 variant strains and their parent strain were
investigated by means of 20 single conidium isolates from each one. The colonies of each
strain obtained were compared with each other macroscopically and microscopically to
detect any further deviation of the variant.
51
Variants of Penicillium expansum
RESULTS
Mass conidia cultures of the treated strains developed quite a number of sectors
originated from the inoculum at the center of the plates, while no sectors were observed in
the mass conidia cultures of the untreated parent strain. Sectoring being considered as the
sign of a mixed culture, it was implied that sectors originated from inoculated variant
conidia. Single conidia were therefore isolated from the strains.
Seven hundred and forty three variant colonies were obtained from the 3800 single
conidium isolates, that is a variation rate of 19.55%, while no variation was detected at all
in the untreated parent strain. Although it is not the purpose of this paper to analyse the
mutagenic effect of the treatment, it can be said that the 1600 single conidium isolates
derived from the 8 strains treated by preservation techniques alone and not subjected to
X-ray have shown an average of 9.44% variant colonies including variants 1 to 6 and
variant 8, while the 2200 single conidium isolates from the other 11 strains incidentally
exposed after preservation to uncontrolled X-rays developed an average of 26.91 %
variants induding variant 1 and variants 4 to 9.
The rates of occurrence of the nine variants and their relative frequency is given in
Table 1.
Descriptions and illustrations (Figs. 1, 3-5) of the single conidium isolates of the parent
strain and of the nine variants were made from MYA2 cultures grown at 24C in the dark
for 7 days. Differences observed on MYA2 cultures grown at 20C in diffuse light and on
Czapek agar cultures grown at 24C in darkness (Fig. 2) are indicated as remarks.
Table 1. Occurrence and relative frequency of variants from untreated and treated
Penicillium expansum MUCL 29412.
Variant
Occurrence number
Occurrence rate (%)
Relative frequency
(%)
From untreated parent strain:

Variant
0/200
0.0
0.0
7.57
0.26
0.16
5.21
0.18
0.13
1.73
2.37
1.92
38.76
1.34
0.81
26.65
0.94
0.67
From treated strains:

Variant 1
Variant 2
Variant 3
Variant 4
Variant 5
Variant 6
Variant 7
Variant 8
Variant 9
288/3800
10/3800
6/3800
198/3800
7/3800
5/3800
66/3800
90/3800
73/3800
8.88
12.11
9.82
Parent strain (MUCL 29412)

Colony: diameter 44 mm; relief plane; depth 1 mm; immersed margin 2 mm wide, hyphae straw yellow,
regular; texture coremiform; colour dull green; exudate colourless as small droplets; soluble pigment none;
reverse yellow green.
Conidioma: stipes 500 x 3.5-4.5 11m, smooth; penicilli 3 branch points, 35-45 x 25-40 Ilffi; rami 2-3, 15-20 x 2.53.5 11m, smooth, appressed; metulae 3-5,10-17 x 2.5-3.51lffi, smooth, appressed; phialides 3-7, 7-10 x 2.5-3.5
Ilffi, ampulliform, smooth, appressed; collula short; conidia mostly ellipsoidal, 3-4 x 2.5-3 Ilffi, smooth.
52
Remarks. Colony grown at 20C in diffuse light strongly coremial, coremia being distinctly zonated. On
Czapek agar at 240C in darkness, diffusely zonate, coremiform to fasciculate, abundant light brown exudate,
reverse yellow to orange. Staining of nuclei in this strain has demonstrated that all conidia are
conspicuously uninucleate.
Figure 1. Colonies on 2 % malt agar at 24C in darkness of variants 1 to 9 (from left to right,
row after row) and the parent strain P. expansum MUCL 29412. A, upper surface and
B,reverse
Variants of
Penicillium expansum
53
Variant 1 (MUCL 30223)
Colony: diameter 44 mm; relief plane; depth 0,5mm; immersed margin 2mm wide, pure yellow, regular;
texture velutinous but with 2 ern diameter central area of white lanose mycelium with delayed sporulation;
colour yellow green; exudate none; soluble pigment none; reverse pure yellow.
Conidioma: stipes 200 x 2-3 ~m, smooth; penicilli 2-3 branch points, (20-)30-35(-45) x 20-25 ~m; rami 1-2,25 x
2.5-3 ~m, smooth, appressed; metulae 3-5,10-17 x 2.5-3 ~m, smooth, appressed; phialides 3-7, 7-10 x 2.5-3
~m, cylindroid ai, smooth, appressed; collula short; conidia mostly ellipsoidal, 3-4 x 2.5-3 ~m, smooth.
Remarks. At 20C in diffuse light, velutinous with no fasciculation, but slightly zonate, with a minute central
outgrowth of white lanose mycelium. On Czapek agar, totally white, plane, furrowed, sterile, reverse white
to pale yellow.
Distinctive characters. The colony is lower and the texture is velutinous. The sporulating
area is yellow green, the colour of immersed mycelium at the margin is pure yellow. No
exudate is produced. The reverse is pure yellow. Conidiophore stipes are shorter and
narrower; some penicilli are biverticillate; terverticillate penicilli are far narrower than
those in the parent strain and there are fewer rami on each stipe, and they are longer and
narrower. Metulae and phialides are also narrower. The phialides are cylindroidal.
Colony: diameter 38 mm; relief plane; depth 1 mm; immersed margin 2mm wide, pure yellow, regular;
texture fasciculate into small coremia; colour dull green; exudate colourless, as small droplets; soluble
pigments none; reverse yellow green.
Conidioma: stipes 300 x 3.5-4.5 ~m, smooth; penicilli 2-3 branch points; (20-)30-35(-45) x 25-40 ~m; rami 1-2,
15-20 x 2.5-3.5 ~m, smooth, appressed; metulae 3-5,10-14 x 2.5-3.5 ~m, smooth, appressed; phialides 3-7,7-10
x 2.5-4 ~m, ventricose, smooth, appressed; collula short; conidia mostly ellipsoidal, 3.5-4.5 x 2.5-3 ~m,
smooth.
Remarks. At 20C in diffuse light, small coremia with distinct zonation. On Czapek agar, zonate and slightly
furrowed, coremiform, reverse orange.
Distinctive characters. The diameter of the colony is smaller; the immersed mycelium at the
margin is pure yellow. The conidiophores are fasciculate into smaller coremia, with
conidiophore stipes shorter, penicilli sometimes biverticillate, rami fewer on each stipe,
metulae shorter, phialides wider and ventricose and conidia somewhat larger.
Colony: diameter 41 mm; relief umbilicate; depth 2-3 mm; immersed margin 1 mm wide, pure yellow,
regular; aerial margin of white lanose mycelium; texture fasciculate at the sporulating central area; colour
dull green; exudate colourless as small droplets; soluble pigment red brown; reverse red brown.
Conidioma: stipes 500 x 3.5-4.5 ~m, smooth; penicilli 3 branch points, 35-45 x 25-40 ~m; rami 2-3, 15-20 x 2.53.5 ~m smooth, apprcssed; metulae 3-5,10-17 x 2.5-3.5 ~m, smooth, appressed; phialides 3-7, 7-10 x 2.5-3.5
~m, ampulliform, smooth, appressed; collula short; conidia mostly subglobose, 2-3.5 x 2.5-3 ~m, smooth.
Remarks. At 20C in diffuse light, zonate but sporulating slowly from the center. No aerial white lanose
mycelium. On Czapek agar, faster growing, zonate and furrowed, granular, sporulating well, reverse red
brown to dark brown.
Distinctive characters. The colony is somewhat smaller in diameter; the relief is umbilicate
and the colony is higher because of its lanose texture. Sporulation from the center of the
colony is delayed, leaving a very wide margin of aerial lanose white mycelium. The
immersed mycelium at the margin is wider and pure yellow. Conidiophores are grouped
in fascicles from aerial hyphae. The colony produces a red brown diffusible pigment and
the colour of the reverse is red brown. Conidia are more globose and their average length
is shorter than those of the parent.
54
Figure 2. Colonies on Czapek agar at 24C in dark of variants 1 to 9 (from left to right, row
after row) and the parent strain P. expansum MUCL 29412. A, upper surface and B, reverse.
Colony: diameter 35 mm; relief plane; depth 0.5 mm; immersed margin 2mm wide, pure yellow, lobate;
texture fasciculate; colour dull green; exudate brownish in droplets; soluble pigment red brown; reverse
yellow green.
Conidioma: stipes 300 x 3.5-4.5 ~m, smooth; penicilli 2-3 branch points, (20-)30-35{-45) x 25-40 ~; rami 2-3,
20-40 x 2.5-3.5 ~, smooth, appressed; metulae 3-5, 10-17 x 2.5-3.5 ~, smooth, appressed; phiaJides
55
3-7, 7-10 x 2.5-3.5 11m, ampulliform, smooth, appressed; collula short; conidia mostly ellipsoidal,
3-4 x 2.5-3 11m, smooth.
Remarks. At 20C in diffuse light, zonate, fasciculate, more paler and slowly sporulating. On Czapek agar,
colony texture granular, grey green, furrowed, red brown soluble pigment, reverse orange to dark brown.
Distinctive characters. The diameter of the colony is smaller and the colony lower. The
outline is slightly lobate and the immersed mycelium at the margin is pure yellow. Brown
exudate and red brown diffusible pigment are produced. Conidiophores are grouped in
fascicules, with shorter stipes, some biverticillate penicilli and some longer rami.
Colony: diameter 37 mm; relief plane; depth 1 mm; immersed margin 2mm wide, straw yellow, regular;
texture coremiform; colour dull green; exudate colourless as large droplets; soluble pigment none; reverse
yellow green;
Conidioma: stipes 600 x 3.5-4.51J.m, smooth; penicilli 2-3 branch points, (20-)30-35(-45) x 25-40 1J.m; rami 2-3,
15-20 x 2.5-3.5 1J.m, smooth, appressed; metulae 3-5, 10-17 x 2.5-3.51J.m, smooth, appressed; phialides 3-7, 7-10
x 2.5-3.5 11m, cylindroidal, smooth, appressed; collula short; conidia mostly ellipsoidal, 3-4 x 2.5-3 1J.m,
smooth.
Remarks. At 20C in diffuse light, similar growth rate to the parent, strongly coremial and sporulating from
the center to the margin. On Czapek agar, slowly growing, zonate and coremiform, ochre to yellow green,
reverse yellow to orange.
Distinctive characters. The diameter of the colony is smaller with exudate in large droplets.
Coremia are larger; the stipes are longer; some penicilli are biverticillate; the phialides are
more cylindroidal.
Colony: diameter 40 mm; relief plane; depth 1 mm; immersed margin 2mm wide, pure yellow, regular;
texture coremiform; colour dull green; exudate colourless, as small droplets; soluble pigment red brown;
reverse yellow green.
Conidioma: stipes 600 x 3.5-4.5 11m, smooth; penicilli 2-3 branch points, (20-)30-35(-45) x 25-40 1J.m; rami 2-3,
15-20 x 2.5-3.5 11m, smooth, appressed; metulae 3-5,10-14 x 2.5-3.51J.m, smooth, appressed; phialides 3-7, 7-10
x 2.5-3.5 1J.m, ampulliform, smooth, appressed; collula short; conidia mostly ellipsoidal, 3-4 x 2.5-3 1J.m,
smooth.
Remarks. At 20C in diffuse light, similar growth rate to parent, strongly coremial and sporulating
throughout. On Czapek agar, fast growing, coremiform in central zones, pale yellow green, intense red
brown soluble pigment, reverse orange red to dark brown.
Distinctive characters. The diameter of the colony is somewhat smaller. The immersed
mycelium at the margin is pure yellow. A red brown soluble pigment is produced. Some
penicilli are biverticillate; the stipes are longer, with shorter metulae.
Colony: diameter 44 mm; relief plane; depth 1 mm; immersed margin 2 mm wide, pale yellow, regular;
texture fasciculate; colour dull green; exudate colorless as small droplets; soluble pigment none; reverse
yellow green.
Conidioma: stipes 500 x 3-41J.m, smooth; penicilli 2-3 branch points, (20-)30-35(-50) x 20-251J.m; rami 1-2,25 x
2.5-3 1J.m, smooth, appressed; metulae 3-5, 10-17 x 2.5-31J.m, smooth, appressed; phiaJides 3-7,7-10 x 2.5-3
1J.m, cylindroidal, smooth, appressed; collula short; conidia mostly subglobose, 2.5-3.5 x 2.5-3.5 1J.m, smooth.
Remarks. At 20C in diffuse light, colony smaller, heavily sporulating throughout and closely zonate. On
Czapek agar, fast growing, strongly zonate, zones differently coloured ochre to yellow green, exudate light
brown, reverse orange.
Distinctive characters. Immersed mycelium in the marginal area is pale yellow. The
conidiophores are grouped in fascicles with some biverticillate and terverticillate penicilli;
penicilli always reduced and sometimes somewhat longer; metulae are less numerous on
56
each stipe, but narrower and eventually longer; metulae and phialides are also narrower.
The phialides are cylindroidal; the conidia are mostly subspherical.
Colony: diameter 35 mm; relief umbonate; depth 6 mm; immersed margin 1 mm wide, pure yellow, regular;
texture floccose; colour white; exudate none; soluble pigment red brown; reverse red brown.
Conidioma: none observed, although a very few conidia have been found.
Remarks. At 20C in diffuse light, mycelium sterile. On Czapek agar, colony white, thick and lanose, reverse
red brown.
0
o o
parent strain
Penicillium expansum MUCL 29412
Figure 3. Conidiophores and conidia of parent strain P. expansum MUCL 29412, on 2% malt
agar at 24C.
57
Distinctive characters. This variant is distinct from the parent strain in all characters. The
colony is smaller in diameter; the immersed mycelium at the margin narrower and pure
yellow. The colony is umbonate and deeper, made of sterile, white, lanose mycelium. No
exudate is produced, but red brown soluble pigment is. The reverse is red brown.
Colony: diameter 36 mm; relief plane; depth 0.5 mm; immersed margin 0.5 mm wide, pale yellow, regular;
texture fasciculate; colour dull green; exudate colourless as small droplets; soluble pigment none; reverse
yellow green.
Conidioma: stipes 500 x 35-4.5 1lID, finely roughened; penicilli 2-3 branch points, (20-)30-35(-50) x 20-25 1lID;
rami 2-3, 15-25 x 2.5-35IlID, smooth or finely roughened, appressed; metulae 3-5, 10-17 x 2.5-35IlID, smooth,
appressed; phialides 3-7, 7-10 x 25-351lID, ampulliform, smooth, appressed; collula short; conidia globose,
3-45 x 3-451lID, smooth.
Remarks. At 20C in diffuse light, zonate and moderately sporulating Conidia globose. On Czapek agar,
slowly growing, yellow green, colony texture very tufty, tufts of white mycelium becoming sporulating,
reverse yellow.
Distinctive characters. The colony is smaller and lower; immersed margin is narrower and
pale yellow. The conidiophores are grouped in fascicles; walls of the stipes and of some
rami are finely roughened. Some penicilli are biverticillate; terverticillate penicilli are
sometimes longer but they are always narrower; rami are also sometimes longer. Conidia
are distinctly globose and larger.
Scanning electron microscopy examination of three day old conidia of the parent strain
and its eigth sporulating variants shows that they are smooth in all cases but size and
shape vary. Variants 2,4,5 and 6, all well sporulating from typically coremial to strongly
fasciculate conidiophores, have acquired conidia somewhat larger than those of the parent
strain but similar in shape (Fig. 6). The less fasciculate variants 7 and 3 have somewhat
smaller conidia, but these are the same shape as the parent strain.
Two variants are noteworthy. Variant 9 shows larger, globose and smooth conidia;
however it is conspicuously fasciculate and does not shed conidial crusts. Variant 1 is not
fasciculate and appears velutinous. Its conidiophores are of a reduced complexity, a
feature also seen in variant 7. It also shows a strong tendency to produce outgrowths of
almost sterile white lanose mycelium similar to the wholly sterile variant 8 (Fig. 7).
Subepidermic inoculations of conidia from the parent and eight sporulating variant made
on Gloster apples has demonstrated the virulence of all the strains; a soft rot of the fruits
soon develops and somewhat small to large coremia emerge at the surface of the rot. The
size of those coremia is similar to their respective size observed on malt agar.
DISCUSSION
The parent strain P. expansum MUCL 29412 showed uninucleate conidia. The absence of
sectors in mass conidia cultures of this isolate, the absence of variant colonies amongst the
200 single conidium isolates and the absence of variation in single conidium subcultures
during three generations demonstrate the genetic homogeneity and stability of the parent
strain cultivated on MYA2 and maintained at 4C. A similar test has been carried out on
50 single conidium isolates from conidia of P. expansum collected on Boscop apple rot. All
single conidium cultures obtained immediately from nature as well as all single conidium
cultures derived from these were typical of the wild type in the first generation.
58
.,.rlanl I (MUCL 302ll)
0 0 0
o 00
0 00
00000 0
00
0
00 Q
a
000000000
0
0
0
0
0
Q
.",.. , , (MUCL ,022"
0
0 C) 0 0 ~
0
0
0
0 0
0
0
800
17
Figure 4. Conidiophores and conidia of variant 1 to 4 of P. expansum MUCL 29412, on 2%

malt agar at 24C.
59
wariult 5 (MUCL ]OZ27)
varillli 6 (MUCL 30228)
00
0 0
0
o00
0
0 0 0
o 0 00 000
0 0
0 0 0
0
00
00
0 0 0000
000000 0
0000
o
0
o OO~ 000 00
0 00 0 0
0 0
0000
00
0 0 0 D 0 0 00
0 0 0 0 0 0 0
0 00
00
Ywnl7 (MUCL 302:19)
00 0
arianl 9 (MUCL lD23J)
Figure 5. Conidiophores and conidia of variant 5 to 9 of P. expansum MUCL 29412, on 2 %

malt agar at 24C.
60
Figure 6. Conidia of the parent strain (a) and variants 1 to 9 (from b to

MUCL 29412 as seen by scanning electron microscopy (x 3800).
i)
of P. expansum
Variants of
61
VELUTINOUS
Figure 7. Schematic representation of the divergence of variants 1 to 9 from the parent strain
P. expansum MUCL 29412 with indication of their common characters.
However it has been reported that large variation may naturally exist in strains from
Penicillium subsection Fasciculata (Samson et al., 1976), and that repeated cultivation on
agar slants can be responsible for variation in Penicillium subgen. Penicillium (Bridge et al.,
1986,1987). In the present case, some of the factors which having induced variation, Le.
preservation techniques such as freeze-drying and frozen-storage at -20 and -196C, as
well as freight control by X-ray in customs, are currently being used.
Nine types of variants have been induced. They differ from the original parent strain
by their cultural characters and some of their microscopic characters. The growth rate can
decrease up to 20% (variants 4, 8, 9). The texture varies from coremial to fasciculate to
velutinous (variant 1) and to lanose (variants 3, 8). This change appears to be correlated to
a delay and a decrease in conidiation (variant 8 sporulated after 7 months). At the same
time the colour of the colony changes from dull green to yellow green (variant 1, 3) and
even white in the lanose, asporogenous mycelium. The colour of the immersed marginal
mycelium varies from pale straw yellow to bright yellow (variants 1, 2, 7). Exudates may
be absent, when present they usually are colourless or brownish (variant 4). A red-brown
soluble pigment can be produced (variants 3,4,6,8).
There are variations in the structure and size of the elements of the conidioma. The
most conspicuous variations occur in the length and width (variants 1, 5, 6) or the
roughness of the conidiophore stipe (variant 9), the reduction of the penicillus (variants 1,
7), the shape of the phialides, that varying from ampulliform to either ventricose (variant
2) or cylindroidal (variants 1,5,7), and the size and shape of the conidia that can become
large and globose (variant 9). A few characters are stable, such as the length of the
phialides and the absence of ornamentation of the conidia. Also the pathogenic virulence
to apples appears unchanged in the sporulating variants.
62
The characters shown to be variable are primarily important in the identification of the
species (Samson et al., 1976; Pitt, 1979; Ramirez, 1982). It also appears that different
characters vary from one variant to another. These observations suggest that characters do
not exist which are sufficiently discriminant either to identify all nine variants as one
species or to differenciate these variants from the parent species and from any other
related species. Indeed when one important character is changed, another may remain
that makes species identification possible.
Variant 9, with rugose conidophore stipes and rami, globose smooth conidia, with a
granular to floccose colony surface and a narrow margin, resembles P. crustosum Thorn
closely. However, variant 9 does not form any powdery or loose crusts of conidia on
MYA2 and is very tufted on Czapek agar. Raper and Thorn (1949) classified P. crustosum
together with P. expansum in the same series Expansa. Fassatiova (1977), comparing
authentic strains of both species, redisposed P. crustosum as P. expansum var. crustosum.
Samson et al. (1976) considered P. crustosum as a synonym of P. cyclopium as P. verrucosum
var. cyclopium, but Stolk and Samson (1985) reverted to maintaining P. crustosum as a
distinct species in the series Expansa. Pitt (1979) also considered P. crustosum as a distinct
species. These opinions indicate that P. crustosum is a taxon very close of P. expansum
Variant 9 of P. expansum appears to corroborate this observation.
Variant 1 has conidiophores that are so short that it appears velutinous; its penicilli
are also reduced and the strain has a strong tendency to form sterile mycelium. On
Czapek agar, it is totally white and sterile, like the lanose variant 8. The character of
colony texture indeed may vary considerably, not only within the species but within the
strain.
Considering the possible variations of taxonomic characters within a species as has
been demonstrated here within a strain, identification is therefore not helped by rigid
dichotomous keys constructed on the basis of typical isolates. To encompass such a
variability within an identification key, a probabilistic computerized matrix should be
constructed on the basis of records of numerous pure strains. This method would allow
consideration as to whether the observed variations are in the range of the natural
variations or if new species or varieties have to be created.
The analysis of the extent of variability of an isolate that might appear to be an
heterogenous population, can be made possible by the observation of single conidium
cultures derivated from the original sample.
This prodedure is applicable to the study of morphological characters of an isolate
and also its physiological or biochemical properties. The production of secondary
metabolites of the nine morphological variants is now investigated by a similar method.
REFERENCES
BRIOCE, P.D., HAWKSWORTH, D.L., KOZAKIEWICS, Z., ONIONS, A.H.S. and PATERSON, R.R.M. 1986.
Morphological and biochemical variation in single isolates of Penicillium. Transactions of the British
mycological Society 87, 389-396.
BRIDGE, P.D., HUDSON, L., KOZAKIEWICS, Z., ONIONS, A.H.S. and PATERSON R.R.M. 1987.
Investigation of variation in phenotype and DNA content between single-conidium isolates of single
Penicillium strains. Journal of General Microbiology 133, 995-1004.
FASSATIOVA, 0.1977. A taxonomic study of Penicillium series Expansa Thorn emend. Fassatiova. Acta
Univ. Carol. BioI., 1974: 283-335.
ONIONS, A.H.S., BRIDGE, PD. and PATERSON, R.R.M. 1984. Problems and prospects for the taxonomy of
Penicillium. Microbiological Sciences 1, 185-189.
63
PIIT, J.1. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
RAMIREZ, C. 1982. Manual and Atlas of the Penicillia. Amsterdam: Elsevier Biomedical Press.
RAPER, K.B. and 1HOM, C. 1949. A Manual of the Penicillia. Baltimore: Williams and Wilkins.
ROBINOW, C.F. 1981. Nuclear behaviour in conidial fungi. In Biology of conidial fungi, Vol. 2, eds. G.T.
Cole and B. Kendrick. New York: Academic Press.
SAMSON, RA., STOLK, A.C. and HADLOK, R 1976. Revision of the subsection Fasciculata of Penicillium
SANSOME, E.R 1947. Spontaneous variation in Penicillium notatum strain NRRL 1249 B21. Transactions of the
British mycological Society 31: 66-79.
- - 1949. Spontaneous mutation in standard and 'GIGAS' forms of Penicillium notatum strain 1249 B21.
Transactions of the British mycological Society 32: 305-314.
STOLK, A.c. and SAMSON, RA. 1985. A new taxonomic scheme for Penicillium anamorphs. In Advances in
Penicillium and Aspergillus systematics, eds. RA. Samson R.A. and J.1. Pitt, pp. 163-192. New York:
Plenum Press.
DIALOGUE FOLLOWING DR. BERNY'S PAPER
BRIDGE: The kind of variation that you have reported here have, in fact, been reported in
Penicillium before, as well as in other fungi. They occur in many fungi without any X-ray,
UV or other treatment whatever. In most of these cases, you are exerting some kind of
selective pressure on the population when you do the isolation. It is difficult to say what
is and what is not a mutant. Most colonies of Penicillium, for example, readily yield
nutritional mutants when we grow them on substrates they do not normally encounter.
More work is reqUired.
PIIT: Variant number 9 looks alot like P. crustosum. Is it possible that this might have been
a contaminant?
HENNEBERT: This is not a contaminant. It comprised 10% (check paper) of the single
conidium isolates. It does look similar to P. crustosum because it does have globose
conidia and rough stipes, but it still rots apples, which P. crustosum does not.
SAMSON: Yes it does!
HENNEBERT: Well, maybe this character is not good enough. But these isolates are derived
from a true, typical P. expansum. These are not contaminants. So, we have to believe that
this "P. crustosum" is a true variant of P. expansum. So the question is, what is the species
concept of P. expansum in this case? The same question arises regarding the velutinous
colony texture. Is this a good enough character to exclude a strain from P. expansum?
Maybe not. And how about the lanose variant? Everyone knows that lanose strains are
degenerate and difficult to identify and classify by morphological methods. So we have
to look at them with other methods, such as secondary metabolites.
But what is important here is that we have all these variants from a single conidium
of P. expansum. It is a question of preservation methods, or perhaps the accidental X-ray
treatment. Some preservation methods we used gave no variants.
My main conclusion is that the species concept should be based on a better study of
variation. The only way to do this is with single conidium isolates.
FASSATIOVA: In some of your strains, you have found rough metulae and phialides, which
are characters of Geosmithia. (Note of the editors: on the slides of the drawings,
64
interrupted lines were showing background phialides giving an impression of a rough

wall. They have been omitted in Figures 3-5 of this paper)
BERNY: No, the phialides were not rough.
SAMSON: However, we have also found several isolates of P. expansum with rough stipes.
Otherwise, they are quite characteristic, and Dr. Frisvad has confirmed this with the
secondary metabolites.
SAMSON: I don't agree that you should key out these deviating forms. When you isolate
from foods or other materials, you will get the wild type. Deviating strains are very rare,
I think. Therefore, if you go to a computer key with these deviating strains, it makes
things quite difficult, but this is not the fault of the key.
PITT: I agree with Dr. Samson. Keys are normally written for fresh isolates. They should
work for people who are isolating from nature. There is no way any key can take into
account variation that is found in cultural conditions. One of the probelms with some of
Raper's descriptions was that they were based on isolates that had been in collections for
a long time. They were therefore difficult to use. Keys should be written for fresh
isolates. They cannot be expected to work for isolates that have been manipulated.
TAYLOR: I wonder if there is correlation between the different kinds of variants and the xray treatment or the preservation methods?
BERNY: There is no correlation.
HENNE BERT: Of the nineteen different preservation methods studied, only eight procedures were used with this particular monoconidial strain. Up to forty percent of the
first generation single conidium isolates were variants. Some of the cultures received
from other culture collections stayed in customs for about one month and were subjected
to X-ray treatments. In these cultures, up to seventy-five percent of the single conidium
isolates were variants. Therefore, one pure culture, stored using different methods over
time, can yield many different variants. This could also happen with extype cultures.
SAMSON: If you start making monoconidial isolates, then you impose another selection on
your type culture.
HENNBERT: You can reselect the original one.
SAMSON: You can also lose the original one.
HENNBERT: Or have lost it.
PITT: Four years ago, we agreed to use a mixed spore inoculum for identifying and
handling Penicillia. This is an important point. The variability in individual conidia of a
haploid genus like Penicillium is quite high. I suspect there may be more than one
genome present in an ordinary Penicillium colony. The way that Penicillium has adapted
so rapidly into man-made environments such a cereals, relates to it having a lot of
variants available. If we make single spore cultures, we are seeing a correct expression of
a part of the genome, but these may be variants that would not normally survive in
nature. Penicillium has the capability of shedding a lot of variants into the environment,
with the possibility that this might allow it to exploit new habitats. Some of the variants
that Dr. Berny has shown us, which consist mostly of mycelium, are most unlikely to
survive in nature. With mass inocula, anastomoses of the germ hyphae result in the
production of a mixed genome representing the sum of the variation in those conidia.
65
PETERSON: In our lab, we routinely compare colony morphology before and after
lyophilization. Changes in colony morphology are very rare in my experience.
SEIFERT: Is it possible that these different variants may represent different vegetative
compatability groups (VCGs)?
TAYLOR: VCG's do occur in anamorphic fungi, as judged by hyphal anastomosis, such as
Fusarium oxysporum. But you wouldn't see these if you made many single conidium
isolates from a single isolate from nature. You would only see them if you made isolates
from different habitats or geographical localities.
CROFT: Regarding the problem of generating variance from a single isolate, I suspect that
in some cases chromosomal rearrangement may be the cause.
67
PENICILLIUM AND ASPERGILLUS IN THE FOOD MICROBIOLOGY

LABORATORY
A.P. Williams
Leatherhead Food R.A.
Randalls Road, Leatherhead, Surrey, I<T22 7RY, UK
SUMMARY
Species of these two genera have long been dominant among the spoilage flora of a range of foods,
especially those with reduced water activity and low pH. As various lobbies seek to reduce levels of
humectant preservatives and acids, such spoilage is likely to increase and, where controlled chill is
used as a substitute, psychrotrophic Penicillia have a particular advantage. Although some species are
ubiquitous, the presence of others can be strongly indicative of particular types of problem (e.g.
EUTotium spp. and marginal moisture abuse). The concept of recognising Indicator and Index
organisms is not unknown in Bacteriology, yet has been slow to be adopted in Food Mycology,
mainly because of the difficulty in identification.
Mycology is advancing rapidly, with clarifications of taxonomy, simplification of keying and new
analytical procedures (e.g. E.L.I.S.A. and lectin binding). It is now time to consolidate those
achievements so that recognition of important species, especially in Penicillium and Aspergillus, and
assessment of their significance, are made available to laboratories that lack the services of an expert
mycologist.
INTRODUCTION
During the last 10 years, Penicillium and Aspergillus have dominated the spoilage
mycoflora of mouldy samples examined in our laboratory. Williams and Bialkowska
(1985) reported that, from 294 samples with visible growth of one or more mould species,
Penicillium accounted for 53.1% ofisolates, Aspergillus and associated teleomorphs 15.1%,
Cladosporium 8.9%, Mucor 8.5%, Rhizopus 4.8% and other genera 9.6%. Of the penicillia,
subgenus Penicillium (90.1 %) was dominant.
The following species lists give details of spoiled foods from which species of
Penicillium and Aspergillus have been isolated, including isolations more recent than 1985.
It is hoped that other laboratories will be encouraged to publish similar lists, so that
further information becomes available on the relationship between species and pattern of
spoilage. This would not only supplement information already available about foods at
risk from particular mould species, but would also highlight those ubiquitous species
whose presence gives no information on likely contamination source.
SOURCES OF PENICILLIUM AND ASPERGILLUS
Penicillia and Aspergilli described below were all isolated either by direct subculture of
visible mould growth on food surfaces or by direct plating of visible mouldy material
from within a food sample. In the following lists, a standard sequence has been adopted
for food types: meat pies and pasties; meat products; cheese (British hard cheese unless
otherwise stated); bread; cakes; jams; nuts; cereals; fruit and vegetables and others. A
tabular form has not been used because the variety of foods is too large. Nomenclature
68
A.P. Williams
follows the conventions of Williams (1990) and, where more than one isolate was obtained
in any category, the number of isolates is given in parenthesis.
Subgenus Penicillium
P. atramentosum: Cheese
P. aurantiogriseum: Pork sausages; slated anchovies; cheese (3); bread (2); palm kernels;
maize (2); muesli; animal feed; peach; apple juice; pickled gherkin; milk shake mix;
pepper.
P. brevicompactum: Cottage pie; meat pie (2); sausage roll; salted anchovies (2); cheese (2);
bread (3); jams (5); maize; grapes; pear; tomato; celeriac; coconut; cabbage; tomato puree;
milk shake mix; pet food; dairy spread.
P. camemberti: Cheese (4).

P. chrysogenum: Cottage pie; Scotch egg; meat pie (3); cooked sausages (2); salted
anchovies; cheese; cottage cheese; bread (5); cake (6); jam (2); cabbage; apple; pet food (2);
milk shake mix; whole egg; mustard; fruit sweets; chocolate biscuit; paprika; tartrazine
solution; suet; dairy spread.
P. commune: Cottage pie; meat pie (9); Scotch egg; sausage roll (3); chicken; sausages (3);
cheese (18); bread (6); Madeira cake (7); jam (2); marrow; banana; orange; lemon; pickled
onion; bay leaves; yoghurt (2); milk shake mix; tartrazine solution (2); dairy spread.
P. coprophilum: Muesli; pet food.
P. crustosum: Sausage; cheese (3); bread (4); cake; jam (3); chestnut; maize (2); raw pastry;
apricot; pear; nectarine; raspberries; peach; tomato (2); apple juice; celeriac; carrot; baked
beans; pickled onions; fruit jelly sweets; ground coffee; pectin; dairy spread.
P. cyclopium: Meat pies (9); sausages (2); cheese (3); bread (6); cake; mincemeat; maize;
rusk; raw pastry (2); canned peaches; suet.
P. digitatum: Lemon (2); orange; tangerine.
P. echinulatum: Sausage roll; bread; jam (2).
P. expansum: Meat pie (2); sausage; cheese (2); Brie cheese; cake (4); walnut; soya meal;
tomato; tomato puree; apple (2); pear (2); pet food; spaghetti; cream; dairy spread.
P. glandicola: Processed potato; pet food.

P. griseofulvum: Rice
P. hirsutum: Pork pie; cheese; bread.
P. italicum: Meat pie; bread; cake; satsuma; lemon; mustard.
P. olsonii: Bread; jam; rusk; tomato (3); orange juice; suet; ground coffee.
P. roqueforti: Fish pate; cheese (15); bread (5); jam (2); biscuit; fruit drink; gherkin;
cucumber; pickled onions (2); fruit wine; pet food.
P. solitum: Meat pie (2); cheese (6); bread (2); processed potato; pet food.
P. verrucosum: Cake.
Penicil/lium and Aspergillus in
the food microbiology laboratory
69
P. viridicatum: Meat pie (2); cheese (4); raw peanuts; maize (2); banana; suet.
Other Penicillia
Talaromyces flavus: Apple juice.

Eupenicil/ium cinnamopurpureum: Jam.
Penicillium citrinum: Bread; raw peanuts; maize; muesli.
P. corylophilum: Bread (2); cake; jam (many); animal feed; pickled onions (2); pepper.
P. fellutanum: Cake; suet.
P. glabrum: Mixed cereal; marrow.
P. islandicum: Suet; pepper.
P. janczweskii: Jam.
P. jensenii: Flour.
P. minioluteum: Pickled onions.
P. miczynskii: Cheese.
P. pinophilum: Animal feed.
P. purpurogenum: Soya meal; onion.
P. rugulosum: Flour.
P. simplicissimum: Pickled onion; food thickener.
P. spinulosum: Meat pie; bread; cake; spaghetti; carrot; pickled onion; onion; fruit drink;
ground coffee.
P. variabile: Chicken paste; onion.
Aspergillus and its teleomorphs

Eurotium amstelodami: Cake (4); jam (3); raw peanuts.
E. chevalieri: Cake; fruit jelly; coconut cream.
E. herbariorum: Cake.
E. manginii: Cake (2).
E. medius: Malt extract.
E. repens: Bread; cake (5); jam (many); rice; paprika.
E. rubrum: Jam; raw peanuts; wheat; fruit drink; malt extract.
Emericella nidulans: Pepper.

Neosartorya fischeri: Raspberry puree.
Aspergillus candidus: Rice; processed potato; pepper.
70
A.P. Williams
A. flavus: Raw peanuts; palm kernels; maize (2); wheat; soya (2); rice; rusk; pet food;
ground coffee; pepper (2); suet; margarine.
A. fumigatus: Bread; nuts; muesli; animal feed; marinated cherries; pepper.

A. niger: Cheese; bread (3); raw peanuts; pistache nuts; cocoa beans; sesame seed; pepper.
A. ochraceus: Fish food.

A. parasiticus: Animal feed.
A. restrictus: Jam (3); rice; malt extract.
A. tamarii: Muesli; pet food; pepper.
A. terreus: Maize; processed potato; animal feed; pepper.
A. ustus: Animal feed; dairy spread.
A. versicolor: Pepper; tartrazine solution.
A. wentii: Raw peanuts; cocoa beans.
IMPLICATIONS OF THE RANGE OF MOLDS ISOLATED
Although the moulds listed above were isolated from food samples that had visually
spoiled, it is inappropriate to comment on their relative incidence, because of the wide
variation in the size of the investigations. For example, although a considerable number of
species have been found growing on pepper, they all came from one episode in which
ground black pepper from a single source had been packed after moisture abuse. The
pickled onion isolates were obtained in a similar manner. On the other hand, isolates from
jams, cheese, bread and meat products were obtained from many samples over a number
of years and are much more representative of the natural spoilage pattern in the U.K.
For practical identification purposes, the number of different species to be separated
presents some difficulty. Of 59 different species identified, 40 were found five times or less
and, of those, 17 were found only once. This means that a microbiologists may well lack
the opportunity to gain familiarity with a considerable number of the species that can
occasionally cause a spoilage problem. In this context, the single isolations of Talaromyces
flavus and of Neosartorya fischeri are noteworthy. Both had caused substantial problems
associated with heat processing and the recognition of these heat-resistant species played
an important part in controlling the spoilage. Where species were isolated frequently,
from different sources, certain trends are evident. Although Eurotium repens continues to
be a major spoilage fungus in jams, Penicillium corylophilum and P. brevicompactum occur
nearly as often. Both of these Penicillia are xerophilic. Perhaps the trend towards
reduction in jam solids, as well as moisture migration during unsatisfactory storage,
account for their regular isolation. Many other Penicillia are ubiquitous e.g. P.
aurantiogriseum, P. commune and P. cyclopium. P. commune appears to have a particular
affinity for protein such as meat and cheese. P. expansum and P. italicum are usually
considered as agents of decay of fruit, yet both were isolated from various sources, P.
digitatum in contrasts was only found on citrus fruit. Among the remaining Penicillia, few
other clear trends were apparent, although P. crustosum was often isolated from fruit and
vegetables, P. olsonii was less rare than the literature would suggest and P. roqueforti was
the frequent spoilage agent of non mould-ripened hard cheeses, as well as growing on
many other foods.
Penicill/ium and Aspergillus in the food microbiology laborafory
71
Aspergilli were, in general, found less often than Penicillia. This is undoubtedly
because the majority of foods submitted to our laboratory have been of high water activity
and intended for chill storage. Aspergillus spoilage usually occurred after accidental
wetting of stable, low water activity foods. The only clear cut affinity was between
Eurotium spp. and spoilage of jams and cakes.
For a food microbiology laboratory that has to deal with mould spoilage, it is
apparent that some species may be expected to occur frequently, others rarely. The
necessity remains to identify them accurately, in order to assess their Significance, to trace
their origins and thus to control and prevent the recurrence of spoilage. Now that
mycology is advancing rapidly, with clarification of taxonomy, simplification of keying
and new analytical procedures (e.g. ELISA and lectin binding) it should be possible to
devise a strategy for investigating mould spoilage of foods. Such an approach might
include a primary screening of isolates to select genera (by ELISA for example); a
secondary range of tests to identify isolates as far as necessary, for example by using
computer-assisted data-sorting and finally a library of information on the physiological
and ecological characteristics of the fungi, to assist in problem solving. Most of the
ingredients needed for this strategy now exist and only await assembly.
REFERENCES
WILUAMS, A.P. 1990. Identification of Penicillium and Aspergillus: computer-assisted keying. In Modern
Concepts in Penicillium and Aspergillus classification, eds. R.A. Samson and J.I. Pitt, pp. 287-292, New
York and London: Plenum Press.
WILUAMS, A.P. & BIALKOWSKA, A. 1985. Moulds in mould spoiled foods and food products. Leatherhead
Food R.A. Research Report No. 527.
3
NOMENCLATURE: CONSERVATION AND STABILITY
OF NAMES OF ECONOMICALLY IMPORTANT SPECIES
75
PROBLEMS AND PROSPECTS FOR IMPROVING THE STABILITY OF

NAMES IN ASPERGILLUS AND PENICILLIUM
D.L. Hawksworth
CAB International Mycological Institute
Ferry LAne, Kew, Surrey TW9 3AF, UK
SUMMARY
The main reasons for instability in names in Aspergl11us and Penicillium are reviewed, emphasizing the
differences between those due to nomenclatural and taxonomic changes. Attention is drawn to the
options now available under the Code for the conservation or rejection of names in the rank of
species, including the conservation of types. Recent international initiatives to improve the stability of
names are summarized, but in the final analysis taxonomic responsibility towards users is crucial.
Rapidly published peer reviews by internationally established panels of specialists may assume
increasing importance in the future.
INTRODUCTION
The stability of names is an emotive subject for both applied biologists and taxonomists,
but for opposing reasons. Applied biologists are becoming increasingly frustrated and
impatient, often refusing to accept changes (Bennett, 1985; Rossmore, 1988), whereas
taxonomists regard any mention of restriction on name changes as a threat to their right to
undertake objective scientific research and publish the results. With increased emphasis
on the relevance of research to practical applications, systematic work which leads to
instability in the vocabulary of applied biology finds difficulty in obtaining support.
Indeed, stability in names is one of the primary demands of the consumers of the products
of taxonomy, that is all users of scientific names. The need for a common language has
been accentuated in recent years by the requirements of computerized bibliographies and
culture information retrieval services, quarantine, and health and safety regulations. Lack
of attention to consumer requirements by taxonomists has been identified as the main
reason why the massive demand for the practical products of systematics is not matched
by the level of resources allocated to the subject (Hawksworth and Bisby, 1988). Perhaps
the greates challenge facing systematists into the next century is the adaptation of their
practices to better satisfy consumer demands and so regain the confidence and respect of
their contemporaries.
In the case of Penicillium Link, only 64 (47%) of the names accepted by Raper and
Thom (1949) were adopted in the monograph of Pitt (1979); the real level of stability will
be even less than these figures indicate as the circumscriptions and typifications of some
of the species names still employed were also changed. The current position is confusing
to users and ways must be found to alleviate this situation (Onions et al., 1984; Samson
and Gams, 1984).
This contribution endeavours to identify the main reasons for such substantial levels
of change, and ways in which these can be minimized under the current International
Code of Botanical Nomenclature (Greuter et al., 1988). Recent international initiatives to
increased nomenclatural and taxonomic stability are reviewed, and action the
Subcommision on Penicillium and Aspergillus Systematics (SPAS) of the International
76
D.L. Hawksworth
Commission on the Taxonomy of Fungi (ICTF) could initiate to limit the possibilities for
future changes in these genera is discussed.
Changes in names can arise in one of two ways: either from the application of the
International Code of Botanical Nomenclature (Le. nomenclatural instability), or from new
scientific evidence and the differing interpretations of taxonomists (i.e. taxonomic
instability). These different situations require disparate solutions, and the problems and
prospects for each category are considered in turn below.
NOMENCLATURAL INSTABILITY
Problems
The International Code of Botanical Nomenclature provides a system of 76 mandatory
rules (Articles), and also Recommendations, aimed at promoting nomenclatural stability.
The Code lays down criteria for effective and valid publication, the ways in which names
are formed, typification procedures, the choice of name when several compete, conditions
under which names can be automatically rejected, etc.
Proposals to modify any provisions in the Code can be made to each International
Botanical Congress; proposals are voted on in a mail ballot, debated and voted on at the
Nomenclature Session of the Congress, and if they gain the necessary substantial majority
(two-thirds is normally required) are incorporated into the new edition of the Code
published after each Congress. Even though the Code was approved at the First
International Botanical Congress held in 1867 (De Candolle, 1867), the number of
proposals continues to be substantial; 349 were made to the 1987 Berlin Congress (Greuter
and McNeill, 1987). Changes in the Code itself are sometimes operative only after the
Congress, but in many cases they are retrospective. Such alterations can consequently
endanger names which were correct under previous editions of the Code.
A major cause of name changes in both Aspergillus Micheli ex Link and Penicillium was
the fundamental retrospective revision of the rules concerning fungi with pleomorphic life
cycles, Art. 59, adopted by the 1981 Congress in Sydney (Voss et al., 1983). Even prior to
this, students of these genera had been reluctant to consider that the names could not be
used for all states (Raper and Thom, 1949; Raper and Fennell, 1965). I quote Bennett (1959,
p. 584): "I have been calling, and will continue to call, both the perfect (teleomorphic) and
imperfect (anamorphic) state of this fungus Aspergillus". Against this background
particularly destabilizing was the decision that even if a species name was proposed
under an anamorphic generic name, if the description and the type included the sexual
ascosporic stage then the name had to be applied to the teleomorph and was no longer
available for the anamorph, the conidial state. The effect this was forecast to have in
genera such as Aspergillus and Penicillium (Hawksworth and Sutton, 1974a,b) was passed
over in favour of simplification of the Code (Weresub, 1979). Samson and Gams (1985)
and Pitt (1979) had to introduce 24 and 15 new anamorph names respectively as a direct
result of this single change; many were for well-known species. While such a massive
number of changes are unlikely to be necessary for this reason again, when a "sclerotium"
proves to be an immature non-ostiolate ascoma in a Penicillium type, the name will no
longer be usable in that genus if the structure was included in the original description.
Changes due to other nomenclatural provisions have had substantially less impact
than those due to the revision of Art. 59. For example, the revision of the starting point for
the nomenclature of conidial fungi from 1 January 1821 back to 1 May 1753 (Demoulin et
al., 1981), only led to the simplification of some author citations in these two genera (e.g.
Hawksworth,1985).
77
Mycologists still all too frequently fail to deposit dried cultures as holotypes for the names
of species described from culture. Living cultures are specifically excluded as acceptable
as the types of names under Art. 9.5, and names not complying with this provision
published after 1 January 1958 are not valid (Art. 37.1). Most important pre-1958 names in
Aspergillus and Penicillium have now been lectotypified by Samson and Gams (1985) and
Pitt (1979) respectively so this should not now threaten names currently in use. Proposals
to change this provision by the International Mycological Association (van Warmelo,
1979) were not accepted. This matter is also of concern to phytologists and yeast
specialists particularly. The debate continues, although a proposal to establish a Special
Committee on living types was unfortunately rejected at the 1987 Congress (Greuter et al.,
1989, pp. 59-60). The value of dried material should not, however, be underestimated; it is
amenable to scanning electron microscopy, analysis of secondary metabolites (Paterson
and Hawksworth, 1985), and DNA extraction and amplification from minute samples can
be expected to be possible shortly.
A greater nomenclatural threat is the discovery of earlier names for accepted species.
In both Aspergillus and Penicillium considerable numbers of names are of uncertain
application; Raper and Fennell (1965) listed 70 in Aspergillus and Pitt (1979) 175 in
Penicillium. Some of these epithets are among the earliest in the genera and could
potentially threaten common species: A. griseus Link, A. virens Link, A. flavescens Wreden,
P. fasciculatum Sommerf., P. glaucum Link, and P. nigrescens Jungh. These could potentially
be resurrected by neotypification, and the First International Penicillium and Aspergillus
Workshop recommended: "That old and unfamiliar names for which no holotype or
material suitable for lectotypification exists are not reintroduced by neotypification other
than in exceptional circumstance" (Samson and Pitt, 1985, p. 455). A proposal to
incorporate this into the Berlin Code received some support but was regretably rejected
(Greuter et al., 1989, p. 46).
Prospects
While the nomenclatural problems surrounding the issue of fungi with pleomorphic life
cycles and occasioned by later starting points have now been clarified and embodied in
the Code, nomenclatural changes due to the taking up of older names (Art. 11.2) remain a
threat. Weresub (1979) counselled that mycologists should not seek too many special
provisions in the Code, and I concur; name changes due to the rule of priority at species
rank concern all groups.
Disadvantageous changes in wellknown family and generic names due to the strict
application of the Code have long been avoidable by invoking the conservation
procedures (Art. 14). At the Sydney Congress in 1981 this possibility was opened up for
the first time for specific names of "species of major economic importance" (Art. 14.2; Voss
et al., 1983). While "case law" needs to develop to define "major economic importance",
species such as Aspergillus fumigatus Fres., A. niger van Tieghem, Penicillium chrysogenum
Thom and P. roqueforti Thom would clearly be expected to fulfil this requirement.
In addition to overcoming threats to well-known names due to priority of publication,
under Art. 14.8 names can be conserved with different types. This provision has not yet
been adopted for any fungal name, but appears to provide a possible mechanism for
avoiding particularly unfortunate changes occasioned by the revised Art. 59, as in the case
of A. nidulans (Frisvad et al., see p. 83-89).
The possibility of widening the scope for the conservation of specific names was
introduced at the 1987 Berlin Congress where it was agreed that cases of rejected
misapplied names previously handled under Art. 69 could be treated in a similar manner
78
D.L. Hawksworth
to those being conserved (Art. 69.3). Art. 69 applies to taxa where significant confusion
might result, but is not confined to species of "major economic importance". This
procedure, and that of conservation on grounds where names are endangered by a strict
adherance to priority, merit greater use for specific names by mycologists than has so far
been the case. Before a name is changed, a responsible taxonomist should always
determine whether the conservation procedures could be invoked. Proposals backed by
an international group of specialists such as the present Workshop or SPAS are more
likely to be favourably received than ones from individuals.
Conservation procedures are lengthy and time consuming. Proposers are required to
publish formal proposals which can then be considered and voted on by the appropriate
Committee. Approval has to await the subsequent International Botanical Congress and
the process can consequently take up to six years, or even longer if the Committee does
not reach a clear majority decision quickly.
The current priority rule and restricted use of conservation procedures for species
names fails to achieve the primary aim of the Code: " ... the provision of a stable method of
naming taxonomic groups, avoiding and rejecting the use of names which may cause error
or ambiguity or throw science into confusion" (Greuter et al., 1988, Preface, p. 1). Since
1985 the matter has been addressed at a series of international meetings and special
working groups. Resultant proposals (Hawksworth, 1988a; Hawksworth and Greuter,
1989) aim at preparing Lists of Names in Current Use for all groups of organisrru. covered
by the Code, and subject to decisions at the next International Botanical Congress in
Tokyo in 1993, when being given a specially protected nomenclatural status at that time.
This initiative was accepted as a part of the International Union of Biological Sciences
(ruBS) scientific programme in 1988 and the Commission on Botanical Nomenclature
(International Association for Plant Taxonomy General Committee) appointed a Special
Committee in March 1989 to initiate the preparation of lists and prepare proposals for the
1993 Congress. The 36,500 accepted generic names are being listed first, together with
trials at species rank for different groups.
This move has been partly inspired by the success of the "Approved Lists of Bacterial
Names" (Skerman et ai., 1980). By that single step and the devalidation of all omitted
names the number of species epithets available for use by bacterial taxonomists was
reduced from about 21,000 to 1,800. Approved names were linked to a revised Code and
procedures established for the registration of newly published names (Sneath, 1986). In
the case of botanical nomenclature, it is not envisaged that names omitted from the Lists
would be devalidated; they would still be available for use so long as existing
requirements for validity and legitimacy were fulfilled, but could not be taken up in
preference to a listed name even if of an earlier date. If adopted, that procedure would in
practice parallel that for the "sanctioning" of names of fungi embodied in the Code since
1981 (Demoulin et al., 1981; Korf, 1982).
It is important to emphasize that the Lists initiatives are concerned with nomenclature
and not taxonomy. Names listed are envisaged as including those necessary to enable
alternative taxonomic schemes to be employed. They can be expected to promote
productive taxonomic work through taxonomists having to spend less time investigating
obscure long-neglected literature and more time collecting data and providing soundly
based schemes. What Cowan said of the former state of bacterial taxonomy applies also to
fungi: "too much attention has been given to nomenclature and too little to the bacteria
themselves, their characters and what they do" (Cowan, 1970, p. 145).
Procedures for the treatment of newly published names are being addressed
separately by a complementary Special Committee on Registration charged with
Problems and prospects for improving the stability of names in Aspergillus and PenicilHum
79
examining various options (Greuter, 1986; Brummitt et al., 1986) also charged with
reporting to the 1993 Congress.
Aspergillus and Penicillium species names are now some of the best catalogued and
typified in the fungi. In the light of the work of the current series of workshops, they are
now approaching consensus systems. They would now be ideal for a pilot study for the
Lists of Names in Current Use initiative, especially in view of their particular economic
importance. The resultant Lists could also be commended for adoption if "sanctioned" by
the International Commission on the Taxonomy of Fungi in the period prior to such
procedures formally being embodied in the Code.
TAXONOMIC INSTABILITY
Problems
Science is progressive, concepts are tested as new data become available, and where
necessary modified to better explain the new observations. Objective taxonomic changes
are therefore to be expected and indeed welcomed by users, Revisions should lead to
clearer understandings of species limits, evolutionary relationships and, more
importantly, increased levels of predictability of functional attributes (e.g. toxin
production, pathogenicity, enzyme and metabolite production, growth parameters).
Inevitably, differences in interpretation of the same data set will occur and often several
stances will be logically justifiable. Variant systems then need testing by new or more
extensive data to see which better fits the evidence. In taxonomy it should not pass
unnoticed that substantial monographs based on large data sets of both specimens and
characters tend to be the most rapidly accepted by peers and also to stand the rigorous test
of time.
Taxonomic instability, in the sense of repeated switching and modification, is
generally associated with poor standards of taxonomic research or independent data sets.
This is especially so when changes are based on studies of few isolates or specimens
(sometimes even wrongly named), and consider very few characters. It is a common
failing of the pioneers of new approaches to consider that their novel techniques or data
sets provides the answer. History generally proves otherwise, and it is prudent to consider
the experience of taxonomists in other groups where particular techniques have already
been employed more extensively (Hawksworth, 1988b).
It would be invidious to single out particular studies in Aspergillus and Penicillium
here, but as a cautionary pertinent example the lichen-forming Ramalina siliquosa (Huds.)
A.L. Sm. may be mentioned. Culberson (1967) recognized six species on the basis of
secondary metabolites. Comprehensive numerical taxonomic studies (Sheard, 1978), and
protein patterns (Mattsson and Karnefelt, 1986) have shown two species to be present, as
already recognized by field observations since the 1860s.
Information on the applicability of some of the newer techniques to mycology is
provided in Jury and Cannon (1989) and Hawksworth and Bridge (1988).
Prospects
In the long-term, the main need for the production of greater taxonomic stability is
funding for major multidisciplinary integrated studies. In the case of the filamentous
fungi, these approaches are being pioneered in Aspergillus (Chapter 5, this volume) and
Penicillium (Bridge et al., 1990), and further through the initiatives of SPAS.
Users are often uncertain as to which of two or more differing taxonomies to follow,
rarely being in a position to assess the merits of each objectively. A case for guidance from
80
D.L.
Hawksworth
international peer groups exists and the ICTF has made a start in this direction. Published
changes are reviewed by the international panel of 11 members that make up the
Commission, and their views published in the series ''Name changes in fungi of
microbiological, industrial, and medical importance" (Cannon, 1986). Authors are less
likely to rush into print if peer reviews can be published widely and rapidly. There have
also been proposals to produce standard lists of names reflecting a single taxonomy
reviewed by internationally appointed peer groups on perhaps a five-yearly basis
(Barnett, 1987). While of great value to users, such proposals remain unpopular as they
could tend to stifle the development of science and delay sound improvements being
widely or quickly adopted. In the case of the generic names of the Ascomycotina, outline
classifications produced since 1982 by an interactive process and revised annually are
proving to be of value (Eriksson, 1989; Hawksworth, 1989; Reynolds, 1989). The twice
yearly journal "Systema Ascomycetum" is devoted to that project, and is being considered
as a model that might be applicable in other groups of organisms.
Most importantly, the standards of training of taxonomists need to be improved.
Mycologists can learn from treatises primarily written for other groups (e.g. Austin and
Priest, 1986; Davis and Heywood, 1963; Heywood and Moore, 1984; Jeffrey, 1982; Stace,
1982) as well as those directed at mycologists (e.g. Hawksworth, 1974). In the case of
Aspergillus and Penicillium, the Recommendations produced by the First International
Penicillium and Aspergillus Workshop (Samson and Pitt, 1985) are of particular value in
promoting good practic; the Second Workshop may wish to consider taking this further.
That Workshop also encouraged the ICTF to prepare a Code of Practice for Systematic
Mycologists which was subsequently drafted, agreed and issued (Sigler and Hawksworth,
1987); Chinese and Russian versions of the ICTF Code have also been prepared in the final
analysis, taxonomic responsibility towards users will remain crucial.
ACKNOWLEDGEMENTS
The proposals made in this paper partly arose from the results of Science and Engineering
Research Counci (SERC) contract no. SO/17/84.
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BRIDGE, P.O., HAWKSWORTH, 0.1., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, R.R.M.,
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DE CANDOLLE, A. 1867. Lois de la Nomenclature Botanique. Paris: Masson and fils.
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problem in the nomenclature of fungi. Taxon 30: 52-63.
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GREUTER,W. 1986. Proposals on registration of plant names, a new concept for the nomenclature of the
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REYNOLDS, D.R 1989. Consensus mycology. Taxon 38: 62-63.
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SHEARD, J.W. 1978. The taxonomy of the Ramalina siliquosa species aggregate Oichenized Ascomycetes>.
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101-105.
SKERMAN, V.B.D., McGOWAN, V. and SNEATH, P.H.A., eds. 1980. Approved lists of bacterial names.
International Journal of Systematic Bacteriology 30: 225-420.
SNEATH, P.H.A. 1986. Nomenclature of bacteria. In Biological Nomenclature Today, eds. W.D.L. Ride and
T. Younes, pp. 36-48. Eynesham, Oxford: IRL Press.
STACE, CA. 1989. Plant Taxonomy and Biosystematics. Second edition. 264 pp. London: Edward Arnold.
VOSS, E.G. et aI., 1983. International Code of Botanical Nomenclature adopted at the Thirteenth
International Botanical Congress, Sydney, August 1981. Regnum Vegetabile 111: 1-472.
van WARMELO, K.T. 1979. Proposals for modifications of the Code of Botanical Nomenclature: IMC2
proposals. Taxon 28: 424-431.
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83
PROPOSALS TO CONSERVE IMPORTANT SPECIES NAMES IN

ASPERGILLUS AND PENICILLIUM
J.e. Frisvad1, D.L. Hawksworth2, Z. Kozakiewicz2, J.I. Pitt3,

e. Stolk4
RA. Samson4 and A.
IDept of Biotechnology
Technical University of Denmark
2800 Lynglly, Denmark
2CAB International Mycological Institute
Kew, Surrey, TW9 3AF, United Kingdom
3CSIRO

North Ryde, N.S. W. 2113, Australia
4Centraalbureau voor Schimmelcultures

Although the Botanical Code has allowed for conservation of generic names for a number
of years, until recently no mechanism existed for protecting important species names. For
example, under the Botanical Code prevailing at the time, Samson et al. (1976) and Pitt
(1979) had no alternative to taking up earlier names for currently accepted species, at least
in cases where it could be shown that well documented neotypes existed. In particular,
Samson et al. (1976) revived P. verrueosum Dierckx for P. viridieatum Westling, while Pitt
(1979) took up P. aurantiogriseum Dierckx 1901 for P. eye/opium Westling 1911 and P.
glabrum <Wehmer) Westling 1893 for P. frequentans Westling 1911. Both Samson et al. (1976;
1977) and Pitt (1979) evaded the issue of the relationship of P. griseoroseum Dierckx 1901
with P. ehrysogenum Thorn 1910, clearly in the hope that a method might eventuate for
saving the obviously threatened P. ehrysogenum. More recent work (Cruickshank and Pitt,
1987) has clearly demonstrated the synonymy of these two species.
In the meantime, a mechanism for conserving threatened specific names has been
added to the Botanical Code. The Sydney Botanical Congress voted to allow conservation
of species where they were "of major economic importance" (Art. 14, Voss et al., 1983).
Consequent on that change, this paper foreshadows a formal proposal to conserve P.
ehrysogenum against P. griseoroseum at the next Botanical Congress. The case for
conservation rests on the importance of P. ehrysogenum as the one species which produces
penicillin, the antibiotic which has revolutionised the treatment of gram-positive bacterial
infections in man and animals. To permit the synonymising of P. ehrysogenum is
unacceptable, indeed unthinkable.
Although recent work has threatened other Penicillium names in current use, e.g. P.
granulatum Bainier 1907, P. claviforme Bainier and P. coneentrieum Samson et al. 1976 (Seifert
and Samson, 1985), these species are of limited importance, and it has been judged
unnecessary, and probably unrewarding, to attempt conservation. Recent taxonomies
have accepted the older names P. glandieola (Oudem.) Seifert & Samson, P. vulpinum
(Cooke & Massee) Seifert & Samson and P. eoprophilum (Berk. & Curt.) Seifert & Samson
respectively (Pitt, 1988; Frisvad and Filtenborg, 1989).
84
J.e. Frisvad et al.
The taxonomy of Aspergillus has not been so thoroughly revised as that of Penicillium in
recent years. However, two obviously threatened names of great importance exist, A. niger
van Tieghem and A. nidulans (Eidam) Winter 1884.
Al-Musallam (1980) pointed out that two older species, A. phoenicis (Corda) Thorn
1840 and A. ficuum (Reichardt) Hennings 1867, accepted by Thorn and Raper (1945) and
Raper and Fennell (1965), were synonymous with A. niger, or at most A. niger represented
a variety of a single earlier species. A clear case exists for conserving A. niger against these
two species, as A. niger is the source of commercial production of citric and other organic
acids around the world, and clearly of major economic importance.
A. nidulans is threatened for a different reason, outlined more fully in the section
below, which relates to the use of holomorphic names in anamorphic genera. The case for
conservation is equally compelling: A. nidulans has been used in a wide range of
significant genetic studies, and adoption of the legitimate name for the anamorph, A.
nidulellus Samson & Gams (1985) would lead only to long term confusion.
Samson and Gams (1985) suggested revival of A. alutaceus Berk. & Curt. 1875 for A.
ochraceus Wilhelm 1877, but later authors (Klich and Pitt, 1988) have not accepted this
change, on the grounds that synonymy of these two species has not been clearly
established. If synonymy becomes accepted, a case for conservation of A. ochraceus may
have to be developed, but the chances of success under the present Botanical Code appear
slim.
Stability in names of Penicillium and Aspergillus may, in the slightly longer term, come
from current proposals to develop lists of protected names (Hawksworth, 1990; see also
the final discussion in these proceedings). However, at this time, conservation of P.
chrysogenum, A. niger and A. nidulans appears to be essential.
The following sections outline proposals which will be placed before the relevant
committees for consideration at the next Botanical Congress in 1993. The formal proposals
will be prepared and submitted for publication in Taxon.
Proposal to conserve the name Aspergillus niger van Tieghem against Aspergillus
phoenicis (Corda) Thorn and A. ficuum (Reichardt) Hennings
The name of the very common and industrially important species Aspergillus niger is
threatened by the older names A. phoenicis (Corda) Thorn and A. ficuum (Reichardt)
Hennings (Al-Musallam, 1980). Conservation of A. niger is proposed because of its great
industrial and economic importance. The earlier names have been used only rarely in
modern publications.
Ustilago phoenicis Corda, Icon. Fung. 4: 9, 1840 = Aspergillus phoenicis (Corda) Thorn, J.
Agric. Res. 7: 14, 1916 = Aspergillus niger var. phoenicis (Corda) AI-Mus allam, Rev. Black
Aspergillus Species: 56, 1980.
Holotype material of U. phoenicis, on fruit of Phoenix dactylifera in PRM. Representative
culture: CBS 126.49 = ATCC 10698 =NRRL 363.
Ustilago ficuum Reichardt, Verh. Zoo1. Bot. Ges. Wien 17: 335, 1867 (issue 1 of volume;
month not determinable exactly). = Aspergillus ficuum (Reichardt) Hennings, Hedwigia 34:
86,1985.
Proposals to conserve important species names of Aspergillus and Penicillum
85
No type material is preserved at W. The identity of this taxon is established by CBS 115.34,
chosen by Raper & Fennell (1965) as a representative isolate. Though the date of the
original publication could not be established with certainty, it appears probable that this
name predates the publication of A. niger by a few months.
Aspergillus niger van Tieghem, AnnIs Sci. nat. Bot., Ser. 5, 8: 240, 1867 (issue 4 of the
volume; month not determinable exactly).
Holotype material is preserved in Pc. Representative culture: CBS 554.65 = ATCC 16888
(designated as "neotype" by Al-Musallam, 1980).
Full descriptions in Raper & Fennell (1965), AI-Musallam (1980), and Klich and Pitt (1988).
A number of additional, later synonyms of A. niger have been compiled by Al-Musallam
(1980).
A. phoenicis was distinguished from A. niger at specific level by Raper & Fennell (1965) and
at varietal level by AI- Musallam (1980), because of smaller, longitudinally striate, slightly
prolate conidia. Recent cryo-SEM pictures of numerous isolates from Aspergillus Sect. Nigri
have shown that striation and tubercles intergrade freely and that in CBS 554.65 a
considerable amount of striation is visible. Thus this distinction represents only a
gradation. Even if the two taxa were separated as varieties within a single species, as AIMusallam (1980) proposed, the nomenclatural consequence is that A. niger must become a
variety of A. phoenicis.
Raper & Fennell (1965) distinguished A. ficuum from A. niger by more rapidly
spreading colonies on Czapek agar and from A. phoenicis by perfectly globose conidia. AlMusallam (1980) included A. ficuum as a synonym of A. niger var. niger.
The taxonomic distances between representative isolates of these taxa were assessed
by AI-Musallam (1980), using cluster analysis involving all available morphological
parameters, after both equal and iterative weighting of characters. Frisvad (unpublished
data) has indicated that A. niger and A. phoenicis possess numerous secondary metabolites
in common, suggesting a lack of separation even at varietal level.
Polonelli et a/. (1985) found that neither immunodiffusion nor two dimensional
immunoelectrophoresis (TDIE) permitted an antigenic differentiation of any of the several
isolates of A. niger, A. phoenicis and A. pulverulentus studied.
Frisvad (unpublished data) also found identical pectinase isoenzyme profiles in the
isolates concerned.
Economic importance.
A. niger is commonly used for the commercial production of citric and other organic acids.
In previous decades it has been used frequently as an indicator for trace elements and in
numerous assays (Domsch et ai., 1980). Its economic value in citric acid fermentation can
be estimated from the fact that world-wide 5850 million litres are produced annually.
Test searches in the BIOSIS databank have shown that in the last decade annually
some 300 references deal with A. niger, 2 with A. phoenicis and 2 with A. ficuum.
86
J.e. Frisvad et al.
Proposal to retain the name Aspergillus nidulans (Eidam) Winter by conservation of

Sterigmatocystis nidulans Eidam with a conserved type specimen
Sterigmatocystis nidulans Eidam, in Cohn, Beitr. BioI. Pfl. 3: 392, 1883; type:- ? France, sine
loc., ex coIl. G. Bainier (IMI 86806 - stat. anam.); typo cons. prop. = Aspergillus nidulans
(Eidam) Winter, Rabenh. Krypt. - FI. 1: 62, 1884.
Aspergillus nidulellus Samson & W. Gams, in Samson and Pitt, Adv. Penicillium and
Aspergillus Syst.: 44, 1985 (1986).
The revision of Art. 59.1 adopted by the XUI International Botanical Congress in 1981
(Voss et al., 1983), endangered many well known species names of anamorphs in
Aspergillus and Penicillium, as already anticipated by Hawksworth and Sutton (1974).
However, that same Congress also amended Art. 14.1 to permit the conservation of
epithets at species rank. Under Art. 14.8, it is further possible to conserve a name with a
type different from that designated by the author or determined by application of the
Code; this provision is not restricted by rank. The familiar fungus Aspergillus nidulans was
first described as Sterigmatocystis nidulans by Eidam (1883). His description included both
the anamorphic and teleomorphic states. His excellent illustrations clearly indicate an
Aspergillus, and the biseriate columnar conidial heads point unequivocally to Aspergillus
nidulans as identified by later authors. Winter (1884) transferred the name S. nidulans to
Aspergillus as A. nidulans (Eidam) Winter, and again the description included both the
anamorphic and teleomorphic states. Later Vuillemin (1927) transferred S. nidulans to
Emericella Berk. & Broome, as Emericella nidulans (Eidam) Vuill. Thus the name
Sterigmatocystis nidulans functions as the basionym for binomials in both Emericella and
Aspergillus.
Under the current Art. 59, names introduced inclusive of both teleomorph and
anamorph are automatically typified by the teleomorph (Art. 59.6) and do not cover the
anamorph alone. Samson and Gams (1985) in addition to the epithet nidulans for the
teleomorph, introduced the new species name Aspergillus nidulellus for the anamorph,
quite correctly under this provision of the Code (Greuter et aI., 1988).
However, Aspergillus nidulans is one of the most familiar names of filamentous fungi
to biologists and in the field of fungal genetics is second only in prominence to Neurospora
crassa. Numerous papers and reviews (approximately 100 per annum) on production,
detection and isolation of mutants, mechanisms of mitotic recombination, arrangement of
alleles and development of chromosome maps, have been published using A. nidulans as
the research tool (e.g. Gossop et al., 1940; Pontecorvo, 1949; Jinks, 1952; Pontecorvo et al.,
1953; Kafer, 1958; Grindle, 1963; Clutterbuck, 1974; Smith and Pateman, 1977). More
recently it has been used as one of the best model systems for the study of eukaryotic gene
expression and its control (e.g. Cove, 1977; Ballance and Turner, 1985; Arst and
Scazzochio, 1985).
Culture collections throughout the world maintain mutant strains of A. nidulans (IMI,
ATCC, CBS, IPO, JCM, NRRL, IMUR, BKMF). Indeed there are many genetics laboratories
which maintain their own A. nidulans genotypes (see Clutterbuck, 1974; 1986), and the
Fungal Genetics Stock Center has 616 genetic variants of this species (Fungal Genetics
Stock Center, 1988).
It has recently been shown that A. nidulans can be genetically engineered to secrete
proteins of bacterial or mammalian origin (Gwynne et al., 1987; Upshall et al., 1987),
potentially a first step towards the use of filamentous fungi for the production of many
important pharmaceutical and industrial processes. It poses an attractive proposition for
the efficient, inexpensive production of valuable and useful proteins. Thus, the name
87
Aspergillus nidulans has been widely used and accepted for more than 100 years by
geneticists and biochemists, as well as by mycologists. The change to A. nidulellus in this
context is highly undesirable, most unlikely to be followed, and will cause confusion and
difficulties in on-line retrieval of data. The above proposal will safeguard this name.
With the proposed typification, the name of the teleomorph cannot be based on
Eidam's epithet. However, in using the epithet in Emericella, Vuillemin (1927: 137) was
clearly basing his decision on the character of the teleomorph. Under Art. 59.6, however,
the name can be regarded as attributed to Vuillemin alone provided that it is neotypified
by the material of the teleomorph, as follows:
Emericella nidulans Vuillemin, C. r. Acad. Sci. Paris 184: 137, 1927; neotype:- ? France, sine
loc., ex colI. C. Bainier (IMI 86806 - stat. teleo.).
The Bainier strain used for the typification of both morphs is that upon which the
diagnosis of this taxon in both its morphs was based primarily by Raper and Fennell
(1965: 497). Further, this same strain was accepted in part as neotype for Eidam's
teleomorphic taxon and in part as holotype for A. nidulellus by Samson and Cams (1985:
44).
Proposal to conserve Penicillium chrysogenum Thorn as the species name for the
principal producer of penicillin.
Penicillium chrysogenum Thorn, Bull. Bur. Anim. Ind. US Dep. Agric. 118: 58, 1910.
Penicillium griseoroseum Dierckx, Annis Soc. Scient. Brux. 25: 86, 1901.
Penicillium brunneorubrum Dierckx, op. cit. 25: 88, 1901.
Penicillium citreoroseum Dierckx, op. cit. 25: 86, 1901
Penicillium notatum Westling, Ark. Bot. 11: 95, 1911
Raper and Thorn (1949) accepted P. chrysogenum and P. notatum as separate species, both
being regarded as penicillin producers. P. citreoseum Dierckx was regarded as a synonym
of P. cyaneofulvum Biourge 1923, and placed in synonymy with P. griseoroseum by Pitt
(1979). Subsequent authors (Samson et a/., 1976; Pitt, 1979; Pitt, 1988) have all accepted P.
chrysogenum, but have placed the other species mentioned, with the exception of P.
griseoroseum, in synonymy.
Raper and Thorn (1949) regarded the description of P. griseoroseum as too meagre to
establish identity and therefore rejected the species; this species, along with P.
brunneorubrum and P. citreoroseum, was also considered doubtful by Samson et a/. (1977).
Pitt (1979) recognised that cultures derived from Dierckx' type of P. griseoroseum still
existed, and on the basis of examination of IMI 92220i retained this species as distinct from
P. chrysogenum, with P. griseoroseum and P. citreoroseum as synonyms. More recently, the
identity of P. griseoroseum with P. chrysogenum has been firmly established by
morphological comparisons (Pitt, unpublished), similar isoenzyme patterns (Cruickshank
and Pitt, 1987) and by identical patterns of secondary metabolites including penicillin,
roquefortine C and meleagrin (Frisvad and Filtenborg, 1989).
Consequently, P. griseoroseum threatens the well-established and economically
important name P. chrysogenum, necessitating conservation against P. griseoroseum. It will
also be necessary to conserve P. chrysogenum against the other Dierckxian names P.
brunneorubrum and P. citreoroseum, considered by Pitt (1979) to be synonyms of P.
griseoroseum.
J.e. Frisvad et al.
88
Economic significance.
P. chrysogenum is the only name for a penicillin-producing species which is in common
use. In past decades the synonymous name P. notatum has also been used extensively in
the penicillin literature, but it has been little used in recent years. Together with a variety
of biological and chemical derivatives, penicillin is still the major fungal compound used
in medicine. A sample search in the BIOSIS data base still produced annual numbers of 56
publications. In addition, P. chrysogenum has been used for the commercial production of
single cell protein and enzyme production. As one of the few common species in
Penicillium subgenus Penicillium which produces no significant mycotoxins, and in view of
its past history of industrial use, further industrial and biotechnological applications can
be anticipated. Stabilisation of the name P. chrysogenum is of great importance.
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AL-MUSALLAM, A. 1980. Revision of the black Aspergillus species. Dissertation, University of Utrecht. 92
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ARST, H.N. and SCAZZOCHIO, e. 1985. Formal genetics and molecular biology of the control of gene
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GRINDLE, M. 1963. Heterokaryon compatibility of closely related wild isolates of Aspergillus nidulans.
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91
NOMENCLATURE STABILITY IN PENICILLIUM AND ASPERGILLUS,

AN ALTERNATIVE VIEW
W.Gams
Centraalbureau VOOT Schimmelcultures
The situation of nomenclatural instability is less deplorable than Hawksworth (1990) has
suggested. As he pointed out, the mere fact that Penicillium and Aspergillus experts meet
and discuss the taxonomy of these genera at regular intervals is the best warranty for
eventual stability.
Hawksworth's main concern is nomenclatural instability. Major changes occurred at
the Sydney Botanical Congress in 1981. The rules of Art. 59 concerning teleomorphanamorph nomenclature were clarified for the first time. Thanks to the efforts of Luella K.
Weresub and G. L. Hennebert, the meaning of the article has become transparent. Liberal
redispositions have become possible and the situation is straight forward. Until 1981 it
was not legitimate to make combinations from anamorphic to teleomorphic genera and
vice versa; now the correctness of such a procedure is only dictated by the kind of fungus
involved. It is exaggerated to state that "teleomorph names are no longer available for
anamorphs", as Article 59 primarily states that teleomorph names are holomorph names,
i.e. they cover the whole fungus, thus also the anamorph. The new ruling implies a small
sacrifice of some Aspergillus and Penicillium names in those cases where ascomata have
been observed from the beginning, if a separate name is found desirable to cover the
anamorph only. In these cases, the expert will use the teleomorph name anyhow in
preference to indicate natural relationships. Use of the same epithets in Aspergillus and
Penicillium is not illegitimate, it is just incorrect, as it comprises the whole fungus and not
only the anamorph, and that is exactly what Thom, Raper and Fennell had intended to do.
A separate name to cover the anamorph only is meaningful in such cases as P. janthinellum
that rarely shows an Eupenicillium javanicum teleomorph, but it has little importance in
other cases where ascomata are regularly found. It is quite likely that the anamorph
epithets introduced by Pitt (1979) and Samson and Gams (1985) will not become popular,
but this is of little concern as the stable teleomorph names are to be used preferentially
anyhow.
Of greater effect on stability are old obscure names. A suggestion that it is good
mycological practice not to resurrect them has been incorporated in the conclusions of the
previous Penicillium workshop, but unfortunately not in the ICTF Code of Practice (Sigler
and Hawksworth, 1987). It is regrettable that even now no ruling exists that efficiently
prevents the resurrection of obscure old names by neotypification. At the Berlin Congress
considerable discussion was spent on Art. 69, that allows rejection of names that have
been widely and persistently used for a taxon or taxa not including the type. A proposal to
remove it from the Code was defeated. While Art. 14.2, newly formulated at Sydney,
92
w. Gams
(conservation of an important name against others) is intended only for species of "major
economic importance", Art. 69 can and should be invoked for many more species.
Long debates have been held at Berlin (Greuter et ai., 1989) on the desirability of indexing
of recognized (listing) and/ or registration of newly published names. The term Indexing
is used for lists like the one in the "Index of Fungi" that are of tremendous value to the
practicing taxonomist. But standard lists now envisaged additionally imply a form of
recognition, or comment on the availability of names for use (comparable to sanctioning).
At Berlin it was quite evident that the great majority of botanists were strongly opposed to
the introduction of any kind of censorship by a registring authority. In the meantime a
special committee appointed by IAPT (Hawksworth and Greuter, 1989) have concluded
that listing of all botanical names is technically feasible by international collaboration and
indeed highly desirable. But there is very little chance that any protected status will be
assigned to registered names at the next Botanical Congress. At present the rules on
fungal nomenclature are made by the International Association of Plant Taxonomy
(IAPT), with a strong input from its Committee for Fungi and Lichens. The IUMS
International Commission on Taxonomy of Fungi (ICTF) is also entering the scene of
taxonomy, but it would be detrimental to taxonomy if the two organizations developed
divergent nomenclatural ideas.
Indexing is very useful, and should be supported on a worldwide basis. On-line
accessible databases are the technical solution to the problem of complete documentation
on all available names. It will also serve nomenclatural stability if the status of all names,
including obscure ones, is elucidated once and for ever. But freezing nomenclature to the
status quo for the sake of stability does no justice to the historical development of our
science; in fact this would mean abolishing all of the philosophy of nomenclature so far
prevailing in taxonomy.
At a Fusarium workshop at Sydney in 1983, Nelson and his colleagues (see Nelson et
al., 1983) proposed recognizing Wollenweber and Reinking (1935) as the starting point for
Fusarium nomenclature. This proposal did not find the support of the community of
experts. Similarly in Penicillium and Aspergillus it would not make sense to sanction the
names used by Thom, Raper, and Fennell at the present day, where dramatic progress is
made in understanding taxonomic structures with a whole range of modern experimental
techniques.
At present the taxonomy of many taxa is under review at both the specific and
infraspecific level. I would like to recommend that in this situation it is better not to
formalise immediately every discovery of taxonomic relevance, such as creation of
infraspecific taxa, new combinations etc., but to wait for support from other related
studies. Self-restraint, especially in the context of resurrecting doubtful old names, cannot
be recommended strongly enough.
Great pressure, some of which appears justified, is now being exerted on taxonomists
to stabilize names in use. But in the present situation it is more important to tell outsiders
patiently what is going on and why all this is necessary rather than to yield to pressure by
adopting standardized names. It is quite likely that considerable consensus on many
Aspergilli and Penicillia will emerge in the next 10-20 years that cannot be anticipated
now. For the time being we must make use of the provisions of the Botanical Code to
conserve important names, as is being considered elsewhere in this Workshop.
With thanks to Prof. D. L. Hawksworth for some improvements, though opinions still
diverge.
Nomenclature stability in Penicillium and Aspergillus
93
REFERENCES
GREUTER, W., MCNEIL, J. and NICOLSON, D. H. 1989. Report on botanical nomenclature - Berlin 1989.
Englera 9: 1-228.
HAWKSWORTH, D.L. 1990. Problems and prospects for improving the stability of names in Aspergilus and
Penicillium. In Modem Concepts in Penicillium and Aspergillus Classification, eds. R.A. Samson and J.1.
HAWKSWORTH, D. L. and GREUTER, W. 1989. Report of the first meeting of a working group on lists of
names in current use. Taxon 38: 142-148.
NELSON, P. E., TOUSSOUN, T. A. and MARASAS, W. F. O. 1983. Fusarium Species, an Illustrated Manual
for Identification. University, Park, Pennsylvania: Pennsylvania State University Press.
PITT, J. I. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London:
Academic Press.
SAMSON, R. A. and GAMS, W. 1985. Typification of the species of Aspergillus and associated teleomorphs.
In Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.!. Pitt, pp. 31-54. New
SAMSON, R. A., STOLK, A. C. and HADLOK, R. 1976. Revision of the subsection Fasciculata of Penicillium
SIGLER, L. and HAWKSWORTH, D. L. 1987. International Commission on the Taxonomy of Fungi (ICTF)
code of practice for systematic mycologists. Mycopathologia 99: 3-7.
WOLLENWEBER, H. W. and REINKING, O. A. 1935. Die Fusarien. Berlin: P. Parey.
94
DIALOGUE FOLLOWING THE PRESENTATIONS BY PROFESSORS HAWKSWORTH

ANDGAMS
HAWKSWORTH: The movement for stabilization of names comes from the International
Union of Biological Sciences (lUBS). It's not really a matter of mycologists isolating
themselves from botany, it's a matter of botanists isolating themselves from biology, both
for nomenclaturalists and users of taxonomy. The work for some botanical groups is
very advanced, for example with the flowering plants. A draft list of generic names in
use for flowering plants will go out this autumn. The moss and hepatic lists will be being
debated by the International Biological Congress in St. Louis later on this summer. The
draft algal list should be finished by the end of the year. The list of fungal genera should
be available at the Fourth International Mycological Congress in Regensberg in 1990.
GAMS: I have no word against the importance of the name Aspergillus nidulans. In fact, we
were reluctant to propose the new name Aspergillus nidulellus. However, I am uneasy
about that conservation proposal. Is there a need to split the anamorph and the
teleomorph in this fungus? All the authors who are talking about Aspergillus nidulans are
in fact talking about the teleomorph, Emericella nidulans. It is not as if the name "nidulans"
is unavailable for the fungus. It's still the right epithet for the holomorph. I doubt that
this proposal will be successful.
NIRENBERG: I think that we are all concerned about what nonspecialists feel about all the
name changes with fungi. Students, plant pathologists, and technical assistants have
problems understanding why we have two different species epithets in one fungus when
we talk about the teleomorph and the anamorph. Why can't we change the rules in the
Code? It would be a lot easier to use the same epithet for the anamorph in the
teleomorph, and just use the oldest one. This would solve many problems.
HAWKSWORTH: The fact is that mycologists went off in their own direction a long time
ago. Phycologists don't have this problem. We have to live with history, or it becomes
too destabilizing now. If we were starting again I would agree with you. Is anyone, apart
from Dr Gams, against the proposal on Aspergillus nidulans?
SAMSON: Do the geneticists have any feeling about this? Is it necessary to conserve
Aspergillus nidulans? You still have the epithet nidulans for the teleomorph. It's not quite
correct according to the rules to use it for the anamorph, but who cares?
MULLANEY: For geneticists, this fungus is just a tool, and I don't think they are too
concerned about why the fungus has this name.
SAMSON: French cheese makers still call Penicillium camemberti by an old name, P.
candidum. Everybody knows what is meant by that name. So, it may be unnecessary to
make such a complicated proposal to protect this name. Penicillium chrysogenum and
Aspergillus niger are different because all mycologists use these names and both are clear
synonyms of earlier names. The formal proposals are necessary for these two cases.
PITT: I agree with Dr Samson. If we do not conserve these names, we are bound to run into
problems later on. But it may also be worth trying A. nidulans as a test case.
(At this point, the proposals were voted upon by the participants. The proposals for
Aspergillus niger and P. chrysogenum received unanimous support. There was one vote
against the A. nidulans proposal).
95
HA WKSWORTH: Does the group wish to make any comments on the problem of
resurrecting old names in Penicillium and Aspergillus? Does the group wish to produce a
list of names that might be later granted protected status, because we are producing a
more stable taxonomy? Without making any final taxonomic judgements, we can make
a list and suggest that these are the names that people should be considering.
SAMSON: You can't just reject a name when there is a very good holotype specimen
available. In Penicillium, there probably are not too many older names that need to be
considered, but in Aspergillus there may be quite a number.
HAWKSWORTH: Linnaeus did this, Fries did this, and there is a general feeling that it may
be time to do this again.
GAMS: This really is relevant to Article 69 cases about which there has been a lot of
misunderstanding. The cases that really concern me are those where no holotype exists.
We should confine our discussion to these cases.
HAWKSWORTH: What is there to discuss? The reality is that people do sometimes take up
old names by neotypification when they should be trying to keep the established name.
This hasn't happened so much with Penicillium and Aspergillus in recent times.
Particularly with generic names, it's clear you should follow the conservation route. The
danger is that if taxonomists and nomenclaturalists don't take this matter into hand
themselves, they will find that other bodies will have done it. For example, a major list
being produced of arthropods of economic importance will be used by all the indexing
services and people preparing reference work worldwide. It's happening with seed
testing, it's happening with plant conservation, and it's the way of the world. The
pressure is very much from the information scientists and legislators, not the
taxonomists and nomenclaturalists. We can't risk isolating ourselves continually from the
users if we want to be funded.
CHRISTENSEN: The main advantage is that it protects against the loss of valuable
information now on hand. If the name changes, we may lose this information when
people forget about the synonymy in years to come. As an ecologist, this worries me.
HAWKSWORTH: This is true.
CHRISTENSEN: As a body of responsible biologists, we should consider this as the primary
rationale for our proposals.
SAMSON: But how much value can you give to this old information? Many of these reports
on particular Penicillium and Aspergillus species are undoubtedly based on
misidentificatons. For many years, the species were not well characterized. There is a
burden of literature with incorrect identifications and this causes a lot of confusion.
HAWKSWORTH: Perhaps we in mycology should think about rejecting a lot more names.
PITT: I have divided loyalties. With Aspergillus and Penicillium, that the best thing we can
do right now is to do the groundwork to stabilize the names. We have made
extraordinary progress in the past four or five years, so perhaps we should be thinking
about producing a list of acceptable names if we have another workshop. There may
very well be consensus on many of these names in four years' time. At that time, I would
like to see this kind of proposal seriously considered by a group such as this.
96
HAWKSWORTH: Who should have the responsibility of compiling such a list that could be
ratified by a third workshop? Perhaps this is the function of the Subcommission on
Penicillium and Aspergillus Systematics. By the time it next meets, we will know what
decisions have been made at the 1993 Botanical Congress concerning such lists.
GAMS: What Dr Pitt may envisage is a publication in 4 or 5 years, perhaps not an
authoritative monograph, but a list of all published names with remarks on their status,
that people will use, without having any nomenclatural status of sanctioning. Penicillium
and Aspergillus taxonomy is obviously more advanced than that of many other fungal
genera. The list concept will at that time not be suitable for many other genera.
HA WKSWORTH: There are large numbers of fungal names that are not catalogued
anywhere, and I suspect that there may also be such names in Aspergillus and Penicillium.
Recently, a zoologist colleague sent me a publication on insects from Greenland with a
list of fungal genera in the appendix, and requested a list of current equivalent names.
There were about 12 genera, none of which had ever been indexed in any fungal
publication.
Final Discussion on the conservation of names.
On Aspergillus nidulans.
GAMS: I agree with the idea of the original CMI proposal that something must be done to
save the use of Aspergillus nidulans. Certainly, geneticists will continue to use this name
whether or not it is formally conserved. Neither the present situation nor conservation is
satisfactory. The name Aspergillus nidulans is used by many authors; this is not quite
correct because it is a holomorph that belongs to Emericella. Editors of reputable journals
are uncertain whether they should impose the use of Emericella nidulans on authors or
not. If Aspergillus nidulans is conserved as proposed, it will have to be done with the
explicit exclusion of the teleomorph. Then, the future user would have an available and
correct name, Aspergillus nidulans, but using this binomial explicitely excludes the
teleomorph. This is not usually the intention. The original description of Eidam, under
the Aspergillus name, included a description of the ascomata. It is difficult to see how we
can now typify this described ascomycete only by its anamorph.
HAWKSWORTH: We're well aware of these problems, but the reality is that people will
continue to use the name Aspergillus nidulans. Therefore, there will still be a
nomenclaturally correct name available for the teleomorph if people wish to use one. The
proposal protects that.
GAMS: I'm not too concerned about a slightly incorrect usage. Databases will have to be
constructed to cope with synonyms anyway. But perhaps editors of journals will be quite
concerned about what to do with such names. Editors of most mycological journals do
not usually impose the use of correct names on their authors.
HENNE BERT: I think it is like a nickname and a proper name. Aspergillus nidulans can be
like a nickname for this anamorph and it won't cause any confusion.
GAMS: But to have a nickname, we can stay with the status quo.
PITT: Even if the proposal is not strictly in accordance with the Code, I think we have to do
something to save this name. This appears to be the simplest route. Several years ago, Dr
Hawksworth and Dr Sutton unsuccessfully attempted to save some of the common
97
names in Aspergillus and Penicillium that included teleomorphs in the protologue. There
is a case here for trying a different approach.
HAWKSWORTH: It would be useful to have this debated in the larger community. After all,
we still can't get people to use names like Acremonium; users still call it Cephalosporium.
The Fusarium-people also have similar problems. Fusarium has te1eomorphs in Gibberella,
but these names are not consistently cited either.
Subcommission on Penicillium and Aspergillus Systematics (SPAS).
HENNEBERT: I would be interested to learn more about the Subcommission on Penicillium
and Aspergillus Systematics (SPAS). Who are the members of this commission? What are
their aims and what are the results? Are there special publications produced?
PITT: SPAS is an organization independent from this workshop. It is currently a
subcommission of the ICTF, the International Commission on the Taxonomy of the
Fungi, which is under the auspices of the Mycology Division of the International Union
of Microbiological Sciences (IUMS). SPAS is a collection of experts governed by the rules
of ICTF and the Mycology Division of IUMS. The aim is to promote the improvement in
and dissemination of information on the nomenclature and taxonomy of Penicillium and
Aspergillus. Selection of members is by the subcommission itself. The present members
are myself as chairman, Dr Klich as secretary, and Dr Samson, Dr Frisvad, Dr
Cruikshank, Dr Mullaney, Mr. Williams, and Dr Onions who is retiring. The
subcommision was formed after the first Penicillium and Aspergillus workshop when it
became clear that some kind of multidisciplinary working group was necessary. Our
work so far has related to clarifying the taxonomy of certain groups of species in
Penicillium where a concensus approach is desirable. Two projects are presently
underway. The first is a study of P. glabrum, P. spinulosum and some closely related taxa.
The second concerns some species in subgenus Biverticillium where both taxonomic and
nomenclatural problems existed. Our work will obviously expand as a result of the
resolutions proposed at this meeting.
Recommendations.
1. That SPAS be encouraged to produce a list of names in Aspergillus and Penicillium in current use,
together with place of publication, dried types or ex-type isolates, for confirmation at the next
Penicillium and Aspergillus workshop, with a view to these being incorporated into the IUBSIIAPT
''List of Names in Current Use" project and granted some special, protected status subject to the
decision of the next International Botanical Congress.
GAMS: I follow your proposal, until the protected status is mentioned. This idea divides
biologists. Would such a list ever be officially recognized? If we include this aim in the
proposal, it may hamper the progress of the project. Personally, I cannot support the idea
of sanctioning names from such a list.
SAMSON: You don't have to sanction anything for this list. We would consider the list at
the next workshop.
HAWKSWORTH: Perhaps this list could be published as part of the proceedings of the next
Penicillium and Aspergillus workshop?
PITI: And if we don't like it at that time, it can just be discarded, after open discussion.
98
HENNEBERT: Is there any precedent for such a list being granted protected status?
HAWKSWORTH: Not yet. Other sample lists are being prepared at the behest of the ruBS
that will be proposed for protected status at the International Botanical Congress in 1993.
One is being made for the Leguminosae on a regional basis around the world. In fact,
ICSU, the International Council of Scientific Unions, the parent body of ruBS, has a body
called CODATA which has a project MSDN, the Microbial Strain Data Network. Last
September, they set up a committee to examine standardization of terminology and
nomenclature in biology. These are mainly information scientists and they have the
funding to do this work. Either the scientists who understand organisms do the work, or
they may get left behind. The CABI Thesaurus includes numerous plant pathogen
names, for example, that is used as a standard by the National Agricultural Library in
the US.
HENNEBERT: Do these lists actually exclude names, such as synonyms? Do you exclude
names of species found only once? What do you call current use?
HAWKSWORTH: The idea is to include any name that people feel might be useful. It isn't a
taxonomic exercise, it is a nomenclatural exercise. If there were differing views on

particular species complexes, you would list all the names that people might choose to
use. Names that had been considered synonyms and were long accepted as such would
not be included. The effect of this would be to reduce the number of names that people
have to consider. We do not intend to follow the bacteriologists in devalidating names
that already exist. You could use names not in the list provided they do not conflict with
names included in the list. It would protect us from the problem of people resurrecting
names by neotypification. It would also save us the problem we now face of having to
conserve names such as Penicillium chrysogenum and Aspergillus niger.
HENNEBERT: Is the rank of the name protected? For example, could the varietal names in
Neosartorya fischeri be raised to species?
HAWKSWORTH: Yes, of course. It is not intended to fossilize taxonomy.

PITT: Would
dropped?
anyone care to second Dr Gams' proposal that the "protected status" clause be
There were no seconders to Dr Cams' proposal. The proposal as given above was accepted by all the
participants; with two dissenters.
2. That SPAS prepare a list of isolates available from service culture collections that can be used as
standards for the TLC detection of secondary metabolites of value in species separation in Penicillium.
This resolution was unanimously accepted.

Miscellaneous.
HAWKSWORTH: I think we should encourage work on the genetics of species in these two
genera so that we will be able to better understand the basis of variation. We don't know
anything about chromosome numbers in these genera, for example. Electrophoretic
methods are now available for separating chromosomes.
99
CHRISTENSEN: I strongly support this. Genetic variation is basic.

SAMSON: We taxonomists should really try to help the geneticists, and make sure they are
working with properly identified isolates.
These sentiments were endorsed by the workshop, but no formal wording was proposed.
4
TAXONOMIC SCHEMES OF PENICILLIUM
103
SPECIATION AND SYNONYMY IN PENICILLIUM SUBGENUS

PENICILLIUM - TOWARDS A DEFINITIVE TAXONOMY
J.I. Pittl and R.H. Cruickshank2

lCSIRO Division of Food Processing
2Department of Agricultural Science

University of Tasmania,
Hobart, Tas. 7000, Australia
SUMMARY
Until a few years ago, taxonomy of asexual, haploid fungi such as Penicillium relied primarily on
morphological or gross physiological characters. Although, in Penicillium, a large measure of
agreement existed, in the final analysis speciation depended on the concepts of the individual
taxonomist. The resulting lack of agreement was nowhere more obvious than in Penicillium subgenus
Penicillium. Recent developments have changed all this. In particular, the introduction of simple
techniques for studying secondary metabolites and improved methods for distinguishing isoenzymes
by gel electrophoresis have resulted in independent methods for assessing species concepts.
Backed by accurate identifications using traditional methods, careful studies using secondary
metabolite profiles and isoenzyme patterns have produced remarkably consistent results both within
and between species. Correlations are so clear that it can now be stated confidently that we are
approaching a definitive taxonomy for subgenus Penicillium.
Of course some new species can be expected to be discovered as unfamiliar habitats are explored,
for example, new species and varieties described from seed stores of marsupials on the U.S. prairies.
Leaving these latter aside because their status has not yet been fully assessed, some 25 well defined
species can be distinguished in subgenus Penicillium.
This paper sets out species in subgenus Penicillium as currently conceived by the authors. For each
species a diagnosis is given, and a list of synonyms where these have been confirmed by studies on
living cultures. Notes on species concepts, ecology and mycotoxin production are also given.
INTRODUCTION
In introducing subgenus Penicillium, Pitt (1979) defined it as including species producing

terverticillate penicilli, and typified it by P. expansum, the type species of the genus. In this
subgenus, he brought together species previously classified by Raper and Thom (1949) in
sect. Divaricata subsects. Lanata, Fasciculata, Funiculosa (in part) and Velutina (in part). This
appeared to provide for a more natural classification. In so doing, he placed in synonymy,
or renamed, 22 species previously recognised by Raper and Thom (1949). However, 17 of
Raper's species remained essentially unaltered, although three of these had to be renamed
to conform with the Botanical Code.
The system devised by Pitt (1979) contrasted rather sharply with the speciation
introduced by Samson et al. (1976) in which 17 species of Penicillium subsect. Fasciculata as
defined by Raper and Thom (1949) were reduced to 8 species and 5 varieties.
Ramirez (1982) reclassification of the genus closely followed the taxonomy of Raper
and Thom (1949) and added little to the picture. However, the differences between Pitt
(1979) and Samson et al. (1976) led to considerable confusion (e.g. Onions et al., 1984). The
major problem lay with the differences in circumscription of species, and that in turn was
J.I. Pitt & R.H. Cruickshank
104
due to the absence of objective methods for judging species concepts in this asexual,
haploid genus.
During the decade since, the situation has changed dramatically. New techniques
have emerged which have enabled the delimitation of species by techniques quite
unrelated to the morphological and gross physiological criteria used by Pitt (1979) or other
contemporary taxonomists. Two techniques in particular emerged: the use of secondary
metabolites and of isoenzymes as taxonomic criteria. Following on initial work by Ciegler
et al. (1973, 1981), Filtenborg and Frisvad (1980) developed the study of secondary
metabolites in Penicillium, and in subgen. Penicillium in particular (Frisvad and Filtenborg,
1983). Although in early work a high degree of correlation between species and secondary
metabolites was often not apparent, refinements in species identifications (e.g. EI-Banna et
al. ,1987) helped to overcome this problem.
At the same time, studies on electrophoretic patterns (zymograms) of certain enzymes
were being undertaken: again a high correlation between species and specific zymograms
was discovered in Penicillium subgen. Penicillium (Cruickshank and Pitt, 1987a, b).
Table 1. Classification of Penicillium Subgenus Penicillium
Section Penicillium
Series Expansa Raper & Thorn ex
Fassatiova
P. atramentosum Thorn
P. chrysogenum Thorn
P. coprophilum (Berk. & Curt.) Seifert &
Samson
P. expansum Link
Series Viridicata Raper & Thorn ex Pitt
P. aethiopicum Frisvad et al.
P. allii Vincent & Pitt
P. aurantiogriseum Dierckx
P. commune Thorn
P. crustosum Thorn
P. echinulatum Raper & Thorn ex
Fassatiova
P. hirsutum Dierckx
P. roqueforti Thorn
P. viridicatum Westling
Series Camemberti Raper & Thorn ex Pitt
P. camemberti Thorn
Series Urticicola Fassatiova

P. brevicompactum Dierckx
P. glandicola (Oudemans) Seifert &
Samson
P. griseofulvum Dierckx
P. OOrdei Stolk
P. solitum Westling
P. verrucosum Dierckx
Section Cylindrospora Pitt

Series Italica Raper & Thorn ex Pitt
P. italicum Wehmer
P. digitatum (Pers.:Fr) Sacc.
P. fennelliae Stolk
Section Coronatum Pitt

Series Olsonii Pitt
P. olsonii Bainier & Sartory
Section Inordinate Pitt

Series Arenicola Pitt
P. arenicola Chalabuda
The superimposition of the information obtained from secondary metabolite patterns and
zymograms on the more traditional taxonomic bases derived from morphology and gross
physiology have shown a dramatic correlation: a correlation so clear cut that it can now be
confidently stated that we are very close to a total comprehension of the biological species
currently known to exist in subgen. Penicillium. This situation would have been
unthinkable at the beginning of this decade. At the same time, this high correlation
showed that morphological and gross physiological properties were effective taxonomic
Speciation and synonymy in Penicillium Subgenus Penicillium
105
features. The major species conceived by Raper and Thorn (1949) and emended slightly, or
where necessary renamed, by Pitt (1979) have been sustained by the more recent evidence
from unrelated sources.
This paper is designed to clarify the taxonomy of subgen. Penicillium. Table 1 provides
a summary of our current thinking, showing sectional classification and accepted species.
The text which follows lists species in alphabetical order, and a list of synonyms. Apart
from a few obligate synonyms, all those included have been grown in culture and
identification established by enzyme electrophoresis. The majority are as listed by Pitt
(1979), but a number of corrections are included. A diagnosis of each accepted species
follows, together with supplementary information on changes in species concepts,
ecology, important secondary metabolites and mycotoxins. The listing of the latter is
intended to cover major metabolites only.
It is believed that the listing in Table 1 and below provides a definitive listing of the
currently known and accepted species. Subdivision of a number of these species into
varieties or chemotypes (Pitt and Hawksworth, 1985) for specialist purposes is possible,
especially if secondary metabolite production is taken as a primary criterion for
speciation. The discovery of further species in Penicillium subgen. Penicillium is to be
expected as more diverse habitats are examined, e.g. the isolates from desert kangaroo rat
and other habitats in the U.S.A. (Frisvad et al., 1987) which have not yet been fully
evaluated. Nevertheless the major speciation now appears to be in place.
MATERIALS AND METHODS
In the course of this study, cultures of types or neotypes of all species of subgen.
Penicillium known to exist in the world's major culture collections were examined. Other
"authentic" cultures from major studies were included. The methods used were those of
Pitt (1979) and Cruickshank and Pitt (1987a, b). A feature of the zymogram technique is
that it was able to effectively classify most old and morphologically deteriorated cultures,
including a number which Pitt (1979) and, earlier, Raper and Thorn (1949) had found great
difficulty in assigning to species. Such a result had been predicted by Paterson and
Hawksworth (1985).
Other major sources of data used to draw the taxonomic conclusions given below
included the studies of species mycotoxin relationships reported briefly by EI-Banna et al.
(1987) and published data of Frisvad (Frisvad and Filtenborg, 1983; Frisvad, 1985, 1986).
Penicillium aethiopicum Frisvad et al. 1989
Colonies growing quite rapidly, texture velutinous to fasciculate; conidia blue
on CYA, dark green on MEA; exudate clear to pale brown, soluble pigment brown;
reverse on CYA orange yellow to yellow brown, on MEA yellow grey. Stipes on CYA
smooth, on MEA usually rough; penicilli terverticillate; conidia spherical to ellipsoidal,
smooth walled. Sometimes growth at 37C. The most distinctive feature of this species is
the presence of definite yellow to brown reverse colours on CYA and MEA.
Morphologically it is best described as intermediate between P. aurantiogriseum and P.
DIAGNOSIS.
viridicatum.
Originally considered to be an unusual P. viridicatum, P. aethiopicum possesses

distinctive secondary metabolite profiles (Frisvad, 1986, as P. viridicatum IV; EI-Banna et
al., 1987, as Sp 1448). The species is also culturally distinct.
CONCEPT.
106
J.1. Pitt & R.H. Cruickshank
P. aethiopicum produces viridicatumtoxin and griseofulvin (Frisvad, 1986; ElBanna et al., 1987).
ECOLOGY. According to Frisvad (unpublished data), this species is primarily found in
African soils.
DESCRIPTION. Frisvad et al. (to be published).
MYCOTOXINS.
Penicillium allii Vincent & Pitt 1989

DIAGNOSIS. Colonies relatively large, velutinous to distinctly coremial; conidia dull green;
exudate and soluble pigment pale yellow; reverse dark brown. Stipes very rough,
usually short, penicilli usually terverticillate, often with rami and metulae rough walled;
conidia spherical and smooth walled.
CONCEPT. P. allii is a recently described species (Vincent and Pitt, 1989). It was recognised
as distinct both morphologically and because of its unique enzyme electrophoretic
patterns (Cruickshank and Pitt, 1987b, as Penicillium sp.).
MYCOTOXINS. Secondary metabolite production has not yet been studied.
ECOLOGY. The known isolates of P. allii have come from the Middle East, and are associated
with garlic (Allium species).
DESCRIPTION. Vincent and Pitt (1989)
Penicillium arenicola Chalabuda 1950
Penicillium canadense G. Smith 1956
Colonies of variable size, texture velutinous to floccose; conidia pale brown;

soluble pigment red brown; reverse brown to dark brown. Stipes smooth to rough,
penicilli terverticillate to irregular, sometimes with 4 or 5 branch levels; conidia spherical
to subspheroidal, smooth to rough.
CONCEPT. The concept of this species remains unaltered from the original.
MYCOTOXINS. Secondary metabolite production is unreported.
ECOLOGY. P. arenicola has been isolated only from soil. It has been reported to be highly
tolerant of copper.
DESCRIPTIONS. Pitt (1979); Pitt (1988).
DiAGNOSIS.
Penicillium atramentosum Thom 1910

DIAGNOSIS. Colonies growing moderately rapidly, texture velutinous; exudate, soluble
pigment and reverse colours on CYA reddish brown. Stipes smooth, penicilli
terverticillate, delicate; conidia spherical, smooth walled.
CONCEPT. The concept of P. atramentosum has remained unaltered since first described.
MYCOTOXINS. No significant secondary metabolites have been reported.
ECOLOGY. This is a rarely reported species, without known significance.
DESCRIPTION. Pitt (1979).
Penicillium aurantiogriseum Dierckx 1901
Penicillium aurantiocandidum Dierckx 1901
Penicillium puberulum Bainier 1907
Penicillium cyclopium Westling 1911
Penicillium brunnioviolaceum Biourge 1923
Penicillium porraceum Biourge 1923
Penicillium martensii Biourge 1923
Penicillium lanoso-coeruleum Thorn 1930
Penicillium carneolutescens G. Smith 1939
Penicillium viridicyclopium Abc 1956
107
Penicillium verrucosum var. eye/opium (Westling) Samson et al. 1976

Penicillium eyelopium var. aurantiovirens (Biourge) Fassatiova 1977
Penicillium solitum, regarded as a synonym of P. aurantiogriseum by Pitt

(1979), is now accepted as a distinct species (Cruickshank and Pitt, 1987b; Pitt and Spotts,
in prep.) P. puberuIum, regarded as a separate species by Pitt (1979), has been placed in
synonymy (Cruickshank and Pitt, 1987a, b); many isolates placed in P. puberulum by Pitt
(1979) are now assigned to P. commune (Pitt et al., 1986).
DIAGNOSIS. Colonies of moderate growth rate, texture velutinous or granular to fasciculate;
conidia blue or grey blue on both CYA and MEA; exudate clear to pale brown, soluble
pigment brown to reddish brown. Stipes smooth or at most very finely roughened on
both CYA and MEA; penicilli terverticillate, a proportion biverticillate in some isolates;
conidia subspheroidal to ellipsoidal, smooth walled. The primary distinguishing features
of this species are the distinctly blue conidial colour and smooth stipes, on both CYA and
MEA.
CONCEPT. The concept of this species, established under the name P. cyclopium by Raper
and Thorn (1949) has been reestablished in recent years (Cruickshank and Pitt, 1987b; Pitt
1988), though in a slightly broader sense. Samson et al. (1976) considered this taxon to be
a variety within their broad species P. verrucosum. Pitt (1979) rejected this, accepted P.
cyclopium as a concept, but took up the earlier name P. aurantiogriseum, and broadened it
by the inclusion of P. martensii, P. commune and P. solitum. Later studies (Cruickshank
and Pitt, 1987a, b; EI-Banna et al., 1987; Polonelli et al., 1987) showed that Pitt (1979) had
confused P. commune with P. puberuIum, and that both P. commune and P. solitum were
separate species. Removal of these species from the concept of Pitt (1979) and Williams
and Pitt (1985) has produced a well defined species (Pitt et al., 1986; Pitt, 1988).
MYCOTOXINS. The principal toxins produced by P. aurantiogriseum are penicillic acid and
verrucosidin (S-toxin; Frisvad, 1986; EI-Banna et al., 1987).
PROBLEMS. The interface between P. aurantiogriseum and P. viridicatum is unclear.
Morphological differences do not tally completely with differences in mycotoxin
production. However, this is a minor difficulty which can be resolved by further study.
ECOLOGY. This is a ubiquitous species, especially in cereals, but also in other foods of a
very wide variety, including fresh and stored fruits and vegetables and fresh and
processed meats (Pitt and Hocking, 1985). P. aurantiogriseum is also the cause of "blueeye" disease of corn in the U.S.A. (Ciegler and Kurtzman, 1970). It is much less
commonly isolated from sources other than foods.
DESCRIPTIONS. Pitt et al. (1986); Pitt (1988).
OTHER SYNONYMS.
Penicillium brevicompactum Dierckx 1901
Penicillium griseobrunneum Dierckx 1901

Penicillium stoloniferum Thorn 1910
Penicillium hagemii Zaleski 1927
Penicillium patris-mei Zaleski 1927
Penicillium brunneostoloniferum Abe ex Ramirez 1982
Penicillium volgaense Beljakova & Mil'ko 1972
Colonies slowly growing, especially on MEA, texture velutinous; conidia green;

exudate clear to reddish brown; soluble pigment reddish brown; reverse yellowish to
reddish brown. Stipes long and broad, smooth to finely roughened, penicilli short and
broad, mostly terverticillate, sometimes quaterverticillate, biverticillate or irregular;
metulae usually apically inflated; conidia usually ellipsoidal, smooth to finely
roughened. P. brevicompactum is a distinctive species, with slower growth on MEA than
DIAGNOSIS.
108
G25N, velutinous texture, green conidia, large usually smooth walled stipes, inflated
metulae and very broad penicilli.
CONCEPT. Pitt (1979) placed P. stoloniferum, a species recognised by Raper and Thorn (1949)
primarily because of "arachnoid" margins, in synonymy with P. brevicompactum.
Otherwise the concept of this species remains as Raper and Thorn (1949) established it.
MYCOTOXINS. The major metabolite produced by this species is brevianamide A, not a
compound of significant toxicity, but a very useful taxonomic marker.
ECOLOGY. Both a food spoilage fungus and an agent of biodeterioration, P. brevicompactum
has been isolated from a very wide variety of habitats, especially dried foods and
decaying vegetation. It is also a weak pathogen on fruits and vegetables (Pitt and
Hocking, 1985).
DESCRIPTIONS. Pitt (1979); Domsch et al. (1980); Pitt and Hocking (1985); Pitt (1988).
Penicillium camemberti Thorn 1906
Penicillium biforme Thorn 1910

Penicillium amdidum Roger upud Biourge 1923.
Colonies slowly growing, deeply floccose; conidia white or pale grey green;
exudate clear; reverse pale, yellow, reddish brown or purple. Stipes smooth or rough,
penicilli terverticillate or irregular; conidia subspheroidal to spherical, smooth walled.
The primary distinguishing features of this species are the persistently white to pale grey
conidia, deeply floccose texture, and recovery only from cheese and cheese related
habitats.
CONCEPT. Raper and Thorn (1949) distinguished P. camemberti from P. caseicola by the
production of grey rather than white conidia. Samson et al. (1977b) and Pitt (1979) placed
P. caseicola in synonymy. Otherwise the concept of P. camemberti is much as first
described.
MYCOTOXINS. It is remarkable that all tested strains of P. camemberti consistently produce
cyclopiazonic acid (EI-Banna et al., 1987). It is equally remarkable that many isolates of
Aspergillus oryzae, another domesticated species, also produce cyclopiazonic acid.
ECOLOGY. As pointed out by previous authors, P. camemberti is a domesticated fungus,
used in cheese manufacture, and is virtually unknown from other sources.
DESCRIPTIONS. Samson et al. (1977b); Pitt (1979); Pitt and Hocking (1985); Pitt (1988).
DIAGNOSIS.
Penicillium chrysogenum Thorn 1910
Penicillium nota tum Westling 1911

Penicillium fluvidomarginatum Biourge 1923
Penicillium rubens Biourge 1923
Penicillium me/eagrinum Biourge 1923
Penicillium chlorophaeum Biourge 1923
Penicillium camerunense Heirn 1949
Penicillium aromaticum forma microsporum Rornankova 1955
Penicillium harmonense Baghdadi 1968
Growth at 25C rapid, absent or occasionally slight at 37C; texture velutinous

to floccose; conidia blue or blue green; exudate, soluble pigment and/or reverse usually
yellow. Stipes smooth, penicilli usually terverticillate; conidia smooth walled, usually
ellipsoidal.
CONCEPT. This species has always been accepted. The concept was broadened marginally
from that of Raper and Thorn (1949) by Samson et al. (1977a) and Pitt (1979) to include P.
nota tum and P. meleagrinum.
DIAGNOSIS.
109
Cruickshank and Pitt (1987b) showed that P. griseoroseum Dierckx 1901 is

probably identical with P. chrysogenum. If this is confirmed by other techniques, P.
chrysogenum will need to be conserved.
MYCarOXINS. P. chrysogenum is the producer of penicillin. It has been reported to produce
mycotoxins occasionally, e.g. by Frisvad (1986) and EI-Banna et al. (1987), but the
significance of these reports remains uncertain.
ECOLOGY. This is an ubiquitous species.
DESCRIPTIONS. Samson et al. (1977a); Pitt (1979); Domsch et al. (1980); Pitt and Hocking
(1985); Pitt (1988).
PROBLEMS.
Penicillium commune Thorn 1910
Penicillium palitans Westling 1911

Penicillium f/tnlOglaucum Biourge 1923
Penicillium aurantiogriseum var. poznaniense Zaleski 1927
Penicillium lanoso-coeruleum Thorn 1930
Penicillium lanosogriseum Thorn 1930
Penicillium lanosoviride Thorn 1930
Penicillium ochraceum var. macrosporum Thorn 1930
Penicillium australicum Sopp ex van Beyrna 1944
Penicillium roqueforti var. punctalum Abe 1956 (nom. invaI.)
Colonies of medium size, texture velutinous to floccose; conidia bluish grey to

dull green on CYA, dull green on MEA; exudate clear to pale brown; reverse usually
pale, occasionally yellow, brown or purple. Stipes finely to conspicuously roughened on
both CYA and MEA, penicilli terverticillate; conidia spherical to subspheroidal, smooth
walled. P. commune resembles P. aurantiogriseum: it differs by consistently green conidial
colours on MEA and having stipes at least finely roughened.
CONCEPT. The concept of P. commune has been badly confused for a long period. Thorn
(1910) regarded it as a common species, but Raper and Thorn (1949) as quite rare, a
floccose species of little significance. Pitt (1979) placed it in synonymy with P. puberulum,
and confused it with P. aurantiogriseum as well. Recent studies on secondary metabolites
(Frisvad, 1983; Frisvad and Filtenborg, 1983; both as P. camemberti W, by enzyme
electrophoresis (Cruickshank and Pitt, 1987b) and a reappraisal of morphology (Pitt et al.,
1987) have clearly shown the validity of Thorn's original concept, and that this is a
common and distinct species.
MYCOTOXINS. The principal mycotoxin produced by P. commune is cyclopiazonic acid
(Frisvad, 1986; EI-Banna et al., 1987). It does not produce penicillic acid or verrucosidin,
metabolites of P. aurantiogriseum as currently circumscribed.
ECOLOGY. Recent studies have shown that this species, which was unrecognised for so
many years, is a common spoilage fungus in foods, feeds and decaying vegetation. It is
also clear now that P. commune is the wild type ancestor of the domesticated species P.
camemberti, used in cheese manufacture (Pitt et al., 1987; Polonelli et al., 1987).
DESCRIPTION. Pitt (1988).
DIAGNOSIS.
Penicillium coprophilum (Berk. & Curt.) Seifert & Samson

Penicillium concentricum Samson et al. 1976
Colonies of moderate size, texture velutinous to fasciculate; conidia dull green.

Stipes smooth walled; penicilli terverticillate; conidia borne as ellipsoids and remaining
so.
DIAGNOSIS.
110
P. concentricum was regarded as a synonym of P. italicum by Pitt (1979). Later

(Seifert and Samson, 1985; see discussion) Pitt agreed that the two species are distinct.
Seifert and Samson (1985) took up the earlier name P. coprophilum for this species.
MycarOXINS. Griseofulvin is the major metabolite produced by P. coprophilum.
ECOLOGY. This species is found principally in dung: according to Seifert and Samson (1985)
it is extremely common in rabbit dung in the Netherlands.
DESCRIPTION. Samson et al. (1976).
CONCEPT.
Penicillium crustosum Thorn 1930
Penicillium crustosum var. spinulosporum Sasaki 1950

Penicillium pseudocasei Abe ex G. Smith 1963
Penicillium farinosum Novobranova 1974
Penicillium terrestre sensu Raper and Thorn (1949)
aurantiogriseum var. poznaniense v. Szilvinyi and P. australicum Sopp ex

v. Beyma, regarded as synonyms of P. crustosum by Pitt (1979), are now considered by
the authors to be synonyms of P. commune. P. verrucosum var. melanochlorum Samson et al.
is a synonym of P. solitum.
DIAGNOSIS. Colonies growing rapidly, fasciculate on CYA, granular to crustose on MEA;
conidia on CYA blue at the margins, dull green elsewhere, on MEA uniformly dull
green. Stipes rough, penicilli large, terverticillate to quaterverticillate; conidia smooth
walled, usually spherical, occasionally ellipsoidal. The most striking diagnostic feature of
this species is the tendency for masses of conidia to be shed by mature colonies on MEA
when jarred.
CONCEPT. The concept accepted here is the same as that of Pitt (1979), and essentially that
of Thorn (1930). Samson et al. (1976) placed P. crustosum in synonymy with P.
aurantiogriseum. SOderstrom and Frisvad (1984) discussed the secondary metabolites of P.
crustosum, but their study included P. commune as accepted here. P. crustosum is now well
accepted as a distinct species (Samson and Pitt, 1985; Frisvad, 1986; Cruickshank and
Pitt, 1987b).
ECOLOGY. Raper and Thorn (1949) regarded P. crustosum as a weak pathogen of fruit, and
uncommon. However Pitt (1979) and Pitt and Hocking (1985) reported that P. crustosum
is a ubiquitous species in foods, compounded feeds and probably also decaying
vegetation in temperate climates.
MYCarOXINS. P. crustosum is the major producer of the neurotoxins known as penitrems.
Penitrem A is a very powerful toxin, responsible for death or brain damage in a variety
of domestic animals (Pitt and Leistner, 1989). Toxicity to man remains uncertain.
DESCRIPTIONS. Pitt (1979); Pitt and Hocking (1985); Pitt (1988).
OfHER SYNONYMS. P.
Penicillium digitatum (Pers.:Fr.) Sacco 1832

None of the 10 synonyms of this species listed by Pitt (1979) are known in
culture.
DiAGNOSIS. Colonies moderately large on CYA, rapidly growing on MEA, texture
velutinous; conidia olive; reverse sometimes brownish. Stipes smooth walled, penicilli at
their most complex terverticillate, but often biverticillate or irregular; conidia borne as
cylinders, in maturity ellipsoidal to cylindroidal, exceptionally large, smooth walled.
This is a distinctive species with olive coloured, very large, elongate conidia.
CONCEPT. The concept of Raper and Thorn (1949) remains unaltered.
MYCarOXINS. No significant secondary metabolites have been reported.
SYNONYMS.
111
Primarily a pathogen of citrus fruits, P. digitatum has been isolated occasionally

from a variety of other habitats.
DESCRIPTIONS. Pitt (1979); Domsch et al. (1980); Pitt and Hocking (1985); Pitt (1988).
ECOLOGY.
Penicillium echinulatum Raper & Thom ex Fassatiova 1977
Penicillium cyclopium var. echinulalum Raper & Thorn 1949 (basionym)

Penicillium palitans var. echinoconidium Abe 1956 (nom. invalid.)
Colonies of moderate size, texture granular to fasciculate; conidia deep green.

Stipes rough, penicilli terverticillate and undistinguished. The primary feature
distinguishing P. echinulatum from other species in sect. Penicillium is the production of
conidia which are spherical and distinctly spinose.
CONCEPT. The concept of P. echinulatum has not changed since the original description by
Raper and Thom (1949).
MYCOTOXINS. No major metabolites are produced.
ECOLOGY. This is a rarely encountered species.
DIAGNOSIS.
Penicillium expansum Link :Fr. 1832
Penicillium aurantiovirens Biourge 1923

Penicillium resticulosum Birkinshaw et al. 19421930
Pitt (1979) listed 14 other synonyms, most previously cited by Raper and
Thom (1949). However, none of these is known in culture. Pitt (1979) regarded P.
aurantiovirens as a synonym of P. aurantiogriseum, an error corrected here.
DIAGNOSIS. Colonies rapidly growing at 25C, fasciculate to coremial; conidia dull green;
exudate and soluble pigment sometimes produced, brown. Stipes smooth, penicilli
typically terverticillate; conidia ellipsoidal, smooth walled.
CONCEPT. Thom (1910) clearly established the identity of Link's original concept, and this
has subsequently remained unaltered. The name P. expansum has been in almost
exclusive use for this species since that time also.
MYCOTOXINS. P. expansum produces patulin, an acutely toxic mycotoxin of some concern in
apple juice, especially that prepared in small operations (Brackett and Marth, 1979).
ECOLOGY. Although known primarily as the cause of a destructive rot of apple and pear
fruits, P. expansum has been isolated from a wide variety of other food and plant
materials. Indeed it is a broad spectrum plant pathogen. It has been found less
commonly away from living tissue.
DESCRIPTIONS. Pitt (1979); Domsch et a/. (1980); Pitt and Hocking (1985); Pitt (1988).
OTHER SYNONYMS.
Penicillium fennelliae Stolk 1969

DIAGNOSIS. Colonies of moderate size, velutinous to floccose; conidia dull green; mycelium
salmon or near on both CYA and MEA; soluble pigment salmon or brown; reverse
orange brown on both CYA and MEA. Stipes smooth, penicilli biverticillate to
terverticillate; conidia borne as cylinders, ellipsoidal to cylindroidal, with walls
spinulose. Colony pigmentation and elongate conidia with spinulose walls make this a
distinctive species.
CONCEPT. The concept is as described by Stolk (1969).
MYCOTOXINS. No information on secondary metabolites is available.
ECOLOGY. This species has been found only from soil, from Zaire.
DESCRIPTIONS. Stolk (1969); Pitt (1979).
112
Penicillium glandicola (Oudemans) Seifert & Samson 1903

Penicillium granulatum Bainier 1905
Colonies slowly growin~ especially on MEA, texture fasciculate to coremiform;

conidia dull green; exudate clear to pale yellow; soluble pigment yellow to deep brown;
reverse usually deep brown. Stipes rough, penicilli terverticillate to quaterverticillate;
metulae sometimes apically inflated; conidia ellipsoidal, smooth walled. Similar to P.
brevicompactum in colony diameters, P. glandicola is readily distinguished by fasciculate
texture and rough stipes.
CONCEPT. The concept of P. glandicola (under the name P. granulatum) as established by
Raper and Thom (1949) remains unchanged. The name was changed to the earlier P.
giandicoia after studies by Seifert and Samson (1985).
MYCOTOXINS. Penitrem A and patulin are sometimes produced (Frisvad, 1986).
ECOLOGY. P. gIandicoia has been isolated from a wide range of habitats, but rather
infrequently.
DESCRIPTIONS. Pitt (1979, as P. granulatum); Domsch et al. (1980, as P. granulatum); Pitt
(1988).
DiAGNOSIS.
Penicillium griseofulvum Dierckx 1901
Penicillium patulum Bainier 1906

Penicillium urticae Bainier 1907
Penicillium fIexuosum Dale apud Biourge 1923
Penicillium griseofulvum var. dipodomyicola Frisvad et al. 1987
Colonies slowly growing, fasciculate to coremiform; exudate clear to pale

yellow, soluble pigment reddish brown. Stipes smooth, penicilli terverticillate to
quaterverticillate; phialides very short, conidia subspheroidal to spherical, smooth
walled.
CONCEPT. Raper and Thom (1949) called this species P. urticae; Pitt (1979) took up the
earlier valid epithet P. griseofulvum. The concept of the species has been unchanged for 40
years or more.
MYCOTOXINS. The principal metabolite produced by P. griseofulvum is the toxic antibiotic
griseofulvin. Patulin and cyclopiazonic acid are produced by some isolates (Frisvad,
1986; El-Banna et al., 1987).
ECOLOGY. A widely distributed species, P. griseofulvum is most commonly encountered as
an agent of biodeterioration. No preferred habitat is obvious.
DESCRIPI10NS. Pitt (1979); Domsch et al. (1980); Pitt and Hocking (1985); Pitt (1988).
DIAGNOSIS.
Penicillium hirsutum Dierckx 1901

OTHER SYNONYMS. P. hordei, considered a synonym of P. hirsutum by Pitt (1979), is now
regarded as distinct (Frisvad, 1986; Cruickshank and Pitt, 1987a, b)
DiAGNOSIS. Growth relatively rapid, colonies usually deep and fasciculate; conidia green;
exudate reddish or violet brown; soluble pigment yellow to orange brown. Stipes rough,
penicilli terverticillate to quaterverticillate; conidia spherical to ellipsoidal, walls smooth
to finely roughened.
CONCEPT. This species was called P. corymbiferum by Raper and Thorn (1949); the earlier
valid name P. hirsutum was taken up by Pitt (1979) without significant change in concept.
MYCOTOXINS. Only minor compounds are known to be produced.
ECOLOGY. A relatively uncommon though widespread species. One specific habitat is as a
pathogen on the bulbs of Uliaceae.
113
Penicillium hordei Stolk 1969

DIAGNOSIS. Colonies growing relatively slowly, texture sometimes floccose on CYA, but
usually with fasciculate areas, often definitely coremial on MEA; mycelium yellow;
exudate orange brown; conidia green. Stipes smooth, penicilli terverticillate; conidia
spherical, small, rough walled.
CONCEPT. The concept of P. hordei erected by Stolk (1969) was not accepted by Pitt (1979),
who regarded this species as a synonym of P. hirsutum. However, he later accepted the
species (Samson and Pitt, 1985).
MYCarOXINS. No significant mycotoxins have been reported.
ECOLOGY. The main reported habitat for this species has been barley seeds in Europe.
DESCRIPI10NS. Stolk (1969); Samson et al. (1976).
Penicillium italicum Wehmer 1894
Penicillium japonicum G. Smith 1963
Colonies growing moderately rapidly on CYA, rapidly on MEA, texture

usually fasciculate to coremial, occasionally velutinous; conidia greyish green; soluble
pigment brown; reverse on CYA usually brownish orange to greyish brown, on MEA
dark brown. Stipes smooth, penicilli terverticillate, sometimes irregular; conidia borne as
cylinders, in maturity ellipsoidal to short cylindroid ai, smooth walled. The main
diagnostic feature of P. italicum is the formation of conidia as cylinders from the
phialides. It also produces dense, fasciculate colonies with green conidia.
CONCEPT. The concept of P. italicum established by Raper and Thorn (1949) was broadened
by Pitt (1979) to include the newly described species P. concentricum Samson et al. P.
concentricum was restored to species status (as P. coprophilum) in later publications
(Samson and Pitt, 1985; Cruickshank and Pitt, 1987a), and the concept of P. italicum
restored to that of Raper and Thorn (1949).
MYCarOXINS. No important secondary metabolites are produced.
ECOLOGY. The primary habitat for this species is as a pathogen of citrus fruits. It has been
reported infrequently from other habitats. Some evidence exists that P. italicum has
evolved from the coprophilic species P. coprophilum.
DESCRIPI10NS. Pitt (1979); Domsch et a/. (1980); Pitt and Hocking (1985); Pitt (1988).
DIAGNOSIS.
Penicillium olsonii Bainier & Sartory 1912.

DIAGNOSIS. Colonies of moderate size, deep but velutinous; conidia dull green; mycelium
white to pale brown; reverse pale yellow to yellow brown. Stipes very long and broad,
smooth walled; penicilli usually terverticillate, multiramulate; conidia ellipsoidal,
smooth walled. P. olsonii is readily distinguished by large stipes bearing up to 6 rami in
well ordered verticils.
CONCEPT. Although this species has sometimes been considered a synonym of P.
brevicompactum, it is quite distinct morphologically. The concept clearly remains as
described and illustrated by the original authors, although now greatly amplified.
MycarOXINS. No secondary metabolites of significance have been reported.
ECOLOGY. P. olsonii remains an uncommonly isolated species, although by no means as rare
as reported by Pitt (1979). It is widely distributed.
DESCRIPI10NS. Pitt (1979); Pitt (1988).
Penicillium roque/orti Thorn 1906
Penicilium roqueforti var. weidemannii Westling 1911

Penicillium grJrgonzolae Biourge 1923
114
Penicillium roquefurti var. viride Dattilo-Rubbo 1938

Penicillium conservandi Novobranova 1974
Colonies rapidly growing, strictly velutinous; conidia at the margins bluish,

elsewhere green; reverse on CYA sometimes dark green. Stipes usually very rough,
penicilli large and terverticillate; conidia large, spherical, smooth walled.
CONCEPf. P. roqueforti has been regarded as a distinct species since it was described. No
consistent differences appear to distinguish strains used in cheese manufacture from
spoilage isolates.
MYCOTOXINS. Two distinct subspecies occur: one typically produces roquefortines and PR
toxin, the other patulin and sometimes penicillic acid (Frisvad, 1986; EI-Banna et al.,
1987). The two subspecies are indistinguishable morphologically.
ECOLOGY. Although more widely known for its role in cheese manufacture, P. roqueforti is
in fact a very widely distributed spoilage fungus, especially on refrigerated products,
and products of low oxygen tension (Pitt and Hocking, 1985).
DESCRIPTIONS. Samson et al. (1977b); Pitt (1979); Pitt and Hocking (1985); Pitt (1988).
DIAGNOSIS.
Penicillium solitum Westling 1911
Penicillium psittacinum Thorn 1930

Penicillium casei var. compactum Abe 1956 (nom. invaJ.)
Penicillium verrucosum var. melanochiorum Samson et al. 1976
Penicillium mali Novobranova 1972 = Penicillium mali Gorlenko & Novobranova 1983.
Colonies small to medium sized, usually velutinous; conidia dark bluish green
to dark green; exudate sometimes produced, clear; reverse on CYA usually pale, on MEA
usually a distinct greyish or brownish orange. Stipes smooth to rough, penicilli usually
terverticillate, but sometimes biverticillate or quaterverticillate also; conidia quite large,
spherical to sub spheroidal, smooth walled.
CONCEPf. Considered to be an uncommon, funiculose species by Raper and Thom (1949),
P. solitum was given recognition as a new variety P. verrucosum var. melanochlorum by
Samson et al. (1976). It was placed in synonymy with P. aurantiogriseum and
unrecognised by Pitt (1979); P. verrucosum var. melanochlorum was incorrectly placed in
synonymy with P. crustosum. Cruickshank and Pitt (1987a, b) revived P. solitum on the
basis of differences in enzyme electrophoretic patterns and morphology. Westling's
concept is now placed on a sound basis.
MYCOTOXINS. Unlike most other common species in Penicillium subgen. Penicillium, P.
solitum does not appear to produce distinctive metabolites or mycotoxins.
ECOLOGY. Because of lack of recognition, the ecology of P. solitum remains largely
unknown. However, unpublished data (L. Leistner and J.I. Pitt) shows that this species is
common in European processed meats. It also has a major niche as a pathogen of
pomaceous fruit (Pitt and Spotts, in prep.).
DESCRIPfIONS. Pitt (1988); Pitt and Spotts (in prep.).
DIAGNOSIS.
Penicillium verrucosum Dierckx 1901

DIAGNOSIS. Colonies slowly growing, especially on MEA, texture velutinous, floccose or
fasciculate; conidia yellow green; exudate clear to pale yellow; reverse on CYA yellow
brown to deep brown. Stipes robust, rough walled, penicilli usually terverticillate, in
some isolates also quaterverticillate, in others also biverticillate or irregular; conidia
usually spherical, smooth walled. Colony appearance is similar to P. viridicatum, but
diameters on CYA and MEA are smaller. Stipes and penicilli are more robust than in P.
viridicatum.
115
Ignored by Raper and Thorn (1949), P. verrucosum was taken up by Samson et al.
(1976) in a very broad sense. Pitt (1979) accepted the species, but with a much narrower
concept. Discussion over the validity of Pitt's concept has now declined, with
corroboration coming from reexamination (Pitt, 1987), enzyme electrophoretic studies
(Cruickshank and Pitt, 1987b) and secondary metabolite profiles (Frisvad, 1986; Pitt,
1987; El-Banna et al., 1987).
MYCOTOXINS. P. verrucosum is the major source of ochratoxin A among the Penicillia. This
toxin is not produced by P. viridicatum (Frisvad, 1986; Pitt, 1987), although this has been
commonly reported in the literature. Some isolates of P. verrucosum also produce citrinin
(Frisvad, 1986; El-Banna et aI., 1987).
ECOLOGY. P. verrucosum is of common occurrence in Scandinavian cereals (Frisvad and
Vuif, 1986, as P. viridicatum Group II), and is the cause of ochratoxin poisoning of both
animals and man in that region. It also occurs in European processed meats (Pitt and
Hocking, 1985). It has not been isolated commonly elsewhere.
CONCEPT.
Penicillium viridicatum Westling 1911

Penicillium olivincroiride Biourge 1923
Penicillium olivicolor Pitt 1979
Penicillium ochraceum Bain. ex Thorn 1930
P. palitans Westling and P. lanosoviride Thorn, placed in synonymy with

P. viridicatum by Pitt (1979), are now regarded as synonyms of P. commune.
DIAGNOSIS. Colonies of moderate size, texture velutinous to fasciculate; conidia yellow
green; exudate clear to pale yellow; sometimes soluble pigment, orange to reddish
brown. Stipes rough, penicilli mostly terverticillate; conidia subspheroidal to ellipsoidal,
smooth walled.
CONCEPT. P. viridicatum became established as a distinct species after acceptance by Raper
and Thorn (1949), and became well recognised. However, Raper and Thorn (1949)
incorrectly placed an earlier species, P. verrucosum, in synonymy with P. viridicatum.
Samson et al. (1976) recognised P. verrucosum, but reduced several other species accepted
by Raper and Thorn (1949) to the status of varieties of it. Pitt (1979) also recognised P.
verrucosum, but in a much narrower sense, and reinstated P. viridicatum in a sense similar
to that of Raper and Thorn (1949). This concept is now gaining acceptance (Frisvad, 1986;
Pitt, 1987).
MYCOTOXINS. As now accepted, P. viridicatum produces the naphthoquinone toxins
viomellein and xanthomegnin. The earlier reports that P. viridicatum produces ochratoxin
A have been shown to be incorrect (Pitt, 1987).
PROBLEMS. A degree of overlap still exists between P. aurantiogriseum and P. viridicatum. See
the discussion under P. aurantiogriseum.
ECOLOGY. Pitt (1979) regarded P. viridicatum as a ubiquitous species. However, although it
is relatively common in cereals in temperate zones, it occurs at quite a low frequency
elsewhere (Domsch et al., 1980; Pitt and Hocking (1985.
OTHER SYNONYMS.
116
J.I. Pill & R.H. Cruickshank
Analytical Key to Penicillium Subgenus Penicillium

1.
Conidia borne as cylinders, with at least a proportion remaining so at maturity ........................................... 4

Conidia borne as ellipsoids or spheroids and remaining so at maturity.......................................................... 2
2.
Conidia en masse brown, penicilli often irregular .............................................................................. P. arenicola

Conidia en masse green or olive; penicilli usually well-defined ........................................................................ 3
3.
Penicilli often with more than 3 rami ..................................................................................................... P. olsonii

Penicilli with no more than 2 rami ......................................................................................................................... 6
4.
Conidia en masse olive.......................................................................................................................... P. digitatum

Conidia en masse green............................................................................................................................................. 5
5.
Mycelium white; conidia smooth walled ............................................................................................ P. italicum

Mycelium salmon or orange; conidia spinose .................................................................................. P. fennelliae
6.
Conidia en masse persistently white or pale grey; isolated from cheese or cheese factory ..... P. camemberti
Conidia en masse blue, green or grey; source inconsequential.. ......................................................................... 7
7.
Colonies on C'fA at 25 0 exceeding 30 mm diam ................................................................................................. 8

Colonies on CYA at 250 not exceeding 30 mm diam ........................................................................................ 22
8.
Stipes on C'fA and MEA smooth walled ............................................................................................................... 9

Stipes often rough-walled, especially on MEA ................................................................................................... 15
9.
Soluble pigment and/or reverse yellow; sometimes growth at 370 ........................................ P. chrysogenum
Soluble pigment and reverse not yellow; no growth at 370.............................................................................. 10
10. Conidia blue on both C'fA and MEA . ....................................................................................P. aurantiogriseum

Conidia green on both C'fA and MEA ................................................................................................................ 11
11. Exudate and soluble pigment red to reddish brown; conidia spherical. ............................. P. atramentosum
Exudate and soluble pigment not red or reddish brown; conidia usually ellipsoidal .................................. 12
12. Conidia dark blue green to dark green, reverse on CYA pale, on MEA greyish to brownish orange .......... .
...................................................................................................................................................................... P.solitum
Conidia yellow green or dull green, reverse on C'fA pale or brown, on MEA pale or dull brown ........... 13
13. Colonies dense and compact, fascicles inconspicuous, corernia absent, conidia yellow green .............. .
.............................................................................................................................................................. P. viridicatum
Colonies fasciculate to coremial, conidia dull green or dark green ................................................................. 14
14. Conidia dull green, colonies deep and fasciculate to coremial, reverse on C'fA pale to orange brown ....... .
..................................................................................................................................................................P. expansum
Conidia dark green, colonies granular to fasciculate, usually with small coremia, reverse on C'fA dark
brown ................................................................................................................................................. P. coprophilum
15. Conidial walls rough or spinose.................................................................................................... P. echinulatum
Conidial walls smooth............................................................................................................................................ 16
16. Conidial colour on C'fA unifonnly blue or greyish blue .................................................................................. 17
Overall conidial colour on C'fA green, although sometimes with bluish marginal areas ........................... 18
17. Conidia dull green on MEA; reverse on C'fA pale to dull brown ................................................. P. commune
Conidia dark green on MEA; reverse on C'fA orange yellow to yellow brown .................... P. aethiopicum
18. Colony reverse on CYA dark brown ........................................................................................................... P. allii
Colony reverse on CYA not dark brown ............................................................................................................. 19
19. Conidia on C'fA green with bluish margins; reverse on C'fA pale or green; conidia usually spherical... 20
Conidia on CYA uniformly green; reverse on C'fA pale or yellow brown; conidia usually not spherical ...
.................................................................................................................................................................................... 21
117
20. Reverse on C'fA pale or brownish; conidia dull green; texture fasciculate on CYA, granular on MEA;
conidia on MEA usually detaching in masses when jarred .......................................................... P. crustosum
Reverse on C'f A often green; conidia dark green; texture velutinous; conidia on MEA not detaching
when jarred ............................................................................................................................................P. roqueforti
21. Conidia yellow green; exudate clear to pale yellow; penicilli strictly terverticillate.............. P. viridicatum
Conidia green; exudate reddish to violet brown; penicilli sometimes quaterverticillate............ P. hirsutum
22. Penicilli broad, often 40 mm or more across the metulae apices; metulae often apically enlarged ............... .
......................................................................................................................................................P. brevicompactum
Penicilli narrow, commonly 30 mm or less across the metulae apices; metulae cylindrical ........................ 23
23. Stipe walls smooth to finely roughened .............................................................................................................. 24
Stipe walls finely to conspicuously roughened .................................................................................................. 26
24. Phialides commonly 45 to 6 ~ long .......................................................................................... P. griseofulvum
Phialides exceeding 6 ~ long .............................................................................................................................. 25
25. Conidia green; mycelium yellow; soluble pigment orange; conidia often rough walled ................ P. hordei
Conidia dark bluish green to dark green; mycelium white; soluble pigment clear; conidia smooth
walled ..........................................................................................................................................................P. solitum
26. Conidia yellow green .............................................................................................................................................. 27
Conidia bluish or dull green to dark green ......................................................................................................... 28
27. Colonies on C'f A and MEA both exceeding 25 mm in diameter ............................................... P. viridicatum
Colonies on CYA and MEA not exceeding 25 mm in diameter. ................................................. P. verrucosum
28. Texture fasciculate to coremial; soluble pigment yellow brown to deep brown; stipes conspicuously
roughened; conidia ellipsoidal. ......................................................................................................... P. glandicola
Texture velutinous to floccose; soluble pigment absent; stipes smooth to roughened; conidia spherical to
subspheroidal.. ......................................................................................................................................................... 29
29. Conidia blue grey to dull green; reverse usually pale on both C'fA and MEA ........................... P. commune
Conidia dark blue green to dark green; reverse pale on C'f A, characteristically greyish or brownish
orange on MEA .......................................................................................................................................... P. solitum
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CIEGLER, A. and KURTZMAN, c.P. 1970. Penicillic acid production by blue-eye fungi on various
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CIEGLER, A., FENNELL, D.I., SANSING, G.A., DETROY, R.W. and BENNETT, G.A. 1973. Mycotoxinproducing strains of Penicillium viridicatum: classif1cation into subgroups. Applied Microbiology 26: 271-278.
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DIALOGUE FOLLOWING DR. PITT'S PRESENTATION

GAMS: In
change?
cultures that have degenerated morphologically, do the isoenzyme patterns also
PITI: Occasionally a band disappears, but in general terms the isoenzyme patterns remain
unchanged. The concentrations of the enzymes may decrease.
121
THE SYSTEMATICS OF THE TERVERTICILLATE PENICILLIA

A.c. Stolk1, R.A. Samson1, J.e. Frisvad2 and O. Filtenborg2
lCentraalbureau voor Schimmelcultures
2Department of Biotechnology
SUMMARY
The species of the terverticillate Penicillia are re-investigated and delimited on the basis of the
morphology of the conidiophores, phialides and conidia. In addition, growth characters and profiles
of secondary metabolites were taken into account for definition of the taxa. In general, the
terverticillate Penicillia represent a biologically homogenous group, but on the basis of their
morphology subdivision into series is proposed. The series and the accepted taxa are discussed briefly
and keyed out dichotomously. A list of the principal mycotoxins produced by each taxon is presented.
Most species can be identified using morphological and cultural characters as observed on Czapek
and 2% malt extract agar, while a more detailed speciation requires the use of a standardized medium
regime including Czapek yeast extract agar, creatine sucrose agar and 5% acetic acid agar.
INTRODUCTION
The Penicillia included in the section Asymmetrica-Fasciculata by Raper and Thorn (1949)
are very important components of the mycoflora of foods (Pitt and Hocking, 1985; Samson
and Van Reenen-Hoekstra, 1988). Many of these species produce potent mycotoxins, and
because of the clear connection between taxa and mycotoxins (Frisvad, 1988), accurate
identifications are most relevant and important (Frisvad, 1989). Three different
approaches to the systematics of Raper and Thorn's sect. Asymmetrica, subsect. Fasciculata
(Samson et al., 1976; Pitt, 1979; Frisvad and Filtenborg, 1983) led to confusion both
nomenclature and species concepts, although Samson and Pitt (1985) have more recently
agreed in may of these. Furthermore, recent biochemical and morphological
reinvestigations in these species (Cruickshank and Pitt, 1987; Frisvad, 1988; Samson and
Van Reenen-Hoekstra, 1988) have resulted in modified species circumscriptions.
In this paper we present a classification of the terverticillate Penicillia based on a
reinvestigation of morphological criteria of cultures mainly grown on Czapek and 2 %
MEA and supplemented with mycotoxin profiles. A dichotomous key to the taxa and a list
of toxins produced by each taxon is provided. This paper includes only the taxa formerly
classified as asymmetric-biverticillate Penicillia, although several terverticillate taxa can
also be found in other subgenera. The taxa described by Frisvad et al. (1987) have not been
treated as they are probably found in very specific habitats, while their proper status is not
yet fully elucidated.
122
A.C. Stalk et a/.
Table 1. Disposition of series of subgenera Furcatum and Penicillium with terverticillate

species
Subgenus FUTcatum
Series Lanosa
Series Chrysogena
Series Urticicola
Subgenus Penicillium
Series Digitata
Series Italica
Series Oxalica
Series Gladiolii
Series Viridicata
Series Expansa
Series Granulata
Series Olsonii
Series CTaviformae
Ex type, authentic and fresh cultures of each species considered were examined on
Czapek agar (Cz) and 2% malt agar (MA) (Samson et al., 1976) and/or malt extract agar
(MEA) (Raper and Thom, 1949) for morphological examinations and on Czapek yeast
autolysate agar (CYA) (Pitt, 1979), Difco yeast extract sucrose agar (YES) (Filtenborg et al.,
1983) and creatine-sucrose agar (CREA) (Frisvad, 1985) for physiological and chemical
examinations. Furthermore the isolates were grown on CYA at 37 C and inoculated into
apples or lemons to test their pathogenicity. Cultures grown on CYA and YES were
examined for profiles of secondary metabolites using the agar plug method (Filtenborg et
al., 1983). The production of particular secondary metabolites was confirmed by highperformance liquid chromatography (HPLC) with diode array detection (Frisvad and
Thrane,1987).
Pitt (1979) classified the terverticillate Penicillia in subgenus Penicillium, including taxa
previously accommodated by Raper and Thorn (1949) as the Asymmetrica sects. Fasciculata
and Funiculosa. We believe that the morphology of the conidiophore and shape of
phialides and conidia justify a more detailed subdivision of the species. (Table 1). A more
detailed subdivision of subgen. Penicillium into varieties new species and chemotypes,
based on physiological characters and profiles of secondary metabolites is briefly
presented by Frisvad and Filtenborg (1990).
Table 2 lists the specific mycotoxins of each accepted species. However, note that in some
species many unknown, - but specific - compounds are present, e.g. in P. olsonii
The systematics of the terverticillale Penicillia
123
Table 2. Accepted species of the terverticillate Penicillia and the production of mycotoxins.
P. atramentosum Thorn
P. aurantiogriseum Dierckx var. aurantiogriseum
P. aurantiogriseum var. viridicatum (Westling) Frisvad et Filtenborg
P. brevicompactum Dierckx
P. camemberti Thorn
P. clavigerum Dernelius
P. commune Thorn
P. coprophilum (Berk. et Curt.) Seifert et Samson
P. crustosum Thorn
P. digitatum (Pers.: Fr.) Sacco
Oxaline
Roquefortine C
Rugulovasine A
Penicillic acid
Verrucosidin
Aurantiamin
Xanthornegnin
Viornellein
Verrucofortine
Cydopenin
Cydopenol
Viridicatin
Viridicatol
Penicillic acid
Viridamine
(Viridic acid)"
Brevianamide A
Verrucofortine
Xanthornegnin
Viornellein
Cydopenin, -01
Viridicatin, -01
Mycophenolic acid
Raistrick phenols
Brcvianamide A
(Botryodiploidin)
Cydopiazonic acid
Penicillin
Roquefortine C
Meleagrin
(Xanthocillin)
(ernodic acid)
(sorbicillin)
PenitrernA
(Roquefortine A)
Cydopiazonic acid
Palitantin
Cydopaldic acid
Rugulovasine A
Roquefortine A
Griseofulvin
Roquefortine C
Meleagrin
Oxaline
PenitrernA
Terrestric acid
Cydopenin, -01
Viridicatin, -01
Roquefortine C
Tryptoquivalins
(cont.)
124
A.C. Stolk et al.

Table 2 (cont.)
P. echinulatum (Raper et Thorn) Fassatiova
P. expansum Unk.
P. fennelliae Stolk
P. glandicola (Oud.) Seifert et Samson
P. griseofulvum Dierckx
P. hirsutum Dierckx
P. hordei Stolk
P. italicum Wehmer
P.lanosum Westling
P. olsonii Bain. et Sartory
P. roqueforti Thorn
P. solitum Westling
P. verrucosum Dierckx
P. vulpinum (Cooke et Massee) Seifert et Samson
Cyclopenin, -01
Viridicatin, -01
Penechins
Patulin
Citrinin
Roquefortine C
Chaetoglobosin C
Penicillic acid
PenitremA
Patulin
Roquefortine C
Meleagrin
Oxaline
Patulin
Griseofulvin
Cyclopiazonic acid
Roquefortine C
Roquefortine C
Terrestric acis
Compactin
Roquefortine C
Terrestric acid
Deoxybrevianamide E
5,6-dihydroxy-4-methoxy2H-pyran-2-one
Griseofulvin
Kojic acid
PR-toxinb
Roquefortine C
Mycophenolic acid
Roquefortine Ab
Mardortinesb
PatulinC
Penicillic acid"
Botryodiploidinc
Compactin
Cyc1openin, -01
Viridicatin, -01
Ochratoxin A
Verrucolone
Citrinin
Patulin
Roquefortine C
Oxaline
Cyclopenin
Viridicatin
Secondary metabolites listed in parenthesis: freqeuncy of isolates producing this metabolite is unknown
bp. roqueforti chemotype I (dark green reverse on Czapek); c P. roqueforti chemotype II (light brown reverse
on Czapek)
The systematics of the terverticillate Penicillia
125
LIST OF ACCEPTED TAXA
The following list outlines the accepted taxa with a short discussion of their placement:
Series LANOSA Stolk et Samson
Adv. Penicillium and Aspergilllus Syst.: 180, 1985
Type species: Penicillium lanosum Westling
P.lanosum was placed as a synonym of P. puberulum by Pitt (1979), while P. kojigenum was
placed as a synonym of P. jensenii. Pitt (1988) placed P. lanosum as a synonym of P.
commune, but we consider it to be a distinct species based both on morphology (Samson et
al., 1976) and secondary metabolites (Table 2). Characteristically P. lanosum produces
terverticillate penicilli with a quite divergent branch. Apart from its morphological
characters, it is the only species in Penicillium producing both kOjic acid and griseofulvin.
Penicillium lanosum Westling 1911

Penicillium kojigenum G. Smith 1961
Series CHRYSOGENA Raper & Thorn ex Stolk & Samson
Series P. chrysogenum Raper & Thorn - Man. Penicillia: 355, 1949 (nom. inval. Arts 21, 36) = Series
Chrysogena Raper & Thorn ex Stolk & Samson in Adv. Penicillium and Aspergillus
Raper & Thom (1949) proposed the series P. chrysogenum-series which included four
species: P. chrysogenum Thom, P. meleagrinum Biourge, P. notatum Westling and P.
cyaneofulvum Biourge. Samson et al. (1977a) observed no significient differences between
the four species, as represented by their type strains and authentic cultures. Consequently
they placed the latter three species in synonymy with P. chrysogenum.
There is general agreement that the ex type of P. griseoroseum Dierckx agrees very well
with the ex type culture of P. chrysogenum Thom. It differs only in producing
predominantly biverticillate penicilli and appears to have deteriorated in culture. The
description of Biourge's (1923) P. griseoroseum was characterized by bi- to terverticillate
conidiophores like those of P. chrysogenum. The species should be regarded as synonyms:
the correct name is P. griseoroseum. However, as at present the name P. chrysogenum is
commonly used in the literature and it is of great industrial significance, so it is proposed
to conserve the name P. chrysogenum (see Chapter 2 on Nomenclature).
The type cultures of P. chrysogenum, and starter cultures of P. nalgiovense used for
mould fermented salami resemble one another closely in morphological respects.
Furthermore they both produce penicillin. P. nalgiovense as it is now used is therefore
regarded as a domesticated form of P. chrysogenum. The ex type culture of P. nalgiovense,
however is a different from the strains used for mould fermented products; the profiles of
secondary metabolites are different: P. nalgiovense NRRL 911 produces nalgiovensin and
nalgiolaxin (Birch and Stapleton, 1967) and other unique compounds, while the
domesticated strains of P. chrysogenum produce penicillin and other specific coumpounds.
Series Chrysogena is classified in the subgenus Furcatum because of included species
produce bi- to quaterverticillate conidiophores with divergent, subterminal and
intercalary branches (metulae). As some P. chrysogenum isolates may develop up to five or
more-stage-branched conidiophores it shows some affinities with the series Urticicola.
However, conidiophores of in the Series Chrysogena are less complicated and the phialides
and metulae are different.
126
A.C. Stolk et al.
Penicillium atramentosum Thom 1910

Penicillium chrysogenum Thom 1910
Penicillium griseoroseum Dierckx 1901

Penicillium citreoroseum Dierckx 1901
Penicillium brunneorubrum Dierckx 1901
Penicillium notatum Westling 1911
Penicillium bacu/atum Westling 1911
Penicillium meleagrinum Biourge 1923
Penicillium eyaneofulvum Biourge 1923
Penicillium roseocitreum Biourge 1923
Penicillium flavidomarginatum Biourge 1923
Penicillium rubens Biourge 1923
Penicillium chlorophaeum Biourge 1923
Penicillium camerunense Heim 1949
Penicillium aromaticum f. microsporum Rornankova
Penicillium harmonense Baghdadi 1968
Penicillium verrucosum var. eye/opium strain ananas-olens Ramirez 1982
Series URTICICOLA Fassatiova
Series P. urficae Raper & Thorn - Man. Penicillia: 531, 1949 (nom. inval. Arts 21, 36) =Series Urficicola
Fassatiova - Acta Univ. Carol. BioI. 12: 324. 1977
Fassatiova (1977) proposed the series Urticicola to accomodate a single species: P. urticae (=
P. griseofulvum). Pitt (1979) emended Fassatiova's concept of the series by adding five
species which like P. griseofulvum are characterized by relatively slow growth on CYA at
25C. However, the added species produce regular, ter- to quaterverticillate penicilli and
thus differ markedly from the complicated, loosely arranged, three- to five-stage-branched
conidial structures, characteristic of P. griseofulvum. Moreover the phialides are quite
different. Most authors (Raper & Thom, 1949; Fassatiova, 1977; Ramirez, 1982) have
classified P. griseofulvum, because of its branched, synnematous conidiophores in Raper &
Thom's subsection Fasciculata Raper and Thom or subgen.Penicillium (Pitt, 1979).
However, in many respects, such as the complicated conidiophores, the divergent
branches and the small metulae and phialides P. griseofulvum differs from species in
subgen.PenicilIium. It is preferred, here to include it in subgen. Furcatum. In fact P.
griseofulvum morphologically is an unique species without close relationships in
Penicillium. Biologically, however, P. griseofulvum is related to the coprophilic,
synnemateous species P. coprophilum, P. glandicola and F. vulpinum. This relationship is
also supported by the similar secondary metabolites produced by all four species:
roquefortine C and patulin are produced by each species, except P. coprophilum which only
produce roquefortine C (Frisvad and Filtenborg, 1989 a). The three-to five-stage-branched
or sometimes even more complex conidiophores are slightly reminiscent of P.
chrysogenum, however. P. griseofulvum differs from species in series Chrysogena in
producing synnematous conidiophores, which are more complicated and irregular in
structure, as well as by the very small phialides and metulae.
In agreement with Raper & Thom (1949) and Fassatiova (1977) Urticicola, as delimited
here, is restricted to only a single species, P. griseofulvum.
Penicillium griseofulvum Dierckx 1901
Penicillium patulum Bain. 1906

Penicillium urticae Bain. 1907
Penicillium flexuosum Dale apud Biourge 1923
127
? Penicillium maltum Hori et Yamamoto 1954

? Penicillium duninii Sidibe 1974
Series DIGITATA Raper et Thorn ex Stolk et Samson
Series P. digitatum Raper et Thorn - Man. Penicillia: 385, 1949 (nom. inva!. Arts 21, 36) = Series Digitata
Raper et Thorn ex Stolk et Samson - Adv. Penicillium and Aspergill/us Syst.: 183, 1985.
Type species: Penicillium digitatum
P. digitatum is a very distinctive species, related to P. italicum only by its cylindrical

conidia and the ability to produce a destructive rot in citrus fruits. It shares no secondary
metabolites with P. italicum. It is excluded from the key provided in this paper, since its
conidiophores are not really terverticillate.
Penicillium digitatum (Pers.: Fr.) Sacco 1881
Penicillium olivaceum Wehmer 1895
Penicillium olivaceum Sopp 1912
Penicillium olivaceum var. norvegivum Sopp 1912
Penicillium olivaceum var. italicum Sopp 1912
Penicillium digitatoides Peyronel1913
Penicillium lanosogrisellum Biourge 1923
Penicillium terraconense RamIrez et Martinez 1980
Series ITALICA Raper et Thorn ex Fassatiova
Series P. italicum Raper et Thorn - Man. Penicillia: 523, 1949 (nom. inva!. Arts 21, 36) = Series Italica Raper et
Thorn ex Fassatiova - Acta Univ. Caro!., Bio!.: 324, 1977 = Series Italica Raper et Thorn ex Pitt - Gen.
Penicillium: 381, 1979.
Type species: Penicillium italicum
Series Italica is represented by one distinctive species, P. italicum. It causes a characteristic

blue rot of citrus fruits. P. italicum is distinguished from P. digitatum by the synnematous
conidiophores, the better developed terverticillate penicilli and greyish, blue green
colonies. White or avellaneous mutants of P. italicum may occasionally be encountered. P.
japonicum G. Smith (= P. digitatum var.latum Abe) is a typical P. italicum, although strongly
coloured and was incorrectly placed in synonymy with P. resticulosum Raistrick et al. by
Pitt (1979).
Penicillium italicum Wehmer 1894
Penicillium aeruginosum Dierckx 1901

Penicillium ventruosum Westling 1911
Penicillium japonicum G. Smith 1963 (= Penicillium digitatum var.latum Abe 1956, nom. invaI.)
Penicillium italicum var. album Wei 1940
Penicillium italicum var. avellaneum Samson et Gutter 1976
Series OXALICA Raper &: Thorn ex Pitt
Series P. oxalicum Raper & Thorn - Manual Penicillia: 376, 1949 (nom. inval. Arts 21, 36) = series Oxalica
Raper & Thorn ex Pitt - Gen. Penicil: 273, 1979
Species of series Oxalica are characterized by well developed penicilli, robust, large
phialides, large cylindrical to ellipsoidal conidia and rapidly growing, velutinous cultures.
They are separated from species from series Digitata by much better developed
conidiophores and from those in series Italica by veluti nous colonies. P. oxalicum is
excluded from the key provided below because the conidiophores are predominantly
128
A.C. Stolk et al.
biverticillate. The two species P. oxalicum and P. fennelliae are closely related
morphologically and consequently are classified together in series Oxalica. The latter series
is placed in the subgenus Penicillium because of the appressed structure of the penicilli.
Ramirez and Martinez (1981) described P. aragonense in terms suggesting a possible
synonymy with P. oxalicum. The type culture of P. aragonense proved to be badly
contaminated, it contained only P. glabrum.
P. sclerotigenum is another species, which is closely related to both P. italicum and P.
oxalicum. Similarities to species in subgenus Penicillium include the ability to produce a
destructive rot in yams, production of roquefortine C and griseofulvin and the appressed
occaSionally terverticillate penicilli.
P. oxalicum Currie et Thom 1915
P. aragonense Ramirez et Martinez 1981

P. asturianum Ramirez et Martinez 1981
P. fennelliae Stolk 1969

Series GLADIOLII Raper et Thorn ex Stolk et Samson
Series P. gladioli Raper et Thorn - Man. Penicillia: 471, 1949 (nom. inval.); ex Stolk et Samson - Adv.
Penicillium and Aspergillus Syst. p. 183. 1985
Penicillium gladioli is the only species classified in subgenus Penicillium which produces
sclerotia. It is closely related to the anamorphs of Eupenicillium crustaceum. Up till now, no
ascospores has been observed in any P. gladioli isolates.
P. gladioli McCulloch & Thom 1928
Series VIRIDICATA Raper et Thorn ex Pitt
Series P. viridicatum Raper et Thorn - Man. Penicillia: 481,1979

Series P. roqueforti Raper et Thorn - Man. Penicillia: 392, 1949
Series P. camemberti Raper et Thorn - Man. Penicillia: 421, 1949
Series P. commune Raper et Thorn - Man. Penicillia: 429, 1949
Series P. terrestre Raper et Thorn - Man. Penicillia: 446, 1949
Series P. ochraceum Raper et Thorn - Man. Penicillia: 475, 1949
Series P. cyclopium Raper et Thorn - Man. Penicillia: 490, 1949
(all nom. invaI., Arts 21, 36)
All species included in series Viridicata produce similar terverticillate, ocasionally

quaterverticillate conidiophores, as well as robust phialides. The conidia of most species
range from globose to subglobose, less commonly ellipsoidal, with walls smooth or nearly
so, except in P. echinulatum, which has echinulate conidia.
Species classfied in series Viridicata are very important spoilage and mycotoxin
producing fungi in food and feedstuffs. They have often been cited in the literature and
consequently a correct identification is important (Frisvad, 1989). Unfortunately the
identification of these species is often difficult, because of great variation within the
species and the presence of morphologically intergrading strains. Raper and Thom (1949)
considerably reduced the number of species accepted by Thom (1930) by regarding many
of them as synonyms. However, they still maintained a great number of closely related
species, which they distinguished from one another by minor differences, such a colony
texture and colour. In an attempt to simplify species determination in these species,
Samson et al. (1976) revived P. verrucosum Dierckx and emended the description, creating
a large variable species, which they divided into six varieties, mainly based on conidial
colour. By the use of colony diameters together with conidial and colony pigmentation to
129
separate species in subgenus Penicillium, Pitt (1979) reinstated some varieties as species.
However, recently novel characters and techniques have been introduced to provide a
more accurate delimitation of these closely related species. Morphological differences
between the species in series Viridicata are limited so more recently, the taxonomic
importance of profiles of secondary metabolites (SMPs) have assisted in delimiting species
in series Viridicata.
A comparative study of morphological and biochemical characters (SMPs) often
proved a correlation between them to be present. For instance even though the
morphological difference between P. verrucosum and P. aurantiogriseum is minute (P.
verrucosum has more distinctly roughened stipes), physiological and biochemical
differences between these two species are significant.
The morphology and physological characters of P. aurantiogriseum and P. viridicatum
are very similar and both taxa can only be distinguished by their secondary metabolites
and conidium colour. Frisvad and Filtenborg (1989) considered them as varieties.
P. aurantiogriseum Dierckx 1901 var. aurantiogriseum
P. aurantiocandidum Dierckx 1901

P. puberulum Bain. 1907
P. conditaneum Westling 1911
P. eyclopiumWestiing 1911
P. aurantiovirens Biourge 1923
P. brunneoviolaceum Biourge 1923
P. martensii Biourge 1923
P. porraceum Biourge 1923
P. aurantioalbidum Biourge 1923
P. johanniolii Zaleski 1927
P. polonicum Zaleski 1927
P. ochraceum Bain. apud Thorn 1930
P. carneolutescens G. Smith 1939
P. viridieyclopium Abe 1956
P. verrucosum Dierckx var. eyclopium (Westling) Samson et al. 1976
P. verrucosum var. ochraceum (Bain.) Samson et al. 1976
P. eyclopium var. aurantiovirens (Biourge) Fassatiova 1977
P. cordubense Ramirez et Martinez 1981
P. aurantiogriseum var. viridicatum (Westling) Frisvad and Filtenborg 1989

P. olivinoviride Biourge 1923
? P. blakeslei Zaleski 1927
? P. stephaniae Zaleski 1927
P. olivicolor Pitt 1979
P. camemberti Thom 1906
? P. album Epstein 1902

? P. epsteinii Lindau 1904
P. rogeri Wehmer apud Lafar 1906
P. caseicola Bain. 1907
P. biforme Thorn 1910
P. camemberti var. rogeri Thorn 1910
P. camemberti Sopp 1912
P. candidum Roger apud Biourge 1923
P. paedlomyceforme von Szilvinyi 1949
P. commune Thom 1910
P. palitans Westling 1911

P. fuscogtaucum Biourge 1923
P. f/avoglaucum Biourge 1923
P. ochraceum var. macrosporum Thorn 1930
130
A.C. Stolk et al.
P.lanosovirideThom 1930
P. psittaanum Thorn 1930

P. australicum Sopp ex van Beyma 1944
P. cyclupium var. album G. Smith
P. roqueforti var. punctatum Abe 1956
P. caseiperdens Frank 1966
P. verrucosum var. album (G. Smith) Samson et al. 1976
P. album (G. Smith) Samson et aI. 1976
P. crustosum Thorn 1930
P. lano50griseum Thorn 1930

P. pseudocasei Abe 1956 (ex G. Smith 1963)
P. terrestre Jensen sensu Raper and Thorn 1949
P. farinosum Novobranova 1974
P. echinulatum (Raper et Thorn) Fassatiova 1977

P. cyc/opum var. echinulatum Raper et Thorn 1949
P. crustosum var. spinulosporum Sasaki 1950
P. palitans var. echinoconidium Abe 195
P. solitum Westling 1911
P. majusculum Westling 1911

P. casei Staub var. compactum Abe
P. mali Novobranova 1972
P. verrucosum var. melanochlorum Samson et al. 1976 = P. melanochlorum (Samson et a/) Frisvad 1985
P. mali Gorlenko et Novobranova 1983
P. roque/orti Thorn 1906
P. aromaticum-casei Sopp 1898

P. vesiculosum Bain. 1907
P. roqueforti var. weidemannii Westling 1911
P. atroviride Sopp 1912
P. roqueforti Sopp 1912
P. virescens Sopp 1912
P. aromaticum Sopp 1912
P. aromaticum-casei Sopp ex Sacc. 1913
P. suavolens Biourge 1923
P. gorgonzolae Weidemann apud Biourge 1923
P. weidemannii (Westling) Biourge 1923
P. stilton Biourge 1923
P. weidemannii var. fuscum Arnaudi 1928
P. biourgei Arnaudi 1928
P. roqueforti var. viride Datilo-Rubbo 1938
P. conservandiNovobranova 1074
P. verrucosum Dierckx 1901
P. casei Staub 1911

P. mediolanense Dragoni et Cantoni 1979
P. nordicum Dragoni et Cantoni 1979; ex Ramirez 1985 (atypically big (and white) conidia)
Series EXPANSA Raper et Thorn ex Fassatiova
Series P. expansum Raper et Thorn - Man. Penicillia: 508. 1949.

Type species: P. expansum
Series Expansa contains only one species: P. expansum, which develops ter- to
quaterverticillate penicilli like those of species in series Viridicata, but differs by
131
predominantly smooth stipes and ellipsoidal conidia. In addition, cultures differ also in
growth rates, zonation and colour.
P. expansum causes a destructive rot of pomaceous fruit, on which it produces
conspicuous synnemata. In fresh isolates,synnemata or strongly fasciculate growth can
also be observed on agar media. P. crustosum and P. solitum in series Viridicata can also can
cause rot of pomaceous fruit, but this is less destructive and limited.
Penicillium expansum Link 1809
P. expansum Link ex S.F. Gray 1821

P. glaucum Link 1822
P. elongatum Dierckx 1901
P. musae Weidemann 1907
P. variabile Wehmer 1913
P. plumiferum Demelius 1923
P. aeruginosum Demelius 1923
P. leucopus (Pers.) Biourge 1919
P. kap-laboratorium Sopp apud Biourge 1925
P. janthogenum Biourge 1923
P. resticulosum Birkinshaw et al. 1942
Series GRANULATA Raper et Thorn ex Fassatiova
Series P. granulatum Raper et Thorn - Man. Penicillia: 539. 1949
Type species: P. granulatum Bain.
Series Granulata contains now five species which may easily be recognized by their
conspicuous synnemata, consisting of a distinct stalk and apical somewhat cylindrical to
subglobose feather-like capitulum. Conidiophores can be mono- or synnematous and are
ter- to quaterverticillate. Species of series Granulata differ from those in series Gladioli by
the presence of well-defined feather-like synnemata and the absence of ascomata and/ or
sclerotia. The production of synnemata is also the distinguishing character from the taxa
included in series Viridicata.
P. coprophilum (Berk. et Curt.) Seifert et Samson 1985
Coremium coprophilum Berk. et Curt. 1869
Stilbum humanum P. Karst. 1888
Pritzeliella caerulea Henn. 1903
Penicillium concentricum Samson et al. 1976
P. glandicola (Oud.) Seifert et Samson 1985

Coremium glandicola Oud. 1903
P. granulatum &in. 1905
P. divergens Bain. et Sartory 1912
P. schneggii Boas 1914
P. hirsutum Dierckx 1901
P. corymbiferum Westling 1911

P. verrucosum var. corymbiferum (Westling) Samson et al. 1976
P. hordei Stolk
P. allii Vincent et Pitt 1989
Series OLSONII Pitt
Series Olsonii Pitt - Gen. Penicillium: 392. 1979
Series P. brevicompactum Raper & Thorn - Man. Penicillia: 404,1949 (nom. inval. Arts. 21, 36)
132
A.C. Stolk et a/.
Type species: P. olsonn Bain. et Sartory
Series Olsonii contains only two species, morphologically closely related: P. olsonii and P.
brevicompactum. They differ mainly in the complexity of their penicilli. In P.
brevicompactum branches are borne singly, though occasionally two or three of them per
branching point may occur, whereas typical penicilli of P. olsonii produce a compact
verticil of up to to six branches developing apically and sometimes also subapically on the
stipe However, detioriorated strains of P. olsonii , such as the ex type of P. volgaense
produce smaller verticils of branches. Penicilli of P. olsonii are sometimes suggestive of the
conidiophores found in section Inordinate, but species of the latter section are
distinguished by phialide shape and brown colony colours.
P. brevicompactum Dierckx 1901
P. griseobrunneum Dierckx 1901

P. stoloniferum Thorn 1910
? P. tabescens Westling 1911
? P. monstrosum Sopp 1912
P. szaferi Zaleski 1927
P. hagemii Zaleski 1927
P. bialowiezense Zaleski 1927
? P. biourgeanum Zaleski 1927
P. patris-mei Zaleski 1927
P. brunneostoloniferum Abe 1956; ex Ramfrez 1982
P. olsonii Bain. et Sartory 1912
P. volgaense Beljakova et Mil'ko

P. breuicompactum var. magnum Ramfrez 1982
Series CLAVIFORMAE Raper and Thorn ex Stolk, Samson et Frisvad

Series P. claviforme - Man. Penicillia: 548, 1949
Series in subgenus Penicillium cum speciebus corerniis vel synnematibus portatis vel stipitites parietibus
levibus.
Species typica: P. vulpinum (Cooke et Massee) Seifert et Samson (= P. claviforme Bain. 1905
Raper and Thorn (1949) placed P. claviforme (= P. vulpinum) and P. clavigerum in P.

claviforme series. Since both species produce well defined synnemata and branched
conidiophores, they included the series in section Asymmetrica subsection Fasciculata. This
assignment was accepted by Samson et al. (1976), Ramirez (1982) and Frisvad and
Filtenborg (1983). Pitt (1979) classified both species in series Duclauxii of subgenus
Biverticillium and regarded P. clavigerum as a synonym of P. duclauxii.
Acuminate phi ali de necks as occur in subgenus Biverticillium were not observed by us
in P. vulpinum and P. clavigerum. P. vulpinum, characterized by long synnemata with
irregularily branched penicilli, occupies an isolated position in Penicillium just as P.
griseofulvum, and interestingly they are both coprophilous, synnematous, with smooth
stipes and ellipsoid smooth conidia. Furthermore they both produce patulin and
roquefortine C. They are clearly different in the structure of the conidiophores and the
phialide morphology, however, and are therefore placed in different series. Synnematous
species in subgenus Biverticillium typically occur on wood or feathers and they produce a
completely different profile of secondary metabolites (Samson et al., 1989).
P. vulpinum (Cooke et Massee) Seifert et Samson 1985

Coremium vulpinum Cooke et Massee 1888
The systematics 01 the terverticillate Penicillia
133
P. claviforme Bain. 1905

Coremium silvaticum Wehmer 1914
P. clavigerum Demelius 1923

KEY TO THE TERVERTICILLATE PENICILLIA
The following key is based on the morphology of fresh isolates grown on MEA and
Czapek agars. For the recognition of some species CREA and 0.5% acetic acid agar (Engel
and Teuber, 1978; Frisvad, 1981) can be very useful. Only in some cases e.g. P.
aurantiogriseum, an analysis of secondary metabolites can be conclusive for the
identification of the taxa. For a more detailed synoptic key, using physiological and
chemical data see Frisvad and Filtenborg (1990).
For the detailed description of the terverticillate species, the reader is referred to Samson
et al. (1976, 1977 a and b), Pitt (1979), Samson and Van Reenen-Hoekstra (1988), Pitt and
Hocking (1985) and Frisvad and Filtenborg (1989).
1.
Sclerotia and/or ascomata present ......................................................................................................................... 2

Sclerotia and ascomata absent ................................................................................................................................ 3
2.
Sclerotia present, no ascospores, stipes strongly roughened, conidia globose to subglobose, 2.2-3.5 1J.ffi,
smooth-walled ......................................................................................................................................... .P. gladioli
Sclerotia and fertile ascomata present, stipes with walls smooth or nearly so ............ Genus Eupenicillium
3.
Conidiophores strictly mononematous or both mononematous and loosely synnematous on MEA .......... 4
Conidiophores strongly synnematous on MEA ................................................................................................. 23
4.
Stipes on MEA with walls smooth or nearly so, conidiophores usually strictly mononematous, but in a
few species both mono- and loosely synnematous ............................................................................................. 5
Stipes on MEA with walls finely to conspicously roughened, conidiophores mononematous and/or
loosely synnematous ............................................................................................................................................... 13
5.
Phialides less than 6 ~m long, with a very short, sometimes inconspicuous neck, conidia grey green,
conidiophore branches strongly divergent, ................................................................................ P. griseofulvum
Phialides more than 6 ~m long, mostly with a distinct neck, conidia in different shades of green,
conidiophore branches slightly appressed or divergent ..................................................................................... 6
6.
Conidiophores, loosely bi- or terverticillate, sometimes more, branches when terminal slightly
appressed or divergent, when subterminal strongly divergent ......................................................................... 7
Conidiophores bi- to quaterverticillate with all elements strongly appressed ................................................ 9
7.
Conidia globose to subglobose, 2-2.8 ~m diam., with walls finely but distinctly roughened,
conidiophores bi- to terverticillate, occaSionally with one or more single subterminal and/or intercalary
branches, stipes with walls smooth or finely roughened .................................................................. P. lanosum
Conidia broadly ellipsoidal to subglobose or globose, 2.5-4 x 2.5-3.5 1J.ffi, smooth-walled, conidiophores
(bi-) ter to quaterverticillate, stipes smooth .......................................................................................................... 8
8.
Cultures blue-green to greyish blue green, reverse on Cz typically yellow, sometimes uncoloured,
production of penicillin, creatine negative .................................................................................. P. chrysogenum
Cultures greyish green to dark green, reverse on Cz orange-red to brownish orange, no production of
penicillin, creatine positive ......................................................................................................... P. Iltrllmentosum
9.
Penicilli short, compact, basically terverticillate, sometimes more complicated because of the septation of
metulae and branches, branches 1-4(6) per verticil, appressed ........................................................................ 10
Penicilli bi- to ter- or quaterverticillate, elongate, not short and compact, branches when present, usually
single ......................................................................................................................................................................... 11
134
A.C. Stolk et a/.
10. Conidiophores very large, with a terminal verticil of up to 6 appressed branches, developing on the apex
but sometimes also on the subapical part of the stipe ......................................................................... P. olsonii
Conidiophores smaller, usually with one asymetrcial ramus and branches short, often simply,
sometimes in a tenninal verticil of 2-3(4) ..............................................................................P. brevicompactum
11. Conidia ellipsoidal, 3-4 x 2.2-3 J.Un, smooth-walled, penicilli ter-(quater) verticillate; causing a rot of
pomaceous fruits ...................................................................................................................................P. exptmsum
~nidia cyli~~rical to strongly ellipsoidal, occasionally to
pyriform, more than 3.S J.Un in length, penicilli

bI-to tervertlollate ................................................................................................................................................... 12
12. Conidiophores mainly mononematous, in marginal areas often synnematous, fonning sometimes welldeveloped loose synnemata, conidia (3.S->4-6.5(-9) x 2.2-3.5 J.Un, smooth-walled, causing a rot of Citrus
fruits .......................................................................................................................................................... P. italicum
Conidiophores strictly mononematous, conidia 3.S-S.5(-7) x 2-2.S(-3), finely roughened, sometimes
appearing slightly striate, not causing a Citrus rot .......................................................................... P. fennelliae
13. Conidiophores mononematous, (bi-) terverticillate, with stipes smooth or finely roughened and
branches divergent, colonies lanose ..................................................................................................... P. lanosum
Conidiophores predominantly mononematous, sometimes also loosely synnematous, ter- (quater)
verticillate with branches usually appressed, especially in fresh cultures, colonies of fresh isolates at the
margin, often granular, sometimes tufted ........................................................................................................... 14
14. Conidia ellipsoidal, 3-4 x 2.3-3 J.Un, smooth-walled, conidiophores in marginal areas synnematous,
especially in fresh isolates, stipes on MEA smooth (sometimes finely roughened); causing a distinct rot
of pomaceous fruits .............................................................................................................................. P. expansum
Conidia globose and subglobose (sometimes slightly ellipsoidal), stipes on MEA finely to definitely
roughened, odour usually mouldy, sometimes causing a limited rot of pomaceous fruit .......................... 15
lS. Conidia small, (2.S-) 2.8-3.2(-3.S) J.Un diam., poor growth on creatine agar ................................................... 16
Conidia larger, 3-4(-S) 11m diam, heavy growth on creatine agar and production of base .......................... 18
16. Colonies on Cz and MEA not exceeding 20 mm diam. in seven days at 25C; no acid on creatine agar,
growth on nitrite agar, producing ochratoxins ............................................................................. P. verrucosum
Colonies on Cz and MEA exceeding 20 mm diam. in seven days at 2SoC, acid production on creatine
agar, no growth on nitrite agar, not producing ochratoxins ...................................... 17 (P. aUTantiogriseum)
17. Conidial areas dull blue-green or bright blue-green, metabolites: penicillic acid, xanthomegnin,
viomellein, and viridicatin ................................................................. P. aUTantiogriseum var. aUTantiogriseum
Con~dial ~reas green, metabolites as var. aurantiogriseum, but with vi~da~in and oc~a.si?nally
breVianarmde ............................................................................................... P. aurantiogrlseum var. vlnd,catum
18. Conidiophores strictly mononematous, with walls of stipes, branches and metulae usually strongly
granular from encrustments, conidia 3.S J.Un diam, smooth-walled; colonies on Cz and MEA spreading
broadly, more than 40 mm diam, at 25C in seven days, green, reverse usually dark green, growth on O.S
% acetic acid .......................................................................................................................................... P. roqueforti
Conidiophores predominantly mononematous, marginal areas often also loosely synnematous, with
stipes finely to strongly roughened on MEA, conidia 3-4(-S) J.Un diam.; colonies on Cz and MEA usually
not exceeding 40 mm diam. at 25C in seven days, reverse not dark green, not growth on 0.5% acetic
acid ............................................................................................................................................................................ 19
19. Conidia with walls strongly roughened ....................................................................................... P. echinulatum
Conidia with walls smooth or nearly so .............................................................................................................. 20
20. Conidial areas white or pale grey green, conidiophores mononematous, colonies often floccose ................ .
.............................................................................................................................................................. P. camemberti
Conidial areas in shades of blue-green, green or greyish green, colonies not floccose ................................ 21
21. Conidial areas dull green to greyish green, forming crusts .......................................................... P. crustosum
Conidia areas blue-green, green or dark green, no crust formation ................................................................ 22
The systematics of the terverticillate PenicilHa
135
22. Conidial areas greyish blue, blue-green or greenish; metabolites: cyclopiazonic acid, cyclopaldic acid,
rugulovasine, palitantin, not causing apple rot ................................................................................ P. commune
Conidial areas dark green or dark blue-green, metabolites: viridicatin, compactins; causing restricted
apple rot ..................................................................................................................................................... P. solitum
23. Colonies strictly synnematous, 24 mm in length, sometimes longer, with a pinkish to reddish stalk and
a clavate green head .............................................................................................................................. P. vulpinum
Conidiophores synnematous, but retaining their individuality, mononematous structures also present 24
24. Synnemata 24 mm high, not differentiated into a distinct stalk and an enlarged head, conidiophores
somewhat irregular, bi to ter-(quater)verticillate borne over nearly the entire length of the synnema,
branches singly, usually divergent, sometimes appressed ........................................................... P. clavigerum
Conidiophores aggregated into distinct, but loosely constructed coremia, consiting of a short, sometimes
inconspicuous stalk and a feathery conidial head, regularly ter- to quaterverticillate, synnemata
interspersed with abundant mononematous conidiophores ............................................................................ 25
25. Conidia ellipsoidal, smooth-walled ...................................................................................................................... 26
Conidia globose, with walls smooth or roughened ........................................................................................... 27
26. Synnemata on MEA, developing in concentric zones with a short white stalk and a green head,
conidiophore elements smooth ..................................................................................................... P. coprophilum
Synnemata on MEA sometimes in concentric zones, strongly feathery, conidiophore elements strongly
roughened ............................................................................................................................................ P. glandicola
27. Conidia distinctly roughened, stipes with walls smooth or finely roughened, mostly associated with
cereals and soil ............................................................................................................................................. P. horaei
Conidia smooth, stipes, branches and metulae strongly roughened, mostly associated with flower bulbs,
onions and vegetables ................................................................................. P. hirsutum (compare also P. allii )
ACKNOWLEDGEMENTS
The authors thank the NATO Scientific Affairs Division (Brussel, Belgium) for the research
grant (0216/86) for international collaboration.
REFERENCES
BIOURGE, P. 1923. Les moississures du groupe Penicillium Link. Cellule 33: 7-331.
BIRCH, A.J. and STAPLEFORD, K.S.J. 1967. The structure of nalgiolaxin. Journal of the Chemical Society C
1%7: 2570-2571.
CRUICKSHANK, R.H. and PITT, J.1. 1987. Identification of species in Penicillium subgenus Penicillium by
enzyme electrophoresis. MycologiJl79: 614-620.
ENGEL, G. and TEUBER, M. 1978. Simple aid in the identification of Penicillium roqueforti Thorn. Growth in
acetic acid. European Journal of Applied Microbiology and Biotechnology 6: 107-111.
FASSATIOVA, O. 1977. A taxonomic study of Penicillium series Expansa Thorn emend. Fassatiova. Acta of the
University Caroliana, Biology. 12: 283-335.
FILTENBORG, 0., J.e. FRISVAD and SVENDSEN, J.A. 1983. Simple screening method for molds producing
FRISVAD, J.e. 1981. Physiological criteria and mycotoxin production as aids in identification of common
asymmetric Penicillia. Applied and Environmental Microbiology 41: 568-579.
- - 1985. Creatine-sucrose agar, a differential medium for mycotoxin producing terverticillate Penicillium
species. Letters in Applied Microbiology 1: 109-113.
--1986. Taxonomic approaches to mycotoxin identification (taxonomic indication of mycotoxin content of
foods). In Modern Methods in the Analysis and Structural Elucidation of Mycotoxins, ed. R.J. Cole, pp.
415457. New York: Academic Press.
- - 1988. Fungal species and their specific production of mycotoxins. In Introduction to food-borne fungi,
eds. R.A. Samson and E. van Reenen-Hoekstra, pp. 239-249. Baarn: Centraalbureau voor
Schimmelcultures.
136
A.C. Stolk et al.
--1989a. The connection between the Penicillia and Aspergilli and mycotoxins with special emphasis on
misidentified isolates. Archives of Environmental Contamination and Toxicology 18: 452-467.
- - 1990. The use of high-performance liquid chromatography and diode array detection in fungal
chemotaxonomy based on profiles of secondary metabolites. Botanical Journal of the Linnean Society 99: 8195.
FRISVAD, J.e. and FILTENBORG, 0.1983 Classification of terverticillate Penicillia based on profiles of
--1989. Terverticillate Penicillia: chemotaxonomy and mycotoxin production. Mycologia 81 (in press)
- - 1990. Secondary metabolies as consistent criteria in Penicillium taxonomy and a synoptic key to
Penicillium subgenus Penicillium. In Modern Concepts of Penicillium and Aspergillus Classification, eds.
RA. Samson and J.1. Pitt, pp. 373-384. New York and London: Plenum Press.
FRISVAD, J.e., FILTENBORG, O. and WICKLOW, D.T. 1987. Terverticillate Penicillia isolated from
underground seed caches and cheek pouches of banner-tailed kangaroo rats (Dipodomys spectabilis).
mycotoxins and other fungal secondary metabolites based on aIkylphenone retention indices and UV-VIS
spectra (diode array detection). Journal of Chromatography 404: 195-214.
PITT, J.1. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Ta/aromyces. London:
Academic Press.
--1988. A Laboratory Guide to Common Penicillium Species. Second edition. North Ryde, N.S.W.: CSIRO
PITT, J.I. and HOCKING, A.D. 1985. Fungi and food spoilage. Sydney: Academic Press.
RAMIREZ, e. 1982. Manual and AtIas of the Penicillia. Amsterdam: Elsevier Biomedical.
SAMSON, RA. and PITT, J.I. 1985. Check list of common Penicillium species. In Advances in Penicillium and
Aspergillus Systematics, eds. RA. Samson and J. I. Pitt, pp. 461-463. New York and London: Plenum
Press.
SAMSON, R.A. and REENEN-HOEKSTRA, E.S. van. 1988. Introduction to food-borne fungi. Third edition.
Baam: Centraalbureau voor Schimmelcultures.
SAMSON, RA., STOLK, A.e. and HADLOK, R. 1976. Revision of the Subsection Fasciculata of Penicillium
and allied species. Studies in Mycology, Baarn 11: 1-47.
SAMSON, RA., HADLOK, Rand STOLK, Ae. 1977a. A taxonomic study of the Penicillium chrysogenum
series. Antonievan Leeuwenhoek 43: 169-175.
SAMSON, RA., ECKARDT, e. and ORTH, R 1977b. The taxonomy of Penicillium species from fermented
cheeses. Antonie van Leeuwenhoek 43: 341-350.
SEIFERT, K.A and SAMSON, R.A 1985. The genus Coremium and the synnematous Penicillia. In Advances
in Penicillium and Aspergillus Systematics, eds. RA. Samson and J. I. Pitt, pp. 143-154. New York and
Londen: Plenum Press.
SAMSON, R. A, STOLK, A.e. and FRISVAD, J.e. 1989. Two new synnematous species of Penicillium. Studies
in Mycology, Baarn 31: 133-143.
THOM, e. 1930. The Penicillia. Baltimore: Williams and Wilkins.
DIALOGUE FOLLOWING DR. SAMSON'S PRESENTATION
In defining and delimiting subgen. Penicillium, I tried to avoid phylogenetic

considerations. The taxonomy is directed towards the user rather than the systematist. A
species like P. oxalicum should not be placed in subgen. Penicillium because it only rarely
produces terverticillate penicilli and so a user would not look for it there.
Regarding the possibility of using the name P. majusculum for what we have been calling
P. solitum, the Code does not indicate priority when two species are described in the
same paper. It's a matter of choosing which species best matches the adopted concept, or
of maintaining usage of a name that is already being used. Raper used the name P.
PITT:
137
soli tum and so this name should be used, especially as the species concept has not
changed.
SAMSON: But the description of P. majusculum fits the concept we are discussing here
better.
STOLK: The original description of P. majusculum states that conidia are a dark green to
black colour, like those of P. melanochlorum. P. solitum was described as having bluegreen conidia. I think P. majusculum is a better name, but you can, of course, use P.
solitum, because both names are valid.
SAMSON: Yes, I agree, we can use the epithet solitum. It is an established name.
139
A REAPPRAISAL OF THE TERVERTICILLATE PENICILLIA USING

BIOCHEMICAL, PHYSIOLOGICAL AND MORPHOLOGICAL FEATURES
P.O. Bridge1, D.L. Hawksworth1, Z. Kozakiewicz1, A.H.S. Onions1,
RRM. Paterson 1, M.J. Sackin2 and P.H.A. Sneath2
leAB International Mycological Institute
Kew, Surrey, TW9 3AF, UK
2Department of Microbiology
University of Leicester
Leicester, LEI 9HN, UK
SUMMARY
Three hundred and forty eight strains of terverticillate Penicillia were examined for 100 physiological,
biochemical and morphological characters. These characters included assessing growth on specific
carbon and nitrogen sources, screening for enzyme production, thin layer chromatography of
secondary metabolites, and scanning electron microscopy of conidia. Test reproducibility and strain
variation were critically considered. Resemblances between each pair of strains were calculated with
Gower's coefficient and the Pattern difference, and dendrograms plotted. Overlap between groups
was calculated and differences between dendrograms were considered in determining 37 species or
species complex clusters. In addition the dendrogram contained a number of ungrouped strains,
mainly single representatives of species included for comparison. In total, 91 % of the strains were
considered grouped.
Additional studies on strain variation and pectinase and amylase isoenzymes were undertaken by
electrophoresis to test the homogeneity of the clusters. This showed that unique banding patterns
were obtained for only two species, and in general a considerable range of patterns were produced for
each species. High resolution melting curves of the DNBA of 14 of the test strains indicated
considerable similarities in the DNA base distributions. Extensive studies on strain variation for all
characters studied showed that although considerable phenotypic variation may occur within both
species and strains, sufficient reliable characters were available to define taxa. Variation within and
between strains could be accounted for by gene or partial chromosome duplication; this hypothesis
was supported by some of the electrophoretic patterns and variations in conidial DNA contents.
This study showed the terverticillate Penicillia to be a group of very similar fungi. Many of the
commonly used taxonomic characters are subjective and can vary significantly within single cultures.
Satisfactory species concepts can be defined by using an integrated multidisciplinary approach and
reliable quantitative identification schemes produced.
INTRODUCTION
The taxonomy of the terverticillate Penicillia has been considerably revised in recent years
(e.g. Samson et al., 1976; Pitt, 1979), but nevertheless areas of uncertainty remains (Onions
et al., 1984; Samson and Gams, 1984). To clarify the systematics and species concepts in the
terverticillate Penicillia, a major integrated multidiSciplinary study was undertaken. This
involved selecting and developing taxonomic characters from morphology, physiology,
biochemistry, and scanning electron microscopy. These characters were then critically
examined for reproducibility and reliability. Additional studies were also carried out on
intra-strain variation and stability (Bridge et al., 1986b). The data were used in a numerical
taxonomy to produce species level clusters in dendrograms (Bridge et al., 1989a). The final
140
P.O. Bridge et al.
clusters were further tested by the construction of a percent positive identification matrix
(Bridge, 1990) and by comparison of electrophoretic amylase and pectinase isoenzyme
patterns. The full methods and results from these studies are published elsewhere
(Paterson, 1986; Bridge et al., 1987; 1989a, b; Paterson et al., 1989a; Kozakiewicz, 1989). This
paper presents an overview of the completed study.
NUMERICAL TAXONOMY
One hundred characters from morphology, physiology, biochemistry and scanning

electron microscopy were selected from over 200 assessed during the study (Bridge, 1985;
Paterson, 1986; Bridge et aI., 1986a, 1987; Kozakiewicz, 1989). Results from these 100
characters were recorded for 348 strains of Penicillium, consisting mainly of terverticillate
species, with some representatives of other subgenera for comparison. Test reproducibility
and strain variation were assessed by including duplicate cultures in the study,
performing tests in duplicate, and repeating tests for up to 80% of the strains at a later
date. Resemblances between strains were calculated with Gower's coefficient (Sneath and
Sokal, 1973) and the Pattern difference (Dp2; Sackin, 1981). The results were clustered by
unweighted average linkage (UPGMA) and single linkage (Sneath and Sokal, 1973) and
presented as dendrograms.
The UPGMA dendrogram from Gower's coefficient was used as the basis of the
taxonomy. This represented the strains in 37 clusters that could be recognised as species or
species complexes at approximately 70% similarity. These clusters contained 80% of the
total number of strains. When single representatives of species and strains included for
comparison were excluded, 91 % of the strains were grouped. A simplified version of this
dendrogram is given in Figure 1. Few of the groupings recovered were entirely surprising
or new, and so only selected taxa will be discussed here.
The P. expansum strains were recovered in three small clusters separately at the top of
the dendrogram. While some differences existed between these groups, they were overall
very similar and so are considered as representing variants of a single species. The P.
aurantiogriseum strains were mainly recovered in one large cluster, although five strains
including the ex-type culture of P. puberulum were recovered as a separate cluster from the
main P. aurantiogriseum cluster. There are no clear-cut differences between this small
cluster and the main P. aurantiogriseum cluster; the name P. puberulum must therefore be
considered synonymous with P. aurantiogriseum. The major area of taxonomic confusion,
including strains named as P. crustosum, P. ochraceum, P. commune or P. palitans, was
recovered as one large cluster. Considerable further work on this area of the study,
including overlap calculations and principal coordinate analysis, suggested that there may
be two very similar taxa here. The oldest reliable name recovered in this cluster was P.
solitum and the main cluster was named as this, with a variety P. solitum var. crustosum.
Strains received as P. chrysogenum or its synonyms were recovered in two separate
clusters. Most of the differences between these clusters were a matter of degree and it was
considered that one cluster represented deteriorated lines of P. chrysogenum. Included in
one of these clusters was the ex-type culture of P. griseoroseum, a name that pre-dates P.
chrysogenum; this placement is in agreement with the findings of Samson et al. (1977) and
Cruickshank and Pitt (1987). However, this strain was in a deteriorated condition and no
original herbarium type material was available, therefore any name change is
inappropriate (see chapter 2, this volume).
A reappraisal of the terverticillate Penicillia

0.'
0.'
0.'
0.'
P. expansum
P. aurantiogriseum
P. soli tum
P. 8ucantiogriseum
P. chrysogcnwn
P. cchinulatum
p.
ali vinoviridc
P. granulatum var. globosum

P. granulatum var. granulatum
P. hirsutum
P. chrysogenum
P. glandicola
var. mononesaatosa
P. camembertii
P. vcrrucosum
P. atramcntosum
Penicillium sp.
P. citrinum
P. brevicompactum
P. raistrickii
P. aurantiogrisewa
var. neoec.hinulatum
P. viridicatum
P. griscoful Yum
Penicillium sp.
P. hirsutum ver.aUB
Corenaial isolates
P. aurantiogriseum
P. ~fivi~:;~oconidium
P. roquet ortii
P. corylophilum
P. c1aviforme
0.'
Figure 1. Simplified UPGMA dendrogram based on Gower's coefficient
141
142
P.O. Bridge et al.
The species P. granulatum and P. camemberti were both recovered as two clusters. In the
case of P. camemberti, these two clusters remained linked together in all of the
dendrograms, differing from each other in their conidial ornamentation and in their
vigour and overall physiological activity. One of these clusters also contained a natural
white-spored mutant of P. solitum. While the conidial ornamentation suggested a distinct
separation into two taxa, the overlap and other data suggested that they were very similar.
These cultures may have been assigned to the single species P. camemberti previously due
to their production of white conidia. The presence of the white mutant further
complicated the interpretation and these two groups have been tentatively retained as a
single taxon. The only other white-spored culture included in the study, P. aurantiogriseum
var. album, was recovered in the smaller of the two P. aurantiogriseum clusters. In the case
of P. granulatum there were sufficient differences between the two clusters to warrant the
creation of a variety, P. granulatum var. globosum.
Strains received as P. atramentosum and P. meleagrinum clustered as three groups in the
UPGMA dendrogram based on Gower's coefficient and merged to form two groups in the
Pattern difference dendrogram. Further investigations of a line of the ex-type culture of P.
atramentosum not included in the study placed this in a different cluster from one regarded
as typical of P. atramentosum by Pitt (1979). Overall, these clusters appear to represent
variants of a single species correctly named as P. atramentosum.
The strains received as P. brevicompactum and P. olsonii clustered together as a single
group. These species could not be separated on any characters and further work (see later;
isoenzyme studies) also failed to separate all but one P. brevicompactum from P. olsonii.
These species should therefore be considered synonymous.
Overall, the numerical taxonomy resolved the strains into satisfactory clusters.
However, additional information can be gained from this approach. The cophenetic
correlation for the UPGMA Gower's coefficient dendrogram is relatively low (0.78) and
the overall test reproducibility was also low (0.82 for repeated cultures). The overlap
calculations showed that although clusters could be regarded as distinct, there was less
confidence in the distinctions between groups of clusters. Many species showed
significant intra-specific variation and in some cases, such as conidial ornamentation in P.
solitum, gradations in characters within the same species were observed. One possible
interpretation of all of these factors would be that the species groups may represent
distinct points within or on a semi-continuous spectrum. Regrettably there is very little
data available on that situation for reliable comparisons to be made here.
VARIATION AND TEST REPRODUCIBILITY
The influence of basal media and additions to basal media were examined as part of the
overall assessment of test reproducibility. The production of both secondary metabolites
and extracellular enzymes can be profoundly influenced by relatively minor changes in
basal medium such as the addition of copper and zinc (Smith, 1949) or the manufacturer
and type of organic ingredients such as peptone (Odds et al., 1978). Recently, the
production of synnemata in the non-fasciculate species P. funiculosum has been
demonstrated on a medium containing tributyltin compounds (Newby and Gadd, 1987).
True synnemata can, however, be induced in many of the fasciculate Penicillia by growth
on a variety of compounds including low levels of pentachlorophenol (see Figure 2). This
demonstrates that the difference between the synnematal species P. daviforme and P.
clavigerum and the fasciculate Penicillia is probably not based on a large structural
143
difference but on a small difference in the control of gene expression; these taxa may
therefore be more closely related than previously recognized.
Variations in test results between different workers in different laboratories is often
due to small differences in the methods used. The ability of Penicillia to grow on sodium
nitrite is an example of this, where different workers have used different concentrations of
nitrite. Sodium nitrite can have a toxic effect which is dependent on pH and concentration,
so variations in these parameters can affect the apparent ability of strains to grow on the
medium (Bridge, 1988).
As a result of the relatively low levels of test reproducibility experienced during the
numerical taxonomy, particularly with certain strains, intra-strain variation was studied.
A number of lines of the same cultures were isolated from single conidia. These lines were
maintained separately and differences in characteristics noted. Overall, minor differences
were apparent in most cases, but in certain strains significant differences in phenotypic
properties were found (Bridge et al., 1986b; Bridge et al., 1987). One possible explanation
for this phenomenon is some kind of genetic rearrangement, perhaps involving
aneuploidy or chromosome duplication and transposition. Further support for this is the
variation in DNA content between conidia of these lines (Bridge et al., 1986c; 1987) and the
description of similar occurrences in other fungi where differences in chromosome size
and number have been demonstrated, such as in Candida (Suzuki et al., 1989).
Figure 2. Induction of synnemata of fasciculate Penicillia by pentachlorophenol (right).
ISOENZYME STUDIES
The electrophoretic patterns of pectinase and amylase isoenzymes were determined for
170 representative strains from the main study using the methods of Cruickshank and
Wade (1980) and Cruickshank and Pitt (1987). There was considerable variation in the
144
P.O. Bridge et al.
intensity of the pectinase patterns from some strains in repeat runs. In many cases
considerable variation was observed in the patterns shown by members of the same
species. The results from the electrophoretic study were analyzed by numerical methods
and the final data set was represented as a dendrogram. The clusters formed in this way
represented strains with the same or similar enzyme banding patterns. These clusters did
not in general correspond to species. All but one of the strains received as P.
brevicompactum gave very similar patterns to those received as P. olsonii, a result similar to
that observed by Cruickshank and Pitt (1987). The single strains of P. funiculosum and P.
roqueforti clustered separately. The other species were represented in more than one
cluster, although some clusters contained only one species (Paterson et al., 1989a).
Interpretation of the differences in isoenzyme patterns between members of the same
species was carried out, and several of the observed differences could be explained by
chromosomal reorganization; this adds further support to the possibility mentioned
above.
DNA BASE CONTRIBUTIONS
High resolution DNA melting curves were obtained for 14 of the strains tested in the main
study, 13 of which were also analysed for isoenzymes (Paterson et al., 1989a, b). The DNA
base distributions obtained indicated considerable similarity between strains at this
fundamental level.
CONCLUSIONS
The application of an integrated multidisciplinary approach combined with numerical

taxonomy techniques and critical studies on test reproducibility has proved to be a very
powerful tool in the systematics of the filamentous fungi. The Penicillia studied here can
be classified in species level groups. Although the fasciculate Penicillia can show
considerable phenotypic variation within both species and strains, species concepts were
firmly established. More detailed further studies have supported the numerical taxonomy
in showing the overall similarity of these fungi, and the suggestion of a genetic basis for
some phenotypiC variation raises possibilities for future studies.
ACKNOWLEDGEMENTS
This work was supported by Science and Engineering Research Council contract
50/17/84 "Systematics of microfungi of biotechnological and industrial importance". We
would like to thank E.M. Oliver, P. Farnell and D. Kavishe for their excellent technical
assistance, and Dr. L.E. Fellows and the Royal Botanic Gardens, Kew, for providing
certain biochemical facilities.
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BRIDGE, P.O. 1985. An evaluation of some physiological and biochemical methods as an aid to the
characterization of species of Penicillium subsection Fasdculata. Journal of General Microbiology 131: 18871895.
- - 1988. A note on use and interpretation of nitrite assimilation tests in Penicillium systematics.
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--1990. Identification of terverticillate Penicillia from a matrix of percent positive test results. In Modern
concepts in Penicillium and Aspergillus classification, eds. R.A. Samson and J.1. Pitt, pp.OO-OO. New York
BRIDGE, P.O., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, R.R.M and
SACKIN, M.J. 1986a. An integrated approach to Penicillium systematics. In Advances in Penicillium and
Aspergillus Systematics, eds R.A. Samson and J.I. Pitt pp. 281-307. New York and London: Plenum Press.
BRIDGE, P.O., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS A.H.S. and PATERSON, R.R.M. 1986b.
Mycological Society 87: 389-396
BRIDGE, P.O., HUDSON, L., HAWKSWORTH, D.L. and BRIDGE, D.A.. 1986c. Variation in nuclear DNA
content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry. FEMS
Microbiology Letters 37: 241-244
BRIDGE, P.O., HUDSON, L., KOZAKIEWICZ, Z., ONIONS, A.H.S. and PATERSON, R.R.M. 1987.
Penicillium strains. JournaI of General Microbiology 133: 995-1004
BRIDGE, P.O., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON R.R.M., SACKIN,
M.J. and SNEATH, P.H.A. 1989a. A reappraisal of terverticillate Penicillia using biochemical,
physiological and morphological features. I. Numerical taxonomy. Journal of General Microbiology (in
press).
- - 1989b. A reappraisal of terverticillate Penicillia using biochemical, physiological and morphological
features. II. Identification. Journal of General Microbiology (in press).
CRUICKSHANK, R.H. and WADE, G.c. 1980. Detection of pectin enzymes in pectin-acrylamide gels.
Analytical Biochemistry 107: 177-18l.
CRUICKSHANK, R.H. and pm, J.1. 1987. Identification of species in Penicillium subgenus Penicillium by
KOZAKIEWICZ, Z. 1989. Ornamentation types of conidia and conidiogenous structures in fasciculate
Penicillium species, using scanning electron microscopy. Botanical Journal of the Linnean Society 99: 273-293.
NEWBY, P.J. and GADD, G.M. 1987. Synnema induction in Penicillium funiculosum by tributyltin
compounds. Transactions of the British Mycological Society 89: 381-384.
ODDS, F.e., HALL, e.A. and ABBOTT, A.B. 1978. Peptones and mycological reproducibility. Sabouraudia 16:
237-246.
ONIONS, A.H.5., BRIDGE, P.O. and PATERSON, R.R.M. 1984. Problems and prospects for the taxonomy of
PATERSON, R.R.M. 1986. Standardized one and twCHiimensional thin-layer chromatographic methods for
the identification of secondary metabolites in Penicillium and other fungi. Journal of Chromatography 368:
249-264.
PATERSON, R.R.M .., BRIDGE, P.O., CROSSWAITE, M.J. and HAWKSWORTH, D.L. 1989a. A reappraisal of
the terverticillate Penicillia using biochemical physiological and morphological features. III. An
evaluation of pectinase and amylase isozymes for species characterization. Journal of General Microbiology
(in press).
PATERSON, R.R.M., KING, G.J. and BRIDGE, P.O. 1989b. High resolution thermal denaturation studies on
DNA from 14 Penicillium isolates. Mycological Research (in press).
PITT, J.1. 1979. The genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
SACKIN, M.J. 1981. Vigour and pattern as applied to multistate quantitative characters in taxonomy. Journal
of General Microbiology 122: 247-254.
SAMSON, R.A. and GAMS, W. 1984. The taxonomic situation in the Hyphomycete genera Penicillium,
Aspergillus and Fusarium. Antonie van Leeuwenhoek 50: 815-824.
SAMSON, R.A., STOLK, A.e. and HADLOK, R. 1976. Revision of the subsection Fasciculata of Penicillium
and some allied species. Studies in Mycology, Baarn 11: 1-47
SAMSON., R.A., HADLOK, R. and STOLK, A.c. 1977. A taxonomic study of the Penicillium chrysogenum
SMITH, G. 1949. The effect of adding trace elements to Czapek Dox medium. Transactions of the British
SNEATH, P.H.A. and SOKAL, R.R. 1973. Numerical Taxonomy. San Francisco: W.H. Freeman and Sons.
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SUZUKI, T., KOBAYASHI, I., KANBE, T. and TANAKA, K. 1989. High frequency variation of colony
morphology and chromosome reorganization in the pathogenic yeast Candida albicans. Journal of General
Microbiology 135: 425-434.
DIALOGUE FOLLOWING THE PAPER BY DR. BRIDGE AND COLLEAGUES
CHRISTENSEN: I have also been concerned about the problem of degenerated cultures. Did
you try your Principal Components Analysis (PCA) using just the newly isolated
cultures.
BRIDGE: We tried to avoid the situation of working with only freshly isolated cultures. The
strains that we receive for identification at CMI vary enormously in quality. In our work,
we tried to use a representative mix of cultures. If you are worried about things like
deteriorated cultures, you can use coefficients such as the pattern coefficient, which has a
component in it that allows you to compensate for the vigour of the strains. For example,
the two P. chrysogenum strains came together easily using the pattern coefficient. Again,
P. puberulum and P. aurantiogriseum came together with the pattern coefficient.
CHRISTENSEN: What percent of the variation was represented in the three axes of the PCA?
BRIDGE: Not an awful lot. About 28%.
CHRISTENSEN: That seems low to me. How do you account for that low figure?
BRIDGE: You are dealing with a reduction of 100 characters down to three dimensions.
This is in effect a large compression of data to fit it into three dimensions, and it is
inevitable that some information will be lost. In this case, it suggests that there is a lot of
variation in a lot of characters. This is a complex situation. If you can reduce a
multidimensional system to two or three dimensions, via PCA, and get 60-70% of the
variability explained, you probably have a simple situation. It is a way of looking at the
data without imposing a hierarchy.
CHRISTENSEN: In ecological studies often 70-80% of the variation is represented in the first
three axes.
BRIDGE: But in ecological studies, one often has nice distinct groups.
PITT: Most of us consider P. crustosum to be a very well-defined species. It surprises me to
see several other strains clustering with it. What did you do with P. italicum?
BRIDGE: We didn't really deal with it in this study, but we had a couple of marker strains
of P. italicum, and they clustered together at the bottom.
SAMSON: But P. italicum is a typical terverticillate species.
PITT: Were some species from subgenus Penicillium left out of the analysis?
BRIDGE: Yes.
PITT: My other concern is your new variety, P. granulatum var. globosum. This species has
ellipsoidal conidia, and it is difficult for me to imagine a variety with spherical conidia. It
must be a different species.
147
BRIDGE: Here we have the problem of having a difference of only one or two characters,
in this case mainly conidial characters. I don't wish to raise something like that to species
status until we are more confident of these differences.
SAMSON: P. glandicola (= P. granulatum) typically has ellipsoidal conidia. We should not
include a variety with globose conidia in the same species.
BRIDGE: But as we see it, the other morphological characters suggest that this is P.
granulatum.
FRISVAD: A lot of your clusters fit nicely with our secondary metabolite groups. But I am
concerned that P. olsonii and P. brevicompactum clustered together, especially as you
included secondary metabolites. We have examined about 200 strains, and P.
brevicompactum always produces mycophenolic acid and the five or so Raistrick phenols.
Brevianamide A is sometimes, but not always, produced. P. olsonii produces some
mystical unknown compounds, but does not produce any of those made by P.
brevicompactum.
PATERSON: A difference of one metabolite may not make much difference in the numerical
analysis.
FRISVAD: Even degenerated strains of P. brevicompactum produce some of these secondary
metabolites.
BRIDGE: If these metabolites were consistently produced, I would expect to see some
difference between the two species, even if it might be within one cluster. However,
there was no evidence of breakdown within this cluster.
FRISVAD: In 1980, I checked all the P. brevicompactum strains listed in the CMI catalogue,
including the type of P. griseobrunneum, and they all produced mycophenolic acid.
FIL TENBORG: What yeast extract did you use for growing your fungi for analysis of
secondary metabolites?
PATERSON: Difco in YES and CYA.
SAMSON: Was all the scanning electron microscopy done with cryofixation?
KOZAKIEWICZ: Cryofixation was used for examining the penicillus. Conidium
preparations were air dried.
Pm: What temperature did you use for doing the isoenzyme patterns.
PATERSON: We used water cooled electrophoresis at 10e.
PITT: This concerns me because Dr. Cruikshank uses 4e. He gets good separation at that
temperature. Perhaps 10 C does not give such good separation.
BRIDGE: We did get good band patterns, but they weren't reproducible within a species.
PATERSON: Well, some strains were reproducible, but others weren't. Proteins don't
denature at 100e.
149
EVALUATION OF THE DIAGNOSTIC FEATURES OF SOME SPECIES OF

PENICILLIUM SECTION DIVARICATUM
D. Fassatiova and A. Kubatova
Department of Botany
Charles University
12801 Praha, Czechoslovakia
SUMMARY
The study was carried out with about 200 isolates mainly from Czechoslovakia and belonging to
Penicillium sect. Divaricatum: P. janczewskii, P. canescens, P. melinii, P. radulatum, P. janthinellum and P.
simplicissimum. The type isolates were compared according to their micro- and macromorphology. The
following are recommended as diagnostical features: the branching of conidiophores, divergence of
metulae and phialides and their length and the character of conidia. The length of phialides is
accurately measured omitting the neck, as neck length can be variable in a single isolate. P. janczewskii
and P. daleae can be distinguished satisfactory on the basis of their micromorphology and colonies. In
both species colonies are very variable in degree of sporulation, in conidial colour and in exudate and
reverse colours. P. granatense is a synonym of P. janczewskii based on our observation of the type
isolate. Isolates of P. canescens have good micromorphological distinguishing features. Isolates of P.
melinii are very similar in micromorphology and colony character to P. radu/atum, which has been only
found once in Czechoslovakia. The type isolate of P. melinii studied during our investigation is
different from the original description and also from our own isolates. A great number of soil isolates
could be identified as either P. janthinellum or P. simplicissimum because their micromorphology is
very similar. They probably represent one species.
INTRODUCTION
During soil isolation of microfungi in Czechoslovakia and in Poland, the more abundantly
occurring representatives of Penicillium sect. Divaricatum were selected on the basis of
colony and conidiophore morphology. We present our own descriptions on colonies
grown on Czapek-Dox agar (CZ) and Czapek yeast extract agar (CYA), incubated at 25C.
Strains of the species P. janczeuJskii, P. daleae, P. canescens, P. simplicissimum, P. melinii, P.
radulatum and P. janthinellum from our region have been deposited in a provisional
collection as 015-0220 and will gradually be transferred into the Culture Collection of
Fungi, CCF, in the Department of Botany, Charles University, Prague. We were able to
compare our isolates with some type cultures from foreign collections.
Penicillium simplicissimum (Dud.) Thom

Syn. Penicillium janthinellum Biourge
CZ: Colonies 30-60 mm diam in two weeks. White, lanose, floccose, raised at centre or depressed. The
colonies becoming in centre pink, salmon, at the margin sometimes yellow. Sporulation in the submarginal
area blue-green or grey-green or inconspicuous. Other colonies velvety or feltlike with yellowish, beige or
ochraceous beige mycelium. Sporulation grey-green and gradually proceeding to the centre though
sometimes only in the submarginal area. Colonies sometimes radially or irregularly sulcate with sparse
shallow or deep furrows. Reverse colourless, intensive pink, orange-ochraceous, yellowish, light green,
salmon, sometimes coral in the centre and at the margin brick coloured. Soluble pigment salmon, light
150
O. Fassatiova & A. Kubatova
....-ft/)
:::
B
lOpm
Figure 1. Penicillium janszewkii Zaleski. A. conidiophores, B. branching patterns of the

penicilli.
Evaluation of the diagnostic features of some species of Penicillium section Divaricatum
151
ochraceous, yellowish, pink, light vinaceous, apricot, scarlet or absent. Unripened cream and soft ascomata
were observed in six isolates after transfer from 3;70C to 25C.
CYA: Colonies 20-58 mm diam in two weeks, lanose, feItlike or velvety. Mycelium white, rosy buff or
pinkish. Sterile margins 2-10 mm wide, white or yellowish. Colonies without radially furrows or radial
sulcate. Sporulation heavy to weak, predominantly in the submarginal area or in the centre, light greygreen, blue-green, grey-brown, yellow-orange, sometimes pinkish or brownish in the centre. Exudate clear,
lemon coloured, yellowish, pinkish brown, yellowish brown, green or absent. Reverse colourless, or rosybuff, golden-yellow at the margin, deep yellow with brown or red centre, yellowish brown, cream-arange.
Soluble pigment pinkish brown, pinkish red or not present. Conidiophores (50-)400-600 x 1.8-3 1llTI, with
stipes smooth or rough. Branching variable, monoverticillate, biverticillate, slightly divergent, with a
cluster of 2-4 metulae terminally and with one or two metulae or branches subterminally. Metulae smooth
or rough 8-23 x 2.3-3 1llTI, sometimes proliferating in a new verticil of phialides. Phialides in clusters of 4-8,
abruptly tapering into a long neck 0.5-3 11m). Dimensions of phialides 6.5-9 x 2.8-3.2 1llTI. Conidia
ellipsoidal or subglobose, very often with a papilla on one end, rough to slightly echinulate, rarely also
smooth, 2.5-3.9 x 2.8-3.21llTI.
P. simpiicissimum has a very broad range in colony habit, but these did not differ
substantially in microstructure. We have compared 53 isolates from Czechoslovakia and 2
isolates from Poland with cultures ex type of P. janthinellum (IMI 40238) and P.
simplicissimum (CBS 280.39). Our results agree with the concept and figures of Stolk and
Samson (1983), who considered P. simplicissimum to be the anamorph of Eupenicillium
javanicum var. javanicum.
The following diagnostic features can be used for this species: a broad spectrum of
colony colour, predominantly in pastel tones in reverse; smooth conidiophores with
terminal divergent verticils of metulae and divergent metulae in subterminal positions.
Conidia ellipsoidal to subglobose, distinctly rough or finely echinulate, often with a
papilla on one end.
Penicillium janczewskii Zaleski - Fig. 1
=P. nigricans Bainier ex Thorn

=P. granatense Ramirez
CZ: Colonies 26-50 mm diam in two weeks, grey-white, slightly raised or depressed in the centre; velutinous
colonies grey-green with sporulation in the centre in dark grey-green or blue-green, at the margin lighter
or vice versa, later becoming dark grey to black. Surface of the colonies smooth or radially sulcate with
furrows shallows or deep. Margin white to yellowish or creamy 1-3 mm wide. Exudate clear, yellowish or
absent. Reverse sometimes lightly sulcate, yellowish, yellowish green, brownish to violet-brown.
Sometimes yellowish, creamish or orange-brown in the centre, olive brown or yellowish at the margin.
CYA: Colonies 30-57 mm diam in two weeks, lanose or velvety, sometimes lanose in the centre and velvety
at the margin. Sporulation weak, colonies white, grey-white, sporulation dense, colonies grey, dark grey,
green, olive-grey, olive-green, black-grey, sometimes with sterile white sectors. White margins 2.5 mm
wide. Radial furrows shallow or deeper. Exudate clear, beige, yellow, creamy, creamy-orange or absent.
Reverse rosy-buff with yellow margin, brownish red with ochraceous margins, brownish orange with
yellowish margin, yellow-brown or pinkish. Sometimes sparse or dense furrows. Conidiophores branched,
30-500 x 1.6-3 I.un, with smooth surfaces. Metulae numbering 2-6 in terminal whorls, very divergent,
apically swollen, 7-15 x 2-2.51llTI, often also borne subterminally. Phialides numbering 6-8, in compact
whorls, ampulliform, 5.4-7.8 x 1.8-2.3 IllTI with a distinct neck, 1-2.3 11m in length. Conidia globose to
subglobose, echinulate, 2.3-3.21llTI.
We compared 42 isolates from Czechoslovakia and three isolates from Poland with an ex
type culture of P. janczewskii (CBS 221.28). P. janczewskii is both in colonies and
microstructure a very distinctive species. Important diagnostic features are: more or less a
152
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::" .
Uj iii
"
10 p.m
Figure 2. Penicillium daleae Zaleski. A. conidiophores, B. branching patterns of the penicilli.
153
lanose colonies often with sterile sectors, sporulation from light grey, grey-green, olivegrey to dark grey or grey-brown; exudate sometimes present, clear; reverse yellow-green,
yellow-brown to brown. Conidiophores have very conspicuous divergent whorls of
metulae (often at right angles), and often further metulae in subterminal positions.
Phialides are very short (without neck 4-5 !Jlll), abruptly tapering to shorter or longer
necks. Conidia globose, dark, typically echinulate. We also examined P. grana tense
Ramirez et al. (lJFM 5965) and found that it was identical with P. janczewkii in all
characters examined.
Penicillium daleae Zaleski - Fig. 2.
cz: Colonies 32-48 mrn diam in two weeks, lanose, velutinous, pinkish creamy, grey-green, brown-green.
Sporulation weak or heavy in centre or at the margin, sometimes in concentric zones, with or without
radial or irregular furrows. Margins with fine furrows white to creamy, 2-2.5 mrn wide, later olive-green.
Exudate clear, yellowish, brownish or brown-orange. Reverse light yellowish brown, pinkish brown,
violet-brown, in the centre pinkish creamy, sometimes soluble pigment present, lightly brownish violet or
brown.
CYA: Colonies 40-45 mm diam in 2 weeks, felted, low, dense, sometimes velvety or with velutinous sectors.
Sporulation greyish olive or greyish creamy, grey or blue-green at the margin. Often lighter or darker
concentric zones. Furrows thin, when present. Exudate clear, amber, ochraceous-brown to dark brown.
Reverse cocoa-brown, dark flesh coloured, maroon or cinnamon brown, exceptionally colourless.
Conidiophores little branched, often with 2-3 metulae terminally and 1-2 metulae subterminally,
sometimes also with 1-2 branches, often monoverticillate. Conidiophores 20-150 x 1.6-31lID, with smooth
stipes. Metulae not very divergent, often unequal length, sometimes concurrent with phialides. Metulae
7.8-23 x 1.8-2.8 1lID. Phialides in verticils of 2-7, weakly divergent or parallel, ampulliform 5.2-8.6 x 2.3-2.5
1lID, with a distinct neck (1.5-31lID). Conidia subglobose, with surface rough, with protuberances or spines
and arranged in bands, 3-3.9 x 2.9-3.21lID.
We compared 40 isolates from Czechoslovakia and 6 from Poland with an ex type of P.
daleae (lMI 89338) and with IFJM 3004. P. daleae is very close to P. janczewskii. When mature
it has brown-grey to creamy-brown colouration of the colonies, exudate is brown or

amber-brown and the reverse has shades of brown or violet-brown. Conidiophores are
less branched and divergent than in P. janczewskii. Conidia are subglobose and have
protuberances or spines arranged in bands.
Penicillium canescens Sopp - Fig. 3.

CZ: colonies 34-50 mrn diam in two weeks, lanose with very weak or heavier sporulation, grey-green,
greyish yellow-green, sometimes with greyish yellow to creamy sectors. Margin white, yellowish, 1-3 mm
wide. Radial furrows rare. Exudate clear, yellowish or absent. Reverse yellowish, orange, yellow-brown,
yellow-orange, orange-red or middle brown. Soluble pigment sparse, yellowish, yellowish brown or light
brown.
CYA: Colonies 43-60 mrn diam in two weeks, lanose, compact, sporulation weak or heavy, grey-green.
Margins white or yellowish, 1-3 mrn wide. Radial furrows dense. Exudate brightly yellow to clear. Reverse
brownish orange, maroon or light brown. Conidiophores very irregularly branched, 2-3 x 3.5 11m wide.
Stipes are slightly sinuous, rough or granular. Metulae terminal, 2-5 in the verticils, also subterminally 9.319 x 1.6-3.1 1lID, with apices somewhat enlarged, and sometimes concurrent with branches.
MonoverticiIlate conidiophores also present. Phialides 3-8 in the verticils, ampulliform 6.3-8.6 x 1.8-2.3 1lID,
with distinct necks (1.6-2.5 1lID). Conidia globose, subglobose, smooth or distinctly rough, 1.9-2.8 IlID in
diam.
o. Fassatiova & A. Kubatova
154
A
10,urn
Figure 3. Penicillium canescens Sopp. A. conidiophores, B. branching patterns of the penicilli.
..." :~.
.~
'.: .:~) ~W
.:~
lOpm
Figure 4. Penicillium melinii Thorn. A. conidiophores, B. branching patterns of the penicilli.
155
156
We compared 8 isolates from Czechslovakia with an ex type culture of P. canescens (lMI

28260). P. canescens has the following important diagnostic features: colonies on CZ lanose,
relatively sparsely sporulating in grey-green or yellow-green; reverse yellow to orangered; conidiophores sinuous, unequally wide, mostly roughened with irregular whorls of
metulae. Conidia are relatively small, ellipsoidal to subglobose, smooth or finely
roughened.
Penicillium melinii Thorn - Fig 4.
CZ: Colonies 30-60 mm diam in two weeks, velvety or powdery, with heavy sporulation, grey-blue-green,
blue-green, in centre brownish grey, margins white, 1-1.8 mm wide, often arachnoid. Radial furrows dense
or sparse, shallow, or absent. Exudate yellow, ochraceous or yellow-orange. Reverse orange, brown, violetbrown, intens yellow, pinkish-orange in the centre, yellow-green, brownish red or greenish brown at the
margin. Soluble pigment orange-yellow, yellowish, brick-red, salmon or orange-green.
CYA: Colonies 33-57 mm diam in two weeks, velvety, heavy sporulating in dark greyish green, dark green,
grey-blue-green or dark olive-green. Margins white, 0.5 mm wide. Sometimes sectors or radial furrows
present. Exudate clear, amber, lightly ochraceous, yellowish or absent. Reverse orange, yellowish orange,
orange-brown or not coloured in the centre, grey-green, grey-brown, yellow-beige at the margin. Soluble
pigment golden-yellow, yellowish brown or rarely absent. Conidiophores 1.9-3.1 11m wide, finely
roughened or rarely smooth, irregularly branched, with terminal whorls of 2-5 metuIae, often of unequal
length, not very divergent, with further subterminal metulae or branches or solitary phialides. Metulae
rough, walled 7.5-18 x 2.2-2.5 !lID. Phialides 5-8 in whorls, ampulliform, 5.6-8 x 1.9-2.5 !lID with or without
a distinct neck (0.6-1.5 !lID). Conidia globose, echinulate or spinose, 2.8-3.8 !lID in diam.
Thirty-nine isolates from Czechoslovakia were compared with an ex type culture of P.

melinii (IMI 40126). P. radulatum G.Smith (CCF 1990), not type, isolated from cysts of
Globodera rostochienses in Czechoslovakia (Novotna and Fassatiova, 1988) showed the same
features in colonies and morphology as P. melinii.
The diagnostic features of P. melinii are as following: typical velvety or powdery
colonies with dark blue-green or brown-grey sporulation and a narrow white margins;
frequent radial furrows, exudate in various tones of yellow, orange to ochre; reverse
orange-brown, also diffusing pigment in similar shades. Conidiophores are
monoverticillate, or with a terminal whorl of metulae, sometimes also with lateral
branches. Metulae are less divergent. Stipes and metulae are often roughened. Metulae are
sometimes concurrent with a whorl of phialides. Phialides taper to short necks. Conidia
are globose, finely echinulate or spinose.
DISCUSSION
In the present study, a number of morphological features were examined mostly in fresh
isolates. The most typical features for diagnosis of the species were selected. The following
microscopical features were preferred: branching of the conidiophore, surface texture of
stipe and penicillus, divergence of metulae and phialides, the length of the phialide neck
and length of the phialides without the neck and the shape, surface and size of the
conidia. The colony character is best described on CZ, but the shades of colonies and other
pigments are more distinctive on CYA.
Our isolates were compared not only with the ex type cultures but also with
descriptions in Raper and Thorn (1949), Pitt (1979), Ramirez (1982) and Stolk and Samson
(1983). From its colony and conidiophore characters P. janthinellum was shown to be a
synonym of P. simplicissimum, as reported by Stalk and Samson (1983). The ex type culture
157
of P. melinii examined has deteriorated, and does not correspond in colony characters or
especially in microscopical features either with the original description or to our isolates.
Pitt (1979) has previously noted this. The conidiophores are very poorly branched and the
dimensions of phialides and conidia are larger.
Further the isolate originally determined as P. radulatum, which was isolated from
cysts of Globodera rostochiensis in Czechslovakia, was compared with our isolates of P.
me/in ii, and we agree with Pitt (1979) that P. radulatum is a synonym of P. melinii. The type
isolate of P. granatense Ramirez et a1. is considered to be a synonym of P. janczewskii.
We assume that this study does not duplicitate the descriptions of the mentioned
species in published monographies. We wanted to verify in a larger set of isolates from
one region the utilisability of selected features for diagnostical purposes.
ACKNOWLEDGEMENTS
The authors wish to their thank Dr. Z. Lawrence (CAB International Mycological Institute,
Kew, UK) and Drs. E. van Reenen-Hoekstra (Centraalbureau voor Schimmelcultures,
Baarn, the Netherlands) for providing us type cultures. They also thank Prof. C. Ramirez
(Thailand Institute of Science and Technological Research, Bangkok) for the isolates of P.
granatense and P. daleae.
REFERENCES
NOV01NA, J. and FASSATIOV'A O. 1988. Three species of the genus Penicillium Link isolated from the
cysts of Globodera rostochiensis Wll. in Czechoslovakia. Ceska Mykologie 42: 90-96.
PITT, J.I. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London;
Academic Press.
RAMIREZ, C. 1981. Manual and Atlas of the Penicillia. Amsterdam; Elsevier Biomedical.
RAPER, K.B. and THOM, C. 1949. Manual of the Penicillia. Baltimore: Williams and Wilkins.
STOLK, A.C. and SAMSON R.A. 1983. The Ascomycete genus Eupenicillium and related Penicillium
anamorphs. Studies in Mycology, Baarn 23: 1-149.
DIALOGUE FOLLOWING DR. FASSATIOVA'S PRESENTATION
CHRISTENSEN: You mentioned a species with very short phialides, only 4-5 11m long. Do
your measurements for phialides include the neck or not?
FASSA nov A: It is very difficult to measure a phi ali de without the neck, so our
measurements include the neck.
SAMSON: Have you found any connection with Eupenicillium? Do any of the species you
worked with produce sclerotia? Do any of these isolates show any connection with
Eupenicillium javanicum?
FASSATIOVA: We have found four strains of P. simplicissimum with sclerotium-like

structures, but have not seen ascospores.
159
REVISION OF PENICILLIUM SUBGENUS FURCA TUM BASED ON

SECONDARY METABOLITES AND CONVENTIONAL CHARACTERS
J.e. Frisvad and O. Filtenborg

2800 LYNGBY, Denmark
SUMMARY
Penicillium subgenus Furcatum contains species that are important in soil ecology and as contaminants
of foods and feedstuffs. Taxa in this subgenus are difficult to identify and a high number of new
species have been described since the monograph of Raper and Thorn in 1949. We have examined all
described taxa in that subgenus using morphological, physiological and chemical characters, with
emphasis on profiles of secondary metabolites. A number of taxa, such as P. megasporum and P.
marneffei, placed in subgenus Furcatum by one or more authors, are transferred to subgenus
Biverticillium. Several species synonymised by Pitt in 1979 are reestablished on the basis of
morphology and profiles of secondary metabolites. All species in subgen. Furcatum produced a high
number of secondary metabolites and nearly all species produced known antibiotics or mycotoxins.
The frequency of mycotoxin production in each species was high, often 100%. Some of the individual
secondary metabolites produced by taxa in subgen. Furcatum, for example roquefortine C and oxaline,
are also produced by taxa in subgen. Penicillium, while mycotoxins such as patulin, penicillic acid,
griseofulvin and citrinin are quite widespread in all subgenera in Penicillium except subgen.
Biverticillium. Proposed synonyms and secondary metabolite profiles of each species are listed.
INTRODUCTION
Penicillium subgen. Furcatum Pitt contains many different species of great importance in
soil ecology, foods and feedstuffs and industrial production of enzymes. A number of
these species have been reported to produce mycotoxins (Frisvad, 1986) and other
secondary metabolites, but misidentified isolates (Frisvad, 1989) have obscured the
connections between taxa in subgen. Furcatum and their mycotoxins.
Many species included in subgen. Furcatum by Pitt (1979) were placed in subgen.
Biverticillium Dierckx (= sect. Biverticillata-Symmetrica; Raper and Thom, 1949) by Raper
and Thom (1949) or Ramirez (1982). These included P. atrovenetum G. Smith, P. novaezeelandiae van Beyma, P. herquei Bainier, P. estinogenum G. Smith, P. paraherquei Abe ex
Udagawa, P. coralligerum Nicot and P. asperosporum G. Smith. Furthermore, Ramirez (1982)
regarded as distinct a number of species synonymised by Pitt (1979). Abe et al. (1983 a,b,c)
revised subgen. Furcatum, emphazising surface ornamentation of conidia; Ramirez (1985)
evaluated several species described since 1979. Fassatiova (1965) and Stolk and Samson
(1983) also evaluated several species placed in subgen. Furcatum by Pitt (1979).
We reexamined these important Penicillia with special emphasis on profiles of
secondary metabolites. A summary of our results is reported below.
J.e. Frisvad & O. Filtenborg
160
Ex type cultures, authentic cultures and several fresh cultures of each taxon allocated to
subgen. Furcatum by different authors were grown on Czapek yeast extract agar (CYA),
Malt extract agar (MEA), yeast extract sucrose agar (with Difco yeast extract) (YES), 25%
glycerol nitrate agar (G25N) (Pitt, 1979) at 25 and on CYA at 37. Profiles of secondary
metabolites were examined by thin layer chromatography (TLC) and high performance
liquid chromatography (HPLC) with diode array detection (DAD) as described by Frisvad
et al. (1989a) and Frisvad and Thrane (1987).
Subgen. Furcatum Pitt is not easy to define and other subgeneric and sectional
arrangements have been proposed (Stolk and Samson, 1985). Morphologically members of
subgen. Furcatum have a strong resemblance to members of subgen. Aspergilloides and
Eupenicillium. All species in these supraspecific taxa are predominantly soil borne fungi.
Table 1. Species placed in Penicillium subgen. Furcatum (Pitt (1979), Abe et al. (1983a) or
Stolk and Samson (1983) but reclassified here in subgen. Biverticillium on the basis of
physiology and profiles of secondary metabolites.
P. acu/eatum Raper et Fennell 1948
P. mirabile Belyakova et Mil'ko 1972
P. allahabadense Mehrotra et Kumar 1962

P. Iwrosum Rai et al. 1969
P. manthae Ramirez et Martinez 1981
P. sabulosum Pitt et Hocking 1985
P. asperosporum G. Smith 1965

=P. echinosporum G. Smith 1962
P. resinae Qi et Kong 1983
P. inflatum StoIk et MalIa 1971

P. marneffei Segretain et al. 1960
P. tardum Thorn 1930

P. elongatum Bain. 1907 (nom. ilIegit.)
P. rugulosum var. atricolum Thorn 1930
P. echinosporum Nehira 1933
P. scorteum Takedo et al. 1934
P. phialosporum Udagawa 1959
P. verruculosum PeyroneJ 1913

P. aculeatum var. apicu/atum Abe 1956
P. minioluteum Dierckx 1901

P. gaditanum Ramirez et Martinez 1981
P. samsonii Quintanilla 1983
These morphological resemblances are supported by biochemical characters. Secondary

metabolites such as citreoviridin, verruculogen, gliotoxin, curvularin, agroclavine,
citromycetin, kojic acid, brefeldin A and spinulosin have been reported from Eupenicillium
(Horie et al., 1985), and subgens. Aspergilloides and Furcatum (Frisvad, 1986). Other
metabolites such as patulin, griseofulvin, penicillic acid, penitrem A, xanthomegnin,
citrinin, mycophenolic acid, roquefortine C, oxaline, isochromantoxin, viridicatumtoxin
and carolic acid are also produced by members of subgen. Penicillium (Frisvad and
Filtenborg, 1989), while secondary metabolites such as mitorubrinic acid, rugulosin,
rubratoxin, vermicellin, glauconic acid, wortrnannin, cyclochlorotine, luteoskyrin and
talaron are exclusively produced by a high number of species in subgen. Biverticillium and
Revision of Penicillium subgenus Furcatum
161
Table 2. Accepted species, synonyms and isolates examined in Penicillium subgen. Furcatum.
1.
P. atrovenetum G. Smith 1956

P. coeruloviride G. Smith 1965
CBS 241.56(T), CBS 243.56, IMI 61836ii, IMI 161964, IMI 103148, CBS 240.65
2.
P. brasilianum Batista 1957

P. paraherquei Abe ex G. Smith 1963
P. skrjabinii Schmotina & Golovleva 1974
CBS 253.55(T), NRRL A-14996, 79S9FC9, Leistner sp. 863, NRRL 5881, FRR 1859,
IMI 297548, CBS 442.65, CBS 349.77, IMI 177905
3.
P. canescens Sopp 1912

P. swiecickii Zaleski 1927
NRRL 910(T), NRRL 918

4.
P. castellonense Ramirez et Martinez 1981
IMI 253791 (T)

5.
P. chalybeum Pitt et Hocking 1985
FRR 2660(T), FRR 2659, FRR 2658

6.
P. citrinum Thorn 1910

Citromyces subtilis Bain. et Sartory 1912
P. subtile (Bain. et Sartory) Biourge 1923
P. aurifluum Biourge 1923
P. fellutanum Biourge 1923
P. implicatum Biourge 1923
P. phaeo-janthinellum Biourge 1923
P. sartoryi Thorn 1930
P. botryosum Batista et Maia 1957
NRRL 184l(T), NRRL 1843, NRRL 783, NRRL3463, IMI 92267, CBS 232.38, IMI 92229ii, CCM F391, 1M! 276863, NRRL 2148
7.
P. coralligerum Nicot et Pionnat
NRRL 346S(T), CBS 1963, CBS 193.72, CBS 290.73

8.
P. corylophilum Dierckx 1901

P. candidofulvum Dierckx 1901
P. obscurum Biourge 1923
P. citreovirens Abe ex Ramirez 1982
NRRL 802m, CBS 320.59, CBS 330.79, NRRL A-23347, CBS 254.37
9.
P. cremeogriseum Chalabuda 1950

P. glaucolanosum Chalabuda 1950
? P. yarmokense Baghdadi 1968
? P. iriense Boretti et al. 1973
P. dodgei Pitt 1979
P. klebahnii Pitt 1979
? P. sajarovii Quintanilla 1982
NRRL 1022, NRRL 3389, CBS 410.69, 1M! 154289

10. P. daleae Zaleski 1927
P. krzemieniewskii Zaleski 1927
IMI89338 (T), NRRL 922, FRR 1914, IMI 228583
(cont.)
J.e. Frisvad & O. Rltenborg
162
Table 2 (cont.)
11. P. dierckxii Biourge 1923
?P. cinerascens Biourge 1923 (a)
P. charlesii G. Smith 1933
P. fellutanum var. nigrocastaneum Abe 1956

P. eben-bitarianum Baghdadi 1968
P. gerundense Ramirez et Martinez 1980
CBS 185.81(T), NRRL 746, NRRL 1887, CBS 415.69, IMI68224, NRRL 778, CBS 304.48
12. P. echinulorudgiovense Abe 1956 (to be validated)
IMI68213(T)
13. P. estinogenum Komatsu et Abe ex G. Smith 1963
P. estinogenum Komatsu et Abe 1956
CBS 31959(T), IMI 304278, IMI304279
14. P. flavidostipitatum Ramirez et Gonzcilez 1984
IJFM 7824(T)
15. P. griseopurpureum G. Smith 1965
CBS 406.65
16. P. herquei Bain. et Sartory 1912
P. luteocoeruleum Saito 1949
NRRL 1040(T), NRRL 3450, CCM F-289, ATCC 46327
17. P. humuli van Beyma 1939
NRRL872(T)
18. P. janczewskii Zaleski 1927
? P. albirlum Sopp 1912
P. kapuscinskii Zaleski 1927
P. nigricans Bain. ex Thorn 1930
P. nigricans var. sulphuratum Abe1956
P. radiatolobatum Lorinczi 1972"
P. grana tense Ramirez et al. 1980
P. murcianum Ramirez et Martfnez1981"
NRRL 919(T), FRR 538, NRRL 2043, NRRL A-23333, CBS 218.28, FRR 74, IMI253795,
IMI 253800, IMI228664, IJFM 7845, FRR 97, ATCC 32023, 1M! 149218, IMI121617, FRR 80, CBS
341.79
19. P.jenseniiZaieski 1927
P. Tivolii Zaleski 1927
P. griseoaz.ureum C. et M. Moreau 1941
? P. corylophiloides Abe 1956
NRRL 909(T), FRR 531, NRRL 906, CBS 325.59, CBS 162.42
20. P. lanosum Westling 1911
P. kojigenum G. Smith 1961
IMI 40224, IMI 90463, NRRL 3442
21. P. maclennaniaeYip 1981
IJFM 7852(T)
163
22. P. manginii DucM et Heirn 1931

P. pedemontanum Mosca et Fontana 1963
P. syriacum Baghdadi 1968
P. atrosanguineum Dong 1973
NRRL 2134(T), IMI 99085, IJFM 7870, CBS 343.52, CSIR 1405, CSIR 1406, FRR 2004,
CBS 380.75, CBS 418.69
23. P. mariaecrucis Quintanilla
CBS 270.83(T), ATCC 48476, Quintanilla 1118, 1022, 1049
24. P. matriti G. Smith 1961
NRRL 3452(T), IMI 96506, CBS 347.61, CBS 188.89
25. P. megasporum Orput et Fennel 1955
P. giganteum Roy et Singh 1968
NRRL 2232, CBS 121.65, CBS 529.65
26. P. melinii Thorn 1930
P. radulatum G. Smith 1957
? P. echinatum Dale 1923
CBS 218.30(T), NRRL 848, IMI 85494, NRRL 3672, IMI 158663, FRR 263
27. P. miczynskii Zaleski 1927
NRRL 1077(T), FRR 1672
28. P. moldavicum Mil'ko et Beljakova 1967
P. kabunicum Baghdadi 1968
CBS 627.67, CBS 409.69
29. P. nalgiovense Laxa 1932
NRRL 911 (ATCC 46455, CBS 138.70, FRR 1647)
30. P. novae-zeelandiae van Beyma 1940
NRRL 2128(T), 1M! 38496, CBS 109.66, FRR 1905
31. P. ochrochloron Biourge 1923
P. biforme var. vitriolum Sato 1939
P. cuprophilum Sato 1939
NRRL 926(T), CCM F-158, FRR 2141, NRRL 924, NRRL 925
32. P. onobense Ramirez et Martinez 1981
CBS 174.81
33. P. oxalicum Currie et Thorn 1915
P. aragonense Ramirez et Martinez 1981
P. asturianum Ramirez et Martinez 1981
IMI 192232(T), IMI 253788, ATCC 32024, CCM F-338, CBS 358.48
34. P. paxilli Bain. 1907
NRRL 2oo8(T), ATCC, 26601, Leistner Sp. 1263
(cont.)
164
Table 2 (cont.)
35. P. piscllrium Westling 1911
P. zonatum Hodges et Perry 1973
NRRL 1075(T)
36. P. pulvillorum Turfitt 1939
P. novae-caledoniae G. Smith 1%5
P. novae-caledoniae var. album Ramirez et MartInez 1981
P. cieg/eri Quintanilla 1982
NRRL 2026(T), lMI 140441, IJFM 7184, IJFM 7673
37. P. rllciborskii Zaleski 1927
P. fagi Martinez et Ramirez 1978
P. CIleTulescens Quintanilla 1983
NRRL 2150(T), CBS 689.77, FRR 1708, Quintanilla 1162, 1155, 1300, 1152, 1147,
IMI 166199, ATCC 32789, CBS 683.77, CBS 685.77, FRR 1695
38. P. rllistrickii G. Smith 1933
P. castellae Quintanilla 1983
NRRL IOO(T), FRR 1821, CBS 215.71, FRR 1576, IMI 137808, Quintanilla 1036, 1349, 1012, 1024
39. P. rolfsii Thorn 1930
NRRL 1078(T), CCM F-337
40. P. rubefllciens Quintanilla 1982
Quintanilla 1133(T)
41. P. sclerotigenum Yamamoto 1955
IMI 68616, IMI 267703
42. P. simplicissimum (Oudem.) Thorn 1930
= Spicaria simplicissima Oudem. 1903
P. janthinellum Biourge 1923
P. raperi G. Smith 1957
P. indonesiae Pitt 1979
NRRL 2016(T), NRRL 2674, CBS 191.67, CBS 346.68, lMI 108033, NRRL 904, ATCC 13154, ATCC
42743
43. P. sizovlle Baghdadi 1968
IMI 140344(T)
44. P. smithii Quintanilla 1982
P. corynephorum Pitt et Hocking
Quintanilla 1097(T), CBS 276.83, FRR 2663, FRR 2676, CBS 349.78
45. P. soppii Zaleski 1927
? P. mIItris-meae Zaleski 1927
P. severskii Schechovtsov 1981
P. michaelis Quintanilla 1982
CBS 226.28(T), NRRL 912, CBS 698.70, CBS 271.73, IJFM 19000, Quintanilla 1150, NRRL A-23325
46. P. steckii Zaleski 1927
P. baradicum Baghdadi 1968
NRRL 2140(T),NRRL 6336, CBS 416.69, CBS 789.70, CBS 993.73, CBS 222.73
165
47. P. vasconiae Ramirez et Martinez 1980

CBS 339.79(T), CBS 175.81, NRRL 721
48. P. velutinum van Beyma 1935
NRRL 2069, FRR 270
49. P. waksmanii Zaleski 1927
P. sumatrense von SziIvinyi 1936
? P. decumbens var. atrovirens Abe 1956
? P. atrovirens G. Smith 1963
NRRL 777(T), IMI 68223, NRRL 779
50. P. westlingii Zaleski 1927
P. chrzaszczii Zaleski 1927

? P. godlewskii Zaleski 1927
? P. charlesii var. rapidum Abe 1956
P. gorlenkoanum Baghdadi 1968
P. damascenum Baghdadi 1968
P. turolense Ramirez et Martinez 1981
IMI 92272(T), IMI 140337, NRRL 903, NRRL 2111, CBS 31859, CBS 176.81, CBS 408.69, CBS
200.86, CBS 552.86
(al Species with a question mark have not been tested for secondary metabolites
These species are morphologically closer to P. canescens than to P. janczewskii.
Talaromyces (Frisvad, 1986 and unpublished results). As an example, P. phialosporum

Udagawa, considered to be a synonym of P. oxalicum Currie & Thom by Abe et al. (1983b),
produced large amounts of mitorubrin and rugulosin, similar to the ex type culture of P.
tardum (Table I), supporting the opinion of Pitt (1979) that P. phialosporum and P. tardum
are the same species.
Because of the form and arrangement of the phialides and metulae, and production of
secondary metabolites typical of subgen. Biverticillium, a number of species have been
excluded from subgen. Furcatum in this study and placed in subgen. Biverticillium (Table
1). Furthermore, the ex type culture of P. griseoroseum Dierckx, placed in subgen. Furcatum
by Pitt (1979), has been shown to be identical with P. chrysogenum in subgen. Penicillium
(Cruickshank and Pitt, 1987; Frisvad and Filtenborg, 1989). The remaining species, listed
in table 2, are confidently placed in subgen. Furcatum. Three taxa, P. victoriae von Szilvinyi,
P. melagrinum var. viridiflavum Abe and P. kabunicum Baghdadi, placed in P. janthinellum
by Pitt (1979), were found not to belong to that species, but could not be placed in other
species listed in Table 2. They will be examined further by HPLC-DAD.
Most species in subgen. Furcatum produced known mycotoxins with additional
antibiotic activity, such as griseofulvin, patulin, penicillic acid, brefeldin A, citrinin,
citreoviridin, kojic acid, mycophenolic acid or asperentin (Table 3). It is interesting to note
that the most abundantly occurring Penicillium species in rhizosphere soil, P. janczewskii
Zaleski and P. westlingii Biourge, consistently produced large amounts of griseofulvin and
citrinin, respectively.
166
A number of common species produced tremorgens such as paxilline (P. paxilli Bainier
and Eupenicillium sheari Stolk et Scott), penitrem A (P. janczewskii and an undescribed
species), verruculogen (P. brasilianum Batista and another undescribed species previously
placed in P. paxilll) or janthitrems (P. piscarium Westling). HPLC-DAD results showed that
P. janthinellum FRR 1893 and P. solitum CBS 288.36 produced verruculogen, but these
isolates did not fit readily in any of the species listed in Table 2.
Table 3. Production of mycotoxins and other secondary metabolites by taxa in Penicillium
subgen. Furcatum.
Taxon
Mycotoxins and secondary metabolites
P. atrovenetum
1. 3-nitropropionic acid (a,b,c)

2. Atrovenetin (c), naphthalic anhydride (a,b,c)
P. brasilianum
1. Penicillic acid (a,b)

2. Viridicatumtoxin (a,b)
3. Verruculotoxin (a,b,c)
4. Verruculogen (a,b,c), fumitremorgin B & C (a,b),
acetoxyverruculogen (c)
5. Paraherquamide (c)
6. Paraherquonine (c)
P. canescens
1. Griseofulvin (a,b,c)
P. castellonense
1. Penicillin
P. chalybeum
P. citrinum
1. Citrinin (a,b,c)
P. coralligerum
P. corylophilum
P. cremeogriseum
1. Brefeldin A (a,b,c)
2. Palitantin (a,b,c)
3. PeniciIlic acid (a,b,c)
P. daleae
1. Asperentin (a,b), 5'-hydroxyasperentin (a,b)
P. dierckxii
1. Carolic acid (a,b,c), carlosic acid (a,b,c), carlic acid (c),
dehydrocarolic acid (a,b,c), carolinic acid (c), viridicatic

acid (c)
P. echinulonalgiovense
P. estinogenum
1. Estin (c)
2. Geodin, dihydrogeodin, erdin (c)
P. flavidostipitatum
P. griseopurpureum
P. herquei
1. Atrovenetin, herqueinone, deoxy- herqueinone,
norherqueinone,herqueichrysin, duc1auxin (c), naphthalic

anhydride (a,b,c)
2. Physicon (c), flavoskyrin (c)
3. Herquline (c)
P. humuli
P. janczewskii
1.
2.
3.
4.
P.jensenii
1. Griseofulvin (a,b)
P.lanosum
1. Griseofulvin (a,b)
2. Kojic acid (a,b,c)
3. Compactin (a,b)
167
Griseofulvin (a,b,c)
Penitrem A (a,b,c)
Amauromine (=nigrifortine) (c)
Penicillic acid (a)
P. maclennaniae
l. Spinulosin (b)
P. manginii
1. Citreoviridin (a,b,c), citreomontanin (c)
P. mariaecrucis
l. Xanthomegnin, viomellein (a,b)
P. matriti
l. Penicillic acid, orsellinic acid (a,b,c)
P. megasprum
l.
2.
3.
4.
5.
6.
PeniciIIic acid (c)

Physicon (c)
Asperphenamate (c)
Megasporizine (c)
Phyllostine (c)
7-hydroxy-4,6-dimethylphtalide (c)
P. melinii
l. Patulin (a) (only in ex type culture)
P. miczynskii
l. Citreoviridin (a,b,c)
2. Citrinin (a)
P. moldavicum
P. novae-zeelandiae
1. Patulin (a,b,c)
P. ochrochloron
P.onobense
l. BrefeldinA
P.oxalicum
l. Roquefortine C (a,b,c)
2. Oxaline (a,b,c)
3. SecaIonic acid D (a,b,c)
P.paxilli
1. Paxillin (a,b,c), dehydropaxillin (a,b), l'-O-acetylpaxillin(a,b)
P. piscarium
1. Brefeldin A (a,b,c)
2. Janthitrem B (a,b,c)
P. pulvillorum
l. Penicillic acid (a,b)
P. raciborski
l. Mycophenolic acid (a,b,c)
P. raistricki
1. Penicillic acid (a,b)

2. Griseofulvin (a,b,c)
P. rollsii
P. rubefaciens
P. sclerotigenum
l. Griseofulvin (a,b)
2. Roquefortine C (a,b)
P. simplicissimum
= P. janthinel/um
1. Xanthomegnin (a,b,c), viomellein, 3,4-dehydroxanthomegnin, semivioxanthin, 7-De-O-methylsemivioxanthin (c)
P. sizovae
1. Agroclavine I, epoxyagroclavine I (c)
(cont.)
168
Table 3 (cont.)
P. smithii
1. Citreoviridin (a,b)
2. Canescin (a,b)
P.soppii
1. Terrein (a,b)
2. Mycochromenic acid (c)
P. steckii
1. Curvularin (a,b,c), dehydroxy-curvularin (a,b,c)

2. 3,7-dimethyl-8-hydroxy-6-methoxy- isochroman
(= isochromantoxin) (a,b,c)
P. vasconiae
P. velutinum
P. waksmanii
P. westlingii
1. Citrinin (a,b,c)
a. Confumed by TLC in two eluents (TEF &: CAp) and two chemical treatments (anisaldehyde spray and
heating, cerium sulphate spray followed by aluminium chloride spray and heating), compared with
standards.
b. Confirmed by reversed phase HPLC and diode array detection, compared with standards.
c. The identity of the original producer has been confumed, but a standard was not available.
Three important problems in the taxonomy of subgen. Furcatum have not been solved
during this work. P. albidum Sopp was revived by Fassatiova (1965) as an earlier name for
the very common species P. janczewskii (= P. nigricans Bainier ex Thorn) because isolates of
these species "showed numerous series of different transitional forms varying from
velvety grey-green and strongly sporing through olive-green and light green to white
colonies with a floccose surface and light sporulation" and "conidia varied from rough to
spiny" (Fassatiova, 1965). We have seen the same variation in P. janczewskii. Furthermore
isolates of P. janczewskii, P. albidum, P. canescens Sopp and P. jensenii Zaleski, together with
their synonyms (Table 2) appear to form something approaching a continuum. All have a
bright yellow or a dark orange to brown reverse on CYA. All these taxa produce
griseofulvin. These taxa probably have a commmon ancestor, but they are sufficiently
different, both morphologically and chemically, to be recognised as three or four species.
Even though the conidia of P. jancZeUJskii (including P. kapuscinskii and the intermediate
forms between P. canescens and P. janczewskii mentioned by Pitt, 1979) may vary from
roughened to spinose, they look quite different from those of P. albidum NRRL 2043
(Ramirez, 1982, pp. 824,825), so the status of P. albidum remains unresolved. However, as
the name P. albidum remains untypified and of uncertain application, it should probably
remain as a nomen dubium. P. murcianum and P. radiatolobatum (Table 2) are
morphologically most closely related to P. canescens, but biochemically to P. janczewskii, as
they produce the same profile of secondary metabolites. Unlike P. canescens, P.
griseopurpureum produce smooth stipes, but the conidia and colony morphology of the two
species are identical, as are the profiles of secondary metabolites. Profiles of secondary
metabolites suggest a classification of these difficult species somewhat at variance with
the major distinguishing features used by Pitt (1979), i.e. stipe and conidium roughening.
Reexamination of this cluster of allied species using a wider range of techniques is
recommended.
Producers of brefeldin A, including Eupenicillium ehrlichii Klebahn and possible
synonyms (Frisvad et al., 1990), P. piscarium, P. onobense Ramirez & Martinez, P.
169
indicated in Table 2. However the conidia of P. piscarium (spinose), E. ehrlichii (finely

roughened) and P. onobense (spirally roughened) appear to be quite different. Again other
taxonomic methods may help in solving this problem. Some morphological variation seem
to be common in abundantly occurring taxa in subgen. Furcatum. The species mentioned
above appear to be related also to E. javanicum (van Beyma) Stolk & Scott and perhaps
more distantly to P. pulvillorum Turfitt, P. mariaecrucis Quintanilla and P. brasilianum
Batista.
Unlike all other isolates of this species and P. radulatum G. Smith, the ex type isolate of
P. melinii produce patulin, so perhaps the name P. radulatum should be revived. Some
other species, including P. atrovenetum G. Smith, P. estinogenum G. Smith and P. westlingii,
the first two included in P. melinii by Pitt (1979), are regarded by us as distinct both
morphologically and biochemically. Of these colourful species we have isolated P.
westlingii and P. estinogenum most often, and always from soil.
P. manginii DucM & Heim and P. soppii Zaleski were included in P. miczynskii Sopp by
Pitt (1979). While we believe that P. manginii, P. miczynskii and P. citreonigrum are certainly
related, differences in morphology and profiles of secondary metabolites indicate to us
that they are separate taxa. P. soppii, on the other hand, is quite different from these three
species, all of which produce citreoviridin (Table 3). All four species are common in soil. P.
smithii Quintanilla, another abundant producer of citreoviridin, is common in European
and Canadian soils (Frisvad, unpublished), but has also been isolated from Indonesian
dried fish (as P. corynephorum; Pitt and Hocking, 1985).
P. coralligerum, P. echinulonalgiovense Abe, P.1anosum Westling, P. raciborskii Zaleski, P.
sizovae Baghdadi, P. steckii Zaleski and P. westlingii were not accepted as separate species
by Pitt (1979), but are all regarded by us as distinct species. The last five species named are
common in soil.
Species in subgen. Furcatum are not usually the most common taxa isolated from food
or feed samples. Exceptions are P. citrinum Thom, which is very commmon in spices and
on vegetables, P. sclerotigenum Yamamoto, which causes a rot of sweet potatoes, and P.
oxalicum, which invades corn. Other species which have been found in foods and feeds
include P. brasilianum, P. corylophilum Dierckx, P. dierckxii Biourge, P. janczewskii, P.
lanosum, P. manginii, P. miczynskii, P. novae-zeelandiae, P. paxilli, P. piscarium, P. pulvillorum,
P. raistrickii G. Smith, P. simplicissimum (Oudem.) Thom, P. smithii, P. soppii and P. steckii.
These species are usually indicators of soil contamination. Apart from P. citrinum and P.
sclerotigenum, they seem to be poor competitors on foods compared to species in subgen.
Penicillium. P. sclerotigenum may be related to the latter subgenus, because its
conidiophores may be terverticillate and it produces roquefortine C. P. oxalicum produces
oxaline and roquefortine C, and may also have an affinity with species in subgen.
Penicillium.
This study has shown that species in subgenus Furcatum are very efficient producers
of secondary metabolites, often with antibiotic or mycotoxic activity, and that all the taxa
are well defined by their profiles of secondary metabolites. Some of the more common
species may vary in the degree of roughening of the conidiophores or conidia, even
though they have nearly the same profile of secondary metabolites. It is recommended
that these taxa should be examined using other biochemical techniques to fix their
taxonomic position and determine their morphological variation.
ACKNOWLEDGEMENTS
We sincerely thank Amelia C. Stolk and Robert A. Samson for advice and many
discussions on the taxa treated in this paper.
170
REFERENCES
ABE, S., !WAI, M. and TANAKA. H. 1983a. Taxonomic studies of Penicillium. Ill. Species in the section
Divaricatum. Transactions of the Mycological Society of Japan 24: 95-108.
ABE, S., !WAI, M. and AWANO, M. 1983b. Taxonomic studies of Penicillium. IV. Species in the sections
Divaricatum (continued) and Furcatum. Transactions of the Mycological Society of Japan 24: 109-120.
ABE, S., !WAI, M. and ISHIKAWA, T. 1983c. Taxonomic studies of Penicillium. IV. Species in the section
Furcatum (continued). Transactions of the Mycological Society of Japan 24: 409-418.
FASSATIOVA, O. 1965. Studies on the variability of Penicillium albidum Sopp emend. and the development
of the conidia. Ceska Mykologie 19: 104-110.
FRISVAD, J.e. 1986. Taxonomic approaches to mycotoxin identification. In Modern Methods in the Analysis
and Structural Elucidation of Mycotoxins, ed. R.J. Cole, pp. 415-457. London: Academic Press.
--1989. The connection between the Penicillia and Aspergilli and mycotoxins with special emphasis on
FRISVAD, J.e. and FILTENBORG, 0.1989. Terverticillate Penicillia: chemotaxonomy and mycotoxin
production. Mycologia 81 (in press).
FRISVAD, J.e., FILTENBORG, O. and THRANE, U. 1989a. Analysis and screening for mycotoxins and other
secondary metabolites in fungal cultures by thin layer chromatography and high-performance liquid
chromatography. Archives of Environmental Contamination and Toxicology 18: 331-335.
FRlSVAD, J.C., SAMSON, R.A. and STaLK, A.C. 1990. Chemotaxonomic evidence for teleomorphanamorph connections in Eupenicillium jlZVllnicum and related species. In Modern Concepts in Penicillium
and Aspergillus Classification, eds. R.A. Samson and J.1. Pitt, pp. 445-454. New York and London: Plenum
Press.
HORlE, Y., MAEBAYASHI, Y. and YAMAZAKI, M. 1985. Survey of productivity of tremorgenic mycotoxin,
verruculogen by EupeniCl1lium spp. Proceedings of the Japanese Association of Mycotoxicology 22: 35-37.
PITT, J.1. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
PITT, J.1. and HOCKING, A.D. 1985. New species of fungi from Indonesian dried fish. Mycotaxon 22: 197208.
RAMIREZ, e. 1982. Manual and Atlas of the Penicillia. Amsterdam: Elsevier Biomedical.
- - 1985. Revision of recently described Penicillium taxa. In Advances in Penicillium and Aspergillus
Systematics, eds. R.A. Samson and J.I. Pitt, pp. 135-142. New York and London: Plenum Press.
RAPER, K.B. and mOM, C. 1949. Manual of the Penicillia. Baltimore: Williams and Wilkins.
STaLK, A.e. and SAMSON, R.A. 1983. The ascomycete genus Eupenicillium and related Penicillium
- - 1985. A new taxonomic scheme for Penicillium anamorphs. In Advances in Penicillium and Aspergillus
Systematics, eds. R.A. Samson and J.1. Pitt, pp. 163-192. New York: Plenum Press.
DIALOGUE FOLLOWING DR. FRISVAD'S PRESENTATION
There are a few species, such as P. westlingii, that I placed. in synonymy because I had
seen only a single isolate. If you have seen more, then I am happy to have the species
reinstated. Again, separation between P. soppii and P. miczinskii is quite welcome now
PITT:
171
that good evidence is available. The relationship between P. corylophilum with the
monoverticillates: I have always considered that there is a continuum between the
monoverticillates and species in section Divaricatum. Separation in The Genus Penicillium
was done for practical reason. The possible conflict with secondary metabolite data does
not concern me.
FRISVAD: I agree with you that there is a continuum. For example, Eupenicillium javanicum
normally has monoverticillate penicilli, but we believe that there is a connection with P.
janthinellum, which often has metulae.
CHRISTENSEN: It seems logical to speculate that these mycotoxins evolved as defense
mechanisms against other species in the soil. Is there any correlation between
mycotoxins and habitats?
FRISVAD: A student of Dr. Gams, Michael Schlag, made a study of microfungi associated
with roots of Pseudotsuga menziesi;. He found many isolates of P. westlingii that all made
enormous amounts of citrinin. Other species were also present. Citrinin has very strong
antibiotic activity and is certain to playa role in soil ecology. But isolates of P. westlingii
from other soils also show the same profile of secondary metabolites. So, this seems to be
related more to the actual species than the particular ecological habitat. There may be
mycotoxins that are produced only in certain habitats, such as those produced by some
of the kangaroo rat isolates, but these were mostly terverticillate Penicillia.
CHRISTENSEN: I'm pleased to see the restoration of several species, such as P. coralligerum
and P. pedemontanum, because we have found these in soil in the US. We have just
finished a survey of Penicillia in Pseudotsuga menziesii forests in the US. It would be quite
interesting to compare this with Mr. Schlag's results.
FRISVAD: In Danish soil samples, we found a continuum between P. canescens and P.
jensenii. Morphologically, these fit P. canescens, but the secondary metabolites were more
like those of P. jensenii than P. janczewskii.
CHRISTENSEN: P. jensenii is a dominant species in sage brush soils in Wyoming. It would
be interesting to compare these isolates with those from other habitats.
PATERSON: In some of the strains you mention only two metabolites. Am I right in
assuming that other metabolites are also produced.
FRISVAD: Well, with HPLC you might see % different metabolites. What we see with TLC
are the largest peaks from the HPLC. So, we might see as many as 15 with two
dimensional TLC. HPLC is not used so much for identification, but for more
fundamental understanding of species relationships.
PATERSON: I'm quite interested in comparing the total profiles, and seeing the percentage
similarity on that basis.
FRISVAD: In some isolates of P. jensenii, P. canescens and P. griseo-purpureum we saw a red
compound, based on its UV spectrum. We don't know what it is and it has quite a
complicated structure. It's a big help if you can find out what such unknown compounds
are, but for now we can only call them compound A, B and C and so on.
PATERSON: Well, that's alright. How do you chose the metabolites to list for each species?
FRISVAD: We first look for mycotoxins and antibiotic substances that we have in our stock
of standards so I can compare the spectra. Then we look at all the others, which provide
172
with broader information. Sometimes, you can obtain identifications by searching

through ultraviolet spectra: that's how we identified compactin.
pm: In my original morphological studies, I saw P. kapusczinskii between P. canescens and
P. janczewskii. I decided that a morphologist would be unable to distinguish these

species. It's interesting that you also saw P. kapusczinskii as a bump in this continuum of
three species. Clearly, they are very difficult to separate using your criteria as well.
Again, with P. manginii and P. miczynskii, I kept them together because a morphologist
would have difficulty separating them, even though there were some differences. I'm
still concerned about the typification of P. simplicissimum. I asked the University of
Leiden for a type, and finding none, I neotypified P. simplicissimum in the sense that
Raper had used. Stolk and Samson subsequently published a drawing of Oudemans
which they accepted as the type in a very different sense from my neotype. In my
opinion, this drawing could just as easily represent a Paecilomyces: after all, this was
described as a Spica ria. Taking up this type destroyed a concept that had been in use for
forty years. I think this was regrettable.
SAMSON: We were reluctant to take up this type. But Oudemans' drawings are very good,
and we have isolated P. simplicissimum from the same area that Koning collected it. The
colour on the drawing also indicated P. simplicissimum. I think that Oudemans would
have recognized a Paecilomyces. He described it as green.
The name P. simplicissimum is very descriptive of the type of penicillus that
Oudemans drew. I think it is also quite regrettable that we have to sacrifice this name.
FRISVAD:
173
THE PENICILLIUM FUNICULOSUM COMPLEX - WELL DEFINED

SPECIES AND PROBLEMATIC TAXA
E.S. Van Reenen-Hoekstra1, J.C. Frisvad2, RA. Samson1 and A.c. Stolk1
lCentrlllllbureau voor Schimmelcultures
2Deparlment of Biotechnology
The Techniclll University of Denmark
2800 Lynghy, Denmark.
SUMMARY
The morphology and production of secondary metabolites of 96 isolates of Penicillium funiculosum and
related taxa have been examined. On basis of both morphology and chemistry P. pinophilum and P.
minioluteum are kept separate. P. varians placed in synonymy with P. funiculosum by Pitt (1979),
proved to be morphologically distinct from P. funicuwsum by its strongly pigmented stipes, cylindrical
conidia and by its secondary metabolite production. Isolates FRR 1714 and CBS 642.68 both ex type of
P. minioluteum proved to be different. Both isolates were believed to represent the original Biourge
isolate no. 60. CBS 642.68 fully fits the original description of the species, but FRR 1714 resembles
Penicillium rubrum sensu Raper and Thorn. P. allahabadense is kept as a separate species and not
included in P. pinophilum, because it differs from P. pinophilum in growth rate at 37"<: and the shape of
conidia. P. diversum is characterized by its poor growth on Czapek agars. The species accepted are
shortly described and illustrated, while isolates of P. funiculosum from major culture collections were
reidentified. A list of mycotoxins and other secondary metabolites specific for each species is given.
INTRODUCTION
Together with Penicillium chrysogenum Thom, P. funiculosum Thom is the most widely used
Penicillium species for biotechnological purposes (Hamlyn et al., 1987). P. funiculosum in
the broad sense of Raper and Thom (1949) is widely used for the production of
biochemicals, such as cellulases and dextranases, but it is not known whether these
products are produced by P. funiculosum, or similar species such as P. pinophilum
Hedgcock or P. minioluteum Dierckx. Pitt (1979) divided P. funiculosum into three taxa, P.
funiculosum, P. pinophilum and P. minioluteum, on the basis of characters such as colony
diameters, colours, texture, stipe length, and penicillus shape. However, species concepts
in P. funiculosum and related taxa remain unclear. To assist in clarifying this problem,
cultures and fresh isolates deposited at the Centraalbureau voor Schimmelcultures were
examined. In addition to this material many cultures from other collections were
investigated together with some recently described taxa belonging to Penicillium section
5implicium Pitt (1979).
In the following paper we present our observations on % strains of P. funiculosum and
related taxa primarily on the basis of morphological structures supplemented with data
on the production of secondary metabolites.
E.S. van Reenen-Hoekstra et al.
174
Table 1. Identity of examined cultures of P. funiculosum sensu Raper and Thom (1949) and
related taxa.
Received as
Collection nr.
Actual identity
P. allahabadense
P. allahabadense
P. aurantiacum
P. diversum
CBS 304.63 (=NRRL 3397)

CBS 441.89
CBS 314.59 (=NRRL 3398)
CBS 320.48 (=NRRL 2121)
NRRL2122
CBS 242.73
CBS 276.58
IMI303611
FRR 977 (=IFO 6580 as P. rubrum)
CBS 330.48 (=NRRL 1768)
CBS 433.62
CBS 631.66 (=IMI 114933)
CBS 104.71
CBS 884.72
CBS 996.72
CBS 272.86 (=FRR 1630)
CBS 195.88 (=NRRL 1159)
IM161383
IMI 61385
IMI63903
IMII04624
IM1132677
IM1134755
IMI134756
IMI142752
IM1163167
IMI170614
IM1211742
FRR883
NRRL 1033
NRRL 1035
NRRL2119
NRRL3114
NRRL3363
NRRL6014
CBS 433.89
CBS 434.89
CBS 435.89
CBS 436.89
CBS 437.89
CBS 438.89
CBS 169.81
CBS 762.68
CBS 170.60
NRRL2127
CBS 252.31
CBS 642.68 (=IMI 89377)
CBS 196.88 (=FRR 1714)
IMI68219
IM1113729
IMI139462
IMI 147406
P. al/ahabadense
P.diversum
P. diversum
P. diversum
P. diversum
P. diversum
P. funiculosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funiculosum
P. funieulosum
P. funiculosum
P. funieulosum
P. funieulosum
P. funieulosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. gaditanum
P. korosum
P.luteum
P. luteoviride
P. minioluteum"
P. minioluteum
P. minioluteum
P. minioluteum
P. minioluteum
P. minioluteum
P. minioluteum
P. allahabadense
P. aurantmeum
P. diversum ch. I
P. diversum ch. I
P. diversum ch. II
P. diversum ch. II
P. d. erythrome/lis
P. d. erythromellis
P. d. pinophilum
P. rubrum
P. pinophilum
P. minioluteum
"P. rubrisclerotium"*
P. minioluteum
P. funieulosum
P. rubrum
"P. rubrisclerotium"*
"P. rubrisclerotium'"
P. pinophilum
P. d. funiculosum
P. funiculosum
P. d. erythromellis
P. funiculosum
P. d. pinophilum
P. funiculosum
P. funiculosum
P. funiculosum
P.sp.l
P. d. resedanum
P. proteolyticum
?
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. funiculosum
P. minioluteum
P. allahabadense
P. pinophilum
Paecilomyces sp.
P. rubrum
P. minioluteum
P.rubrum
P. rubrum
P.rubrum
P. rubrum
175
The Penicillium funicu/osum complex
P. minioluteum
P. minioluteum
P. minioluteum
P. minioluteum
P. pinophilum""
P. pinophilum'"
P. pinophilum
P. pinophilum
P. pinophilum
P. pinophilum
P. proteolyticum"
P. purpurogenum
var. rubrisclerotium"
P. purpurogenum
P. purpurogenum
P. rademiridi
P. rubicundum
P. samsonii
P.samsonii
P. varians
P. varians
P. varians
P. varians
P. varians
P. varians
P. zacinthae
P.rubrum
IMI178519
CBS 442.89
CBS 443.89
CBS 444.89
CBS 329.48
CBS 631.66
FRR 1397
FRR 1487
CBS 439.89
CBS 440.89
CBS 303.67 (=NRRL 3378)
P. pinophilum
P. pinophilum
P. pinophilum
P. pinophilum
P. pinophilum
P. proteolyticum
CBS 270.35 (=NRRL 1064 =NRRL 1142)
P. pinophilum
CBS 365.48
"P. rubrisclerotium"
CBS 436.62
CBS 140.84
CBS 34259 (=IMI99723 =NRRL 3400)
CBS 137.84 (=Q1032)
CBS 138.84
CBS 386.48 (=IMI40586 =NRRL 2096)
CBS 884.71
CBS 885.71
CBS 272.73
ATCC20150
ATCC28070
CBS 178.81 (=IMI285805)
"P. rubrisclerotium"
?P. diversum
P. rubicundum
P. minioluteum
P. minioluteum
P. varians
P. funiculosum
P. funiculosum
P. d. funiculosum
P. d. minioluteum
P. dtrinum ch. II
P. al/ahabadense
P. minioluteum
P. minioluteum
P. minioluteum
P.rubrum
"P. rubrisclerotium" is based on P. purpurogenum var. rubrisclerotium CBS 365.48. This strain still
produces dark reddish brown sclerotia (Stoll<, 1973). The "culture ex type" NRRL 1064 = CBS 270.35
may have been a contaminant because it resembles P. pitwphilum, which never produces sclerotia.
.. These strains are listed under P. funiculosum in the CBS catalogue (1987)
Isolates of P. funiculosum and related species examined are shown in Table 1. Isolates were
grown on CZ, CYA, 2% MEA, and YES agars (Samson and Van Reenen-Hoekstra, 1988) at
25C and on CYA and MEA at 37C. They were examined after one week for
morphological, physiological and chemical characters. Colony colours are described
according to Ridgway (1912).
For the analysis of secondary metabolites, the isolates were examined using the agar
plug method (Filtenborg et al., 1983), while representative strains, including ex type
cultures, of all species were examined by high-performance liquid chromatography
(HPLC) with diode array detection (Frisvad and Thrane, 1987).
Descriptions of the recognized taxa with short discussions about their taxonomic
placement are given. The isolates examined and their identity are summarized in Table 1.
The isolates of P. funiculosum and allied species produced a large number of secondary
176
Table 2. Production of mycotoxins and other secondary metabolites by species related to P.

funiculosum.
Species
Secondary metabolites
P. allahabadense
Mitorubrinic acid, mitorubrin
P. aurantiacum
P. diversum
Alternariol monomethylether"
Austinol* and isoaustin
Diversonols
Lichexanthone
P. funiculosum
Secalonic acid D* (traces)
P. minioluteum
Spiculisporic acid*
Minioluteic acid
Secalonic acid D*
Mitorubrinic acid, mitorubrin,
Mitorubrinol, mitorubrinol-acetat
P. pinophilum
Vermiculin
Vermicellin
Mitorubrinic acid, mitorubrin,
Mitorubrinol, mitorubrinol acetate
P. rubicundum
Skyrin
P. varians
Recognized as mycotoxins.
metabolites. The profiles of secondary metabolites of the following species were specific
(Table 2). P. pinophilum, P. minioluteum and P. rubicundum have secondary metabolites in
common, while P. rubicundum and P. allahabadense also had secondary metabolites in
common with P. islandicum, P. variabile and related species (Frisvad, unpuhl. data).
DESCRIPTIONS
Penicillium funiculosum Thorn - Fig. 1-2.
Penicillium funiculosum Thorn (sensu stricto}-Bull. Bur. Anim. Ind. US Dept. Agric. 118: 69, 1910
On MEA colony attaining a diameter of 30-52 mm in one week at 25C; floccose, cottony, funiculose, often
with well developed funicles. Mycelium white, salmon to buff, pinkish, or pale yellowish. Sporulation grey
green, yellow green (Tea Green, Celandine Green; Ridgway PI. XLVII). Reverse pale, yellow-orange,
ochraceous, pinkish, cinnamon, salmon or dark brown-red. Exudate absent or a few hyaline drops present,
soluble pigment lacking.
On CZ 15-30 mm diam after one week; on CYA 25-45 mm diam; on YES 30-45 mm diam.
At 37C on MEA good growth, colony reaching a diameter of 20-45 mm, sporulating; on CYA 21-38 mm
diam.
The Penicillium funiculosum complex
177
0
0
()
\ 10 pm
\J
\)
CJ
()
a
0
o
o
C)
<:)
l00~lll
Figure 1. P. funiculosum CBS 272.86 (Neotype). Conidiophores and conidia.
E.S. van Reenen-Hoekstra et a/.
178
Figure 2. [a-c1 P. funiCIIlosum CBS 272.86 (Neotype); [a] funicles with conidiophores and
conidia. (x350); [bod conidiophores and conidia (x1200); [doe] CBS 884.71 (xl2OO)
Conidiophores on MEA arising both from the funicles and the substrate. Stipe length ranging from 25-150
from the funic1es the shorter ones are dominant if borne from the substrate the longer ones are
dominant, not or very slightly pigmented, smooth walled or sometimes very finely rough. Typically
biverticillate, sometimes branches below the metulae may be present. Metulae 3-5, adpressed, measuring 810(12) x 2-3 J.lIIl. Phialides acerose, 10-12 x 2 ~m. Conidia varying from subglobose to ellipsoidal, up to
cylindrical (1,5)2-3 J.lIIllong, hyaline, mostly smooth-walled.
IJ.lIl, if borne
179
Isolates examined:
Culture ex neotype: CBS 272.86 = 1M! 193019 = FRR 1630 from Lagenaria vulgaris, India 1975, J.S. Chohanrather short stipes, typical of the species.
NRRL 1033, unrecorded source, Miss Bottomley, Pretoria, S. Mrica-larger stipes compared to the neotype
and more or less pigmented. Conidia ellipsoidal to fusiform with rather thick walls.
NRRL 1035, from G. Smith, London-morphology comparable with the neotype.
FRR 833, isolated from pasture grass, Qld., Australia, 1971, M.D. Connole-typical of the species.
CBS 884.71 and CBS 885.71, from air Indonesia, 1971, originally identified as P. varians-intermediates
between P. funiculosum and P. varians, pigmentation much less than in P. varians, chemically the same as P.
funiculosum.
1M! 142752, from air, Egypt, 1969-representative isolate of P. funiculosum

IMI 170614, from soil in rhizosphere of Arachis hypogaea, India 1972-representative strain, somewhat
restricted growth, pigmentation not as strong as mentioned by Pitt (1979).
Singh 65a (=CBS 433.89), 69 (=CBS 434.89), 70b (=CBS 435.89), BOa (=CBS 436.89), 90 (=CBS 437.89), and 92
(=CBS 438.89) ex Zea mays, India 1987, K. Singh-representative isolates.
IMI 99723 = CBS 342.59 = NRRL 3400, ex type of Penicillium rubicundum Miller et al. from soil, Georgia, USA,
1956.
CBS 314.59 = IMI 99722 = ATCC 13216 ex type P. aurantiacum Miller et aI. from cultivated soil, Georgia, USA
1955, A.A. Foster.
In Penicillium funiculosum sensu lato as mentioned by Raper and Thom (1949) also P.
minioluteum and P. pinophilum are included. Thom (1910, 1930) characterised P.
!uniculosum having broadly spreading floccose, tufted, greyish-green colonies and
conidiophores with appressed metulae and short stipes, 20-80(100) Ilm. Pitt (1979)
neotypified P. funiculosum which is accepted here. However, we disagree with Pitt (1979)
in placing P. varians, P. aurantiacum and P. rubicundum in synonymy with P. funiculosum. P.
varians differs by its dark pigmented stipes and small cylindrical conidia. P. aurantiaceum
differs in having larger and narrow ellipsoidal conidia, similar to those of P. variabile. The
isolates of this species, all derived from the type, are now in a relatively poor condition
and the production of secondary metabolites is also poor. Besides the morphological
differences also the secondary metabolite profiles were distinct from P. funiculosum. P.
rubicundum Miller et al. was considered by Pitt (1979) as a synonym of P. funiculosum. The
CMI strain of this species is in a good condition and produces several characteristic
secondary metabolites, different from those of P. funiculosum.
P. minioluteum and P. pinophilum are considered separate species here. P. minioluteum
has more or less restricted colonies with striking yellow mycelium, fails to grow at 37"C or
only shows micro-colonies and has longer, often coloured stipes. P. pinophilum has a
greater number of diverged metulae and also larger stipes compared to P. funiculosum.
Besides the morphological characters also differences in secondary metabolite patterns
justifies to keep both P. minioluteum and P. pinophilum separate from P. funiculosum.
Some isolates of P. funiculosum have been reported to produce mycotoxins such as
wortmannin (Haefliger and Hauser, 1973) and spiculisporic acid (Mantle, 1987), but these
isolates probably have been misidentified. The isolate of P. funiculosum NRRL 3363 which
was claimed to produce wortmannin is a representative of P. proteolyticum Kamyschko.
180
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"'"
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0
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00
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Figure 3. P. varians CBS 386.48 (Type). Conidiophores and conidia.
181
Penicillium varians G. Smith - Fig. 3-4
Penicillium varians G. Smith in Trans. Br. mycol. Soc. 18: 89-90, 1933.
On MEA colony attaining a diameter of 28-34 mm in one week at 25C; cottony and funiculose, especially in
the central part. Mycelium whitish to pinkish. Sporulation pale grey-green (Tea Green, Ridgway PIXLVII).
Reverse dark green, with or without salmon or buff coloured areas. Exudate and soluble pigment lacking.
On CZ reaching a diameter of 14-18 mm, with a thinner colony than on MEA; on CYA 15-23 mm diam,
strongly funiculose with salmon-pinkish mycelial ropes; On YES 25-37 mm diam.
At 37C on MEA and CYA good growth, colony diameter of 25-30 mm.
Conidiophores on MEA arising in right angles from the mycelium. Stipes short 25-100 JUn x 2-2,5(3) JUn,
typically darkly pigmented, smooth to finely rough-walled, septate. Penicilli typically biverticillate, but
monoverticillate cOnidiophores also present. Metulae dominantly 2-3, strongly ad pressed, 10-15 x 2-2.5 JUn,
darkly pigmented. Phialides acerose, 10-15 x 1.5-2 JUn. Conidia cylindrical, later becoming more or less
ellipsoidal, 2-3 JUn long, hyaline, smooth walled.
Isolates examined:
Culture ex type: CBS 386.48 =IMI 40586 =FRR 2096 =NRRL 2096 =ATCC 10509 =IFO 6112, ex cotton yarn,
Great Harwood, GB, 1927, G. Smith-all strains are similar and show all characteristics of the species.
Figure 4. P. varians CBS 386.48 (Type). [a-b]Conidiophores and conidia (x1200).
182
P. varians is characterised by its strongly pigmented conidiophores and small smoothwalled cylindrical conidia. These pronounced features could not been observed in
representative isolates of P. funiculosum. The illustrations by Smith (1933) show the type
strain in its original well-developed form. It may have deteriorated during years of
subculturing but most characters are still preserved.
When examined by HPLC-DAD, P. varians produces some unique secondary
metabolites not shared with other species of subgen. Biverticillium.
Penicillium pinophilum Hedgcock - Fig. 5-6.
Penicillium pinophilum Hedgcock apud Thorn, BulL Bur. Anim. Ind. US Dept. Agric. 118: 37, 1910.
? P. purpurogenum var. rubrisclerotium Thorn 1915
On MEA colony attaining a diameter of 35-40(45) mm in one week at 25C; cottony, more or less floccose
with small funic1es. Mycelium pale yellow, often dominating colony appearance. Sporulation in blue green
colours (Artemisia-Celandine, Ridgway PlXLVII). Reverse yellowish to brown (Vinaceous Buff, Ridgway PI.
XL). Exudate hyaline to yellowish, soluble pigment absent.
On CZ reaching a diameter of 15-25 mm. On CYA 25-35 mm diam. At 3TC on MEA and CY A good
growth reaching 35 mm diameter with good sporulation.
Conidiophores on MEA arising from aerial mycelium on the small funicles. Stipes vary from 50-240 x 253!J.II1, smooth-walled. Metulae (3)5 to 8(10), often vesiculate with more than 5 metulae, and divergent, 10-14
Ilm x 2.5-3 !J.III. Phialides acerose, (8)10-12(15) x 2-25 Ilm. Conidia subglobose to ellipsoidal 25-3 Ilm long
also larger ones present, smooth or finely roughened, more or less thick walled.
Representative isolates examined:

Culture ex neotype: CBS 631.66 from PVC, France, DSM, 1944
CBS 33Q.48 = IMI 40235 = ATCC 10446 = NRRL 1768, from soil, USA 1941-chemically atypical and good
producer of duc1auxin.
CBS 170.60
luteum.
= IMI 87160 = ATCC 9644, unknown source, Harvard USA 1944, originally identified as P.
NRRL 1064 = NRRL 1142 = CBS 270.35, from Zea mays, USA LB. Lockwood, 1935-all three strains similar.
IMI63903, from soil Mexico, 1956, J. Nicot, originally identified as P. funiculosum-representative of species.
IMI 211742, from unknown source, Mass. USA, E.G. Simmons-originally identified as P. funiculosum, but its
long stipes, the shape of the conidia and the yellow mycelium are entirely characteristic of P. pinophilum.
Singh 92a (=CBS 439.89) and 93 (=CBS 440.89), from Zea mays, India 1987, K Singh.
Pitt (1979) deSignated IMI 114933 = CBS 631.66 as neotype of P. pinophilum, because the
original isolate had lost most of the important characters. His neotypification is accepted
here. It is doubtful whether P. purpurogenum var. rubrisclerotium is a synonym of P.
pinophilum: Raper and Thorn (1949) mentioned that there was little to suggest that NRRL
1064 (= CBS 270.35) was representative of the original concept of P. purpurogenum var.
rubrisclerotium, and indeed we have never observed sclerotia in any strain of P. pinophilum.
Another problem is that NRRL 1064 has more ellipsoidal conidia, pink aerial mycelium
and a more floccose colony than P. pinophilum, but these may just be insignificant
variations. All other isolates of P. purpurogenum var. rubrisclerotium (CBS 365.48, NRRL
1132, CBS 884.72, FRR 1095, IMI 61363, IMI 61385, IMI 147406, IMI 104624) are unique
morphologically and chemically and should be described as a new species.
Reported producers of dextranase could be included in P. pinophilum (CBS 170.60), P.
pinophilum (CBS 330.48) and P. purpurogenum var. rubrisclerotium (NRRL 1132). Only CBS
183
330.48 was found to produce large amounts of the mycotoxin duclauxin, so other isolates
of P. pinophilum may be the most suited producers of dextranase.
o
o
0
0 0
0
0
()
....
()
d)
0
0
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100 fJ.ffi
Figure 5. P. pinophilum CBS 631.66 (Neotype). Conidiophores and conidia
184
Figure 6. P. pinophilum CBS 631.66 (Neotypel. Conidiophores with diverging metulae. [a]
CBS 170.60; [b) CBS 631.66 (Neotypel; [cod] CBS 440.89 (all xl2(0).
185
()
OJ
0
C)
11100,""
'GO
~v
aD.
(J
00
DO
0
0 c
b
Figure 7 [a-b]. P. allahabadense CBS 304.63 (Type). Conidiophores and conidia with remnants
of the connectives. Mter prolonged incubation synnema development can be observed (b).
[c]. P. zacinthae CBS 178.81 (Type). Conidiophores and conidia.
186
Penicillium allahabadense Mehrotra et Kumar - Fig. 7.
Penicillium allahabadense Mehrotra et Kumar, Can. J. Bot. 40: 1399, 1%2.

P. korosum Rai et aI., Antonie van Leeuwenhoek 35: 430, 1969
P. zacinthae Ramirez et Martinez, Mycopathologia 74: 167, 1981
On MEA colony attaining a diameter of 25 mm in one week at 25 C; strongly funiculose, in central part also
floccose, and after a prolonged incubation period becoming synnematous. Mycelium white to yellowish.
Sporulation olive to blue green (Slate Olive to Artemisia Green, Ridgway PI. XLVII). Reverse yellowish to
cream. On Czapek agars somewhat smaller colony diameter,20-23 mm. Colony morphology more or less
the same as on MEA. At 37 C on MEA very restricted growth, 3-10 mm diam. funiculose and rich
sporulation. Conidiophores biverticillate sometimes irregularly branched. Stipes up to 250 x 3 jJm, smooth
walled. Metulae 2-5, appressed. Phialides with a narrow tapering neck, 8-11 x 2-2.5 jJm. Conidia ellipsoidal
to somewhat fusiform, often apiculate at one side or with a remnant of the connective still visible, 3-4 jJm
long, smooth-walled (occasionally more or less finely roughened).
Culture ex type P. allahabadense: CBS 304.63 = NRRL 3397 = FRR 3397 = ATCC 15067, isolated from soil,
Allahabad, India, B.S. Mehrotra, 1962-NRRL 3397 is in a good condition.
CBS 178.81 = IMI 253805 = ATCC 48474, ex type of Penicillium zacinthae, isolated from leaves of Zacintha
verrucosa, Alicante Spain, 1979, e. Ramirez.
IBT TK06 (=CBS 441.89) ,isolated from ground coriander seeds, 1989, J.e. Frisvad.
CBS 762.68 ex type of P. korosum: from rhizosphere Brassica campestics var. toria, IN. Rai-in poor condition,
showing little growth at 3;>Oe.
P. korosum and P. allahabadense were included in the synonomy of P. pinophilum by Pitt

(1979). However, both these species grow slowly at 37C and the conidia are more
ellipsoidal than in P. pinophilum. The isolate NRRL 3397 of P. allahabadense is in a good
condition, and descriptions and drawings made when accessioned at CBS showed that it
is a separate species. P. korosum grows poorly at 37C and has degenerated, but
biverticillate penicilli can still be observed. P. zacinthae shows all the characteristics of P.
allahabadense and should be regarded as a synonym. These conclusions are strongly
supported by secondary metabolite production.
Penicillum minioluteum Dierckx - Fig. 8-9.
Penicillum minioluteum Dierckx, Ann. Soc. Scient. Brux. 25: 87, 1901
Penicillium gaditanum, Ramirez & Martinez, Mycopathologia 74: 163-171,1981
Penicillium 5amsonii Quintanilla, Mycopathologia 91: 68-78, 1985.
On MEA colony attaining a diameter of 15-20 mm in one week at 25C; more or less velvety with a raised
central area at first, becoming lanose to funiculose after two weeks. Mycelium white and yellow. Sporulation
olive to dark green (Leaf Green to Pois Green, Ridgway PI. XLI). Reverse orange to pinkish red (Japan Rose,
Ridgway PI. XXVIIO.
On CZ 5-8 mm in one week (about 10 mm after two weeks), with white to bright yellow mycelium and
dark green sporulation. Reverse orange to dark red brown.
On CYA reaching up to 15 mm in one week. Mycelium white, pinkish or orange-yellow. Soluble pigment
red, diffusing into agar. Exudate present as hyaline to pinkish drops. Reverse in orange shades to dark redbrown (Indian Red, Ridgway PI. XXVIO.
On Yes up to 25 mm, mycelium pinkish to yellow. Reverse red to dark red. (Dragon's blood Red to
Pompeion Red, Ridgway Pl. XIII). At 37"C no growth on MEA and CYA.
Conidiophore stipe 100-250 x 3 jJm, hyaline sometimes more or less pigmented, thick-walled, smooth.
Metulae 3-5(7) appressed, measuring 10-12 x 2.5-3 jJm. Phial ides acerose, 10-12 x 2-3 jJm. Conidia ellipsoidal
to subglobose, sometimes more or less pointed or 2.5-3 jJm, often thick-walled, smooth to finely rough,
brownish.
187
Q()
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0 \)
00
C)
0
0
0
10 pm
()
()
0
oC)
0
C)
0
0
C)
Figure 8. [a-bJ. P. minioluteum (Neotype). Conidiophores and conidia; [aJ.CBS 642.68;

[bJ IMI89377
188
Figure 9 [a-dl. P. minioluteum Conidiophores and conidia; [a-bl.CBS 642.68 (Neotype);

[c-dl CBS 996.72 (all x1200).
189

Culture ex neotype: CBS 642.68 =IMI 89377, unknown source, P. Biourge (no. 60).
CBS 169.81 = IMI 253792 = ATCC 42230. ex type P. gaditanum Ramirez & Martinez, from air, Madrid, Spain,
1978.
CBS 137.84 = Q1032 = IMI 327872 ex type P. samsonii Quintanilla, from apples damaged by insects
Valladolid, Spain. The typical yellow mycelium is lost in CBS 137.84, but it is still very striking in Q1032 and
CBS 138.84.
CBS 138.84 (= Q1283), received as P. samsonii Quintanilla, the same source as CBS 137.84
CBS 104.71, from Tulipa sp., the Netherlands
CBS 9%.72, from jute sugar bags, the Netherlands
P. minioluteum differs from P. funiculosum by its more compact and rather restricted
colonies, the bright yellow to orange mycelium and the dark green to olive green
sporulation. At 37C on MEA and CYA no growth or only microcolonies up to 2 mm
diameter were observed. P. funiculosum generally shows more spreading colonies with
whitish to pinkish mycelium and grey green sporulation. At 37C on MEA and CYA P.
funiculosum grows well. Chemically P. minioluteum and P. funiculosum are different (Table
2).
The culture FRR 1714, which Pitt (1979) used for the description of P. minioluteum
differs markedly from CBS 642.68, although both strains were believed to represent the
original Biourge isolate no. 60. CBS 642.68 showed a more floccose and compact colony
with yellow to orange mycelium and dark green sporulation and on most media an
yellow or brownish red reverse. FRR 1714 grows more rapidly and thinner (especially on
MEA) often with red transparant areas, showing ropes in the central part after a
prolonged incubation and with a greenish transparant reverse with red. The conidia are
more fusiform than in CBS 642.68. The correct desposition of P. minioluteum sensu Pitt
must be determined, but the isolate FRR 1714 morphologically and chemically resemble
NRRL 1062, which Raper and Thorn (1949) used to define P. rubrum. However, this isolate
is not the orginal type culture and therefore a more detailed study of isolates belonging to
P. rubrum sensu Raper and Thorn will be carried out in a future study.
The type cultures of P. gaditanum and P. samsonii morphologically and chemically
resemble P. minioluteum and are here considered synonyms of P. minioluteum.
Penicillium diversum Raper & Fennell- Fig. 10
Penicillium diversum Raper & Fennell- Mycologia 40: 539, 1948.

? Penicillium rademirici Quintanilla - Mycopathologia 91: 69-78, 1985.
On MEA colony attaining a diameter of 20-30 mm in one week at 25C; velvety, plane in concentric zones,
sporulating area alternating with submerged mycelium. Mycelium whitish to yellow. Sporulation olive to
dark green (Slate Olive to Vetiver Green, Ridgway PI. XLVII), Grayish-Olive (RidgwayPI. XLVI) or Storm
Gray (Ridgway PI. LII). Reverse uncoloured or yellowish (Naples Yellow Ridgway PI. XVI). Exudate absent
or sometimes present as hyaline drops.
On CZ restricted and poor growth, 2-5 mm in diam. Sporulation sparse, grey-green.
On CYA restricted growth attaining a diameter of 5 mm. Sporulation poor, grey green. Reverse buff to
brownish.
On YES agar, a diameter of 5-9 mm. Sporulation grey or light mouse gray (Mouse Gray Ridgway PI. U).
Reverse brownish. At 3~C on MEA 20-25 mm in diam., on CYA 2-5 mm diam.
190
00
lOpm
CJ
00
0
()
0
()
aO
0
00
Figure 10. P. diversum CBS 320.48 (Type). Conidiophores and conidia.
Conidiophores on MEA bome from submerged or from basal mycelium. Stipes long, 150-300 (350) x 2.5-3
~. Phialides with a long tapering neck, 8-10 x 1.5-2.5 ~. Conidia subglobose to ellipsoidal 2-3 ~,hyaline,
smooth to finely rough walled.
Isolates examined:
Culture ex type: CBS 320.48 = NRRL 2121 = ATCC 10437 = lMI 40579 = IFO 7759 = QM 1921, from mouldy
leather, USA-at preser.t CBS 320.48 often shows many monoverticillate conidiophores, but typical
biverticillate conidiophores were observed when the isolate was deposited, designated here as chemotype I.
NRRL 2122, from soil, Sweden, restricted growth at 37 C on MEA, chemotype I.
CBS 242.73, from lung Rana macrodon, 1972 - rough striate conidia, designated here as chemotype II.
CBS 276.58 from Beta vulgaris, 1958, chemotype II.
CBS 140.84 ex type of P. rademirici Quintanilla = Q 1248, from air under willow trees in the bank of Duero
river, Herrea (Valladolid), Spain.
191
P. diversum is characterized by its poor growth on Czapek agar. Cultural and

micromorphological characters are similar to those of P. rademirici Quintanilla, which also
grows weakly at best on Czapek. In CBS 140.84, the type of P. rademirici, we have observed
only biverticillate structures, not branches as drawn and described by Quintanilla (1985).
Also the striking yellow to orange yellow mycelium is no longer present. Quintanilla
(1985) placed the species between P. primulinum Pitt and P. islandicum Sopp. The type
strain of P. primulinum at the CBS has lost the original yellow mycelium and looks now
identical with P. diversum, but the two taxa differ significantly in their production of
secondary metabolites. P. rademirici also differs from P. diversum in secondary metabolites
produced and perhaps should be considered as a distinct species.
ACKNOWLEDGEMENTS
REFERENCES
FIlTENBORG, 0., FRISVAD, J.e. and THRANE, U. 1983. Simple screening method for molds producing
myrotoxins and other fungal serondary metabolites based on alkylphenone retention indices and UV-VIS
spectra (diode array detection). Journal of Chromatography 404: 195-214.
HAEFLIGER, W. and HAUSER, D. 1973. Isolierung und Strukturaufklarung von ll-Desacetoxywortmannin. Helvetica Chimica Acta 56: 2901-2904.
HAMlYN, P.F., WALES, D.s. and SAGAR, B.F. 1987. Extracellular enzymes of Penicillium. In Penicillium and
Acremonium. Biotechnology Handbooks I, ed. J.F. PEBERDY, pp. 245-284. New York and london:
Plenum Press.
MANTLE, P.G. 1987. Serondary metabolites of Penicillium and Acremonium. In Penicillium and Acremonium.
Biotechnology Handbooks 1. ed. J.F. PEBERDY, pp. 161-243. New York and london: Plenum Press.
PITT, J.1. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London and
New York: Academic Press.
QUINTANIllA, J.A. 1985. Three new species of Penicillium belonging to subgenus Biverticillium Dierckx,
isolated from different substrates. Mycopathologia 91: 69-78
RAMIREZ, e. and MARTINEZ, A.T. 1981. Four new species of Penicillium isolated from different substrates.
Mycopathologia 74: 163-171.
RIDGWAY, R. 1912. Color standards and color nomenclature. Washington, De.
RAPER, K.B. and C. THOM. 1949. Manual of the Penicillia. Williams and Wilkins, Baltimore.
SAMSON, R.A. and REENEN-HOEKSTRA, van E.S. 1988. An introduction to food-borne fungi. Third
Edition. Baam: Centraalbureau voor Schimmelcultures.
SMITH, G. 1933. Some new species of Penicillium. Transactions of the British Mycological Society 18: 88-91.
STOlK, A.e. 1973. Penicillium donkii sp. nov. and some observations on sclerotial strains of Penicillium
funiculosum. Persoonia 7: 333-337.
THOM, e. 1910. Cultural studies of species of Penicillium. Bulletin of the Bureau of Animal Industry United
States Department of Agriculture 118: 1-109.
THOM, C. 1930. The Penicillia. Williams and Wilkins, Baltimore.
192
E.S. van Reenen-Hoekstra
et al.
DIALOGUE FOLLOWING THE PRESENTATION OF THE PAPER BY ELLEN VAN

REENEN-HOEKSTRA
CHRISTENSEN: Did I notice an isolate called P. diversum var. aureum or P. primulinum?

SAMSON: Yes, I believe this is a good species. Dr. Pitt also included an isolate under that
name that has been called P. marneffei. The type isolate of P. marneffei is in poor condition,
but recently we have obtained some twenty new isolates from bamboo rats and AIDS
patients and it seems to be a very good species. It is similar to P. primulinum.
PITT: I am pleased to see that you might resurrect P. allahabadense. The extype isolate was
badly deteriorated, and I had little choice but to place it in synonymy. I still am
unprepared to regard P. aurantiacium and P. rubicundum as separate species. They are
quite closely related to P. funiculosum. Regarding P. minioluteum, I have only just realized
the error that I made. The neotype isolate on which I based my concept of P. minioluteum
came from the CMI collection and was clearly quite different from the CBS strain of the
same isolate. This is regrettable but can easily happen. I tried to obtain the best isolates I
could in the mid 1970's, but I failed in this case. P. minioluteum sensu Pitt will have to be
given a different name.
SAMSON: This is a real problem. We also discovered that the CBS strain of the extype of P.
solitum is quite different from the real type. It's very important to make sure that the
cultures we work with match the original descriptions.
HENNEBERT: This is worth emphasizing. Many of the strains of Biourge's isolates kept in
other culture collections show a lot of sectoring. When I discovered this, I started making
single conidium isolates from Penicillium cultures. I often find three or four different
colony morphologies can be derived from a single strain. Single spore cultures from
extype strains should be carefully studied by traditional morphological methods and
new techniques, such as secondary metabolites, to establish their identities. The other
problem is that the descriptions of older authors, such as Dierckx, were made from
totally different culture media than we now use. They used gelatin, not agar. Biourge
added some agar to solidify the gelatin. In my lab, we are now studying the type strains
of Biourge, the neotypes for Dierckx's species, using the original culture media. We
eventually hope to provide a comparative morphology to allow a better comparison with
the original descriptions.
193
IDENTIFICATION OF PENICILLIUM SPECIES ISOLATED FROM AN

AGRICULTURAL LOESS SOIL IN GERMANY
H.I. Nirenberg and B. Metzler
Institut fUr Microbiologie
Biologische Bundesanstalt fUr Land- und Forstwirtschaft
1000 Berlin 33 Dahlem, FRG
SUMMARY
Of more than 5000 fungal isolates which were recovered from an agricultural loess soil in Germany,
20% were Penicillium and were identified as 15 species. One species close to P.janthinellum of unclear
identity is described and discussed in detail.
INTRODUCTION
Since April 1987 a soil protection program has been carried out at the Biological Research
Center for Agriculture and Forestry (BBA) in the Federal Republic of Germany. Five
institutes have investigated the long-term impact of an intensive use of pesticides and
fertilizers on chemical residues and soil biology of a loess soil in the vicinity of
Braunschweig. Winter wheat, winter barley, and sugar beets have been grown in rotation.
For seven years the production intensity and cropping system for each plot has been
constant.
The Institute of Microbiology at BBA has analysed the fungal and algal soil flora in the
cropping systems of two different production intensities. This paper is mainly concerned
with the method of qualitative and quantitative assessment of Penicillia and especially
with the taxonomy of a Penicillium species, which proved to be related to P. janthinellum
and difficult to identify.
Soil samples from the upper 5 em of loess soils were taken every two months during a
period when winter barley was grown (5.10.87-8.8.88). The recommended soil washing
technique (Gams and Domsch, 1967) imposes serious problems if applied to loess soil
because of its tiny particle sizes. More than 95% of particles are less than 200 IJlIl. in
diameter (Feyk and Pretsch, 1988). The remaining bigger particles are not considered to be
representative for the soil. Therefore we used the following method: subsamples were
homogenized by shaking and sieving, then soil particles of 0.5 to 0.63 mm diameter are
taken and placed individually on SNA (Synthetischer Niihragar, Nirenberg, 1981) in
plastic Petri dishes (120 per sampling date). Bacterial growth was suppressed by three
antibiotics (chlortetracy cline 10 mg/l, dihydrostreptomycin-sulphate 50 mgt!, penicillin
G 100 mg/l). Incubation was at 20C, the first five days in darkness, then the following
week under continuous near ultraviolet light (to induce sporulation of certain fungi). Petri
dishes were then stored at 20 to 25C under natural day/night lighting cycles for an
additional four weeks. Plates were examined under the microscope after one, three and six
H.1. Nirenberg & B. Metzler
194
weeks. Fungi were identified. during this time, if necessary after isolation. Species were
counted. only once per soil particle.
As Penicillium species usually sporulate rapidly, they can be transferred to other SNA
plates after the first five days. On this medium the following species were determined: P.
canescens Sopp, P. melinii Thom, P. janczewskii Zaleski, P. expansum link, P. griseofulvum
Dierckx, P. brevicompactum Dierckx, P. glabrum (Wehmer) Westling (P. frequentans), and P.
restrictum Gilman & Abbott. P. hordei Stolk and an unknown Penicillium species under
discussion below, could often be identified on the original plate. The other Penicillia were
cultured for species determination as recommended by Raper and Thom (1949) and Pitt
(1979).
RESULTS
From 720 soil particles, 5734 fungi were counted belonging to 62 genera and 148 species.
Penicillium accounted for 20% of the isolates, representing 15 species (Table 1). The most
frequent were P. janczewskii, P. melinii, P. hordei, P. canescens, and an unidentified species.
A clear influence of the seasons could not be determined.
Table 1. Penicillia found in the soil samples (treatments summarized)
Sampling date
Numbersof particles
examined
P. aurantiogriseum
P. brevicompactum
P. canescens
P.expansum
P.glabrum
P. griseofulvum
P. hordei
P. janczewskii
P. janthinellum
P. jensenii
P. meliniip
P. restrictum
P. rugulosum
P. simplicissimum
P. spec. "trimorphum"
5-10-87 1-12-87
1-2-88
18-4-88
13-6-88
8-8-88
Exi
120
120
120
120
120
120
720
4
0
6
2
0
0
32
81
5
0
66
3
1
2
12
0
0
25
9
0
0
12
80
3
0
61
1
0
2
15
2
0
17
3
1
1
8
63
0
0
57
2
0
1
14
0
3
14
2
0
0
19
58
0
0
32
2
0
0
13
0
2
21
7
2
1
37
81
0
1
42
3
0
0
15
6
0
19
11
0
10
32
52
0
0
41
3
0
2
7
12
5
102
34
3
12
140
415
8
1
299
14
1
7
74
On SNA, most of the Penicillium species produced more or less velutinous growth;
mycelial and reverse colours as well as soluble pigments were usually not pronounced.
Those Penicillia which could be recognized after 7 days on this medium are listed below
in the order of decreasing growth rate. Characterisation on SNA included. features visible
under the low power microscope.
P. expansum link: Fast and loosely growing colonies, forming concentric rings, penicilli
tervertcillate, conidia densely packed in columns, bright green.
Identification of PenicilHum species isolated from an agricultural loess soil in Germany
195
P. griseofulvum Dierckx: Conidiophores more or less terverticillate, varying from very

simple to quite complex, crowded at the inoculation point, conidial masses greyish green.
P. hordei Stolk: Conidiophores soon forming typical fascicles, often with a drop of exudate,
often crowded at the inoculation point. Penicilli terverticillate, conidial masses brownish
green.
P. melinii Thom: Spreading broadly, penicilli irregular (divaricate), conidia in columns,
brownish green.
P. janczewskii Zaleski: Penicilli divaricate, conidial chains disordered, colonies appearing
dark grey.
P. canescens Sopp: Growing more restrictedly, penicilli divaricate, delicate ramification
and conidial columns, blue green.
P. restrictum Gilman & Abbott: Growing very restrictedly, very short monoverticillate
conidiophores.
Penicillium species: Conidiophores long and more or less monoverticillate or medium long
with one or two metulae; conidial chains divergent, coloured Isabelline; on short
conidiophores especially after prolonged cultivation brownish conidia in false heads (Figs.
1 a-c). On the original plate usually identifiable after three weeks.
Since this fungus is hard to classify, a detailed description according to the model of Pitt
(1979) is given:
01A, 25C, 7 days: Colonies 30-40 mm diam, radially sulcate, surface texture floccose, low; mycelium white;
margins entire, low, rarely deep; conidiogenesis light, inconspicious; no exudate; no soluble pigment;
reverse pale.
MEA, 25C, 7 days: Colonies 30-50 mm diam, surface floccose, plane, margins entire, low or subsurface;
mycelium white, sometimes with yellowish sectors; conidiogenesis inconspicous to greyish green; no
exudate, no soluble pigment, reverse pale.
G25N, 25C, 7 days: Colonies 0-6 mm diam; other characters as on 01A.
CYA, 5C, 7 days: Growth varying from germination to colonies of 1-2 mm. 3~C, 01A, 7 days: Usually no
growth, sometimes germination and formation of a rnicrocolony of large vesicles (Fig. 1d).
Conidiophores borne from surface or aerial hyphae, stipes variable in length 15-300(-500) x 2-3 J1Ill, flexible
to stiff, more or less thin walled, smooth; penicilli mostly monoverticillate or with one, rarely 2-3 metulae,
sometimes irregular; metu1ae more or less divergent, usually 10-15 x 2-2.5 J1Ill; phialides in verticils of (1-4)5-8, cylindroidal to flask shaped, 8-12(-15) x 2.5-3 J1Ill (Fig.1e); conidia broadly ellipsoidal to apiculate 3.5-4
x 2.5-3 J1Ill, smooth walled, sometimes faintly rugulose, born in long disordered chains, or in false heads
when formed on short conidiophores with divergent phialides near the surface of SNA (Fig. 1c), in the latter
case with brown pigment.
DISCUSSION
The unidentified Penicillium species, which we provisially will name: "P. trimorphum" is
clearly a member of Penicillium series Janthinella because of its long collula and divaricate
penicilli tending to be monoverticillate. In Table 2 a synoptic key is given for
representative strains and their respective species descriptions.
The new species resembles most closely P. ochrochloron Biourge sensu Pitt in its smooth,
broadly ellipsoidal apiculate conidia of 3.5-4 Ilm length; in the phialides with long
cylindrical collula, the penicilli with only few metulae, and the quite rapid growth.
196
H.I. Nirenberg & B. Metzler
Neither species grows at 37C, both show sparse conidiogenesis and colouration.
However, "P. trimorphum" grows at SoC on CYA better than P. ochrochloron, and much
slower on G25N. Moreover, we have never found a strain with rugose conidiophores,
which can be present in P. ochrochloron. The major problem is that the original description
of Biourge (1923) and the description by Pitt (1979) differ in the evidently pronounced
Figure 1 a-e. "Penicillium trimorphum", a. Long and slender type of conidiophore,

monoverticillate with long disordered conidial chains; grown on SNA; 125 x, b. Medium
long type of conidiophores, divaricate, with disordered conidial chains; grown on SNA; 125
x, c. Short type of conidiophores with conidia forming false heads; after one month on SNA;
125 x, d. Extraordinary vesicles among the substrate mycelium on Czapek agar; 750 x, e.
Nearly monoverticillate penicilli, phialides with long collula, conidia smooth, ellipsoidal;
grown on SNA; 1500 x
Identification of Penicillium species isolated from an agricultural loess soil in Germany
197
roughness of the conidia and the conidiophores in Biourge's description. Therefore, it is

hard to follow Pitt's neotypification base dof an isolate from C. Thorn. An isolate, which
more closely fits Biourge's description exists in the collection of the BBA (no. 64184).
Another similar species proposed by Samson (pers. comm.) is P. cremeogriseum
Chalabuda (1950). The identity is here rejected because the type culture (CBS 223.66) of P.
cremeogriseum produces smaller conidiophores and smaller, rather globose conidia which
are definitely rugulose. In addition it has a different growth on CYA.
P. janthinellum Biourge seems less closely related to "P. trimorphum" because of its
globose rough conidia, more branched conidiophores and the vivid colouration of the
cultures. As discussed by Pitt (1979), neotypification of this species is not unequivocal.
After examination of Pitt's neotypification of P. ochrochloron, we propose that it be
rejected on the grounds that it is inconsistent with the protologue of Biourge (1923) and
therefore a new species "Penicillium trimorphum" with P. ochrochloron sensu Pitt non
Biourge as a synonym. Another neotype for P. ochrochloron Biourge should be selected;
strain BBA 64184 represents a possible choice.
Table 2. Synoptic key with critical characteristics of the disr.ussed Penicillia
Species
P. janthinellum
P. cremeogriseum
"P. trimorphum"
(BBA6S203)
P. ochrochloron
(Biourge, 1923)
P. ochrochloron
(Pitt, 1979)
P. ochrochloron
O?
(Raper & Thorn, 1949)
P. ochrochloron
(BBA64184)
P. janthinellum
(Biourge, 1923)
P. janthinel/um
(Pitt, 1979)
(Raper & Thorn, 1949)

(Chlabuda, 1950)
P. cremeogriseum
(ex-type CBS 223.66)
Explanation of abbreviations and signs: +: with the property indicated, 0: without this property, :
property less pronounced, -: not specified
ACKNOWLEDGEMENT
This study was partly supported by a grant of the German "Bundesminister fill Forschung
und Technologie" to Prof. Dr. W. Sauthoff. We are also grateful to Mrs. Marlene Katz for
her skillfull technical assistance.
198
H.I. Nirenberg & B. Metzler
REFERENCES
BIOURGE, P. 1923. Les moisisssures du groupe Penicillium Link. La Cellule 33: 7-331.
CHALABUDA, T.V. 1950. Species novae e genere Penicillium Unk. Botaniceskie Materialy Otdela Sporuuych
Rastenij/Akademija Nauk SSSR 6: 168.
FEYK, M. and PRETSCH, K. 1988. Bodenkundliche Spezialkartierung des BBA-Versuchsstandortes Ahlum

bei Wolfenbuttel. Hannover: Niedersiichsisches Landesamt fur Bodenforschung.
GAMS, W. and DOMSCH, K.H. 1967. Beitriige zur Anwendung der Bodenwaschtechnik fUr die Isolierung
von Bodenpilzen. Archiv fUr Mikrobiologie 58: 134-144.
NIRENBERG, H.I. 1981. A simplified method for identifying Fusarium spp. occurring on wheat. Canadian
Journal of Botany 59: 1599-1609.
pm, J.I. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London:
Academic Press.
RAPER, K.B. and mOM, c. 1949: A manual of the Penicillia. Blatimore: Williams and Wilkins
DIALOGUE FOLLOWING DR. NIRENBERG'S PRESENTATION
Pm: Dr. Nirenberg, I would place your unknown species, without too much hesitation, in
Penicillium janthinellum. This species, in my work and in all previous works, is a variable
species. It's quite possible to have a pale reverse and with many monoverticillate
penicilli. Someday, someone may be able to split this species, but I can't do it on
morphological grounds.
NIRENBERG: We have at the same time found P. janthinellum from the same sand that was
treated with sulphuric acid and it is a completely different species. If you compare the
microscopic features, such as the metulae, and see the differences in colony differences at
37"C and SoC, it is really not the same fungus. We are less sure of the distinction from P.
ochrochloron or P. simplicissimum. Our isolate has certain similarities with your concept of
P. ochrochloron. We have a P. ochrochloron in our culture collection that matches the
original description perfectly. It has the rugose conidia and stipes and therefore, I really
question whether one should have neotype strains that are morphologically degenerated
and no longer showing the original morphology. The P. ochrochloron that we received
from your culture collection has conidia that are up to S ~ long, whereas the original
description has them 4 urn long.
CHRISTENSEN: As a community ecologist, I would ask if you have compared this with
what has been called P. simplicissimum in the literature?
NIRENBERG: Yes. We have found P. simplicissimum in the same soil, and it has compact
metulae, rugose stipes and the conidia are much more rounded. In the isolates of our
unidentified species, conidia are not globose or subglobose but strongly apiculate. In 14
day old cultures, the conidia are really dark brown.
CHRISTENSEN: I was quite interested in your total list, because these are species that I
associate with grassland soils, P. restrictum, P. canescens and others. Did you also find
Paecilomyces lilacinus or Fusarium species?
NIRENBEG: Yes. Of course, we found lots of Fusarium species. Paecilomyces lilacinus and P.
marquandii were both common, particularly the latter. The soil was once a grassland soil,
but initially and later mustard were grown there. The species spectrum has changed
somewhat, but the Penicillia have not.
5
TAXONOMIC SCHEMES OF ASPERGILLUS
201
CHEMOTAXONOMY AND MORPHOLOGY OF ASPERGILLUS

FUMIGATUS AND RELATED TAXA
J.e. Frisvadl and RA. Samson2

SUMMARY
Many isolates of Aspergillus fumigatus and related taxa in Aspergillus sect. Fumigati were examined
morphologically and for profiles of secondary metabolites. The isolates of secondary metabolite
production by A. fumigatus was very homogeneous: tryptiquivalins, fumigaclavine A, verruculogen,
other fumitremorgins and fumagillin were produced by all isolates tested, Fumigatin and sulochrin
like metabolites were produced, less frequently, while gliotoxin and helvolic acid were not detected.
A. brevipes, A. viridinutans, A. duricaulis and A. unilatera/is, produced unique profiles of secondary
metabolites different from those above. A. brevipes and A. viridinutans produced viriditoxin, while A.
duricaulis produced cyclopaldic acid and chromanols 1, 2 and 3. It is concluded that species of
Aspergillus sect. Fumigati are well defined, both morphologically and by their profiles of secondary
metabolites.
INTRODUCTION
Aspergillus fumigatus Fres. is a widespread thermophilic species. Its importance as a

pathogenic fungus has been stressed repeatedly (Schonheyder, 1987). It is also allergenic
(Cole and Samson, 1984; Wilken-Jensen and Cravesen, 1984) and produces several
mycotoxins (Cole and Cox, 1981; Turner and Aldridge, 1983) (Table 1).
A. fumigatus, A. brevipes G. Smith, A. duricaulis Raper & Fennell, A. unilateralis Thrower
and A. viridinutans Ducker & Thrower are classified in Aspergillus sect. Fumigati (Cams et
a/., 1985) (= the Aspergillus fumigatus group; Raper and Fennell, 1965) together with
anamorphs of Neosartorya species. A. fumigatus and N. fischeri (Wehmer) Malloch & Cain)
var. fischeri produces fumitremorgin A, Band C and verruculogen (Nielsen et aI., 1988),
while A. duricaulis and N. quadricincta, have been reported to produce cyclopolic acid or
similar metabolites (Turner & Aldridge, 1983). These data suggest that Neosartorya and
Aspergillus sect. Fumigati are related both morphologically and biochemically.
The identity of several varieties of A. fumigatus (Samson, 1979) is still uncertain and
their mycotoxin production has been little investigated. In addition, little data exists on
secondary metabolite production A. fumigatus isolates from different habitats. Clinical
isolates often differ quite markedly from food-borne or soil isolates. This paper compares
morphology and secondary metabolite production by isolates of A. fumigatus from
different sources, and by other taxa in sect. Fumigati.
J.e. Frisvao & A.A. Samson
202
Table 1. Secondary metabolites reported from Aspergillus fumigatus and related species
A. fumigatus
Gliotoxin (Waksman and Geiger, 1944)
HelvoJic acid (Chain et al., 1943)
AgrocIavine, chanocIavine, elymocIavine and festuc1avine (Spilsbury and Wilkinson, 1961)
Fumagillin (Tarbell et al., 1960)
Fumitoxin A, B, C and D (Debeaupuis and Lafont, 1978)
Fumigaclavine A, Band C (Cole et al., 1977)
Fumigatin and spinulosin (Anslow and Raistrick, 1938a, b)
Small phenols (Turner, 1971; Turner and Aldridge, 1983)
Fumitremorgin A and B (Yamazaki et al., 1971), verruculogen and TR-2 (Cole et al., 1977)
Tryptiquivaline C-H, I, and J, L, M (Yamazaki et al., 1976, 1977, 1978, 1979)
2-chloro-l,3,8-trihydroxy-6-methylanthrone, AO-l and 2-chloro-l,3,8-trihydroxy-6hydroxymethylanthrone (Yamamoto et al., 1968),
Monomethylsulochrin (Turner, 1965)
Trypacidin (Balan et al., 1965)
2,6-dihydroxyphenylacetic acid (Turner and Aldridge, 1983)
4-carboxy-S,S'-dihydroxy-3,3'-dimethyldiphenyl ether (Yamamoto et al., 1972).
A. viridi-nutans
Viriditoxin (Weisleder and Lillehoj, 1971)
4-acetyl-6,8-dihydroxy-S-methylisocoumarin (Aldridge et al., 1966),
4-acetyl-6,8-dihydroxy-S-methyl-3,4-dihydroxyisocoumarin and 4-acetyl-3,8-dihydroxy-6methoxy-S-methyl-3,4-dihydroisocoumarin (Turner and Aldridge, 1983).
A. duricaulis
Cyclopaldic acid, 3-O-methyl-cyclopolic acid and the related Chromanol 1,2 and 3 (Brillinger et
al., 1978; Achenbach et ai., 1982 a & b).
For morphological examination isolates were grown on 2% maltextract agar, oatmeal agar
and Czapek agar and incubated for 7 days at 25 and 37 C. For secondary metabolite
analysis, isolates were grown on Czapek yeast extract agar (CYA), Difco yeast extract
sucrose agar (YES), Sigma (Y-4000) yeast extract sucrose agar (SYES), and Blakeslee malt
extract agar (MEA) (Samson and Pitt, 1985) for two weeks at 25 C. Three Petri dishes, one
of CYA, YES and SYES were placed in a Stomacher bag and extracted with
chloroform/methanol (2:1) for 3 min on a Colworth Stomacher. Further procedures were
as described by Frisvad and Thrane (1987). The resulting extracts were analyzed by high
performance liquid chromatography (HPLC) with diode array detection DAD as
described by Frisvad and Thrane (1987) and the production of particular secondary
metabolites was confirmed by thin layer chromatography (TLC) viewed under UV light
before and after 50% sulphuric acid treatment (Frisvad et al., 1989).
Aspergillus fumigatus is morphologically a quite variable species, as documented by the
varieties described since 1965 (De Vries and Cormane, 1969; Samson, 1979). Varsney and
Sarbhoy (1981) concurred with Samson (1979) in placing nearly all of these varieties in
synonomy with A. fumigatus, but upgraded A. fumigatus var. acolumnaris as A. acolumnaris
(Rai et al.) Varsney & Sarbhoy to species status. HPLC analysis of extracts of isolates from
203
Chemotaxonomy and morphology of Aspergillus fumigafus and related taxa
Table 2. Production of mycotoxins and other secondary metabolites by members of

Aspergillus sect. Fumigati.
A. breuipes
A. duricaulis
? A. duricaulis
A. fumigatus
A. fumigatus
CBS 118.53
CBS 481.65
IMI217288
CBS 113.26
CBS 132.54
CBS 192.65
CBS 143.89
CBS 144.89
CBS 145.89
CBS 146.89
CBS 147.89
CBS 148.89
CBS 149.89
CBS 150.89
CBS 151.89
CBS 152.89
CBS 153.89
CBS 154.89
+ - - + + -
- - - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + -
W n n u u s u
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + +
+
+ + +
- - +
- - +
+ + +
+ - +
+ + +
+ + +
+
+ + +
+ + +
+ + +
- - +
- - +
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
- - - - + -
var. acolumnaris
CBS 457.75
+ + + + -
+ + -
+ -
phialiseptus
CBS 542.75
+ + + + -
+ + -
+ -
+ + -
+ -
+ + -
+ -
+ + -
+ -
+ -
A. fumigatus
var.
A. fumigatus
var. ellipticus
CBS 407.65
var. sclerotiorum
CBS 458.75
CBS 283.66
CBS 126.56
CBS 127.56
IMI240496
A. fumigatus
A. unilateralis
A. viridinutans
(? mixed)
1.
2.
3.
4.
5.
6.
7.
8.
+ - -
viriditoxin
cydopaldic acid, 3-O-methyl- cydopolic
acid, chromanoll, 2 & 3
ruguIovasine
canesin-Iike metabolite
tumitoxins
fumagillin
verrucologen, fumitremorgin C
tryptiquivalins
9.
10.
11.
12.
13.
14.
15.
16.
trypacidin
monomethylsulochrin
FUA
FUB
tumigatin
tumigaclavine A
terrein
mycophenolic acid
different habitats showed that A. fumigatus is a very homogeneous species with respect to
profiles of secondary metabolites. Nearly all isolates produced fumigaclavine A,
fumagillin, fumitoxins, trypacidin, monomethylsulochrin, several tryptoquivalins,
verruculogen, fumitremorgin C and metabolites of two chromophore families, named
here FUA and FUB. A. acolumnaris produced the same secondary metabolites as other
isolates of A. fumigatus. Synonymy with A. fumigatus, as concluded by Samson (1979) is
confirmed. The only chemically different isolate of those identified as A. fumigatus, CBS
153.89, produces many metabolites in common with Neosartorya fischeri var. fischeri.
J.C. Frisvad & A.A. Samson
204
A. fumigatus var. sclerotiorum is also different. It produces several metabolites in common

with A. viridi-nutans IMI 240496, which may be a mixed culture.
Table 2 lists metabolites produced by the isolates examined. Metabolites from clinical, soil
and food isolates of A. fumigatus are very similar, indicating that at least biochemically this
is a homogeneous species.
SI2II2I
4 N.
Tischerl
IBT 3023
4121121
3121121
2121121
1121121
:l
a:
121
-1121121
-2121121
-3121121
-4121121
-SI2II2I
1121
2121
T1 me
2
(m1 n. )
A.
3121
Tumlgatus CBS
113.26
4121
Fig. 1. Comparison of HPLC traces of Neosartorya fischeri var. fischeri and Aspergillus fumigatus. Note
that retention times are about 0.75 min delayed for N. fischeri. The alkylphenone indices were the same.
Metabolite 1 is fumitremorgin C, 2 is verruculogen, 3 is fumitremorgin B, 4 is fumitremorgin A, 5 is
fumigaclavine A, 6 is FUA, 7 is trypacidin, 8 is monomethylsulochrin, 9 is FUB and 10 is fumagillin.
The other species in Aspergillus sect. Fumigati, A. brevipes, A. duricaulis, A. unilateralis

and A. viridinutans produced distinct secondary metabolites, with few in common with A.
fumigatus. Considerable variability was observed. Metabolites from the two isolates
identified as A. duricaulis differed (Table 1). IMI 217288 produced rugulovasine A and a
canescin-like metabolite. Canescin has been reported to be produced by Neosartorya
fischeri var. glabra (Samson et al., 1989). Growth rates and morphology also indicated that
1M! 217288 was correctly classified as an anamorph of N. fischeri var. glabra. Apart from
viriditoxin, as reported by Weisleder and Lillehoj (1971), A. brevipes and A. viridinutans did
not have any secondary metabolites in common.
Possible connections between Aspergillus sect. Fumigati and Neosartorya are also of
interest. Fig. 1 shows a comparison of HPLC traces of N. fischeri IBT 3023 and A. fumigatus.
Most isolates of both species produce fumitremorgins, verruculogen and some
tryptoquivalins, but differ in the capacity to produce several other known and unknown
secondary metabolites. Tryptoquivalins, trypacidin and cyclopaldic acid, metabolites from
three separate biochemical pathways, were found in species both with or without a
teleomorph state (Table 3). Frisvad (1989) considered that species with more than three
Chemotaxonomy and morphology of Aspergillus fumigatus and related taxa
205
Table 3. Production of mycotoxms and antibiotics in Neosartorya and Aspergillus secl
Fumigati.
Species
Mycotoxins and antibiotics
A.breuipes
A. duricaulis
A. fumigatus
Viriditoxin
Cyclopaldic acid, chromanol 1, 2, and 3
Fumagillin, fumitoxins, fumigaclavine A, fumigatin, fumitremorgin A, B,
& C, gliotoxin, monomethylsulochrin, trypacidin, tryptoquivalins,
FUA,FUB, helvolic acid
Mycophenolic acid
Viriditoxin
Helvolic acid
FUA, trypacidin, tryptoquivalins
FUA, trypacidin, viridicatumtoxin
FUB, fumitremorgin A, B, C,verruculogen, tryptoquivalins
FUB
Mevinolins
FUA, cyclopaldic acid
FUA, terrein, tryptoquivalins
Canadensolide
A. unilateralis
A. viridinutans
N.aurata
N.aureola
N. fennelliae
Nfischeri
N. glabra ch. I
N. glabra ch. II
N. quadricincta
N. spinosa
N. stramenia
secondary metabolite families in common are identical, so these results suggest that
Neosartorya and Aspergillus sect. Fumigati are a natural group. These fungi share secondary
metabolites only with species in Aspergillus subgen. Clavati (tryptoquivalins) and subgen.
Versicolores sect. Terrei (terrein). The discovery that Neosartorya fennelliae synthesises the
tetracycline-like mycotoxin viridicatumtoxin was unexpected, as it has only been found to

date in Penicillium aethiopicum and P. brasilianum. However, several other mycotoxins are
produced by species in both these genera (Frisvad, 1986).
Brief descriptions follow of the morphology and secondary metabolites of the species
studied.
Aspergillus fumigatus Fresenius - Beitrage zur Mykologie: 81, 1863
A. fumigatus var. ellipticus Raper et Fennell 1965

? A. anomalus Pidoplichko et Kirilenko 1969
A. fumigatus var. acolumnaris Rai et al., 1971
? A. fumigatus var. a/bus Rai, Tewari et Agarwal 1974
? A. fumigatus var. fulvoruber Rai, Tewari et Agarwal 1974
? A. fumigatus var. griseobrunneus Rai et Singh 1974
A. fumigatus var. sclerotiorum Rai, Agarwal et Tewari 1971
A. phialiseptus Kwon-Chung 1975
See also Raper and Fennell (1965)
A. fumigatus is morphologically somewhat more variable than described by Raper and

Fennell (1965). Conidia may be nearly smooth-walled occasionally, phialides may be
septate (A. phialiseptus) and heads may be somewhat radiate (A. fumigatus var.
acolumnaris). The ex-type isolate of A. fumigatus var. ellipticus seems distinct because of its
smooth walled, ellipsoidal conidia, but chemically it is identical with A. fumigatus var.
fumigatus. Kozakiewicz (1989) raised the var. ellipticus to species level as A. neoellipticus on
the basis of differences of conidial ornamentation. This taxonomy is not followed, because
206
J.e. Frisvad & R.A. Samson
the great variation seen in numerous A. fumigatus isolates. Kozakiewicz (1989) based her
conclusions only on one isolate. All recently described varieties of A. fumigatus produce
similar secondary metabolites to the species and should be synonymised. Clinical isolates
were often more floccose, with less conidia, but could not be separated from the ex type
isolate or other isolates from soil or feedstuffs, by techniques used here.
Aspergillus brevipes G. Smith - Trans. Brit. Mycol. Soc. 35: 241, 1952
Short and heavy walled stipes and finely spinulose conidia make A. brevipes
morphologically distinct. It is also characterized by its vesicles borne at an angle to the
stipe, as in A. viridinutans and A. duricaulis, a reddish brown reverse on CYA, coloured
vesicles and phialides and dark blue green conidia. It produced three families of
secondary metabolites (as determined by HPLC DAD). Weisleder and Lillehoj (1971) and
Cole and Cox (1981) reported that A. brevipes produces viriditoxin. The observations were
based on NRRL 576 (= NRRL 4365) which is A. viridinutans and A. brevipes (S. Peterson,
pers. comm.)
Isolates examined: CBS 118.53 (ex type) from soil, Australia, G. Smith
Aspergillus duricaulis Raper & Fennell- Gen. Aspergillus: 249, 1965
A. duricaulis differs from A. brevipes by conspicously echinulate conidia and weakly
coloured reverse on CYA. No secondary metabolites are synthesised in common with
other species in Aspergillus sect. Fumigati, except cyclopaldic acid, also produced by
Neosartorya quadricincta. Cyclopaldic acid, chromanol I, 2, and 3 and other unknown
secondary metabolites, are produced. This taxon differs in many respects from A. brevipes
and therefore we do not follow Kozakiewicz (1989) who synonymize these species.
Isolates examined: CBS 481.65 (ex type) from soil, Buenos Aires, Argentina, J. Winitzky.
Aspergillus unilateralis Thrower - Aust. J. Bot. 2: 356, 1954
A. unilateralis is distinguished by phialides clustered on one side of the vesicle, echinulate
conidia, a slow growth rate and a dark reverse on CYA. The two strains investigated
produced several unique secondary metabolites. One of them produced small amounts of
mycophenolic acid.
Isolates examined: CBS 126.56 from soil, Australia and CBS 283.66 from a similar source.
Aspergillus viridinutans Ducker & Thrower - Aust. J. Bot. 2: 357, 1954
A. viridinutans also produces vesicles borne at an angle to the stipe. The species is
distinguished by thin-walled, sinuous stipes. Vesicles are uncoloured, conidia are pale
blue green and only finely roughened, and conidium production is quite weak compared
to the other species in the section. This species produces viriditoxin.
Isolates examined: CBS 127.56 from rabbit dung, Frankston, Australia and IMI 280490
from Zambia, J.N. Zulu, perhaps mixed with A. fumigatus.
ACKNOWLEDGEMENTS
The authors thank the NATO Scientific Mfairs Division (Brussel, Belgium) for the research
Chemotaxonomy and morphology of Aspergillus fumigatus and related taxa
207
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209
EXOCELLULAR POLYSACCHARIDES FROM ASPERGILLUS

FUMIGATUS AND RELATED TAXA
J.P. Debeaupuis1, J. Sarfati1, A. Goris2, D. Stynen2, M. Diaquinl
and J.P. Latgel
1Unite de Mycologie
Institut Pasteur
75724 Paris Cedex 15, France
2Eco-Bio/Diagnostics Pasteur
3600 Genk, Belgium
SUMMARY
Exocellular antigenic (EP) material excreted into the culture medium and composed of proteins and
polysaccharides was studied in order to characterize isolates of various species of the Aspergillus
{umigatllS group (Aspergil/llS sect. Fumigati).
The EP produced by forty five strains of the A. {umigatllS group (including teleomorphs of the genus
Neosartorya) were analysed. The polysaccharide moiety of the extracellular fraction varies from 10 to
65% of the total lyophilized product. Galactose, mannose, and glucose are the main constituents of the
EP of all strains studied. Their proportions vary considerably from one strain to another (galactose 4
to 58%, mannose 11 to 61%, glucose 4 to 68%). In spite of these variations, pathogenic and nonpathogenic strains A. {umigatus sensu stricto or teleomorphs and anamorphs cannot be separated on
the basis of monosaccharidic composition. All isolates tested reacted positively with one monoclonal
antibody directed against A. {umigatus galactomannan indicating the presence of galactofuran in all
EP studied. The ELISA results with the rabbit antiserum indicated that A. brevipes, A. duricaulis, A.
unilateralis are different from A. {umigatllS and suggested the presence of specific sugar linkages in the
EP of these species.
INTRODUCTION
Polysaccharides produced by zoopathogenic fungi possess antigenic properties and are

important in the immune response of the host during fungal infection (Huppert, 1983).
The galactomannans represent one of the most widely distributed classes of
polysaccharides among fungi and have been described as integral components of the
mycelial cell wall (Johnston, 1965) as well as exocellular extracts (Gorin and Spencer,
1968). The majority of studies on the structure and antigenic properties of galactomannans
have involved polysaccharides which have been either chemically extracted from cell wall
preparations of mycelium or spores (Barreto-Bergter and Travassos, 1980; Barreto-Bergter
et al., 1981; Webster and McGinley, 1980), or from cytoplasmic material after total
disruption of the mycelium (Bennett et al., 1985). Galactomannans, with molecular weights
greater than 10,000 daltons, can be also released during growth (Preston et al., 1969;
Gander et al., 1974; Notermans et al., 1987).
During growth, several Aspergillus species have been shown to produce exocellular
polysaccharides containing galactomannan (Azuma et al., 1971; Bardalaye and Nordin,
1977; Reiss and Lehmann, 1979; Barreto-Bergter and Travassos 1980; Gorin and Spencer,
1968). Recently, we have isolated an exocellular antigen which is actively released during
210
J.-P. Debeaupuis et al.
vegetative growth. The polysaccharide portion of this antigen is a galactomannan linked

to protein and polyhexosamines (Latge et al., 1988).
Suzuki and Takeda (1975) have demonstrated an immunological cross-reactivity of
galactomannans extracted from the cell wall of Aspergillus fumigatus, A. niger, Trichophyton
rubrum and Hormodendrum pedrosoi against H. pedrosoi serum. It was postulated that
galactosyl groups on these antigens may be immunodominant as removal of these
residues, which are acid-labile, decreased the reactivity with rabbit antiserum. More
recently, Bennett et al. (1985) confirmed that galactofuranosyl groups of a galactomannan
isolated from A. fumigatus mycelium were immunodominant.
In contrast to the results of Suzuki and Takeda (1975), Notermans and Heuvelman
(1985) and Notermans and Soentoro (1986) reported that the exocellular polysaccharides
produced by various mould species may be genus specific. Cross reactivity occurred only
between the closely related genera Penicillium and Aspergillus.
In the following study, we evaluated the variability of the chemical and antigenic
composition of the exocellular material released by different isolates of A. fumigatus and
taxonomically related species with emphasis on the polysaccharide components of the
exoantigen.
MATERIAL and METHODS
Fungal isolates and culture methods.

Forty five isolates of A. fumigatus and related taxa from Aspergillus sect. Fumigati and
Neosartorya were used for this study (Table I). Isolates were maintained on malt extract
agar slants. Conidia were harvested and inoculated into 150 ml flasks containing 50 ml of
sterilized, liquid glucose (2%) peptone (1 %) medium. Cultures were incubated at 25C on
a rotary shaker at 120 rpm. The duration of cultivation, due to different growth rates,
varied among the isolates (Table I).
Purification of the exoantigen.
Culture filtrates (two flasks per isolate) were precipitated with 4 volumes of ethanol
overnight at 2-4e. After centrifugation (10 min at 3000 g at 4C) precipitates were washed
(2x) with ethanol and freeze dried.
Chemical analysis.
Hexoses were analysed by the phenolsulfuric acid method (Dubois et al., 1956) and
proteins by using the Bio-Rad technique based on the method of Bradford (1976).
Hexosamines were evaluated after acid hydrolysis (8 N HCI, 2 hours at 100C), using the
reaction of Elson-Morgan with p-dimethylaminobenzaldehyde (Ashwell, 1966).
Monosaccharide analysis.
Ethanol precipitates (EP) were hydrolysed and methylated in 0.5 M methy1chloride
overnight at 80e. Resulting methylglycoside residues were trifluoroacetylated with a
mixture of trifluoroacetic anhydride and dichloromethane (1:1) twice, for 5 min at 150C in
a sand bath. Chromatographic analysis was performed on a DI200 (Delsi Instruments,
Suresnes, France) gas liquid chromatograph (GLC), at an oven temperature gradient
between llOC and 240C (2C per min), using a column (3 m x 3 mm) of OV 210 on
Chromo sorb WHP (Zanetta et al. 1972). Meso-inositol was used as an internal standard.
Exocellular polysaccharids of Aspergillus fumigatus and related taxa
211
Table 1. Strains of Aspergillus fumigatus and related taxa used in this study.
Nomenclatures
CBS"
Others
118.53
481.65
148.89
153.89
154.89
2458.56
113.26
143.89
152.89
146.89
132.54
192.65
542.75
145.89
147.89
150.89
151.89
149.89
144.89
457.75
487.65
458.75
283.65
126.56
2172.88
B617B
IFAS.19
IFAS.20
1028
152. El
2109
2140
DUV.IP
P8
152.W3
B614A
DAL.lP
2404.90
127.56
466.65
105.55
106.55
598.74
599.74
NHL2951
NHL2952
544.65
404.67
111.55
112.55
483.65
297.67
135.52
941.73
NHL2948
NHL2949
498.65
Species
Duration of
culture (h)
Nx for figures
164
164
114
89
89
120
114
89
89
89
66
89
89
140
89
66
89
96
89
66
164
140
114
114
140
114
94
140
140
94
137
137
137
137
92
92
164
140
164
168
160
164
137
137
236
AB
ADI
AD2
AFI
AF2
AF3
AF4
AF5
AF6
AF1
AF8
AF9
AFI0
AFll
AF12
AF13
AF14
AF15
AF16
AFt 7
AFA
AFE
AFS
AUt
AU2
AVI
AV2
NAU
NAI
NA2
NFEI
NFE2
NFE3
NFE4
NFlI
NFl2
NFGl
NFG2
NFSI
NFS2
NQl
NQ2
NSI
NS2
NST
Aspergillus brevipes
A. duricaulis
A. duricaulis
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
- A. fumigatus
A. fumigatus
- A. fumigatus
A. fumigatus
A.fumigatu
A.fumigatu
- A. fumigatu
- A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
- A. fumigatus
A. fumigatus var. acolumnaris
A. fumigatus var. ellipticus
A. fumigatus var. sclerotiorum
A. unilateralis
A. unilateralis
A. viridi-nutans
A. viridi-nutans
Neosartoria aurata
N. aureola
N. aureola
N. fennelliae
N. fennelliae
N. fennelliae
Nfennelliae
Nfischeri
Nfischeri
N. fischeri var. glabra
N. fischeri var. spinosa
N. quadricincta
N. quadricincta
N. spathulata
N. spathulata
N. slramenia
CBS: Centraalbureau voor schimmeicultures, Baarn, The Netherlands

- Strains isolated from human pathogenic lesions
212
J.-P. Oebeaupuis et al.
Binding of leetins.
EP were diluted (1 ~g ml of total hexose) in 0.05 M carbonate buffer pH 9.6 and coated on
wells of microtitration plates (Greiner, Bischwiller, France). Plates were incubated for 1
hour at 37x then overnight at 4C. Wells were washed 5 times with phosphate buffer
saline containing 0.1 % Tween 20 (pBST). The washed wells coated with exoantigen were
treated with peroxidase labelled lectines, diluted in PBST containing 1% bovine serum
albumin. Concanavaline A (ConA-perox type VlSigma, St Louis, MO, USA) was diluted at
1.10-3 and Wheat Germ Agglutinin (WGA-perox, Sigma) was diluted at 1.10-2. After
incubation 1 hour at 3~C, the plates were washed as before. Peroxidase was revealed by
H2~ and orthophenylene diamine hydrochloride (OPD) in 0.05 M citrate buffer at pH 5.2.
The reaction was stopped by adding 50~ of 3.6 N sulfuric acid.
Monoclonal antibodies.
The monoclonal antibodies were produced according to the procedure described by
Stynen et al. (1989). Basically Loul c rats were immunized with the supernatant of
homogenized A. fumigatus mycelium. Spleen cells of the immunized animal were fused
with IR 983 F rat plasmacytoma cells as described by Bazin (1987). Interesting hybridoma
cultures were cloned three times by limiting dilution. Antibody EB-A2, an IgM, was
selected because of its specificity for galactomannan. It was produced in vivo in AMORAT
rats and purified on an affinity column with an allotype specific murine monoclonal antirat Ig (Bazin, 1987). EB-Y8, a rat monoclonal IgM, specific for lipopolysaccharides of
Yersinia enterocolitica 0:8 (Stynen et al., 1989) was used as a control antibody.
Rabbit antiserum.
Antiserum was raised in New Zealand White rabbits using EP from A. fumigatus isolate
CBS 143.89 injected subcutaneously. In the two first injections the EP was mixed with
Freund's complete adjuvant and following injections with Freund's incomplete adjuvant.
The working dilutions of the antiserum in EUSA were 1.10-3 to 5.10-4.
Enzyme-linked Immunosorbent Assay (ELISA).
Precipitates (EP) were diluted and coated on microtiter plates, incubated and washed as
described previously for lectin binding studies. Samples (sera from immunized or control
rabbits, or rat monoclonal antibodies) diluted in PB5-Tween-BSA were applied to washed
microtiter wells. After incubation 1 hour at 37C and washing 5 times with PBST, the
peroxidase conjuguates (Ab anti IgG Rat-peroxidase, Sigma or anti IgG H+L-peroxidase,
Bio-Sys) were added, followed by incubation and staining by OPD as described above.
Inhibition.
Inhibition studies were carried out using the ELISA method. Inhibitors were pools of EP
from either A. fumigatus isolates or N. /ischeri, N. fischeri var. glabrata or N. fischeri var.
spinosa diluted in PBS (20 ~g/ml). Inhibitors were incubated overnight at 3~C with rabbit
antisera (immunized and control) (1:1) and added to washed EP coated wells prepared as
described previously. After incubation for 1 hour at 37C and washing 5 times with PBST,
the peroxidase conjuguate (Ab anti IgG H+L-peroxidase, Bio-Sys) was added, followed by
incubation and staining by OPD as described above.
213
Table 2. Composition of exoantigen from isolates of Aspergillus fumigatus and related taxa
Nomenclatures Genus and Species

CBS 118.53
CBS 481.65
2172.88
CBS 148.89
CBS 153.89
CBS 154.89
2458.56
CBS 113.26
CBS 143.89
CBS 152.89
CBS 146.89
CBS 132.54
CBS 192.65
CBS 542.75
CBS 145.89
CBS 147.89
CBS 150.89
CBS 151.89
CBS 149.89
CBS 144.89
CBS 457.75
CBS 487.65
CBS 458.75
CBS 283.65
CBS 126.56
2404.90
CBS 127.56
CBS 466.65
CBS 105.55
CBS 106.55
CBS 598.74
CBS 599.74
NHL2951
NHL2952
CBS 544.65
CBS 404.67
CBS 111.55
CBS 112.55
CBS 483.65
CBS 297.67
CBS 135.52
CBS 941.73
NHL2948
NHL2949
CBS 498.65
A. duricaulis
A. duricaulis
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumiga tus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumiga tus
A. unilateralis
A. unilateralis
A. viridi-nutans
A. viridi-nutans
Neosartorya aurata
N. aureola
N. aureola
N. fennelliae
N. fennelliael
N. fennelliael
N. fennelliael
Nfischeri
N.fischeri
N. quadricincta
N. quadricincta
N. spathu/ata
N. spathulata
N. strarnenia
Hexosamines
4
5
6
10
7
20
37
10
10
23
14
36
20
25
35
33
30
31
24
23
6
14
11
9
7
28
4
7
33
40
3
7
5
43
18
9
8
17
29
15
4
25
0
0
4
Percentages of
Proteins
25
16
25
31
30
34
54
61
45
56
51
42
57
40
48
51
521
52
54
51
38
24
47
40
20
57
40
23
43
37
38
31
34
5
30
29
37
33
42
54
25
34
0
0
21
Hexoses
71
79
69
59
63
45
9
29
45
22
36
23
23
35
17
15
8
17
22
25
56
62
42
51
73
15
56
69
24
23
60
52
51
51
52
62
55
50
29
31
72
41
n
n
75
0
I
o
,
0,
,n,
0 0 0
",9,D,
AMMMMMMMMMMMMMMMMMMMMMM~~~~~~~WWWWWWWWWW~~W~~
B 1 2 1 2 3 4 5 6 7 8 81011121314151617 A E 5 1 2 1 2 U 1 2E1 E2ElE411 12G1G25152 1 2 1 2 T
10 , 00
Figure 1. Amino sugars of EP from Aspergillus fumigatus and related taxa. A. Total hexosamines according to the Elson-Morgan reaction (jJ.glmg EP;
B. Relation between hexosamine levels (Ilg) and WGA-peroxidase binding (o.d.) for 1mg EP. Filled box: A. fumigatus isolate
OoS
1.S
Ut
, ,Ill,
AMMMMMMMMMMMMMMMMMMMMMM~~~~~~~WWWWWWWWWW~~~~~
B 1 2 1 2 l 4 :I 6 7 8 81011121314151617 A E S 1 2 1 2 U 1 2E1E2E3E411 12G1C25152 1 2 1 2 T
o ,mJ,llil,I;", liiV;I,M" .I,I..!,/.I,UI,M,H,t;','H,D.I,I..I,H,E.',M,I.',I. I.I. I,t.',M,U!..I,ffil,I.I,M,U,IjiJ,M,I';',U,lUH,J.,!,lj;',M,M,Eij!,LI,
10
15
20
25
3D
40
35
:10
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"Eiii
sa
(!)
c...
.jo.
II
000
I
I
I
I
00
I
10 1
11 0 1
I
10 1
0
I
00 0 00
I
I I
I
I
0
00
00
00
00
91011121:514151617 A E S 12 12 U 1 2E1E2E:5E-41112G1G2S1S2 1 2 12 T
910"121:514151617 A E S 12 12 U 1 2E1E2E:5E-41112G1G2S1S2 1 2 12 T
1 2 1 2 :5 -4 5 67
Figure 2. Relative composition of EP from Aspergillus fumigatus and related taxa. A. Proteinslhexoses ratio; B. Amino sugars/neutral sugars ratio.
Filled box: A. fumigatus isolates.
I ~I
I ~~--I-+---t-+-
A~M~N~N~~~M~~~N~~~N~~~~~~MMAA~AA~~~~~~~~~~~~~~~
1212:5 -4567
::::r
'"o~
I~
Ql
(1)
c.
Ql
a.
CD
er
OJ
~.
'"c:-
<0
~
~
co
l>
5:
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er
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"8
c:
m
x
o
A~M~~~~~~~~~~~~~~~~~~~~~~MM~~AA~~~~~~~~~~~~~~~
II
Ql
'--rJlIOI
ID I 0
-4
,I,
Figure 3. Composition of the polysaccharide moiety of the EP from A. fumigatus and related taxa. Percentages of galactose (A), fucose (A, in ADl),
mannose (B) and glucose (C).
MMMM~~M~~~~M~~M~~~~~~~~~~~~~~~WWWW~~~FWW~~~~~
1 Z 1 Z ~ 4 5 6 7 8 8 1011 12 1l 14 1~ 16 17 A E S I 2 1 2 U 1 2 El E2 E3 E4 1 Z 1:1 g 51 52 1 2 1 2 T
il.,I,.,I,.,.,.,I,.,I,.,.,IJI,I,I,.,.,IJI,.,I,III,I,.JI,IJlIJI,.,.,.,I,IJI,I,I,IJI,1l
MMM~~~~~~~MM~~MM~~~M~~~~~~~~~~WWWW~~~~WW~~~~~
I 2 I 2 ~ 4 5 6 7 8 8 lU 11 12 1l 14 1~ 11 17 A E 5 1 2 I 2 U 1 2 EI E2 E3 E4 1 21:1 g 51 52 1 2 1 2 T
A,
!U'i.Dn~,~,~nnn~,~~,n.~m~,~~n,~,~Dm~,~~,~,~m~nn~,~~nn n
C.)
,...
iii"
-g
eo
ceo
III
c:
:b
!'-
OJ
.' . '
o
a a
Figure 4. Relation between ConA-peroxidase affinity to EP and mannose levels of A. fumigatus and related taxa. Filled box: A. fumigatus isolates.
1 12 3 5 678 91011121314151617 II E S 1 2 U 1 2E1E21112C1C2S1S2 1 2 1 2 T
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
O+-~~-+~~~+-~~-+~~~+-~~-+~~~+-~~-+-r~~+-~~-+-r~
0.5
1.5
2.5
;'.5
;,
m
)(
-oJ
D>
Cl.
(I)
1(iI
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g.
:::>.
:::T
D>
"8
er
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2"
1m
I
1II1II.
II
II1II
II
I.....
Figure 5. Binding of monoclonal Rat antibody specific of galactofuran (A) and polyclonal Rabbit antiserum specific of A. fumigatus (B) to EP from
different isolates of A. fumigatus and related taxa. A. Monoclonal Rat antibody EB-A2; B. Polyclonal Rabbit anti EP (CBS 143.89) antiserum. Results
are expressed as the difference between optical densities obtained with the positive and the control antisera.
1 2 1 2 -' ... 5 6 711 &1011121;'1 ... 151617 A E 5 1 2 1 2 U 1 2E1E2E;'UI112C1G25152 1 2 1 2 T
BMMAFI>FAFI>FI>FAFI>FAFI>FI>FAFI>FAFI>FAFAFAFI>FAFAF~~~~NANANAIf"If"If"If"If"II'If"If"If"II'NQNQNSNSNS
B ADADAF /IF AF AF AF AF AF AF I>FAF I>F AFAF AF AF AF I>F I>F AF AFAUAUAVAVNANANMF If"If" II' 11'11' 11'11' If"II'NQNQN5NSN5
1 2 1 2 -' ... 56711 &101112131 ... 151617 A E 5 1 2 1 2 U 1 2E1E2E-'UI112C1G25152 1 2 1 2 T
o III.
m.z
UL+
116
IL8
1.2
1.4.
1.6
C.2
D....
D.6
D.B
1.2
1....
1.6
f\)
'",...
9a.
(ii'
-g'"
0-
tt>
tt>
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(Xl
219
RESULTS
Production of exoantigens.
All the isolates studied produced exoantigens when grown in the peptone glucose media,
except Neosartorya spathulata (2 isolates tested). Relative amounts of material released
during growth varied among isolates. Most of the A. fumigatus isolates produced the
highest levels of exocellular material after 2 to 3 days growth (Table 1). The majority of
species had to be grown for 5 to 7 days to obtain a precipitate, however, the growth of N.
stramenia was very slow and ten days growth was required to produce a precipitate
sufficient for chemical analysis.
General composition of culture filtrates.
The main constituents of all EP from broth filtrates were amino acids, hexosamines and
neutral sugars (Table 2). A. fumigatus isolates produced significatively higher amounts of
proteins and hexosamines than most other species (Table 2). The exception was N. aureola,
which produced exocellular material containing 30-40% hexosamines (Fig. lA).
Hexosamine production was more irregular in A. fumigatus than protein production. In
Fig. 1B the comparison between total hexosamine produced by the various moulds
according to both the Elson-Morgan reaction and the binding of WGA, specific to 2acetamide, 2-deoxy oligosaccharides to exocellular filtrates are summarised. Results
suggest that the hexosamine moiety in the filtrates is a heteropolymer of hexosamines: Nacetyl-hexosamines. The level of WGA binding compunds are lower in most A.fumigatus
than in the other taxa. Additionally, A. fumigatus isolates produced the largest quantities
of hexosamines, but levels varied from 5 and 50% of the total dry weight of the precipitate.
A. fumigatus produced higher levels of proteins but a lower percentage of neutral sugars
(15 to 25% of the total precipitate) than other fungal species which produced EP
containing 35 to 75% hexoses (Fig. 2).
Sugar composition of the EP.
Figure 3 shows the results of the GLC analysis of monosaccharide composition of the
exopolysaccharide moiety in the EP. All isolates studied produced galactose, mannose and
glucose, and one isolate, A. duricaulis (CBS 481.65), produced fucose. Galactomannan was
the major component of the exopolysaccharide, especially for A. fumigatus. Levels of
galactose were significant in all species studied, with the exception of A. duricaulis, one
isolate of which produced 5% fucose while producing only 4% galactose. Fucose was not
found in other species tested isolates. Hydrolysis of exopolysaccharides with O.OIN HCl
caused a significant reduction (76 to 97%) in the galactose content of the EP suggesting
that galactose moiety was mainly composed of galactofuranosyl units. Glucose present in
the EP could result also from nonspecific hydrogen binding of the monosaccharides to the
EP. Such an hypothesis is strenghtened by the significant decrease of the glucose content
observed in EP from A. fumigatus (isolates CBS 148.89, CBS 113.26, 152.89) after 0.01 N Hel
hydrolysis. In these isolates the concentration of glucose, accounting for up to 30% of the
polysaccharidic moiety of the EP became to 10% after Hel 0.01 N hydrolysis. A similar
decrease in the glucose content was observed after 0.01 Hel hydrolYSiS of EP from
Neosartorya aurata,N. aureola, N. quadricincta which were characterized by a high glucose
concentration (Fig. 3).
With the A. fumigatus isolates, binding of exocellular precipitates with eonA were
relatively low but similar for all the isolates of this species (figure 4). ConA binding was 2
to 3 time more important in some of the other taxa.
J.-P. Debeaupuis et a/.
220
Table 3. Binding of the different EP to 4 different rabbit antisera (expressed as optical

density obtained by EUSA)
Optical Density
Nomenclature
Species
(CBS)
118.53
481.65
148.89 153.89
154.89
113.26143.89 152.89
146.89
132.54 192.65 542.75145.89 147.89
150.89
151.89
149.89 144.89
457.75
487.65
458.75
283.65
127.56
466.65
105.55
106.55
598.74
599.74
544.65-404.67-111.55 -112.55 -483.65-297.67-135.52
941.73
498.65
duricaulis
fumigatus
fumigatus
fumigatus
fumigatus
fumigatus
fumigatus
fumigatus
Afumigatus
A fumigatus
A fumigatus
A fumigatus
A fumigatus
Afumigatus
Afumigatus
Afumigatus
Afumigatus
A fumigatus var. acolumnaris
A fumigatus var. ellipticus
A fumigatus var. sclerotiorum
A unilateralis
A viridi-nutans
Neosartorya aurata
N. aureola
N. aureola
N. fennelliae
N. fennelliae
Nfischeri
Nfischeri
N. quadricincta
N. quadricincta
N. stramenia
A
A
A
A
A
A
A
A
II
III
0.372
0.260
0.612
0.534
0.626
0.932
1.470
1.044
1.303
1.390
1.426
0.843
1.403
1.611
1.540
1.598
1.548
1.451
0.817
0.939
0.672
0.351
0.814
0.334
0.985
0.743
0.844
0.628
1.119
1.100
0.605
0.810
1.168
1.230
0.425
0.%2
0.394
0.242
0.261
0.149
0.127
0.084
0.152
0.093
0.129
0.099
0240
0.113
0.113
0.125
0.167
0.114
0.112
0.120
0.147
0.216
0.086
0.207
0.256
0.318
0.192
0.339
0.382
0.203
0.216
0.242
0.152
0.248
0.195
0.222
0.173
0.312
0.232
0.262
0.255
0.259
0.133
0.890
0.107
0.359
0.463
0.235
0.318
0.309
0.569
0.133
0.406
0.626
0.322
0.441
0.280
0.461
0.398
0.212
0.290
0.249
0.411
0.203
0.380
0.386
0.258
0.415
0.321
0.509
0.306
0.211
0.298
0.291
0.264
0.256
0.255
0224
0244
0.138
0.176
0.152
0.252
0.387
0272
0.416
0.347
0.667
0219
0.406
0.406
0.505
0.609
0.474
0.589
0.573
0.267
0.366
0.224
0.580
0.216
0.531
0.524
0.287
0.467
0.448
0.412
0.351
0.248
0.527
0.315
0276
0.346
0.250
: Anti 1028 (CBS 143. 89) EP antiserum

II : Negative antiserum (control)
III : Anti 1028 EP antiserum adsorbed with EP from isolates of Afumigatus (-)
IV : Anti 1028 EP antiserum adsorbed with EP from isolates of N fischeri and related varieties (--).
Antigenic activities of EP.

As shown in Fig. SA, monoclonal antibody EB-A2, which is specific of galactofuran,
reacted with EP from all species studied except A. unilateralis CBS 481.65, which produced
only 4% galactose.
221
The polyclonal antiserum from rabbit immunized with A. fumigatus EP (CBS 143.89) was
more discriminatory than the monoclonal antibody, reacting strongly with all A. fumigatus
isolates. Variable responses against other species were obtained. A. brevipes, A. unilateralis,
A. duricaulis, N. aureola, and N. stremenia gave a lower reaction. N. fischeri and A.
viridinutans gave a potent response, similar to A. fumigatus (Fig. 5B).
Inhibition. The results of the ELISA inhibition are summarized in the Table 3.
Polyclonal rabbit serum was inhibited by pools of EP from isolates of A. fumigatus, N.
fischeri and related varieties. Antigenic community between these two species was
confirmed by the high inhibition (approximately 100%) obtained with the two types of
adsorbed antisera.
DISCUSSION
Identification of A. fumigatus is important because it is one of the most important
mycopathogen associated with immunocompromised patients. This comparative study
aimied to determine if chemical and/or antigenic properties of the exocellular material
produced by A. fumigatus could be used to delineate this species from non-pathogenic
Aspergillus and other related taxa. The results indicate extensive homology among the
species examined. Galactofuranosyl groups were detected in all EP examined and cannot
be considered to be species specific. In agreement with Notermans et al. (1987, 1988) who
reported antigens common to various Aspergillus and Penicillium species, we have found
here that the immunodominant exopolysaccharide present in A. fumigatus is also present
in related species as well as in A. flavus, A. nidulans and A. niger (unpubl. data).
Study of the types of linkages in the polysaccharide fractions is now desirable, especially
for several isolates of A. fumigatus which showed unusual features (isolates CBS 148.89,
CBS 153.89, CBS 154.89,2458.56) and for other taxa which are most different (A. duricaulis,
A. unilateralis, N. au rata, N. stramenia).
None of the chemical or antigenic criteria investigated in this study are species
specific. However, the conjunction of several parameters can be helpful in defining species
in the A. fumigatus group. As we have seen A. fumigatus is characterized by 1) an abundant
EP with high proportions of proteins, 2) a heteropolymer of amino sugars, mostly
hexosarnines, 3) a moderate reactivity to ConA, and 4) a strong antigenic response to a
polyclonal antiserum. These conclusions are based on a study of sixteen A. fumigatus
isolates but only one or two from other taxa. More are needed.
From our results, sugars in the EP provide less effective separation than the protein
moiety (Hearn et al., 1989). However, the identification of specific sugar linkages of the
exocellular polysaccharides may lead to the selection of monoclonal antibodies more
specific that the one we used, which was produced from a very crude antigen.
Intracytoplasmic, carbohydrate containing molecules, should be also studied and
compared to exocellular polysaccharides, as common antigenic reactions have been
demonstrated between these two antigenic sources (Hearn, 1988).
ACKNOWLEDGEMENTS
The project at Eco-Bio was partially funded by I.W.O.N.L.
222
J.-P. Debeaupuis et al.
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225
BIOTYPING OF ASPERGILLUS FUMIGATUS AND RELATED TAXA BY

THE YEAST KILLER SYSTEM
L. Polonelli, S. Conti, L. Campani and F. Fanti
istituto di Microbiologia
Universita deg/i Studi di Parma
Parma, Italy
SUMMARY
The yeast killer system has proven to be an effective procedure for intraspecific differentiation of
filamentous fungi. This biotyping method may have an useful application as an epidemiological
marker in outbreaks of mycotic infections, especially when other approaches are not readily available.
The opportunistic pathogen Aspergillus fumigatus Fresenius is the most common etiologic agent of the
different clinical types of human aspergillosis. Strain differentiation of either morphologically
identified A. fumigatus or related species as well as related teleomorphs by the yeast killer system is
described.
INTRODUCTION
Aspergillus fumigatus is the most common etiologic agent of human aspergillosis. The
species is an ubiquitous saprophyte in nature. In man, the respiratory tract is the normal
portal of entry as the small diameter of the conidia allows them to reach and germinate in
the pulmonary alveolar spaces. Complications may occur in immunocompromised hosts,
ranging from allergic bronchopulmonary aspergillosis to aspergilloma and, by
hematogenous spreading, invasive aspergillosis.
Hospitals provide a striking concentration of patients receiving immunosuppressive
therapy (Aisner et ai., 1976; Allo et ai., 1987; Arnow et aI., 1978; Attah and Cerruti 1979;
Brandt et aI., 1985; Fanti et aI., 1989; Glotzbach, 1982; Kyriakides et al., 1976; Mawk et aI.,
1983; Meyer et ai., 1973; Opal et al., 1986; Petheram and Seal 1976; Ross et al., 1968; Simpson
et al., 1977; Tack et al., 1982; Viollier et aI., 1986; Weems et al., 1987; Wellens et aI., 1982).
Aspergillus nosocomial infections have emerged as a major cause of morbidity and
mortality in hospitalized patients during the last two decades. A six year report (19701975) from the Centers for Disease Control (CDC) revealed an increase of 158% in the
incidence of nosocomial aspergillosis (Fraser et aI., 1979). The estimate should be
considered quite conservative since invasive aspergillosis often is diagnosed or
misdiagnosed only at postmortem examinations.
Nosocomial mycoses provide an intolerable discrepancy between the extraordinary
progress in the management of seriously ill patients and the very slow advancements
made in elucidating the pathogenesis and dynamics of fungal infections. More efforts
need to be directed toward clinical research in medical mycology for diagnosis,
prevention and infection control. Differentiation of biotypes of fungi responsible for
human mycoses may represent a powerful tool in the investigation on the epidemiology
of fungal outbreaks as well as in correlating associations among different pathology
characters such as virulence, morphology and serology.
226
L. Polonelli et al.
Table 1. Aspergillus sect. Fumigati isolates tested for sensitivity to killer

yeasts.
Study
number
2
3
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Species
Aspergillus fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A.fumigatus
A. brevipes
A. fumigatus var. acolumnllris
A. fumigatus var. e/lipticus
A. fumigatus
A. unilateraiis
A. viridinutRns
Neosartorya aurata
N. aureola
N. aureola
N. fenne/liRe
N. fenne/liae
N. fenne/liae
N. fennel/iRe
N. fischeri var. fischeri
N. quadricincta
N. spathulatR
N. spathulatR
Collection
number
CBS 113.26 [1]

CBS 132.54
CBS 143.89
CBS 144.89
CBS 145.89
CBS 146.89
CBS 147.89
CBS 148.89
CBS 149.89
CBS 150.89
CBS 151.89
CBS 152.89
CBS 153.89
CBS 154.89
CBS 192.65
CBS 118.53
CBS 457.75
CBS 487.65
CBS 542.75
CBS 458.75
CBS 283.66
CBS 127.56
CBS 466.65
CBS 105.55
CBS 106.55
CBS 598.74
CBS 599.74
CBS 410.89
CBS 411.89
CBS 544.65
CBS 404.67
CBS 111.55
CBS 112.55
CBS 483.65
CBS 297.67
CBS 135.52
NHL2948
NHL2949
[1] CentraaIbureau vaar Schimmelcultures, Baarn, The Netherlands
In this study we report the application of a biotyping system to A. fumigatus isolates based
on their differential sensitivity to the action of selected killer yeasts. The system, which
proved to be very effective in the differentiation of cultures of filamentous fungi (Polonelli
et al., 1987) has been extended to comparisons with other species of Aspergillus sect.
Fumigati as well as related Neosartorya species.
Biotyping of Aspergillus fumigatus and related taxa by the yeast killer system
Table 2. Yeast killer strains used for studying the sensitivity of

Aspergillus sect. Fumigati isolates to killer toxins.
Colk
Species
Collection
Number
K 1
K 2
K 3
K 4
K 5
K 6
K 7
K 8
K 9
K 10
K 12
K 17
K 18
K 19
K 20
K 22
K 23
K 24
K 25
K 26
K 27
K 28
K 29
K 30
K 31
K 32
K 33
K34
K 35
K36
K 37
K 44
K 48
K 49
KSO
K 51
Pichia sp.
Pichiasp.
P.anomaia
P.anomaia
P.anomaia
P. californica
P. canadensis
P. dimennae
P. mrakii
P. kIuyveri
P. lrimundalis
P. lrimundalis
Pfabianii
P. holstii
P. subpelliculosa
Candida guilliermondii
C. rnaltosa
P. spartiniae
P. nonfermentans
P. carsonii
P. farinosa
P. guilliermondii
C. pseudotropicalis
C. pseudotropicalis
C. pseudotropicalis
P. kIuyveri
P. kluyveri
P. memlJranaefadens
P. kIuyveri
P.anomaia
Sturnm(1)
Stumm
UM(2)
CBS (3)
Ahearn (4)
Ahearn
Ahearn
Ahearn
Ahearn
Stumm
Ahearn
CBS
Ahearn
CBS
CBS
UCSC(5)
UCSC
UCSC
UM
CBS
1034
1035
3
5739
Um866
WC40
WC41
WC44
WC51
1002
WC38
5642
WC45
4140
5767
0
0
0
200
810
185
2031
241
254
330
5F
6F
10F
llF
25F
255
IFOI267
2359
2360
0
1
P. mrakii
Kluyveromyces lactis
K. /actis
K. /actis
Kfragilis
Kfragilis
CBS
CBS
UP (6)
UP
UP
UP
UP
UP
UP
UP
UCSC
Gunge(7)
CBS
CBS
UP
UP
(1) C. Stumm, University of Njimegen, Njimegen, The Netherlands, (2)

Istituto di Igiene, Universita' di Milano, Milan, Italy, (3) Centraalbureau voor
Schimmelcultures, Baarn, The Netherlands, (4) D.G. Ahearn, Georgia State
University, Atlanta, Georgia, USA, (5) Istituto di Microbiologia, Universita'
Cattolica del Sacro, Cuore, Rome, Italy, (6) Istituto di Microbiologia,
Universita' degli Studi di Parma, Parma, Italy, (7) N. Gunge, Kumamoto
Institute of Technology, Kumamoto, Japan
227
L. Polonelli et al.
228
Isolates examined. The 38 isolates of the Aspergillus sect. Fumigati investigated in this study
were provided by the Centraalbureau voor Schimme1cultures and are listed in Table 1. All
of them were selected for comparative evaluation by other investigative procedures, being
reported elsewhere in these proceedings (Samson and Pitt, 1990).
The yeast isolates used in this study have previously been shown to have killer
activity towards a number of potentially sensitive isolates (Polonelli and Morace, 1986).
The 36 recognized killer yeasts were grouped into triplets to make the recording of their
activity easier by using a conventional code. All of them are maintained in our culture
collection. Many were obtained from different international sources (Table 2).
Media.
Yeast Extract Peptone Dextrose Agar (yeast extract 1%, peptone 2%, glucose 2%, agar 2%)
(Difco Laboratories, Detroit, Michigan, U.S.A.) buffered at pH 4.5 and added with
methylene blue (0.003%) was used in the studies on the killer system. Sabouraud Dextrose
Agar (Difco) was used as the growth and maintenance medium for the the killer yeast and
sensitive fungal isolates.
Figure 1 Differential activity of the yeast isolates Pichia anomala UM 3 (K3), Pichia anomala
CBS 5739 (K4) (killer activity), Pichia sp. Stumm 1034 (Kl) and Pichia sp. Stumm 1035 (K2)
(no killer activity) on Aspergillus fumigatus isolate CBS 146.89. Left: view of the front plate.
Right: view of the bottom plate.
Biotyping by the yeast killer system.

The differentiation of the Aspergillus sect. Fumigati isolates into biotypes by the yeast killer
system was carried out according to the procedure of Polonelli et al. (1987). Briefly, seven
day old mycelial cultures grown on Sabouraud Dextrose Agar slants, were scraped to
obtain a suspension of approximately 20,000 conidia/ml in distilled sterile water. One ml
of the suspension was mixed with 20 ml of the buffered medium maintained at 450C and
poured into a Petri dish. After cooling a heavy loopfool of each killer yeast grown on
229
Sabouraud Dextrose Agar for 48 hours at 250C was streaked on the surface of the agar.
The plates were then incubated at 25oC.
Reading and interpretation of the results.
The culture plates were read after two weeks. The killer effect was considered positive
when a clear zone of inhibition surrounded the streak of killer yeast (Figure 1). A code
was used to record the combined effect of the killer yeasts grouped into triplets for
distinguishing purposes. The triplets were then further coded, for ease of use, by numbers
(Table 3).
Table 3. Coding modalities of the activity of each

triplet of selected killer yeasts used for the
differentiation of Aspergillus sect. Fumigati
isolates.
1st
killer
2nd
killer
3rd
killer
Code
+
+
+
+
+
+
7
6
5
4
3
+
+
+
+
+
2
1
RESULTS
Most of the Aspergillus sect. Fumigati isolates tested proved to be sensitive to the action of
at least one killer yeast (Table 4). Only, four of them (A. fumigatus var. ellipticus CBS 487.65,
Neosartorya fischeri var. fischeri CBS 404.67, N. fischeri var. glabra CBS 112.55, N. fischeri var.
spinosa CBS 483.65) appeared to be resistant to all of the killer yeasts used in this study.
The pattern of sensitivity was recorded according to the adopted conventional code
(Table 5). Among the 15 species and varieties investigated the yeast killer system was able
to differentiate 15 different biotypes (Table 6). As expected, the largest number of different
biotypes (seven) was detected among the A. fumigatus isolates which were the most
numerous of these studied.
The different biotypes varied significantly in killer yeast sensitivity. Some biotypes
proved to be sensitive to many killer yeasts (Le. biotypes G, J, K, L, 0,) while another
(biotype E) was sensitive only to Pichia guilliermondii CBS 2031 (killer yeast 28). Different
species and varieties grouped into the same killer biotype with no apparent relatedness to
conventional identification. Also different biotypes occurred within the same species or
variety.
A broad spectrum of killer biotypes was also detected among the Neosartorya isolates
tested. With the possible exception of N. spathulata, all the species with more than one
tested isolate, were differentiated into biotypes.
K 98
K 10
K 12
i K 17
,~ K 18
K 19
, K 20
I K 22
I K 23
" K 24
K 25
K 26
K 27
K 28
K 29
K 30
K 31
K32
K 3J
K 34
K J5
K 36
K 37
K 44
K 48
K 49
K 50
K 51
, K
K
I K
i K
"
!~
~ Code
II Killer
I 2I 3I I I 5I 6I 7I 8I
study nullber (see table I)
ft.spergillus isolates
9 110
Ill! 12 III ! 11 115 ! 16 ! 17 118 119 I 20
Aspergillus isolates
nlnlnlHI~IHlnlulBlm
Aspergillus Isolates
Table 4. Sensitivity of Aspergillus sect. Fumigati isolates to killer yeasts.
3tJ!2
I 33 I 34 I 35 I 36 137 138
Aspergillus isolates
study "mber (see table I)
'"o
:-
Q)
::l
(5
231
Table 5. Biotyping of Aspergillus sect. Fumigati isolates according to their

sensitivity to yeast killer toxins
Numerical killer system biotype
Study
code
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
473
473
473
473
473
473
473
473
473
473
473
473
000
473
473
773
473
000
472
000
473
000
473
473
773
473
473
473
473
773
000
000
000
000
000
001
473
473
300
300
300
300
300
300
300
300
300
300
300
300
000
300
300
300
300
000
300
000
300
000
320
320
320
300
300
300
300
320
000
000
000
000
000
000
300
300
410
010
410
010
010
010
010
010
010
010
030
010
010
010
010
030
030
000
474
010
010
010
630
630
630
030
030
030
030
630
000
010
000
000
010
010
430
430
Alphabetical killer
system biotype
000
006
000
000
000
006
000
000
000
006
067
006
000
067
000
026
000
000
026
000
000
000
000
000
000
000
000
006
006
000
000
000
000
000
000
000
006
006
B
A
C
C
B
C
C
C
B
D
B
E
F
C
G
H
I
E
C
E
K
K
L
H
H
M
M
L
I
E
I
I
E
N
0
0
DISCUSSION
The definition and terminology of intraspecific variation in fungi is poor and often
confusing. As Odds (1985) pointed out, the categories of subspecies, variety, subvariety,
form, and biotype are inextricably bound to concepts of taxonomy. However, his
difinitions do not exactly coincide with those of epidemiology. "Strain" is the most
common term used by medical mycologists for intraspecific variation. "Biotype" is
primarily used by epidemiologists interested in the correlation between individual strains,
their habitats and mode of transmission.
232
L. Polonelli et al.
A condition for an effective biotyping system is that it is independent from other

unrelated identification tests so that a cumulative information may be acquired by
evaluating all of the different characters. Moreover, a biotyping system should be a
reasonable compromise between test simplicity and effectiveness in discriminating among
the largest possible number of isolates to be processed.
The yeast killer system has proved able to match these expectations, by differentiating
the numerous isolates of sect. Fumigati studied into biotypes regardless of morphological
characteristics and, therefore, of the species. Biotypes were randomly distributed among
the different isolates: that the same biotype may refer to different species, and as well
different biotypes may occur within the same species. There was no obvious correlation
with morphological criteria of identification.
In our hands, the yeast killer system had already proven to be a flexible and reliable
method for investigating cases of presumptive nosocomial infections caused by A.
fumigatus isolates (Fanti et al., 1989). This study confirms that the system can be easily
applied to related species and varieties of their anamorphic or telemomorphic characters.
The multidisciplinary approach to the characterization of the same isolates of sect.
Fumigati used in this study (i.e. study of secondary metabolites including mycotoxins,
extracellular polysaccharides, water soluble components by SDS PAGE and Western
Blotting other than morphological studies) being reported elsewhere in these proceedings
(Samson and Pitt, 1989) could throw more light upon the intra and inter specific linking of
Aspergillus sect. Fumigati.
Table 6. Grouping of killer biotypes among teleomorph and anamorph of Aspergillus sect.
Fumigati isolates.
Alphabetical killer code

A B C D E F G H I J K L MN 0
Number of tested isolates

species and variety
A. brevipes
A. fumigatus var. acolum7lllris
A. unilateralis
A. viridinutans
Neosartorya aurata
N.aureola
N. fennel/iae
N. fischeri var. glahra
N. quadricincta
N. spathula/a
16
1
1
1
1
1
1
1
2
4
2
2
2
1
2
Total tested isolates
38
1
1
2
1
1
1
1
1
2
4
233
REFERENCES
AISNER, J., SCHIMPFF, S.C, BENNETI, J.E., YOUNG, V.M and WIERNIK,P.H. 1976. Aspergillus infections
in cancer patients: association with fireproofing materials in a new hospital. Journal of American Medical
Association 235: 411-412.
ALLO, M.D., MILLER, J., TOWSEND, T. and TAN, C. 1987. Primary cutaneous aspergillosis associated with
Hickman intravenous catheters. New England Journal of Medicine 317: 1105-1108.
ARNOW, P.M., ANDERSON, R.L. MAINOUS, P.D. and SMITH, E.J. 1987. Pulmonary aspergillosis during
hospital renovation. American Review of Respiratory Diseases 118: 49-53.
ATIAH, C.A. and CERRUTI, M.M. 1979. Aspergillus osteomyelitis of sternum after cardiac surgery. New
York State Journal of Medicine 79: 1420-1421.
BRANDT, S.J., THOMPSON, R.L. and WENZEL, R.P. 1985. Mycotic pseudoaneurysm of an aortic bypass
graft and contiguous vertebral osteomyelitis due to Aspergillus. American Journal of Medicine 79: 259-262.
FANTI F., CONTI, S., CAMPANI, L., MORACE, G., DETIORl, G. and POLONELU, L. 1989. Studies on the
epidemiology of Aspergillus fumigatus infections in a university hospital. European Journal of Epidemiology
5: 8-14. FRASER, D.W., WARD, J.I., AJELLO, L, and PLIKATYS, B.D. 1979. Aspergillosis and other
systemic mycoses: the growing problem. Journal of the American Medical Association 242: 1631-1635.
GLOTZBACH R.E. 1982. Aspergillus terreus infection of pseudoaneurysm of aortofemoral vascular graft with
contiguous vertebral osteomyelitis. American Journal of Clinical Pathology 77: 224-227.
KYRIAKIDES, G.K., ZINNEMAN, H.H., HALL, W.H., ARORA, V.K., UFTON J.. DE WOLF, W.E. and
MILLER, J. 1976. Immunologic monitoring and aspergillosis in renal transplant patients. American Journal
of Surgery 131: 246-252.
MAWK, J.R., ERICKSON, D.L., CHOU, S.N. and SELJESKOG E.L. 1983. Aspergillus infections of the lumbar
disc spaces. Journal of Neurosurgery 58: 270-274.
MEYER, R.D .., YOUNG, L.S. and ARMSTRONG, D. 1973. Aspergillosis complicating neoplastic disease.
American Journal of Medicine 54: 6-15.
ODDS, F.C. 1985. Biotyping of medically important fungi. In Current Topics in Medical Mycology, eds. M.R.
McGinnes, p. 155-171. New York: Springer Verlag.
OPAL, S.M., ASP, A.A., CANNADY, P.B., MORSE, P.L., BURTON, L.J. and HAMMER, D.A. 1986. Efficacy
of infection wntrol measures during a nosocomial outbreak of disseminated aspergillosis associated with
hospital construction. Journal of Infectious Diseases 153: 634-637.
PETHERAM, I.S. and SEAL, R.M.E. 1976. Aspergillus prosthetic valve endocarditis. Thorax 31: 380-390.
POLONELU, L and MORACE, G. 1986. Reevaluation of the yeast killer phenomenon. Journal of Clinical
POLONELU, L., DETIORl, G., CATTEL, C. and MORACE, G. 1987. Biotyping of mycelial fungus cultures
by the killer system. European Journal of Epidemiology 3: 237-242.
ROSS, D.A., ANDERSON, D.C. MACNAUGHTON, M.C. and STEWART, W.K. 1968. Fulminating
disseminated aspergillosis complicating peritoneal dialysis in eclampsia. Archives of Internal Medicine 121:
183-188.
SAMSON, R.A. and PITI, J1. eds. 1990. Modem Concepts in Penicillium and Aspergillus Classification. New
SIMPSON, M.B., MERZ, W.G., KURLINSKI, S.P. and SOLOMON, M.H. 1977. Opportunistic mycotic
osteomyelitis. Bone infections due to Aspergillus and Candida species. Medicine Baltimore 56: 475-481.
TACK, K.L., RHANE, F.S., BROWN, B. and THOMPSON, R.C. 1982. Aspergillus osteomyelitis: report of four
cases and review of the literature. American Journal of Medicine 73: 295-300.
VIOLLlER, A.F., PETERSON, D.E. DE JONGH, C.A., NEWMAN, K.A., GRAY, W.C., SUTHERLAND, J.C.,
MOODY, H.A. and SCHIMPFF, S.c. 1986. Aspergillus sinusitis in cancer patients. Cancer Philadelphia 58:
366-371.
WEEMS, J.J. jr. ANDERMONT, A., DAVIS, B.J. TANCREDE, C.H., GUIGET, M., PADHYE, A.A.,
SQUINAZI, F. and HARTONE, W.J. 1987. Pseudoepidemic of aspergillosis after development of
pulmonary infiltrates in a group of bone marrow transplant patients. Journal of Clinical Microbiology 25:
1459-1462.
WELLENS, F., POTULIEGE, c., DEUVART, F.E. and PRIMO, G. 1982. Aspergillus osteochondritis after
median sternotomy. Combined operative treatment and drug therapy with amphotericin B. Thoracic and
Cardiuoascolar Surgery 30: 322-324.
235
ANALYSIS OF COMPONENTS OF ASPERGILLUS AND NEOSARTORYA

MYCELIAL PREPARATIONS BY GEL ELECTROPHORESIS AND
WESTERN BLOTTING PROCEDURES
V.M. Hearn1, M. Moutaouaki12 and J.-P. Latge2
1Mycological Reference lJlboratory
Central Public Health lJlboratory
London NW9 5HT, UK
2Unite de Mycologie
Institut Pasteur
75724 PARIS, Cedex 15, France
SUMMARY
Twenty three isolates of Aspergillus spp. including 16 of A.fumigatus and 14 of Neosartorya spp. were
analysed by 5D5-polyacrylamide gel electrophoresis and immunoblotting. Qualitative and
quantitative differences were observed among species and individual isolates especially in their
reactivity towards human IgG. Grouping of many A. fumigatus isolates and their relation to
Neosartorya isolates is indicated by the protein profiles seen with mycelial and culture filtrates on 50SPAGE and on denaturing polyacrylamide gels.
INTRODUCTION
The taxonomic usefulness of protein profiles obtained by electrophoretic separations has

often been demonstrated for fungal genera, including Aspergillus (Sorenson et al., 1971).
More recently, the frequency of occurrence of two cell sap components in a limited
number of Aspergillus species, including A. fumigatus, A. flavus, A. fischeri and A. fennelliae
has been investigated by two-dimensional electrophoresis (Piechura et al., 1985). In the
work reported here, we have compared molecular composition and associated
immunogenicity of several Aspergillus and Neosartorya species in an attempt to further
characterize these closely related fungi.
Proteins and glycoproteins of water soluble fractions of mycelial preparations,
together with ethanol precipitates of culture filtrates, were separated by electrophoresis in
polyacrylamide gels. The Coomassie Blue staining patterns of both intact molecules and
molecular subunits were compared in nondenaturing gels and sodium dodecylsulphate
containing gels, respectively. Possible variation among the different species in molecules
possessing catalase activity was also examined. Finally, the antigenic reactivity of both
Aspergillus and Neosartorya species was determined by combined SDS-PAGE and Western
blotting procedures. Blots were probed with serum from patients with antibodies to A.
fumigatus antigens, as measured by ELISA.
Species examined.
The origin of the Aspergillus and Neosartorya species studied are shown in Table 1.
V.M. Hearn
236
et at.
Table 1. Isolates of Aspergillus and Neosartorya species
Reference
Collection
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
N. fischeri var.glabra
N. fennel/iae
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
CBS 111.55
CBS 599.74
CBS 127.56
CBS 404.67
CBS 487.65
CBS 544.65
CBS 598.74
CBS 118.53
CBS 941.73
CBS 457.75
CBS 483.65
CBS 297.67
CBS 112.55
CBS 135.52
CBS 481.65
CBS 466.65
CBS 192.65
CBS 498.65
CBS 105.55
CBS 283.65
1028
DUV.IP
CBS 458.75
152.WS
IFAS.20
B614B
P8
CBS 481.65
NCPF2140
B617B
CBS 542.75
CBS 132.54
NCPF2109
CBS 106.55
DALIP
IFASl9
152.EI
CBS 113.26
CBS 126.56
NHL2951
NHL2952
2172.88
2458.56
A. viridinutans
Nfischeri
A. fumigatus var. el/ipticus
Nfischeri
N. fennelliae
A. brevipes
N. quadricincta
N. quadricincta
A. duricaulis
N. aurata
A. fumigatus
N. stramenia
N. aureola
A. unilateralis
A. fumigatus
A. fumigatus
A.I umigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. duricaulis
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
N. aureola
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. unilateralis
N. fennelliae
N. fennel/iae
A. duricaulis
A. fumigatus
a. SDS-PAGE; b. Western blots.
samples studied
a+b
a
b
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Analysis of water soluble components of Aspergillus and Neosartorya mycelial preparations
237
Preparation of fractions.
The isolates were grown in 2% glucose, 1% peptone broth at 2SC. The time of growth
varied with the isolate (Debeaupuis et al., 1989). Mycelium was separated from the culture
medium by paper filtration and washed extensively with water. Culture filtrates were
treated with 4 volumes of ethanol overnight at 4C. After centrifugation, precipitated
material (EP) was washed with ethanol, resuspended in water and stored at -20C. Water
soluble fractions were prepared from the fungal mycelium by disruption in SOmM
ammonium bicarbonate at SoC in an MSK Braun cell homogenizer with 1 mm diam glass
beads for approximately 4-S min. Soluble constituents were separated from the insoluble
residue by centrifugation at 100,000 g for 1 hr. Supernatants were concentrated
approximately 5-fold by overnight dialysis at 4C against polyethylene glycol 6000 to give
the water-soluble (WS) fraction of each mycelial preparation (Hearn and Mackenzie, 1979).
Protein analysis.
The total protein of each fraction was determined using the Bio-Rad (Munich, G.F.R.) dye
binding protein assay with bovine serum albumin as a standard. Measurement was done
directly on WS fractions, whereas EP fractions were homogenized by ultrasonication and
boiled S min in IN NaOH before protein measurement.
Polyacrylamide gel electrophoresis (SDS-PAGE).
Samples were subjected to electrophoresis in a Bio-Rad Mini Protean TM II apparatus (8.2
x 10 x 0.07Scm) using a separating gel of 12.S% polyacrylamide, essentially according to
Laemmli (1970). Samples (25 Ilg of protein) were diluted 1:1 in 'denaturing buffer' (2%
SDS, 10% glycerol, S% mercaptoethanol and a trace of bromophenol blue in 64
mMTris/HCI buffer, pH 6.8) and boiled for 3 min. A Tris-glycine tank buffer (pH 8.3) was
used. Electrophoresis was carried out at a constant current of 30mA (Hearn et al., 1989).
Molecular weight (mol.wt.) protein standards (Gibco/BRL SARL, France) were run in
parallel.
Protein and glycoprotein detection.
Separated components were stained for protein in 0.1 % Coomassie Brilliant Blue R-250 in
2S% methanol and 7.S% acetic acid for 1 hr at room temperature and destained in the
same solvent without dye (Dzandu et al., 1984).
Electroblotting.
The e1ectrophoretically separated proteins and glycoproteins were electro-transferred to
nitrocellulose membranes (Hybond C, Amersham Int., U.K) in a transblotting chamber
(LKB 200S Transphor), using the method of Towbin et al. (1979).
Antigenic reactivity of WS preparations.
The free binding sites on the membrane were saturated with phosphate buffered saline,
pH 7.2, containing O.lS% Tween 20 (PBS-T) for 1 hr at room temperature. Two separate
pools of human serum samples (highly reactive in an EUSA for A. fumigatus antibodies)
were incubated at dilutions of 1:600 or 1:800 in PBS-T for 2 hr at room temperature. Blots
were then exposed to rabbit anti-human IgG conjugated to peroxidase (Dako, Denmark) at
1:1000 dilution in PBS-To Antigen/antibody complexes were localised by staining for
peroxidase activity with 3,3'-diaminobenzidine as substrate for a maximum incubation
time of 10 min.
V.M. Hearn et a/.
238
Electrophoresis in native gels.

PAGE was done with WS extracts in an LKB 2001 Vertical Electrophoresis Unit, initially in
7.5% but finally in gradient 5-15% polyacrylamide gels (16 x 18 x O.lcm). Samples for
analysis containing 30 ~g protein were applied in 10% glycerol and a trace of
bromophenol blue in 64 mM Tris/HCI buffer, pH 6.8; SDS and mercapto-ethanol were
omitted from the system. The tank buffer was again Tris-glycine at pH 8.3. Electrophoresis
was done at 30 rnA per gel. High mol. wt. protein standards for use in native gels (Sigma
Chemical Co.), were run in parallel. Following electrophoresis, gels were stained for the
presence of protein and glycoprotein moieties as described above.
Detection of catalase activity.
The ferricyanide negative stain of Woodbury et al. (1971), was used to locate bands
containing catalase following PAGE in native gels, as described by Wayne and Diaz (1986).
The loading of each fraction was based on the amount necessary to optimize the
separation of the multiple catalase bands present in many of the preparations.
RESULTS
SDS-PAGE analysis.
All WS preparations were subjected to SDS-PAGE; the separated components were
stained for protein with Coomassie Blue (Fig. 1). Each sample contained an array of
molecules which ranged in apparent mol. wt. from 10 kDa to approximately 100 kDa.
Some extracts contained low concentrations of additional components with apparent mol.
wts between 100-200 kDa. The profiles obtained showed qualitative and quantitative
differences among samples.
MW
(kDa)
11
12
13
14
15
16
17
18
200974-'
6843-
257-
18,414,3-
Figure 1. SDS-PAGE on a 12.5% gel of water soluble fractions from sample Nos. 11-18,
stained for protein with Coomassie Blue. Mol. wts of protein standards are shown in the left
margin.
239
Fewer protein bands were detected with Coomassie blue in the ethanol precipitate of the
culture filtrates than in the WS fractions. Most of the protein bands had apparent mol. wts
less than 67 kDa. With one exception (No. 43), all A. fumigatus isolates displayed similar
protein profiles, with three major bands with apparent mol. wts between 50 and 30 kDa
(Fig. 2). The profiles obtained with one isolate of each A. unilateralis and A. duricauIis (No.
39 and 42, respectively), were closely related to the typical A. fumigatus pattern, whereas
the other isolate of A. unilateralis (No. 20) was quite different.
MW
(kDa)
97'4-
25,7-
974-
68-
257Figure 2. SDS-PAGE on 12.5% gel of ethanol precipitates of the culture filtrates (20l1g
protein per well) stained for protein with Coomassie Blue. Mol. wts of standards are shown
in the left margin. Arrows indicate recurring bands.
All the isolates of Neosartorya fischeri studied produced patterns very similar to those of A.
fumigatus. In contrast, the patterns obtained with the N. fenneIIiae and N. au rata EP s
differed from those shown by A. fumigatus and N. fischeri.
Reactivity of extracts towards Aspergillus-positive sera.
Two ELISA-positive human serum pools showed very different binding patterns when
exposed to the WS fractions of the isolates examined. Thirty six from a total of thirty seven
isolates tested gave a strong reaction with multiple bands in the 50-200 kDa range with
human serum pool 'a' (Fig. 3). The number of components of mol. wt. <50 kDa which
showed antigenic reactivity towards A. fumigatus antibodies were relatively few and the
V.M. Hearn et sl.
240
Figure 3. Water soluble fractions from sample Nos. 11-18 separated by SOS-PAGE and
transferred to nitrocellulose for probing with anti-Aspergillus antiserum. Mol. wts of protein
standards are shown in the left margin.
MW
(kDa)
31
12
19
15
974-
68-
43-
257184- _
143Figure 4. Ethanol precipitate separated by SOS-PAGE and transferred to nitrocellulose for

probing with anti-Aspergillus human antiserum (10-3 dilution). Mol. wts of standards are
shown in the left margin. The arrow~ indicates a dominant., recurring band at 17 kOa.
241
band staining was much less intense by comparison with high mol. wt. moieties. Only one
A. duricaulis (No. 15) showed a very weak binding with A. fumigatus IgG. When another
human serum pool (pool 'b') was tested in the same system, 10 of 36 isolates were only
weakly reactive, including five isolates of A. fumigatus (results not shown). Twenty four
from a total of twenty six isolates tested gave a positive reaction when their EP fractions
were separated by SD5-PAGE and probed with a pool of human antisera after transfer to
nitrocellulose. The number of protein bands which showed antigenic reactivity toward A.
fumigatus antibodies was relatively small and never exceeded 15 bands. Most of the bands
were in the 10-90 kDa range, with a major antigen of mol. wt. of 16-18 kDa (Fig 4). Based
on the presence or absence of this antigen band, two groups of species could be
differentiated (Table 2). These results confirmed the grouping made on the basis of the
Coomassie Blue protein staining of SDS-PAGE of EP: A. fumigatus, N. fischeri and N.
aureola could be distinguished from A. viridinutans, A. duricaulis, A. unilateralis, A. brevipes,
N. fennelliae and N. quadricincta.
Table 2. Presence or absence of the 16-18 kDa antigen band inA. fumigatus and related taxa.
Presence
Absence
A. fumigatus: isolates 36, 31,43,27,26,17,10,23

A. fisheri: isolates 6, 1, 13, 12, 11
N. aureola: isolates 19, 34
N. straemenia: isolate 181
A. viridinutans: isolate 3
A. lIrevipes: isolate 8
A. duricaulis: isolates 15,42
A. unilateralis: isolate 20
N. fennel/iae: isolates 7, 21
N. quadricincta: isolates 9, 14
N. aurata: isolate 161
1 overall
reactivity of the blot with human pool was very low
Analysis on native gel.

All WS preparations were examined following electrophoresis in native gels. In most
samples Coomassie Blue stain revealed a number of bands, with a wide range of mol. wts
(Fig.5). A few preparations showed a minimal number of Coomassie Blue stained bands
(viz., N. stramenia, A. unilateralis and two A. fumigatus). Most A. fumigatus isolates (12 of
16, three only in trace amounts) showed the presence of a double band with mol. wts near
100 kDa. Exceptions were A. fumigatus Nos 32 and 36, where this double band appeared to
be absent. Of the other species tested, only A. fumigatus var. ellipticus and N. fischeri var.
glabra possessed double bands with mobilities indistinguishable from those of A.
fumigatus. A smaller number of A. fumigatus isolates (No. 21, 26, 27, 30, 31, 35, 37 and 38)
shared three additional bands, as did A. fumigatus var. ellipticus (Fig. 5).
Analysis of catalase band patterns on native gels.
Staining for catalase activity after electrophoresis on native gels showed the presence of at
least one catalase component in all WS preparations. Most A. fumigatus isolates produced
two major bands, one with approximate mol. wt above and one below 272 kDa called slow
and fast bands, respectively. The slow band was consistently present and its mobility
appeared constant in all A. fumigatus isolates; the fast component was absent from sample
V.M. Hearn et al.
242
21 26 27 29 30 31 33 35 37 6
11 12 13
8 10 14 38
MW
(kDa)
Figure 5. PAGE on a native gel (5-15% gradient) of water soluble fractions: from left to right,
9 isolates of A. fumigatus, 9 other species, 1 isolate of A. fumigatus and 1 other species. The
gel was stained for protein with Coomassie Blue. Arrows indicate the most commonly shared
bands (->-, and other shared bands (- present in the different species. Mol. wts of bovine
serum albumin (monomer and dimed are shown in the left margin.
No. 36 and its mobility showed isolate to isolate variation (Fig. 6). One sample (No. 25)
gave a catalase pattern which differed markedly from all other A. fumigatus isolates tested.
Several A. fumigatus WS preparations contained additional, minor catalase bands, but
these components showed no constant pattern. Some other species also produced two
major catalase bands but they usually showed different mobilities from those seen in A.
fumigatus. However, A. fumigatus var. acolumnaris (No. 10) and N. fischeri var. glabra (No.
13), produced two catalase bands of similar mobility to these of A. fumigatus. The two
isolates of N. fischeri (Nos. 4 and 6), also contained two catalase bands with mobilities
similar to A. fumigatus, but were only minor components. These results are summarized in
Table 3 and Fig. 6.
243
Table 3. Catalase band patterns seen with Aspergillus and Neosartorya species
A. fumigatus isolates
with 2 major catalase
bands
17
21
22
24
26
27
29
30
31
A. fumigatus isolates Other species with 2 major bands

with >2 catalase bands
17
22
24
26
27
29
30
35
37
32
33
35
37
38
TOTAL
14
2 (N. fennelliae)
5 (A. fumigatus var. e/lipticus)
7 (N. fennelliae)
10 (A. fumigatus var. acolumnaris)
13 (N. fischeri var. g/abra)
20 (A. unilateralis)
23 (A. fumigatus var. sclerotium)
DISCUSSION
Separation of WS preparations of Aspergillus and Neosartorya species by SDS-PAGE

showed very complex protein patterns, when visualized with Coomassie Blue. Since
differences among these preparations were readily apparent, the method offers the
potential of determining protein homologies (Le., the number of common protein bands)
among isolates and species of Aspergillus and Neosartorya. However, the multiplicity of
constituent molecules present in each fraction could make it difficult to assess the
reproducibility of the gel electrophoresis method and the batch to batch variation of the
samples. Because the number of protein bands detected is lower, EP profiles obtained by
SDS-PAGE may prove more suitable than WS extracts to cluster isolates or species. In
addition EP bands lie in the 10-80 kDa range where separation of proteins is better than in
the high mol. wt. range.
The antigenic patterns of these WS preparations, examined by immunoblotting,
varied considerably with the two pools of human serum tested. Some isolates (20 out of
36) bound strongly to antibodies present in each of the two pools; with others (9 of 36), the
binding profile changed from strong (for serum pool 'a') to weak (for serum pool 'b').
Thus, heterogeneity among immunogens was readily detectable. More precise
information may be obtained by using antibody specific to certain immunodominant
antigens, as indicated by the profiles achieved with EP preparations.
Electrophoresis of the intact molecules gave a much simplified protein pattern, with
some evidence of recurring components in A. fumigatus isolates. Analysis showed two
closely associated bands with mol. wts. in the 100 kDa range. These were present in 12
out of 16 WS preparations, when monitored in both 7.5% and gradient polyacrylamide
gels.
Enzymes, including catalase, have been shown by several workers to possess
antigenic activity when tested with anti-Aspergillus antisera (Tran van Ky et al., 1968;
244
V.M. Hearn
et at.
MW
(kDa)
Figure 6. PAGE on a native gel (5-15% gradient) of water soluble fractions: from left to right,
9 isolates of A. fumigatus, 9 other species, 1 isolate of A. fumigatus, 1 other species. The gel
was stained for catalase by the ferricyanide method. Arrows indicate the 2 major catalase
components. Mol. wts of urease (trimer and hexamed are shown in the left margin.
Schonheyder and Andersen, 1984). PAGE revealed 1-5 bands with catalase activity when
Aspergillus and Neosartorya species were examined. Catalase band patterns of A. fumigatus
isolates showed a measure of similarity, with one major exception (No. 25). No
characteristic pattern was obvious among other species, but numbers tested were small. In
this study, two batches of A. fumigatus No. 33 (NCPF 2109) were monitored for catalase
activity; one batch contained significantly higher levels of both the fast and slow
components, at comparable protein concentrations. In a separate study where ten batches
of this isolate were analysed, 3 of 10 batches appeared to lack the fast catalase component
(Hearn, unpublished). Thus batch variation can represent an important factor in such
analyses and requires investigation in order to fully interpret the results obtained.
245
Four parameters have been used to analayse relatedness among the Aspergillus sect.
Fumigati and Neosartorya. While A. fumigatus isolates showed a tendency to cluster, some
isolate differentiation was evident. Tests which compare constituent molecules of WS on

native gels plus associated catalase activity, in addition to protein and antigenic patterns,
of EP tended to align N. fischeri and N. aureola with Aspergillus sect. Fumigati.
ACKNOWLEDGEMENT
The authors wish to express their thanks to Dr. c.K. Campbell, of the Mycological
Reference Laboratory, for helpful discussion in the preparation of the manuscript.
REFERENCES
DEBEAUPUIS, J.P., SARFATI, J., GORIS, A., STYNEN, D., DIAQUIN, M. and LATGE, J.P. 1989. Exocellular
polysaccharides from Aspergillus fumigatus and related taxa. In Modem Concepts in Penicillium and
Aspergillus Classification, eds. R.A. Samson and J.1. Pitt, pp. 209-223. New York and London: Plenum
Press.
DZANDU, J.K., DEH, M.E., BARRATT, D.L. and WISE, G.E. 1984. Detection of erythrocyte membrane
proteins, sialoglycoproteins and lipids in the same polyacrylamide gel using a double-staining technique.
Proceedings of the National Academy of Sciences, U.S.A. 81: 1733-1737.
HEARN, V.M. and MACKENZIE, D.W.R. 1979. The preparation and chemical composition of fractions from
Aspergillus fumigatus wall and protoplasts possessing antigenic activity. Journal of General Microbiology
112: 35-44.
HEARN, V.M., GRIFFITHS, B.L. and GORIN, P.A.J. 1989. Structural analysis of water-soluble fractions
obtained from Aspergillus fumigatus mycelium. Glycoconjugate Journal 6: 85-100.
LAEMMLI, U.K. 1970. Oeavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 227: 680-685.
PIECHURA, J.E., KURUP, V.P., FINK, J.N. and CALVANICO, N.J. 1985. Antigens of Aspergillus fumigatus.III.
Comparative immunochemical analyses of clinically relevant aspergilli and related fungal taxa. Clinical
and Experimental Immunology 59: 716-724.
SCHONHEYDER, H. and ANDERSEN, P. 1984. IgG antibodies to purified Aspergillus fumigatus antigens
determined by enzyme-linked immunosorbent assay. International Archives of Allergy and Applied
Immunology 74: 262-269.
SORENSON, W.G., LARSH, H.W. and HAMP, S. 1971. Acrylamide gel electrophoresis of proteins from
Aspergillus species. American Journal of Botany 58: 588-593.
TOWBIN, H., STACHELIN, T. and GORDON, J. 1979. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National
Academy of Sciences, U.S.A. 76: 4350-4354.
TRAN van KY, P., BIGUET, J. and VAUCELLE, T. 1968. Etude d'une fraction antigenique d'Aspergillus
fumigatus support d'une activite catalasique. Consequence sur Ie diagnostic immunologique de
l'aspergillose. Reoue d'Immunologie 32: 37-52.
WAYNE, L.G. and DIAZ, G.A. 1986. A double staining method for differentiating between two classes of
mycobacterial catalase in polyacrylamide electrophoresis gels. Analytical Biochemistry 157: 89-92.
WOODBURY, W., SPENCER, A.K. and STAHMANN, M.A. 1971. An improved procedure using
ferricyanide for detecting catalase isozymes. Analytical Biochemistry 44: 301-305.
246
V.M. Hearn et al.
DIALOGUE FOLLOWING THE FOUR PRESENTATIONS ON ASPERGILLUS

FUMIGATUS
HEARN: We were unable to distinguish between pathogenic and nonpathogenic isolates of

Aspergillus fumigatus using our antisera. In fact, all the isolates we tested reacted with the
antiserum for A. fumigatus. We realize now that the antiserum may be reacting with
sugars and that these might be common not only in Aspergilli, but in other fungi as well.
Upon reflection, if we had taken a different approach to the problem, perhaps we would
have had better results. H we had removed the carbohydrate moieties from the outer
surfaces of the walls and raised the antibodies to the underlying protein parts of the
molecule, perhaps the resulting serum would have been more specific.
LATGE: One problem we faced was that we had twenty isolates of Aspergillus fumigatus,
and only one or two strains of the other species. Would it be possible to get more strains
of the other species?
SAMSON: Unfortunately, many of the isolates of these other species in culture collections
are misidentified, and they are very rare in any case.
CHRISTENSEN: An obvious idea would be to return to the type locality. For example, A.
brevipes was originally isolated from tundra soil on Mt. Koskiusko in Australia. It would
be fun to go back there and try to find it.
HEARN: H these species are so rare, do we really have to worry about them?
SAMSON: For our purposes, no.
CHRISTENSEN: It seems to me that they may not be rare. Perhaps we just haven't been
looking in the habitat in which they are most abundant.
SAMSON: Ecologists or morphologists may be interested in seeing more isolates. But when
you are considering medical importance, then A. fumigatus is sufficient.
CHRISTENSEN: Aspergillus nutans, A. parvulus and A. kanagawaensis are species that have
pinkish conidia, but they are otherwise very similar to the species of sect. Fumigati. An
Indian worker also reported a tan-coloured A. fumigatus. Have these been considered in
these studies?
FRISVAD: No, but it would be interesting to see them.
CHRISTENSEN: A. nutans also produces nodding conidiophores and so do A. brevipes and
A. unilateralis.
SAMSON: I was unable to obtain that tan-coloured mutant from India.
PATERSON: What is the taxonomic significance of species of Aspergillus producing some of
the same secondary metabolites as Penicillium species?
FRISVAD: H you have two species, one in Penicillium, the other in Aspergillus, with globose
conidia, you would not say they are closely related only for that reason. I feel the same
way about secondary metabolites. H you are working with smaller groups of taxa, then
the secondary metabolites have taxonomic significance. For example, emodin is
produced by P. islandicum, but it is also produced by rhubarb.
CHRISTENSEN: Would the collaborators in this project be prepared to subject their
information to Principal Components Analysis (PCA)?
247
FRIsVAD: There are some programs available to do that, of course.
BRIDGE: If you are going to do PCA, you have to be certain that the correlation between
the organisms in Euclidian. Immunological data is ideal for this, because it is
representative of a distance matrix.
CHRISTENSEN: Is there suitable immunological data that could be used?
POLONELLI: We have tried to correlate the killer yeast data with the immunological data
we have from Western blot analysis from antibodies against Candida albicans.
Apparently, there was a real correlation between the two different biotyping approaches.
We don't know why. We have investigated cases of sexually transmitted infection and
some cases of nosocomial infection from mother to newborne baby with both systems.
Biotyping with the killer yeast system and the Western blot analyses using monoclonal
antibodies corresponded.
249
TAXONOMY OF ASPERGILLUS SECTION RESTRICTA
J.1. Pitt1 and R.A. Samson2

lCSIRO
2Centraalbureau roor Schimmelcultures
005terstraat 1, 3742 SK &am, The Netherlands
SUMMARY
The taxonomy of Aspergillus Section Restricti (the Aspergillus restrictus Group) has been reviewed and
revised on the basis of morphological taxonomy, a bibliographic review and a herbarium search.
Three species are accepted: Aspergillus caesiellus Saito, A. penicillioides Spegazzini and A. restric/us G.
Smith. A. conicus Blockwitz and A. gradlis Bainier are of uncertain application. A. i/aconicus, also
studied, is not related to species in this Section. A key and descriptions are included.
INTRODUCTION
Presumably because of their xerophilic character, Aspergillus restrictus and related species
were regarded by Thorn and Raper (1945) as a Series within the "Aspergillus giaucus
Group", now more commonly known as the genus Eurotium. Raper and Fennell (1965)
raised this Series to "Group" status as the "A. restrictus Group". Gams et al. (1985) renamed
this set of related taxa as Aspergillus Section Restricti in accordance with the Botanical
Code.
Taxonomy within this Section has remained unclear. The best known species, A.
restrictus G. Smith 1931 has the lowest priority under the Code of any of the species
included by Raper and Fennell (1965). Perhaps the most common species, and certainly
the most xerophilic, is A. penicillioides Spegazzini 1894. It grows poorly if at all on
commonly used (high water activity) media, is often overlooked and infrequently
recognised.
Other species in Sect. Restricti are A. caesiellus Saito 1904, A. conicus Blockwitz 1914
and A. gracilis Bainier 1907. A. itaconicus Kinoshita 1931 was also examined, because Thorn
and Raper (1945) included it in their concept of the A. giaucus Group. Each of these is a
rarely encountered and little known species.
A. conicus, A. gracilis and A. penicillioides were neotypified by Samson and Gams
(1985). However, the picture is confused by the fact that isolates used as a basis of the
descriptions of A. gracilis and A. penicillioides by Thorn and Raper (1945) were both placed
in A. restrictus by Raper and Fennell (1965). There is also doubt about the accuracy of the
neotypifications of A. conicus and A. gracilis.
This paper is intended to clarify the taxonomy of these species.
250
J.I. Pi" & R.A. Samson
Cultures.
Representative isolates were examined from A. caesiellus, A. gracilis, A. conicus, A.
penicillioides, A. restrictus and A. itaconicus. Cultures were obtained from the CBS, FRR,
NRRL and ATCC collections.
Media.
Isolates were examined on Czapek yeast extract agar (CYA), malt extract agar (MEA), 25%
glycerol nitrate agar (G25N; Pitt, 1979); and also 20% sucrose Czapek yeast extract agar
(CY20S) and malt yeast 50% glucose agar (MY50G; Pitt and Hocking, 1985). All cultures
were examined after 7 days incubation at 25C, and on CYA at 3~C as well.
Capitalised colour names and adjacent numbers in brackets are from the Methuen
"Handbook of Colour" (Kornerup and Wanscher, 1978).
A bibliographic search and a check on all available herbarium material was also
conducted.
RESULTS
Examination of two strains of the extype isolate of A. itaconicus (CBS 115.32 and FRR 161)
showed that morphology and growth rates were inconsistent with placement in
Aspergillus Sect. Restricti. Growth rates, especially on CY20S and G25N, were much faster
than observed for other species considered here. Vesicles were large and spherical, clearly
indicating that Raper and Fennell (1965) were correct in placing this species in a section
distant from Sect. Restricta.
Other isolates examined formed a cohesive pattern of growth rates and microscopic
morphology which indicated a close relationship, and a natural series. Two distinct
clusters of isolates were observed; one was homogeneous, the other could be split into two
species on the basis of a variety of minor features. Hence three species in all are
recognised here.
Naming of these species was not so simple. The homogeneous cluster included a
variety of isolates which had been identified as A. penicillioides. Examination of the packet
labelled "A. penicillioides" from Spegazzini's herbarium by one of us (R.A.S.) revealed no
useful information, other than that Spegazzini had clearly intended A. penicillioides to be
spelled that way, rather than "A. penicilIoides" (Fig. 1), a variant which has been in common
use for many years (Raper and Thorn, 1945; Raper and Fennell, 1965). Neotypification,
based on concepts with which the authors were in close agreement (Pitt and Hocking,
1985; Samson and van Reenen-Hoekstra, 1988) was the obvious procedure for stabilising
this name in the sense in which it is in common use. This was carried out by Samson and
Gams (1985), who designated Herb.1MI 211342 (= CBS 540.65, WB 4548) as neotype.
It is notable that Raper and his coworkers' concept of A. penicillioides was not so clear
as it has become in more recent times. NRRL 151, regarded by Raper and Thorn (1945) as
"fit satisfactorily" and similar to Thorn 4197.3 "agreed to by Spegazzini" was not
mentioned under this species by Raper and Fennell (1965), but placed under A. restrictus.
Isolates in the second cluster were regarded as representative of A. caesiellus, A.
conicus, A. gracilis and A. restrictus by various previous authors and collections. Of these,
only A. caesiellus and A. restrictus were represented by cultures derived from types. One
Taxonomy of Aspergillus section Restricta
251
taxon in this cluster was represented by the oldest name, A. caesiellus, which was therefore
accepted as a distinct species.
Of the other three names, considerable doubt exists about the validity of A. conicus
and A. gracilis, despite the neotypification of Samson and Gams (1985). Using a name
suggested by Blockwitz, Dale (1914) published A. conicus, based on her description of "a
Penicillium" in Dale (1912), but without further description. According to Thom and Raper
(1945), Blockwitz (1929) expressed a lack of confidence in Dale's usage of his name.
Nevertheless, Raper and Thom (1945) accepted it. In the absence of a type, and in view of
the obvious confusion over what this species represented, we see no reason to continue its
usage. In consequence we reject A. conicus, as of uncertain application.
A. gracilis was described and illustrated by Bainier (1907) in terms which are not
clearly indicative of a species belonging in Sect. Restricta. Indeed, A. gracilis may equally
well have been related to A. fumigatus. NRRL 145 (= Thom 4246), used as a basis of the
description of A. gracilis in Thom and Raper (1945: 139), was later "believed to represent an
exceptionally slow-growing A. restrictus". On the basis of these inconsistencies, the
neotypification of A. gracilis by Samson and Gams (1985) is rejected. Like A. conicus, this
species is also regarded as of uncertain application.
The valid name for the taxon under discussion at this point is therefore A. restrictus, a
widely used name over a number of years, though almost certainly confused with A.
penicil/ioidesin much of the literature.
Figure 1. Original handdrawing and writing by C. Spegazinni on the folder of the holotype
of Aspergillus penicillioides (herb. LP).
252
J.I. Pitt & A.A. Samson
TAXONOMY
Key to the species in Aspergilli sect. Restricti

1. Conidia borne as cylinders; at maturity usually cylindrical to doliiform (barrel shaped) ............................. 2
Conidia borne as ellipsoids; at maturity usually ellipsoidal to subspheroidal ...................... A. penicillioides
2. Colonies on CY20S at 7 days not exceeding 20 mm in diameter ..................................................... A. restrictus

Colonies on CY20S at 7 days exceeding 20 mm in diameter ........................................................... A. caesiellus
Aspergillus caesiellus Saito - J. Fac. Sci. CoIl. Imp. Univ. Tokyo 18: 49, 1904 - Fig. 2.
Aspergillus gracilis var. sartoryi Batista et al. - Mycopath. Myco!. App!. 8: 196,1957.
Figure 2. Aspergillus caesiellus. A. Colonies on CYA and MEA, 7 days. B. Conidial heads, x
750. C. Conidia, x 1875.
253
Colonies on C'fA 6-12 mm in diameter, dense, usually umbonate, often irregularly wrinkled, of very close,
velutinous texture; mycelium white; conidial heads sparse to numerous, Greenish Grey to Dark Green (26DF2-3); reverse pale to dull green. Colonies on MEA 10-16 mm in diameter, low and plane, sparse to
moderately dense, velutinous or lightly funiculose; mycelium inconspicuous, white; conidial production
moderate to heavy, Dark Green (26F3-4); reverse pale to very deep green. Colonies on G25N variable,
usually 4-12 mm in diameter, often irregular in shape, dense, surface velutinous to somewhat floccose or, in
some isolates, mucoid; condia absent to abundant, in the latter case dark green; reverse colourless to grey
green. Colonies on CY20S showing optimal development, 20 to 30 mm in diameter, plane or lightly
wrinkled, dense, velutinous to floccose-funiculose; mycelium sometimes conspicuous in overgrowths,
white; conidial production heavy, Dark Green (26-27F3-5); reverse pale to deep green. Colonies on MY50G
12-18 mm in diameter, plane, sometimes with irregular margins, often floccose; mycelium white; conidial
production absent or moderate, in the latter case Greenish Grey (26-27D-E2), paler than on the other media;
reverse pale to pale green. Sometimes slow growth on C'fA at 37C, with colonies up to 6 mm in diameter,
of white mycelium.
Conidiophores borne from surface hyphae, developing optimally on C'f20S, stipes varying widely with
isolate, measuring (25-)50-150(-300) x 4-7(-9) fJIIl, with thin, smooth, colourless walls, sometimes septate or of
irregular diameter; vesicles pyriform to funnel-shaped, 10-161J1ll in diameter, bearing phialides only, often
confined to more or less flattened apices; phialides crowded, but seldom numerous, commonly 6-8llm long;
conidia borne as cylinders and often swelling in diameter only when separated from the phialide by 4-6
younger conidia, at maturity ellipsoidal to doliiform, (4-)5-6(-9) x 3-41J1ll, with spinose walls.
Typification. Herb. IMI 172278, derived from Saito's type, was designated as lectotype by
Samson and Gams (1985). Cultures derived from type include IMI 172278 and CBS 470.65.
Distinctive features. A. caesiellus and A. restrictus are readily distinguished from A.

penicillioides by more vigorous growth on CYA and MEA, and more fundamentally by the
method of formation of conidia: conidia in A. caesiellus and A. restrictus are borne as
cylinders, and only slowly become more round, while those of A. penicillioides are borne as
ellipsoids, and rapidly become ellipsoidal to subspheroidal.

A. caesiellus and A. restricus are closely related. A. caesiellus grows more rapidly on
CY20S than A. restrictus. Colonies of A. caesiellus usually exceed 20 mm in diameter on
CY20S, while those of A. restrictus rarely do. A. caesiellus produces somewhat larger
conidia and, in the isolates examined here, shorter, less numerous phialides. The isolates
of A. caesiellus examined grew at 37C, though weakly. It is not known if this is a
consistent character.
Occurrence. This has been a quite rarely recognised species, as it has probably been
confused with A. restrictus. An understanding of its distribution must await better
recognition.
Isolates examined. CBS 470.65 & NRRL 5061 (= FRR 3697), ex type, K. Saito; CBS 116.55 &
NRRL 4745 & ATCC 11969 (= WB 4745, FRR 3696), ex type of A. gracilis var. sartoryi Batista
et al., laboratory contaminant, Brazil, A. C. Batista; NRRL 2652 (= FRR 3695), source
unknown; FRR 2176, from dried chili, Papua New Guinea, A.D. Hocking.
Aspergillus restrictus G. Smith - J. Textile Inst. 22: 115,1931- Fig. 3.

Aspergillus restrictus var. B G. Smith, J. Textile Inst. 22: 115,1931.
Penicillium fusco-flavum Abe, J. Gen. App!. Microbio!. 2: 64, 1956.
Colonies on C'fA 6-12 mm in diameter, sulcate or wrinkled, low, dense and velutinous; mycelium
inconspicuous, white; conidial heads often poorly formed, sparse to numerous, in the latter case Dull Green
(26-27C-E3); reverse pale to very dark green. Colonies on MEA 6-12 mm in diameter, occasionally smaller,
similar to those on CYA or centrally raised, conidial production heavy, but heads poorly formed, coloured
Dull Green to Dark Green (27C-FS); reverse usually pale. Colonies on G25N 10-14 mm in diameter, plane or
254
umbonate, usually similar to those on MEA, but heads well formed, producing long columns of conidia
when mature; reverse pale or sometimes dark green. Colonies on CY20S 16-20 mm in diameter, generally
similar to those on G25N apart from slightly more rapid growth. Colonies on MY50G 12-16 mm in diameter,
plane or umbonate, with aerial growth and conidial production usually sparse, coloured Greenish Grey to
Dull Green (27C-D3); reverse pale. No growth on CYA at 3~.
Conidiophores borne from surface hyphae, developing optimally on CY2OS, stipes 75-200 J.l.m long,
sometimes sinuous, with thin, colourless, smooth walls, enlarging from the base gradually, then more
abruptly to pyriform vesicles; vesicles 10-15 J.Lm in diameter, fertile over the apical hemisphere, or less,
bearing phialides only; phialides crowded, 8-10 J.Lm long; conidia borne as cylinders, in long appressed
columns, adhering in liquid mounts, when mature nearly cylindrical or doliiform, 4.0-55 J.Lm long, with
rough walls.
Figure 3. Aspergillus restrictus. A. Colonies on CYA and MEA, 7 days. B. Colonies on CY20S
(left) and MY50G (right), 14 days. C. Conidial heads, x 750. D. Conidia, x 1875.
Taxonomy of Aspergillus section Resfricta
255
Typification. Samson and Gams (1985) designated Herb. IMI 16267 as lectotype of A.
restrictus. Cultures ex type include 1MI 16267, CBS 117.33, CBS 541.65, NRRL 154 and
ATCC 16912.
Distinctive features. See A. caesiellus above.

Occurrence. A. restrictus is the most commonly recognised species in Sect. Restricta, but it
seems likely that it has often been confused with A. caesiellus and A. penicillioides. It is
widely distributed in cereals and other dried foods (Pitt and Hocking, 1985).
Isolates examined. CBS 541.65 & NRRL 154 (= CBS 117.33, FRR 3689, 1M! 16267, ATCC
16912), ex type, from mouldy cloth, United Kingdom, G. Smith; CBS 118.33 & NRRL 148
(= FRR 3726, IMI 16268) ex type of A. restrictus var. B, from cotton fabric, United Kingdom,
G. Smith; CBS 331.59 & FRR 1173 (= 1MI 68226), ex type of Penicillium fusco-flavum Abe,
from ?soil, Japan, S. Abe; NRRL 158 (= FRR 3692), received as A. conicus; NRRL 159 (= FRR
3693), received as A. conicus; NRRL 160 (= FRR 3694), received as A. conicus; NRRL 156 (=
FRR 3690), received as A. gracilis; FRR 1992, from dried split peas, Australia, A.D. King;
FRR 2967 and FRR 2973, both from Indonesian dried fish, K.A. Wheeler.
Aspergillus penicillioides Spegazzini - Revta La Plata Univ. Fac. Agron. Vet. 2: 246, 1896Fig. 4.
Aspergillus vitricola Ohtsuki, Botan. Mag., Tokyo 75: 436, 1962.
Aspergillus glaucus var. tonophilus Ohtsuki, Antiques and Art Crafts Japan 13: 22, 1956.
Colonies on CY A up to 5 nun in diameter, but sometimes only microcolonies, of white mycelium only.
Growth on MEA usually limited to microcolonies, occasionally colonies up to 5 mm in diameter fonned,
similar to those on CYA. Colonies on G25N 8-14 nun in diameter, plane or centrally raised, sometimes
sulcate or irregularly wrinkled, texture velutinous or lightly floccose; mycelium usually inconspicuous,
white; conidial production moderate, heads typically radiate, uncommonly in loose columns also, coloured
Dull Green to Dark Green (27C-F8); reverse pale to dark green. Colonies on CY20S varying from
microcolonies up to 10 nun in diameter, similar to those on CYA, occasionally some dull green conidial
production but conidiophores poorly fonned; reverse pale. Colonies on MY50G 10-16 nun in diameter, plane
or umbonate, relatively sparse, velutinous to floccose; conidial production moderate, Greyish Green to Dull
Green (27C-D3); reverse pale. No growth on CYA at 37"<:.
Conidiophores bome from surface or aerial hyphae, showing optimal development on G25N or MY5OG,
stipes 150-300(-500) ~ long, sometimes sinuous, with thin, smooth, colourless walls, enlarging gradually
from the base, then rather abruptly to pyriform to spathulate vesicles; vesicles mostly 10-20 ~ in diameter,
usually fertile over two thirds of the area, bearing phialides only; phialides (7-)8-11 ~ long; conidia bome
as ellipsoids, at maturity ellipsoidal to subspheroidal, 4.0-5.0 ~ in diameter or in length, with spinose
walls.
Typification. In the absence of type material, Samson and Gams (1985) designated Herb.
1M! 211342 as neotype of A. penicillioides. Cultures ex neotype include CBS 540.65, WB
4548 and FRR 3722.
Nomenclature. Although usually written "A. penicil/oides", Spegazzini called this species "A.
penicillioides". This is also more correct orthographically (Stearn, 1966).
Distinctive features. Apart from its more xerophilic character, resulting in smaller colonies
on the more dilute media than the other species, A. penicillioides produces conidia as
ellipsoids rather than cylinders.
256
Figure 4. Aspergillus penicillioides. A. Colonies on CYA and MEA,. 7 days. B. Colonies on

CY20SC (left) and MY50G (right), 14 days. C. Conidial heads, x 750. C. Conidia, x 1875.
Occurrence. In our experience (Pitt and Hocking, 1985; Samson and van Reenen-Hoekstra,
1988), A. penicillioides is quite a common species in many kinds of dried foods and related
habitats, but it has frequently been overlooked or misidentified. It is readily missed,

because it requires media of reduced water activity for efficient enumeration or isolation.
For example Hocking (1981) reported high counts of A. penicillioides from capsicums and
other dried foods when dichloran 18% glycerol agar was used as the enumeration
medium, but complete failure in detection using dichloran rose begal chloramphenicol
agar, a medium of water activity much closer to those usually used in routine isolation
work.
Christensen (1955) reported the frequent isolation of A. restrictus from grains stored just
above the safe moisture limit, and described the damage inflicted on the grain. From
details in his paper, it is clear that that he was referring to A. penicillioides.
257
Isolates examined. CBS 540.65 & ATCC 16910 (= FRR 3722, IMI 211392, WB 4548), ex
neotype, from human skin, Brazil, D. Borelli; ATCC 16905 (= FRR 3734, IMI 108298), ex
type of Aspergillus vitricola, from binocular lens, Japan, T. Ohtsuki; ATCC 14567 (= FRR
3735, IFO 6529), from binocular lens, Japan, T. Ohtsuki; CBS 116.26 & NRRL 151 (= FRR
151), from sugar cane product, Louisiana, U.S.A., Owen; CBS 539.65 & ATCC 16906 (=
FRR 3725, WB 4962), from a gun firing mechanism, R. Emerson; ATCC 26634 (= FRR
3733), from spoiled rice, Japan, H. Ito; CBS 118.55 (= FRR 3720), from man, Netherlands;
FRR 2766 and FRR 2855, both from Indonesian dried fish, Australia, K.A. Wheeler; FRR
2177 and FRR 2188, both from dried chili, Papua New Guinea, A.D. Hocking; numerous
isolates from dry habitats including house dust (Samson and van der Lustgraaf, 1978),
furniture, human skeletons etc.
ACKNOWLEDGEMENTS
The authors thank the curators of the culture collections and herbaria mentioned in the
text for providing cultures and type specimens.
REFERENCES
BAINIER, G. 1907. Mycotheque de l'Ecole de Pharmacie. XII. Bulletin trimestrielle de Societe de Mycologique de
France 23: 90-93.
BLOCHWITZ, A. 1929. Die Gattung Aspergillus. Neue Spezies, Diagnosen, Synonyme. Annales Mycologici 27:
205-240.
DALE, E. 1912. On the fungi of the soil. Annales Mycologici 10: 452-477.
-1914. On the fungi of the soil. TI. Annales Mycologici 12: 33-62.
CHRISTENSEN, C.M. 1955. Grain storage studies. XVITI. Mold invasion of wheat stored for sixteen months
at moisture contents below 15 percent. Cereal Chemistry 32: 107-116.
GAMS, W., CHRISTENSEN, M., ONIONS, A.H.5., PITT, J.I. and SAMSON, R.A. 1985. Infrageneric taxa of
Aspergillus. In Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 5562. New York and London: Plenum Press.
HOCKING, A.D. 1981. Improved media for isolation of fungi from foods. CSIRO Food Research Quaterly 41:
7-11.
KORNERUP, A. and W ANSCHER, J.H. 1978. Methuen Handbook of Colour. London: Eyre Methuen.
Academic Press.
pm, J.I. and HOCKING, A.D. 1985. Fungi and Food Spoilage. Sydney: Academic Press.
RAPER, K.B. and FENNELL, D.I. 1965. The Genus Aspergillus. Baltimore, Maryland: Williams and Wilkins.
SAMSON, R.A. and GAMS, W. 1985. Typification of the species of Aspergillus and associated teleomorphs. In
Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 31-54. New York
SAMSON. R.A. and LUSTGRAAF, B. van der. 1978. Aspergillus penicilloides and Eurotium ha/ophilicum in
association with house-dust mites. Mycopath%gia 64: 13-16.
SAMSON, R.A. and VAN REENEN-HOEKSTRA, E.S. 1988. An Introduction to Food-borne Fungi. Baarn,
Netherlands: Centraalbureau voor SchimmelcuItures.
STEARN, W.T. 1966. Botanical Latin. New York: Hafner Publishing Company.
THOM, C. and RAPER, K.B. 1945. A Manual of the Aspergilli. Baltimore, Maryland: Williams and Wilkins.
259
ISOENZYME PATTERNS IN ASPERGILLUS FLAVUSAND CLOSELY

RELATED SPECIES
R.H. Cruickshank1 and J. I. Pitt2
1Department at Agricultural Science
University at Tasmania
Hobart, Tas. 7000
2CSIRO
Division at Food Processing
SUMMARY
Polyacrylamide gel electrophoresis was used to examine several kinds of exoenzymes from a number
of isolates of Aspergillus f/avus, A. parasiticus, A. oryzae, A. sojae, A. tamarii and A. nomius. Enzymes
studied were pectinases, ribonucleases, amylases and proteases. Methods used were similar to those
previously published, with some additional media.
Very well defined patterns were obtained for each enzyme and substrate system. A. f/avus, A.
parasiticus, A. tmnarii and A. nomius produced distinct patterns. However, A. oryzae produced patterns
very similar to those of A. f/avus, and A. sojae to those of A. parasiticus. Consequently, isoenzyme
patterns could not be used to distinguish the domesticated species from their wild types.
INTRODUCTION
Patterns of isoenzymes produced by electrophoretic techniques (zymograms) have proved

to be of considerable taxonomic value with species in Penicillium subgenus Penicillium
(Cruickshank and Pitt 1987 a, b). Continuing interest in the taxonomy of the
mycotoxigenic and fermentation species in Aspergillus sect. Flavi prompted examination of
the same techniques with important species in that series.
The taxonomy of Aspergillus sect. Flavi has had some controversial aspects recently.
Kurtzman et al. (1986) reduced the important aflatoxin producing species A. parasiticus to
the status of a subspecies of A. flavus. The food fermentation species A. oryzae and A. sojae
were reduced to the status of varieties. Klich and Pitt (1985, 1988) and others have argued
that such action is both taxonomically unsound and unacceptable for practical reasons:
taxonomic separation of potent toxin producers and fermentation cultures at species level
is essential.
This paper reports a study on some species in Aspergillus sect. Flavi by studies of
patterns of enzyme electrophoresis.
Fungi.
Almost 130 isolates from the FRR collection at CSIRO Division of Food Processing, North
Ryde, were examined. The isolates were selected from the large range studied by Klich
and Pitt (1988) and included the species A. flavus, A. parasiticus, A. oryzae, A. sojae, and
R.H. Cruickshank & J.I. Pitt
260
tamar ii, plus the newly described species A. nomius (Kurtzman et ai., 1987).
Conventional taxonomy was carried out by standard methods (Raper and Fennell, 1965)
and newly developed criteria (Klich and Pitt, 1988). Isolates for enzyme electrophoretic
studies were examined blind, i.e. without any indication of conventional taxonomic
results.
A.
Enzyme electrophoresis.
Methods for the production, electrophoresis and detection of enzymes were generally as
described by Cruickshank and Pitt (1987), but with minor changes and additions. Pectic
enzymes were produced and examined as described. To extend the range of pectic
enzyme detection to include lyases active at high pH, separate gels were incubated for 1
hr in 0.05 M tris-HCl buffer, pH 7.5, containing 4 mM CaCI2, before staining with
ruthenium red.
Amylase and ribonuclease were produced in a decoction from 10% fresh weight of
potato in distilled water, before detection as described. Protease was also produced in this
potato decoction. It was examined in 10.25% acrylamide (2.5% bisacrylamide) gels
containing 0.05% glycinin. Glycinin was extracted from defatted soybean flour using a
method based on that of Smith and Circle (1938). After electrophoresis, gels were
incubated for 1 hr in 0.1 M acetate buffer, pH 5.0, then stained for 2 hr in 0.1 % crocein
scarlet. Excess stain was removed by changes of distilled water.
RESULTS
The 129 isolates could be placed in four major groups by pectic zymogram evidence
obtained under acidic conditions (Fig. 1). and under alkaline conditions in the presence of
calcium ions (Fig. 2). This grouping was supported by characteristics of acid protease
zymograms (Fig. 3). One group coresponded with A. tamarii, a second with A. parasiticus,
and a third with A. flavus. The fourth group included isolates which were classifiable
morphologically as A. flavus but were atypical of that species, in that both B and G
aflatoxins were produced, a characteristic of A. parasiticus (Klich and Pitt, 1988). These
isolates belonged to A. nomius (Kurtzman et ai., 1987).
1 2
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Figure 1. Pectic zymograms of gels obtained under acidic conditions (0.1 M malic acid).
Numbers refer to isolates in Table 1.
Isoenzyme patterns in Aspergillus flavus and closely related species

Table 1. Isolates used in the production of figures
Lane
Isolllfe'1
Species
Source
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ATCC269501
MRC2080
VOR32
ATCC26804
MRC3425
VDR4
ATCC26768
MRC200
VOR3
ATCC 10196
MRC4243
VOR18
ATCC24109
MRC1174
VOR8
ATCC22546
MRC1318
VOR23
ATCC28540
MRCl%
VOR20
ATCCI5546
IMII90557
A. tamarii
A. tamarii
A. tamarii
A. parllSiticus
A. parllSiticus
A. parllSiticus
A. parllSiticus
A. parllSiticus
A. parRSiticus
A. oryzae
A.flavus
A.flavus
A.flavus
A.flavus
A.flavus
A. flavus
A.flavus
A.flavus
A. fIavus
A. oryzae
A.flavus
A. nomius
A. nomius
Black pepper, USA

Dried bean, Mozambique
Sunflower seed, Swaziland
Peanut,USA
Oats, S. Africa
Sunflower seed, S. Africa
Dried sausage, Poland
Com, Mozambique
Soil, S. Africa
Pine board, USA
Apricot kernel, S. Africa
Sunflower seed, Namibia
Black pepper, USA
Sorghum, S. africa
Sunflower seed, Lesotho
Corn,USA
Maize malt, S. Africa
Sunflower seed, S. Africa
Buckwheat, Japan
Cassava, Mozambique
Fenugreek seed, S. Africa
Wheat, USA
Turmeric, india
a Culture collections: ATCC, American Type Culture Collection, Rockville, MD, USA; IMI, CAB!
International Mycological Institute, Kew, Surrey, UK; MRC, National Research Institute for Nutritional
Diseases, Tygerberg, South Africa; VDR, W. B. van der Reit, Pretoria, South Africa.
I 2
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Figure 2. Pectic zymograms of gels incubated for 1 br in 0.05 M tris-HCI buffer, pH 7.5,
containing 4 mM CaCI2. Numbers refer to isolates in Table 1.
261
262
10 11
12
13
14 15 16
17 18 19 20 21
22 23
Figure 3. Acid protease zymograms. Numbers refer to isolates in Table 1.
The amylase zymograms of A. flavus (Fig. 4) and the ribonuclease zymograms of A.

parasiticus and A. flavus (Fig. 5) provided evidence of subgrouping. For example, the A.
parasiticus isolates could be placed in two subgroups by the Rf of their fast-migrating
ribonuclease isoenzymes, as in lanes 4-6 and 7-9 in Fig. 5. These subgroups did not
correspond with specific morphological properties, so far as could be determined, or, in A.
flavus, with the presence or absence of aflatoxin production.
The isolates of A. sojae examined grouped with A. parasiticus and those of A. oryzae
with A. flavus. One isolate of A. oryzae, MRC 1%, gave aberrant results (lane 20 in each
zymogram). The reason for this is not understood.
10 11 12 13 14
15 16 17
18
19 20 21 22 23
Figure 4. Amylase zymograms. Numbers refer to isolates in Table 1.
9 10 11 12 13 14 15 16 17 18
19 20 21
263
22 23
Figure 5. Ribonucelase zymograms. Numbers refer to isolates in Table 1.
DISCUSSION
This study has shown that the major species in Aspergillus sect. Flavi: A. flavus, A.
parasiticus, A. tamarii and A. nomius can be effectively differentiated on the basis of enzyme
electrophoretic patterns. The correlation with morphological taxonomy, as previously
determined by Klich and Pitt (1988), was absolute for the 129 isolates studied. This
provides independent evidence of the effective nature of the taxonomic criteria selected by
Klich and Pitt (1988) for differentiating A. flavus from A. parasiticus, particularly spore
surface texture. Moreover, the correlation between enzyme electrophoretic patterns and
secondary metabolite production is also absolute for the 129 isolates: A. flavus produces
aflatoxin Bl and B2 or cyclopiazonic acid, or both, while A. parasiticus produces aflatoxin
Gl and G2 as well as Bl and B2, but never cyclopiazonic acid.
Correlations shown between electrophoretic patterns, secondary metabolite
production and morphological taxonomy in Penicillium subgen. Penicillium are also
evident in the Aspergillus section studied here.
The anomolous isolates reported by Klich and Pitt (1988) to have the morphology of
A. flavus but to make G aflatoxins have been shown to belong to the newly described
species A. nomius, which is clearly distinct. A. nomius isolates have the morphology of A.
flavus, except that sclerotia, produced by some isolates, are smaller and vertically elongate,
not spherical like those of A. flavus. This morphological distinction correlates with their
distinctive isoenzyme patterns and the production of both B and G aflatoxins.
Enzyme electrophoretic patterns do not enable differentation of A. flavus and A.
oryzae, or A. parasiticus and A. sojae. It has already been established (Kurtzman et ai., 1986;
Klich and Pitt, 1988) that these two pairs of species are very closely related. However, as
has been argued previously (Klich and Pitt, 1988), a very strong case exists for maintaining
separate species for the food fermentation Aspergilli, regardless of the closeness of those
relationships. Most isolates of A. orYZile, and all known of A. sojae, can accurately be
described as domesticated fungi. Taxonomy and the world in general is best served by
maintaining separate species names for such important fungi.
264
REFERENCES
CRUICKSHANK, R.H. and PITT, J.1. 1987a. The zymogram technique: isoenzyme patterns as an aid in
79: 614-620.
KLICH, M.A. and PITT, J.1. 1985. The theory and practice of distinguishing species of the Aspergillus flavus
group. In Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.1. Pitt, pp. 211-220.
- - 1988. Differentiation of Aspergillus flavus from A. parasiticus and other closely related species.
KURTZMAN, CP., SMILEY, M.J., ROBNE1T, CJ. and WICKLOW, D.T. 1986. DNA relatedness among wild
and domesticated species in the Aspergillus f1avus group. Mycologia 78: 955-959.
KURTZMAN, C.P., HORN, B.W. and HESSELTINE, CW. 1987. Aspergillus nomius, a new aflatoxinproducing species related to Aspergillus f1avus and Aspergillus tamarii. Antonie van Leeuwenhoek 53: 147-158.
RAPER, K.B. and FENNELL, D.1. 1965. The Genus Aspergillus. Baltimore, Maryland: Williams and Wilkins.
SMITH, A.K. and CIRCLE, S.J. 1938. Peptization of soybean proteins - extraction of nitrogenous constituents
from oil-free meal by acids and bases with and without added salts. Industrial and Engineering Chemistry
30: 1414-1418.
DIALOGUE FOLLOWING DR. PITTS PRESENTATION
SAMSON: Doesn't your data prove, in fact, that Kurtzman et al. were right to lump these
species together? If your data show that the species are so very closely related, doesn't
this support their point of view?
PITT: Our data doesn't necessarily support their point of view, but it doesn't contradict it
either. Aspergillus flavus and A. parasiticus are really quite distinct species. Klich and Pitt
(1988) showed how to separate these species qUickly and accurately with the microscope.
There are very important differences between these species, and we must keep them
separately. I have recently been carrying out a study on the physiology of toxigenic fungi
and sorting out what we know about water relations, temperature relations and pH.
Most of the papers in the literature that talk about A. flavus and the production of
aflatoxin in relation to environmental conditions are really talking about A. parasiticus.
The critical difference is that A. flavus only produces B aflatoxins. A. parasiticus produces
both B and G aflatoxins. Most people who have reported on the physiology of A. flavus
have in fact been working with A. parasiticus, and this is clear from their reports of which
aflatoxins were produced. The literature, in fact, is very confused. Concerning A. oryzae
and A. sojae, the question of whether they are the same as A. flavus and A. parasiticus is, in
practical terms, irrelevant. Regulatory authorities and the food industry must have
distinct names for the species that are important in food fermentations.
PATERSON: Could you explain why the two A. oryzae isolates looked so different in their
pectinase patterns?
PITT: No. Dr. Cruikshank did not regard the differences as significant. I might add that
the correlation between the isoenzyme patterns and types of mycotoxin produced was
absolute.
CHRISTENSEN: What is the source of A. nomius?
SAMSON: It was isolated from mouldy wheat and diseased alkali bees.
265
PITT: It has sclerotia that are indeterminate and kind of ant hill shaped. Some isolates
don't make these sclerotia, and they look just like A. flavus. However, they make Band G
aflatoxins like A. parasiticus. It was described by Kurtzman et al. (1987) and is a good
species.
I remember seeing such a culture in Raper's collection. How does A. nomius
differ from A. Ieporis. It is characterized by columnar heads and also has indeterminate
sclerotia.
CHRISTENSEN:
FRISV AD: A.
species.
Ieporis produces lots of beautiful secondary metabolites and is a distinct
leporis is quite a characteristic species in sage brush grasslands in

Wyoming, as are Artemesia and antelope.
CHRISTENSEN: A.
Pm: I've never seen A. Ieporis. I'm interested in aflatoxin production, not in dung.
A.Ieporis has a ubiquinone Q-1O system. In contrast, most of the other species
in section Flavi have a Q-1O(H2) system. I think A. /eporis should be excluded from the
section Flavi.
SUGIYAMA:
6
COMPUTER-ASSISTED IDENTIFICATION OF
PENICILLIA AND ASPERGILLIA
269
COMPUTER APPLICATIONS IN PENICILLIUM AND ASPERGILLUS

SYSTEMATICS
M.A. Klich
United States Department of Agriculture
Southern Regional Research Center
New Orleans, Louisiana 70179, USA
SUMMARY
Computer technology is having an impact on several major areas of concern to Penicillium and
Aspergillus systematists. Use of Database Management Systems and other computer programs is
allowing curators of culture collections to readily obtain and distribute information on strains, species
and other taxa. Numeric classification systems require computers to analyse the empirical data on
which they are based. Computers are also being used in fungal identification systems. The need for a
multiuser database system for Penicillium and Aspergillus systematics is discussed.
INTRODUCTION
At the Penicillium identification workshop held by Dr J. I. Pitt and myself in Athens,

Georgia, USA in 1985, the need for an improved or alternate identification system for this
genus became obvious. The idea of developing a computer-assisted key to the common
species of Penicillium was conceived at that time. Since computer keys had not been
developed for many filamentous fungi, and users were not familiar with them, we
decided to simply computerize a key form that most mycologists are familiar with, a
synoptic key. I wrote the program in FORTRAN, using data from Pitt's books (Pitt, 1979;
Pitt, 1985) and files, information in other published descriptions (e.g. Raper and Thorn,
1949; Samson et al., 1976), data gathered by participants in the workshop, and data
gathered in my laboratory. This provided information on several isolates from each
species, grown at more than one location. The data dearly demonstrated that many
characters, including colony diameter, colour and texture, varied when a single isolate
was grown at different times and in different locations. Several microscopic
characteristics, however, were very consistent. These characters were emphasised in
PENKEY.
Most of the isolates used in the initial development of PENKEY had been held in
collections for some years. To obtain information on more recent isolates, PENKEY was
distributed on an experimental basis to researchers and university personnel in Australia
and the USA. Users were asked to return completed data sheets both for isolates for which
PENKEY provided logical identifications and for ones which it did not. Data sheets were
then checked against cultures in one of our laboratories. Data were also compiled on fresh
isolates sent directly to us for identification. Variability, especially in macroscopic
characters, remained high. However, when PENKEY was used in the Penicillium and
Aspergillus identification workshop taught by us in August, 1988 in Japan, about 80% of
identifications were correct. By this time, the ultimate need for a databank/ database with
information from many isolates grown at many locations was obvious. From such a
database, systems could be devised wherein probabilities of occurence for the various
character states, such as a colony diameter, could be utilized. Such a database cannot be
270
M.A. KUch
created in two or three years. So while we continue to offer identifications in return for
completed data sheets for the various versions of PENKEY, time exists to consider
methods for establishing the best possible Penicillium database system. The rest of this
paper is dedicated to background, thoughts and ideas that may be useful in creating
computer-based systems for Penicillium and Aspergillus systematics.
Computers and computer usage have evolved rapidly since the first commerical
computers were developed in the 1950s (Gore and Stubbe, 1983). At that time the
hardware contained vacuum tubes and software programming was difficult and very
tedious. By the mid 1960s vacuum tubes were replaced by transistors and computer
languages such as FORTRAN and COBOL were introduced. These mainframe computers
were large and expensive, and for the most part, data were processed at central locations
using punched cards. As integrated circuits were developed, smaller computers became
feasible, leading to an explosion in the development of minicomputers and
microcomputers since the late 1970s. Computers are now relatively inexpensive and
accessible to virtually everyone in developed countries. With the introduction of 'user
friendly' software packages, a computer user no longer needs to have any real
understanding of computer programming. Of special interest to those of us needing to
organize large volumes of data has been the development of data base systems in which
data, entered once, may be retrieved for many purposes. Commercial software packages
called Data Base Management Systems (DBMS) are now available and contain programs
to create, access and update databases.
It should be noted that coevolution of computer technology and the use of computers
in Aspergillus and Penicillium systematics or in other disciplines has not been
straightforward. A strong human factor exists in the decision to use computers. The
feeling that computers might take over peoples' jobs, and the inherent tendency to avoid
the unfamiliar, have delayed the acceptance of computers by many potential users. Until
recently, computer software was not 'user friendly' and potential users had to rely on the
expertise of computer scientists.
An amazing parallel exists in peoples' perceptions of computer science and
systematics. Both disciplines have a large public service component. Both computer
scientists and systematicists must develop usable systems for non-experts. Both sciences
have been accused of using jargon indecipherable to those outside the discipline and
maintaining an aura of being an exclusive priesthood. In short, some of us have not been
'user friendly'. Both disciplines have an inherent subjectivity such that even experts in the
field sometimes disagree. Both disciplines are dynamic. New computer systems seem to
appear with great frequency which is confusing to casual computer users. Users of fungal
systematics often become disgruntled at simple name changes let alone new taxonomic
systems!
In spite of the problems in using computers, their potential utility has not been
disregarded by systematists. There are three main areas of computer application in
systematics: bookkeeping, e.g. cataloging curatorial, bibliographic and biogeograpic
information; classification, e.g. numerical taxonomy; and identification. I will briefly
review relevant work in each area and then discuss potential applications to an Aspergillus
or Penicillium database system.
BOOKKEEPING
TAXIR, written for mainframe computers in the 1960s, was one of the earliest computer
systems for organization of data on organisms. In the U.K. it became known as EXIR.
Computer applications in Penicillium and Aspergillus systematics
271
TAXIR has a flat file structure which means that the information must be in a form similar
to a two-dimensional table. This is an efficient and rapid system, but not suited for data
structures that cannot be construed as flat files. Various solutions to this problem have
been devised. TAXIR has a limit of 112 characters per state and problems such as more
than one authority, etc. are difficult to solve.
Adey et al. (1984) used EXIR to develop a database for the Vicieae (the tribe of
legumes including vetches and peas), consisting of five database files (nomenclatorial,
morphological, geographical, curatorial, and chemical). Programs associated with EXIR
include EXIRPOST, used to tabulate retrieved material or to prepare it for input into other
application programs, while CONFOR is used to create printed descriptions and
SYNONYMS produces nomenclatoriallists.
Anderson et al. (1976) used a series of FORTRAN programs to assemble data on
microscopic soil fungi on the computer so that these data could be used at a later date in
published monographs. The information included reference lists, taxonomic,
physiological, and ecological data. The information from these files was used to create the
excellent reference book "Compendium of Soil Fungi" (Domsch et ai., 1980).
Many curatorial databases have been developed for individual herbaria and culture
collections. Wetmore (1979) used a DBMS called SYSTEM 2000, and keypunched cards to
computerize the herbarium at University of Minnesota. The commercial packages
Datastar, Supersort, and Wordstar were used in a system developed at the University of
Surrey for the culture collection there (Bryant, 1983). Wolff et al. (1982) used dBASE II to
create a culture collection database at the University of Waterloo, Ontario, Canada. The
New York Botanical Garden uses dBASE ill PLUS for its record keeping (Thiers, 1988).
Larger microbial database systems have included the Canadian Fungal Collection
Database (National Research Council of Canada, Halifax); the Microbial Culture
Information Service (Department of Trade and Industry, UK) which includes
physiological and biochemical data useful to industry; the Microbial Strain Data Network
(UK); and the World Data Center on Microorganisms sponsored by the World Federation
of Culture Collections (RIKEN, Saitama, Japan). These systems are coordinating several
collections and making collection information more readily accessible to outside users.
Recently, the Microbial Information Network Europe (MINE) has been developed for
microbial culture collections in Europe (Gams et al., 1988). The main function of MINE is
to provide a standardized access system for information in the major European culture
collections. The curators of the institutions participating in MINE have agreed to a general
format for computerized strain data which thereby can easily be exchanged. In this
hierarchical system (using BASIS software), a strain record is created for each strain, and a
species record to which strains may be linked contains more general data on the species.
MINE also contains files for synonyms and for teleomorphl anamorph connections. Input
into this system is strictly standardized and data is validated before being entered into the
database. The system also includes a security system to prevent unauthorized use.
NUMERICAL TAXONOMY
It is perhaps unfortunate that the first introduction of many taxonomists to the use of
computers in taxonomy was "Principles of Numerical Taxonomy" (Sokal and Sneath,

1963). Several negative comments at the beginning of the book on the training,
background and intelligence of taxonomists could not have endeared the authors to many
taxonomists at the time. Perhaps these comments helped engender a negative attitute
toward the proposed methods. The 1963 book was also idealistic in what numerical
272
M.A. Klich
taxonomy could accomplish. McNeill (1984) summarized the perceptions of early

numerical taxonomy as a four step process: 1) select a large number of attributes; 2)
measure the attributes on the population in question; 3) feed the data into the computer
(black box); and 4) obtain THE objective classification. By the time when Sneath and Sokal
published their second book (Sneath and Sokal, 1973), numerical taxonomists were much
less idealistic.
In the ten years between 1963 and 1973, 13 papers were published which used
numerical taxonomic techniques on filamentous fungi. Some studies supported existing
taxonomic interpretations. For instance, a study of Helminthosporium and closely related
taxa supported the separation of Drechslera, Bipolaris and Stemphylium as distinct genera
(Ibrahim and Threlfall, 1966). However, the results of other studies were often
unacceptable, even to the authors. Kendrick and Weresub (1966) used the methods
suggested by Sokal and Sneath (1963) in a study of the resupinate Basidiomycetes. They
noted in their abstract that "using haphazardly assembled, equally weighted characters
results in a haphazard classification, at least in the Basidiomycetes." The discussion of this
method in the paper itself was even less kind. Sneath and Sokal (1973, p. 420) responded
to this criticism by stating again that phenetic techniques will not necessarily yield the
same relationships as traditional methods.
Some taxonomists did not realize immediately that the purpose of numerical
taxonomy was to construct classes rather than to help taxonomists assign indiviuals to
previously established classes. There has been a continuing conflict between proponents
of phylogenetic classifications and hierarchical (phenetic) classifications. Computer based
systems are not intended to reflect evolution (Sneath and Sokal, 1973). However, as Gower
(1975) states, "Evolutionists ask that, if the hierarchies are not estimates of phylogeny and
usually cannot be used in diagnostic keys because they are polythetically based, what use
are they?" Numerical classification has not lived up to its promise of providing
unambiguous and respected methods for constructing classifications, because, according
to Heywood (1984), there is no one 'true' or 'correct' classification. Different classifications
will be produced when different methods are used.
A different kind of criticism of numerical classifications is that they are so frequently
concordant with conventionally produced classifications that they are not worth the effort
involved to produce them (Heywood, 1984). This argument could be interpreted as either
indicating that the previous classifications were essentially correct or that it has not been
possible to eliminate subjectivity in the numerical techniques.
The concept of unweighted characters in the Sokal and Sneath (1963) method was
another heavily criticised aspect of numerical taxonomy (Hogeweg, 1976; Kendrick, 1965).
Various methods have been developed to give more weight to some characters than to
others. To Sokal and Sneath (1963), this process was seen as adding subjective factors into
the numerical taxonomic system. Kendrick and Proctor (1964) found that in unweighted
systems similarities or differences in secondary characters could outweigh similarities or
differences in primary characters, leading to anomalies in the relationships between the
taxa as measured by the similarity coefficients. In Aspergillus and Penicillium, for instance,
presence of metulae would be considered a primary character, and size, shape, or number
of metulae secondary characters. In an unweighted analysis, the similarity coefficient for
species with metulae might be artificially high or low because of similarities or differences
in the secondary characters. In their study, Kendrick and Proctor (1964) gave additional
weight to primary characters, and were able to construct a satisfactory dendrogram for the
Hyphomycetous species they were examining. They did not define 'satisfactory' but
apparently meant a system close to that derived by standard taxonomic methods.
Co~uter
applications in Penicillium and Aspergillus systematics
273
One benefit of numerical taxonomy has been that it has forced taxonomists to reappraise
their methodology. For the most part, taxonomists have accepted the fact that there is an
inherent subjective component in this discipline, but at the same time acknowledge that
empirical analysis of data is a vital part of developing modern classification systems.
Frank and Zwanziger (1988) summed up the situation well: "the initial enthusiasm for
numerical methods has smoothed down; the main reasons are both misinterpretation of
the aims and exaggerated expectations in the infallibility of the mathematical apparatus."
They suggest that taxonomists remain aware that computers are only an aid in taxonomy:
computers can only make suggestions, and should never dictate solutions.
IDENTIFICATION
Part of taxonomy is a service industry. Once a classification system has been established, it
must be communicated in the most efficient way possible to users. Computers can help to
define characters most appropriate for identification keys and even write identification
keys based on information in a classification system. Payne and Preece (1980) reviewed
the theory and practice of keys and key development as well as the statistical theory
involved in creating efficient keys of all kinds.
Several systems are available for generating keys. Hall (1970) created a dichotomous
key system in which the couplets were chosen by the computer from a data matrix. At the
time this required a mainframe computer. Johnston (1980) used the computer to generate
polyclaves (punch card systems) from data matrices. Rhoades (1988) has developed a
taxonomic database which will generate synoptic keys and has constructed such keys to
several groups of fungi.
Kendrick (1972) used computer graphics to write a key to the didymosporous
Hyphomycetes. In this ingenious key, images appear on the screen and the user picks one.
After a series of choices, the final image contains the name of the fungus, a diagram of its
salient features and a brief written description. Margot et al. (1984) devised an on-line
identification program for mushrooms. Written in FORTRAN, this key is based on a data
matrix and is essentially synoptic.
One of the earliest large computer-assisted mycological identification keys was to the
yeasts (Barnett and Pankhurst, 1974). In this key, 52 physiological tests were used to
identify 434 species of yeasts. The species were divided into eight groups, and then a
dichotomous key was generated to the species within each group. The six physiological
tests used to divide the species into eight groups were selected based on their separation
coefficient values. The character which best divided the species into two equal groups had
the highest separation coefficient. The dichotomous key for each group was then
generated by the computer, using the characters with the lowest levels of intraspecific
variability (Barnett and Pankhurst, 1974).
The first major review of biological identification systems using computers was
published in 1975 (Pankhurst, 1975). It was generally agreed that computer-stored
dichotomous keys had little advantage over written dichotomous keys. However, several
dichotomous keys had been generated by the computer using 'recursive algorithms',
which repeatedly divided the taxa into two mutually exclusive subsets, with each
application producing one couplet in the key. Discriminant analysis, which involves
weighting characters by their ability to help distinguish between two taxa or clusters of
taxa, was already being used to aid identification procedures involving taxa with many
overlapping characters. The disadvantage of discriminant analysis is that it requires the
274
M.A. Klich
observer to record a large number of characterisitics per specimen. Cluster analyses and
similarity coefficients of various sorts were also being used at that time.
Several problems were recognized with the computer techniques (Pankhurst, 1975).
Taxonomists were often not concerned with empirical analysis, so were often inconsistent
in their species descriptions, leaving out characters which would be necessary for
developing computer-based identification systems. There was apparently a feeling at the
time that if computers could perfonn a task, they would do it better than humans. Also,
many taxonomists were unfamiliar with the capabilities and limitations of computers in
their field. Now, many taxonomists are familiar with these limitations. Species
descriptions in recent works tend to be more detailed, and most people are much more
realistic about use of computers because computers are more a part of daily life.
PREVIOUS USE OF NUMERICAL METHODOLOGIES IN STUDIES OF ASPERGILLUS
AND PENICILLIUM SYSTEMATICS
A variety of approaches to Penicillium and Aspergillus taxonomy have made use of

numerical methodology. Cluster analyses have been used to study relationships based on
long chain fatty acids in both Aspergillus and Penicillium. Seventeen species of Penicillium
fell into three major groups, but the groups bore no resemblance to any known taxonomic
scheme (Dart et ai., 1976a). The same was true for cluster analyses of long chain fatty acids
for 14 species of Aspergillus, although intraspecific variation in Eurotium amsteiodami and
A. niger was quite low (Stretton et ai., 1976; Dart et ai., 1976b). When the Aspergillus study
was repeated at different temperatures, the clusters were quite different, with only two
major clusters, one of which contained all four Eurotium species (Lee et ai., 1977). In a
study of both Aspergillus and Penicillium it was concluded that the two genera could not be
separated from one another by differences in their long chain fatty acid composition (Lee
et ai., 1976).
In her revision of the black-spored Aspergilli, AI-Musallam (1980) conducted

numerical analyses on 78 isolates related to A. niger. In unweighted and weighted analyses
based on 28 morphological features, characters having the highest significance in forming
the clusters were very similar. These included colony diameter on Czapek's medium,
conidial head color, conidial ornamentation, stipe length and vesicle diameter. In each of
the two analyses, two major clusters and several minor clusters (subclusters) were formed.
The major clusters were recognized as species (A. niger and A. foetidus) and the subclusters
as varieties and formae. A key to these taxa was written using the characters of
importance from the numerical analyses.
To better understand the koji moulds used in Oriental fermentations, Murakami
(1971) used both numerical and traditional methods to examined 406 isolates from
Aspergillus sect. Fiavi, including 306 strains of koji moulds. His numerical analysis was
based on 20 features, each of which had three character states, some of which were
qualitative (e.g. none, little, much) rather than empirical. Significant characters were
established, including conidial diameter, conidial wall texture, pink conidial color on
anisaldehyde medium and anisic acid medium, reverse coloration, production of
aflatoxins, kojic acid and sclerotia. After these and other characters were tested in the
laboratory, the most consistent characteristics were considered to be most suitable for use
in identification keys. Many of the above characteristics, as well as presence or absence of
metulae and wrinkling of the colony reverse, were used to write a key to the koji moulds.
Christensen (1981) generated similarity coefficients (C) from data in a synoptic key to
members of Aspergillus sect. Flavi to assess species relationships. The most closely related
Computer applications in Penicillium and Aspergillus systematics
275
species were A. toxicarius and A. flavus (C= 0.96). She suggested that A. toxicarius is an
occasionally biseriate A. parasiticus. Others have also considered A. parasiticus and A.
toxicarius to be synonymous (eg. Klich and Pitt, 1985). A. parasiticus was also found to be
quite similar to A. sojae (C= 0.81). However, A. oryzae was 'comparatively distinct' from A.
flavus (no value given).
Klich and Pitt (1985) also studied Aspergillus sect. Flavi. Based on discriminant analysis
on morphological features of 62 isolates, five characters were judged most satisfactory for
separating A. flavus, A. oryzae, A. parasiticus, A. sojae, and A. tamarii. These were colony
colour; conidial size and surface texture; vesicle diameter; conidiophore length; and
presence or absence of metulae. Several of these characters were similar to those used by
Murajkarni (1971) to distinguish koji moulds.
The Subcommission on Penicillium and Aspergillus Systematics (SPAS) of the
International Commission on Taxonomy of Fungi is currently completing its first study.
Isolates of the four closely related species P. glabrum, P. spinulosum, P. purpurescens, and P.
montanense were examined for standard morphological features, secondary metabolites,
isoenzymes, and DNA restriction fragment length polymorphisms. Species differentiation
by the various approaches was in fairly good agreement. To determine the best
discriminating characters, the morphological data were analyzed using discriminant
analysis. In the preliminary analyses, conidial wall texture, maximum diameter on CYA
and minumum on G25N (Pitt, 1979), minimum phi ali de width and maximum vesicle
diameter were the most useful characters. Currently, a larger sample is being analyzed to
test and refine the model.
A numerical taxonomic study of Penicillium subgen. Penicillium is currently being
conducted at the CAB International Mycological Institute (CMI) (Bridge et al., 1985). This
large undertaking, involving the acquisition and analysis of 195 characters for each of 300
isolates, will be reported elsewhere in these Proceedings (Bridge et al., 1990).
In the past few years, several computer assisted identification systems have been
developed. PENKEY (Klich and Pitt, 1988) is a computer assisted synoptic key to common
Penicillium species. PENNAME (Pitt, 1990) is a dBASE III Plus computer key to the same
set of species. A key to Penicillium subgen. Penicillium has been developed in Apple BASIC
for the food industry (Williams, 1990), and a further key to this subgenus (Bridge, 1990) is
also presented in these proceedings.
TAXONOMIC DATABASE SYSTEMS FOR ASPERGILLUS AND PENICILLIUM
Development of a multiuser /multicontributor database systems for Aspergillus and

Penicillium would faciltiate research efforts on these two genera. First they would allow us
to attain a greater understanding of intra- and inter-specific variability so that future
classification and identification systems may be based, as much as possible, on empirical
data. Second, these database systems would serve as focal points for cooperative or
coordinated efforts among researchers at different institutions. Crovello et al. (1984)
pointed out that systematics has suffered from the lack of coordination among
institutions. A shared system would prevent duplication of efforts, provide data for
discussion, and provide taxonomic researchers with more information than anyone
researcher alone could obtain. As new biochemical and genetic methods are developed
they will provide additional tools for taxonomy, and these can easily be integrated into
database systems.
What should be the characteristics of the ideal Aspergillus or Penicillium database
system? First it should be easy to create and update files as the systems expand. It should
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MA Klich
be easy to create and combine subsets of data from different files in the system. It should
be amenable to statistical manipulation, key generation, etc. The information and current
updates should be easy to transfer among users and capable of integration into other
database systems such as those of culture collections. Files or subsets of files should be
easy to print. Finally, the system should be inexpensive to run.
Several major decisions will be faced by the potential creators and users of such a
database before commencing the work. One need only thumb through a recent computer
magazine or a review such as "Databases in Systematics" (Allkin and Bisby, 1984) to
realize that there are a number of potential Database Management Systems from which to
choose. The strengths and weaknesses of these relative to the objectives and desirable
characteristics of the Aspergillus and Penicillium systems should need to be carefully
considered. Decisions would need to be made on the characters to be entered. Although it
is not difficult to add characters to some systems, a well defined intial set would reduce
complications later.
The technical difficulties are probably minor compared to the practical problems in
setting up a taxonomic database system to be used by participants from many institutions.
How would the work be funded? Who would have access to the system? How would use
of the system be acknowledged in publications? How often would the system be updated,
and how would the updates be distributed? Perhaps these and other practical
considerations can be discussed during the final session of this meeting.
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AL-MUSALLAM, A. 1980. Revision of the black Aspergillus species. Ph.D Thesis. Utrecht, Netherlands:
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ALLKIN, R. and BISBY, F. A., eds. 1984. Databases in Systematics. Orlando, Florida: Academic Press.
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DART, R. K., STRETTON, R. J. and LEE, J. D. 1976a. Relationships of Penicillium species based on their longchain fatty acids. Transactions of the British Mycological Society 66: 525-529.
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DOMSCH, K. H., GAMS, W. and ANDERSON, T. H. 1980. Compendium of Soil Fungi. London: Academic
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FRANK, H. M. and ZWANZIGER, H. 1988. Macrochemical color reactions of macromycetes. VI. Cluster
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Orleans, Louisiana: privately published.
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279
PENNAME, A NEW COMPUTER KEY TO COMMON PENICILLIUM

SPECIES
J.I. Pitt
CSIRO Division of Food Processing
SUMMARY
Problems in identifying Penicillium species are well recognised. Fundamentally, these result from a
lack of completely stable characters of taxonomic value. Conventional dichotomous keys use a
relatively few characters, and hence are sensitive to variation in them. The synoptic key is inherently
more effective for this difficult genus, but the large number of species makes synoptic keys unwieldy
to use. Designed to overcome this problem, PENNAME is a new computer based synoptic key to 68
common Penicillium, Eupenicillium and Talaromyces species. It is designed to be used with "A
Laboratory Guide to Common Penicillium Species" (Pitt, 1988), but can be of value with other modem
taxonomies as well. PENNAME is "user friendly", accepting keyed in data on colony and microscopic
characters directly on screen. Output in the current experimental version consists of a list of species,
with the number of characters by which each differs from the test isolate. Later versions are intended
to be "stand alone", with simple taxonomic treatments of common species included.
A fundamental problem in the identification of Penicillium species is that nearly all

characters of taxonomic value lack complete stability. Conventional dichotomous keys use
a relatively few characters, and hence are sensitive to variation in those characters. A
second problem, common to most fungal genera, is that data available is often incomplete,
or questionable, making dichotomous keys cumbersome and difficult to use. The synoptic
key (Leenhouts, 1966) is inherently more effective for difficult genera, but the large
number of species in Penicillium makes a synoptic key unwieldy to use.
Designed to help overcome this problem, PENNAME is a new computer key to
Penicillium species. At present, it will key the 52 most common species of the genus,
together with 10 Eupenicillium and 6 Talaromyces species, but in time it will be expanded to
accommodate all currently accepted species. At present, it is designed primarily for use
with the second edition of ''Laboratory Guide to Common Penicillium Species" (Pitt, 1988),
and it keys the species included in that work. However, it will be of value as an adjunct to
any modern taxonomy, provided the media, general methods and plating regime of Pitt
(1979) are used, i.e. CYA, MEA and G25N at 250, plus CYA at 50 and 370, and incubation
for 7 days.
PENNAME is basically a synoptic key, and operates by comparing input data with
standard information contained in the program's databases. In this respect PENNAME is
similar to PENKEY (Klich and Pitt, 1988). Like PENKEY, PENNAME is a "stand alone"
program, and it can be run on any IBM PC (registered) or compatible computer, using MSDOS (registered) or similar operating system.
Unlike PENKEY, which is written in FORTRAN, PENNAME is written in dBase III
Plus (registered) software, and compiled in Clipper (registered). This leads to a number of
advantages. PENNAME also has several interesting features relating to the data input
280
J.I. Pitt
system, the primary data output, and various secondary choices. These are described in
more detail below.
Data input.
Input of data into PENNAME is particularly simple. Raw data on colony diameters and
pigmentation, together with microscopic features of the unknown isolate are input
directly onto a screen entry form. The PENNAME program then interacts directly with
this data, interpreting it in a form in which it can be compared with standard data. The
program calculates certain other parameters, i.e. metulae to phialide length ratio, and
conidial length to width ratio, and uses these data also. Depending on responses to
questions in the data input, PENNAME may call for supplementary data on a second
screen entry.
Primary output.
The primary output provided by PENNAME is a list of eight species names, which the
program considers to be the most likely identifications for the unknown. Beside each
species name is displayed a "count" figure, which represents the number of characters by
which the unknown differs from the standard description of that species. PENNAME is
quite a stringent key: a count of 0 indicates a high probability that the unknown has been
correctly classified, while a count greater than 4 indicates a high probability of a
mismatch.
Secondary output.
The user then has the choice of looking more closely at the characters by which the
unknown differs from a particular species. Entry of a three letter code for the species at the
appropriate prompt produces a display of entered characters or data ranges which do not
match those of the chosen species, and a display of the corresponding characters or data
ranges which the chosen species does exhibit. Up to four such sets of differing characters
will be displayed. Although designed to be used with the most closely matching species
shown on the primary output display, any species may be compared with the unknown
by entering the appropriate three letter code. The codes are mostly the same as the first
three letters of each species name, but have been modified where two species have the
same first three letters. A complete list of the codes is provided with notes on use of the
key.
For reasons of space, the secondary output is rather cryptic for characters which involve
numerical ranges. However, an explanation is provided with the notes to PENNAME, and
usage of the key will rapidly lead to familiarity.
Probability figure.
Accompanying the secondary output is a "probability figure" designed to indicate the
likelihood of the unknown being the species selected for comparison in the secondary
output. It is emphasised that this figure is based on the input data, and cannot for obvious
reasons take into account inaccurate raw data. The figure is based on calculations which
relate both to the number of species with a low "count" figure, and a comparison of data
ranges input for the unknown with standard ones for the selected species. It is also
emphasised that the probability figure can only be taken as a guide at present.
Penname, a new computer key to common Penicillium species
281
Species diagnosis.
PENNAME also can provide brief diagnoses of all the included species. The user can
indicate a wish to see a diagnosis of a particular species, again by typing in the three letter
code for the species at the appropriate prompt.
Advantages of PENNAME.
As with all synoptic key systems, data entered into PENNAME may be as much or as little
as is available. This provides great flexibility in use, including applications other than
determinative taxonomy. However, In the author's opinion, PENNAME possesses several
additional advantages over similar keys now available:
1. PENNAME is up to date, being based on Pitt (1988).
2. The databases used may be readily modified by the program originator, to allow
updating of "standard" information, changes in species names, or the addition of
extra species, as required.
3. Data entry is simple and direct, with no user manipulation necessary. The
program calculates further useful data from the input also.
4. Most of the data to be entered is simply acquired, and requires little or no
knowledge of Penicillium taxonomy. In particular, lack of certainty about
penicillus type will rarely produce a misleading output. For example, although the
experimental version displayed at this Workshop asks the user to differentiate
where possible between penicilli belonging to subgen. Furcatum and subgen.
Biverticillium, later versions will not. PENNAME will permit quite effective
determinations for many species when no penicillus type is specified at all. Later
versions will take advantage of this by suggesting a first run where doubt on
penicillus type exists, and a second more definitive run based on the output from
the first.
5. PENNAME allows considerable interaction with the user. The secondary output
enables the user to quantitatively compare data from an unknown isolate with any
number of known species. Diagnoses may also be checked directly within the
program. It is believed this user interaction will be of great value.
Detailed notes on the use of PENNAME will accompany the software, which may be
obtained on a 5.25 inch disc from the author.
REFERENCES
KUCH M. A. and pm, J. I. 1988. A computer-assisted synoptic key to common Penicillium species and their
teleomorphs. New Orleans, Louisiana: privately published.
LEENHOUTS, P.W. 1966. Keys in Biology. A survey and a proposal of a new kind. Proceedings Koninklijke
Nederlanse Akademie van Wetenschappen, Series C, 69: 571-596.
Academic Press.
- - 1988. A Laboratory Guide to Common Penicillium Species. 2nd ed. North Ryde, N.S.W.: CSIRO
283
IDENTIFICATION OF TERVERTICILLATE PENICILLIA FROM A MATRIX

OF PERCENT POSITIVE TEST RESULTS
P.D. Bridge
CAB International Mycological Institute
Ferry Lane, Kew, Surrey, TW9 3AF, UK
SUMMARY
The fifty-seven most discriminatory characters from an integrated multidisciplinary numerical
taxonomy were used to produce a percent positive matrix for 37 species and species groups of
terverticillate and similar penicillia. Identification of unknowns against the percent positive matrix
was determined by two methods: the first gave a normalized probability score and the second
calculated the Modal Likelihood Fraction (MLF), which was based on both the highest probability
attained and the theoretical highest probability. The matrix and identification procedures were tested
with the results from over 100 known and unknown cultures. In most cases a normalized probability
score of 0.99 or better to the correct taxon was obtained. The MLF scores obtained were very low,
reflecting the high level of variation in most taxa; 10-9 appeared to be a suitable cutoff point, aU except
one isolate giving values in excess of this to the correct taxon.
The merits of this quantitative identification scheme are discussed, and the selection of the most
suitable identification score is considered.
INTRODUCTION
The use of percent positive tables and computer programs to produce computer assisted
identification matrices is well established in microbiology (Sneath, 1978; Willcox et ai.,
1980; D'Amato et ai., 1981). The detailed methodology of identification matrices is well
described elsewhere, and methods also exist for assessing their performance and
discrimination (Sneath and Sokal, 1973; Willcox et ai., 1973; Willcox et ai., 1980; Sneath,
1980 a, b, c). Unlike keys, identification from a percent positive matrix uses all of the
available information and so the effect of a single erroneous result or an atypical isolate is
minimised. Most computer assisted systems will operate on the data available for an
unknown and so can provide identifications from incomplete data sets (e.g. Sneath,
1979a).
The results from an integrated multidisciplinary numerical taxonomy of terverticillate
penicillia which defined 37 taxa with 100 characters were used to produce an
identification matrix (Bridge et al., 1989a, b). The full data set was examined for the most
discriminatory characters with the programs CHARSEP and DIACHAR (Sneath, 1979b,
1980a) and a matrix containing 57 characters for the 37 taxa was produced. The
identification scores obtainable for the most typical member of each group were
determined with the program Mosrryp and overlap between groups was estimated with
the program OVERMAT (Sneath, 1980b, c). These last two procedures suggested that the
groups within the matrix were distinct on the characters included and that typical isolates
should emerge with high identification scores. The full matrix has been published
elsewhere (Bridge et al., 1989b) and this contribution considers some particular features of
the data and the identification coefficients.
284
P.O. Bridge
IDENTIFICATION COEFFICIENTS
A large number of different identification coefficients have been proposed for use with
identification matrices. Coefficients can be based on the similarity of an unknown to the
taxa, the distances of unknowns from the taxa,. or the probability of the unknown
belonging to the taxa; for full details see Sneath and Sokal (1973) or Willcox et al. (1980).
Probability based coefficients are generally used, and values in the percent positive table
are considered to be the probabilities of properties being recorded for the taxa. An
important consideration in the use of probability coefficients and matrices is that they can
be used to calculate the likelihood of an unknown belonging to anyone of the taxa
included. They assume however that the different taxa are distinct and that the unknown
belongs to a taxon included in the matrix.
A number of formulae are available for calculating identification scores from
probabilities, the most generally used being the Willcox score, which is based on Bayes'
theorem (see Willcox et al., 1973):
P(U) P(tlU)
P(U I t) = LU P(U) P(t I U)
where p(U I t) is the probability for taxon U on t characters; P(U) is the prior probability for
taxon U and P(t I U) is the probability that a member of taxon U will show the characters t.
This theorem gives a relative or normalized probability by dividing the final best
absolute likelihood by the sum of the scores to all taxa. This method acts as a correction if
some taxa have extremely variable character states and also indicates if there is only little
difference between the best and other identifications. A further feature of Bayes' theorem
is that it includes values for prior probalitities, so that final scores can be weighted by the
likelihood of isolating a particular taxon (see Sneath and Sakal, 1973; Willcox et al., 1980).
This is particularly useful if the data matrix contains both commonly and rarely isolated
taxa. In practice, however, it is difficult to accurately estimate prior probabilities in
advance and so they are usually set equal for all taxa and cancel out.
Identification scores based on Bayes' theorem are to a certain extent dependent upon
the other taxa in the matrix. As a result, if an unknown does not belong in any group in
the matrix, it may still give a high identification score if it is significantly less unlikely that
it belongs to one taxon as compared to the others. One way of controlling this is to take
account of any mis-matches in characters, so that an unknown that gave a high
identification score but only poorly matched to that taxon would be noticed.
Identification scores that do not consider the other taxa directly in their calculation can
also be useful in these circumstances. One example is the Modal Likelihood Fraction
(MLF; Dybowski and Franklin, 1968), which is the ratio of the absolute likelihood that an
unknown belongs to a taxon and the absolute likelihood that would be obtained for the
most typical isolate on the characters used.
DETERMINATION OF IDENTIFICATION
The identification matrix was used with the program MATRIX to identify a collection of
52 previously identified isolates and 51 unidentified isolates. One isolate remained
unidentified from this set and one isolate was identified to the correct species but to an
incorrect variety. The criteria used in the program MATRIX were the Willcox
identification score and the MLF. Correct identifications were made when the Willcox
285
score was greater than 0.9 and the MLF was greater than 10-9 In six cases the
identification score was less than 0.9 to the correct group, but the MLF score was
satisfactory (Bridge et al., 1989b).
The Willcox identification score has been used in bacteriology and the value used to
determine a "good" identification has varied in practice from 0.85 to 0.999 (Willcox et al.,
1973; Feltham and Sneath, 1982; Williams et al., 1983; Priest and Alexander, 1988). The
performance of the Penicillium matrix is within this range, and is therefore considered
satisfactory. However, several authors have pointed out the dangers of relying solely on a
single Willcox score for identification (e.g. Feltham and Sneath, 1982; Priest and
Alexander, 1988). The program MATRIX also gave an MLF score and listed any possibly
incorrect test results.
The MLF score was introduced by Dybowski and Franklin (1968), but they did not
suggest any cut off level for accurate identifications. The most typical member of a taxon
would give a score of 1, while atypical members could give very reduced scores. The
results from the Penicillium matrix showed that in this case there appeared to be a natural
cutoff value at about 10-9, although most clear cut identifications were made when the
MLF was greater that 10-6 (see Bridge et al., 1989b). However, a number of factors can
affect the final value of an MLF score. MLF is dependent on the score obtained for the
most typical member of the taxon: if this organism does not exist in practice, this would
reduce the MLF. IT a taxon consisted of a group of organisms that show significant
variation in the characters used, then further isolates could be expected to be some
distance from the most typical, and so again the MLF would be reduced. One significant
advantage with the MLF score was that with the Penicillium data it acted to some extent as
an indicator for excluded taxa. Strains belonging to some Penicillium species not included
in the matrix were tested. The Willcox identification scores were often quite high and
could have indicated an erroneous identification. However, the MLF scores were very low
indeed in the order of 10-19 or less.
PERFORMANCE WITH REDUCED SETS
Unlike a traditional identification key, an identification from a matrix does not normally
emphasize particular characters (Sneath, 1978). As a result an identification matrix
procedure can be used with a reduced data set. This was tested with the Penicillium matrix
by attempting to identify a number of strains chosen at random with a variety of reduced
data sets. The data used in the construction of the Penicillium identification matrix were
taken from a multidisciplinary study and included characters from physiological,
biochemical and SEM techniques in addition to morphological characters (see Bridge et al.,
1989a; 1989b). Test reduction to produce reduced data sets was performed by excluding
blocks of characters, such as all metabolite characters or all conidial characters, to give
data sets that varied from 57 to 15 characters. Although the detailed performance of the
matrix varied depending upon the isolate used, correct identification was possible with
the MLF score and the Willcox identification score. As examples, some results from two
isolates are given in Table 1. PP280 was a well characterized isolate used in the original
numerical taxonomy, and strain IMI 154241 was a line of the ex-type culture of P.
granulatum not included in the main study. These results showed that identification to a
particular group was not necessarily dependent upon one type of the character, and that
different character sets were in general complementary. This would be expected if the taxa
were accurately described by all of the tests.
286
P.O. Bridge
Table 1. Identification performance with reduced data sets.

(i) Penicillium hirsutum PP 280
set of characters used

All
Spore characters excluded
Spore and metabolite characters excluded
Physiology and SEM
Plate and slide morphology
Plate morphology
number of
tests
identification score
to corred taxon
MLFscoreto
correct taxon
57
41
28
23
21
15
0.9999998
0.9997911
0.8587269
0.9398831
0.9971092
0.9985357
4.9xlO-5
4.7xl0-3
5.5xlo-3
4.7xl0-4
4.4xl0-4
0.105
number of
tests
identification score
to corred taxon
MLFscoreto
correct taxon
57
41
28
23
21
15
0.9999982
0.9913316
0.9942101
0.7245676
0.9914898
0.8101497
(ii) Penicillium granulatum IMI 154241
set of charaders used

All
Spore characters excluded
Spore and metabolite characters excluded
Physiology and SEM
Plate and slide morphology
Plate morphology
l.4xl0-4
5.9xl0-4
0.493
0.493
0.119
0.243
CONCLUSIONS
This is the first report of probability based identifications in the filamentous fungi, and the
initial results are very encouraging. The identification matrix for the terverticillate
penicillia and the associated computer programs can successfully identify unknown
cultures at species level. Pitt (1985) suggested that 70-80% of isolates of Penicillium from
common sources were readily identifiable using gross morphological and cultural
features. This figure was substantially improved on in the current tests of the percent
positive matrix (Bridge et al., 1989b), although it is recognized that many more strains
need to be studied for a complete evaluation. The performance of the identification
procedure is ultimately a test of the classification embodied within it. Here the results
supported the species concepts developed by Bridge et al. (1989a).
In this case, the Modal Likelihood Fraction proved to be a more reliable measure of
identification than the usual Willcox identification score. However, the MLF has been
used in few practical systems prior to this work, and so there is little additional experience
for comparison. As the MLF is derived from the probabilities involving a single taxon and
its most typical member, similar results may be obtained with one of the coefficients based
on taxon radius (Sneath and Sokal, 1973).
This investigation into matrix performance with reduced data sets has been very
encouraging. Although further investigation is needed, the results suggest that in most
cases basic reliable identifications can be made with reduced data sets consisting of mainly
gross morphological and cultural characters. However, the more critical characters will be
needed in some cases.
287
ACKNOWLEDGEMENTS
This work was supported by Science and Engineering Research council contract 50/17/84
"Systematics of microfungi of biotechnical and industrial importance". I would like to
thank Prof. P.H.A. Sneath and M.J. Sackin, Department of Microbiology, Leicester
University, for their considerable help and discussions in developing the matrix.
REFERENCES
BRIDGE, P.D., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, R.R.M.,
SACKIN, M.J. and SNEATH, P.H.A. 1989a. A reappraisal of the terverticillate penicillia using
biochemical, physiological and morphological features I. Numerical taxonomy. Journal of General
Microbiology (in press)
BRIDGE, P.O., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, RR.M. and
SACKIN, M.J. 1989b. A reappraisal of the terverticillate penicillia using biochemical, physiological and
morphological features II. Identification. Journal of General Microbiology (in press)
D'AMATO, RF., HOLMES, B. and BOTTONE, E.J. 1981. The systems approach to diagnostic microbiology.
CRC Critical Reviews in Microbiology 9: 1-44
DYBOWSKI, W. and FRANKLIN, D.A. 1968. Conditional probability and identification of bacteria: a pilot
study. Journal of General Microbiology 54: 215-229
FELTHAM, R.K.A. and SNEATH, P.H.A. 1982. construction of matrices for computer-assisted identification
of aerobic gram- positive cocci. Journal of General Microbiology 128: 713-720
pm, J.1. 1985. A Laboratory Guide to Common Penicillium species. North Ryde, N.S.W.: CSIRO Division of
Food Research
PRIEST, F.G. and ALEXANDER, B. 1988. A frequency matrix for probabilistic identification of some bacilli.
Journal of General Microbiology 134: 3011-3018
SNEATH, P.H.A. 1978. Identification of microorganisms. In Essays in Microbiology, eds. J.R Norris and
M.H. Richmond, pp. 10/1-10/32. Chichester: John Wiley and Sons.
- - 1979a. BASIC program for identification of an unknown with presence-absence data against an
identification matrix of percent positive characters. Computers and Geosciences 5: 195-213
--1979b. BASIC program for character separation indices from an identification matrix of percent positive
characters. Computers and Geosciences 5: 349-357
- - 1980a. BASIC program for the most diagnostic properties of groups from an identification matrix of
percent positive characters. Computers and Geosciences 6: 21-26
- - 1980b. BASIC program for determining the best identification scores possible from the most typical
examples when compared with an identification matrix of percent positive characters. Computers and
Geosciences 6: 27-34
- - 198Oc. BASIC program for determining overlap between groups in an identification matrix of percent
positive characters. Computers and Geosciences 6: 267-278
SNEATH, P.H.A. and SOKAL, R.R. 1973. Numerical Taxonomy. San Francisco: W.H. Freeman and Sons
WILLCOX, W.R., LAPAGE, S.P., BASCOMB, S. and CURTIS, M.A. 1973. Identification of bacteria by
computer: theory and programming. Journal of General Microbiology 77: 317-330
WILLCOX, W.R., LAPAGE, S.P. and HOLMES, B. 1980. A review of numerical methods in bacterial
identification. Antonie van Leeuwenhoek 46: 233-299
WILLIAMS, S.T., GOODFELLOW, M., WELLINGTON, E.M.H., VICKERS, J.c., ALDERSON, G., SNEATH,
P.H.A., SACKIN, M.J. and MORTIMER, A.M. 1983. A probability matrix for identification of some
Streptomycetes. Journal of General Microbiology 129: 1815-1830
289
IDENTIFICATION OF PENICILLIUM AND ASPERGILLUS: COMPUTER

ASSISTED KEYING
AP. Williams
Leatherhead Food R.A.
Randalls Road, Leatherhead, Surrey, KT22 7RY, UK
SUMMARY
Ouring the last few decades, practical identifications schemes for bacteria and yeasts have abandoned
the use of dichotomous keys, in favour of the recognition of profiles of reactions typical of the
organisms sought. Examples of such profile schemes include the A.P.1. bacterial and C.O.M.P.A.S.S.
yeast identification systems. Identification by profile has several advantages over dichotomous keys,
including the use of a constant range of criteria, the capacity to incorporate variable results and ready
adaptation for speedy use on a microcomputer. Additionally, the principles of such identification
schemes are readily understood by microbiologists who only occasionally need to identify moulds.
Now that more data on physiological characteristics of common moulds are available to supplement
existing morphological information it is possible to construct identification keys that take into account
various aspects of the whole organism. This presentation discusses ways of simplifying the
identification of species of Penicillium subgenus Penicillium and Aspergillus.
INTRODUCTION
Although the following discussion is related primarily to the identification of species of

Penicillium and Aspergillus, the intention is to encourage improved identification schemes
for any suitable fungal genera. Penicillium subgenus Penicillium and Aspergillus have been
selected because of their dominance of the spoilage mycoflora of many types of food,
especially processed food. Additionally, subgenus Penicillium (species with terverticillate
penicilli) includes species that are among the most difficult to distinguish by conventional
methodology.
First, we must decide whether improved identification schemes are needed and, if so,
who needs them. Certainly the professional mycologist is very likely to be familiar with
the characteristics of moulds within a given area of interest and is more likely to be
concerned with the taxonomic implications of the differences between those moulds. On
the other hand the Quality Assurance or Control Microbiologist in industry, who rarely
has had any formal mycological training, is occasionally faced with a mould spoilage
problem. He then needs to know the significance of the species present, those properties
of the mould that have given rise to the problem such as heat resistance, psychrotolerance,
xerotolerance etc., the likely source of the contamination and the means to control it.
Although accurate identification remains fundamental, the extent to which detailed
speciation is necessary will be much more subjective. For example, where survival of heatresistant spores is suspected, it is obviously desirable to recognise species in Byssochlamys,
Neosartorya and Talaromyces that might be implicated. On the other hand, few would wish
to speciate the occasional spoilage isolate of Phoma from a dairy product, because no
further useful information would be gained.
290
A.P. WiIUams
The busy microbiologist in industry has a number of requirements of any identification

scheme, whether bacteriological or mycological. First and most important he must be told
clearly what test to do and what to record. The criteria used must have reproducibility
and should, as far as possible, be easy to observe and should not involve scarce or
prohibitively expensive equipment. Second, the result, when obtained, should be easily
placed into context. When a significant species has been recognised, it must be easy for the
user to find examples of recorded habitats and known problems associated with that
species. Equally, where a species is ubiquitous and unlikely to indicate a special problem,
this also should be recognisable.
For those charged with the task of compiling practical identification schemes, the onus
is on them to provide clear and unambiguous instructions for use and to supply a greater
quantity of interpretive information than has hitherto been considered necessary or
desirable.
THE MODERN APPROACH TO SELECTION OF IDENTIFICATION CRITERIA
Few, if any, mycologists would question that the taxonomic basis for the recognition of
fungal species should be morphological. The problem in practical terms remains that
identification by morphology alone requires a degree of specialist training that is not
available to most microbiologists. On the other hand, in recent years much supplementary
physiological and metabolic information has been acquired for moulds that are of
economic significance, including Penicillium and Aspergillus (Bridge et al., 1985; Frisvad,
1981; Pitt, 1979; Pitt and Hocking, 1985). In some cases such information has already been
incorporated into practical identification keys (Pitt, 1979; Pitt and Hocking, 1985) although
the basis of separation has remained either dichotomous or synoptic keying.
Most microbiologists are, however, more familiar with identification routines where a
fixed range of tests is carried out on a bacterium or yeast that has already been
presumptively identified to some extent (e.g. the A.P.1. bacterial identification schemes).
Now that much more information is available about a variety of the characters possessed
by many mould species, it is possible to simplify their identification by using a similar
approach and building an overall identification strategy.
As in bacteriological analysis, identification can be broken into several stages, all of
which are greatly enhanced by use of a well-programmed laboratory computer. Having
obtained a culture that requires identification, the first stage, is to use a routine that will
select the correct database to be used and indicate the test results that will be needed. This
has an exact analogy with the first stage of the bacterial identification system used in
Cowan and Steel's Manual for the Identification of Medical Bacteria (Cowan, 1974). for
example, in Penicillium subgenus Penicillium, a relatively small range of conidial colours
(white, grey, brown, blue and shades of green) needs to be recorded and teleomorphs are
absent. On the other hand, in Aspergillus, a wider range of conidial colours is found and
ascospore characteristics are used to distinguish several species. The second stage in
identification is to carry out growth tests under standard conditions and to record the
information requested by the instructions. The information recorded would include
growth rate measurements, colony colours, substrate utilisations and microscopic
characteristics. Having recorded the information and identified by comparison with a
database prepared for all relevant taxa, the final stage is to present the user with
information about the mould identified. This, although the most subjective stage of the
operation, is also the most important in terms of the overall usefulness of the
identification. Data, supplied at this stage or sources indicated, must include information
Identification of Penicillium and Aspergillus: computer-assisted keying
291
about incidence, significance in the environment and relevant details on physiological and
nutritional requirements.
The schemes described below are intended for use with isolates of Penicillium
subgenus Penicillium and foodborne species of Aspergillus. The range of media used and
characters recorded are the minimum needed to distinguish most common isolates. It is,
however, accepted that better identification criteria may soon be available and that their
incorporation into any identification scheme would be desirable, as would any accepted
changes in the names of recognised taxa
CONSTRUCTION OF A COMPUTER KEY
Perhaps the major power of a computer key is its ability to analyse a range of characters
simultaneously, and thus to reduce the errors that result from inaccurate or missing data,
when dichotomous keys are used. Additionally, a standard amount of information about
each species is used in each identification. The princile of building profiles is widely used
in other microbiological disciplines. For example a Pseudomonas would not be primarily
recognised by a dichotomous key, but would be defined by the profile "Gram negative,
non-sporing, catalase positive, oxidase positive rod, attacking sugars oxidatively". Profiles
are also the basis of the A.P.1. bacterial and C.O.M.P.A.S.S. yeast identification schemes.
Another powerful feature of the use of profile keys is the flexibility possible in the
construction of the internal database. Many characteristics (either positive of negative)
will be clear cut and easily incorporated. Others, however, may be less easy to read or
subject to variability. Examples from our own culture collection include isolates of
Penicillium aurantiogriseum with consistently smooth stipes; dairy isolates of Penicillium
roqueforti with abnormally slow growth rates and isolates from several species, which
show unusually roughened conidia. It is easy to adapt the database so that the less
common character also scores as a positive and does not adversely affect the
identification.
In the identification schemes described below, all information entered at the keyboard
is taken into account in making the identification. For any characteristic, agreement
between the unknown and each species in the database is awarded a score of one; a
characteristic that is very rarely found scores 0.5 and a clear disagreement scores zero. It
would be a simple matter to add a weighting factor to emphasise key characteristics, but
this has not been found to be necessary in the present context.
IDENTIFICATION SCHEMES FOR PENICILLIUM SUBGENUS PENICILLIUM AND FOR

COMMON SPECIES OF ASPERGILLUS
Cultural methods
All cultures are inoculated as triple points on appropriate media and incubated in air at
25C for 7 days. Media used are listed in Table l.
After incubation, a fixed number of characteristics is recorded for the isolate to be
identified. These are listed in Table 2.
A.P. Williams
292
Table 1. Media Used in Identification Schemes
Penicillium
Aspergillus
Czapek yeast agar (CYA)

(Pitt, 1979)
Malt extract agar (MEA)
(Pitt, 1979)
Yeast extract sucrose agar (YES)
(Frlsvad,1983)
Creatine sucrose agar (CREA)
(Frisvad,1983)
Nitrite sucrose agar (NSA)
(Frlsvad,1983)
Czapek yeast agar (CYA)

(Pitt, 1979)
Malt extract agar (MEA)
(Pitt, 1979)
25% glycerol nitrate (G25N)
(Pitt, 1979)
Table 2. Cultural Characteristics Used for Identification
Penicillium
Aspergillus
Colony diameter on CYA

Colony diameter on MEA
Colony diameter on YES
Strength of growth on CREA
AcidonCREA
Strength of growth on NSA
Conidia on CYA
Reverse colour on CYA
Reverse colour on YES
Texture of stipe surface
Texture and shape of conidia
Shape of penicillus
Conidial crusts on MEA
Colony texture on CYA
Colony diameter on CYA

Colony diameter on MEA
Colony diameter on G25N
Conidial colour on CYA
Presence of c1eistothecia or sclerotia
Vesicle shape
Conidial shape
Texture of conidial surface
Phialides or metulae + phialides
Ascospore ornamentation
The computer keys.

Both keys operate in the same manner. For each unknown isolate to be identified, the
results are entered into the computer via a simple menu and are converted into a data
string comprising fourteen subunits (Penicillium) or ten subunits (Aspergillus). The data
string contains each positive result in a unique position that depends on the answer given
to each question. This is compared sequentially with the master strings in the database (23
for subgenus Penicillium or 22 for Aspergillus), which have been compiled by examination
of fresh isolates and published data (Pitt, 1979; Frisvad, 1981, 1985a,b; Pitt and Hocking,
1985). Tables 3 and 4 give the names of species included in the keys and sources in the
literature that contain the nearest equivalent standard description. Agreement with each
species in the database is calculated as a percentage, and results are sorted by the
computer into descending numerical order.
293
Identification of Penicillium and Aspergillus: computer-assisted keying
Table 3. Species of Subgenus Penicillium in the Key
Current name
Original name (source)
P. atramentosum
P. aurantiogriseum
P. brevicompactum
P. camemberti
P. chrysogenum
P. commune
P. copruphilum
P. crustosum
P. cyclupium
P. digitatum
P. echinulatum
P. atramentosum
P. puberulum
P. brevicompactum
P. camemberti
P.expansum
P. glandicola
P. griseofulvum
P. hirsutum
P. hordei
P. italicum
P.olsonii
P. roqueforti
P. solitum
P. verrucosum
P. viridicatum
P.vulpinum
P. chrysogenum
P. commune
P. concentricum
P. crustosum
P. aurantiogriseum
P. digitatum
P. echinulatum
P. expansum
P. granulatum
P. griseofulvum
P. verrucosum var. corymbiferum
P. hordei
P. italicum
P.olsonii
P. roqueforti
P. verrucosum var. melanochlorum
P. verrucosum
P. viridicatum
P. claviforme
(1)
(1)
(1)
(1)
(1)
(2)
(2)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(2)
(2)
(1)
(1)
(1)
(2)
(1)
(1)
(1)
(1) Pitt 1979

(2) Samson el aI. (1976)
After analysis, the identification is displayed on the screen and the percentage agreement
with that species is stated. There is an option to scan the percentage agreements for all
other species in the database and to display details of any tests that failed to agree. A
printed Result Form can be made, if required.
CONCLUSION
The two identification schemes that have been briefly described are intended to make the
task of mould identification easier for the non specialist. The criteria used to make the
identifications have been chosen, initially, because they work, but it is hoped that
improvements may be made as better criteria become known.
A.P. Williams
294
Table 4. Species of AspergillliS in the Key
Neosartorya fischeri
Eurotillm amstelodami
Eurotium chevalieri
Eurotium herbariorum
Eurotium repens
Eurotium rubrum
Emericella nidulans
Aspergillus candidus
Aspergillus clavatus
Aspergt7lus flavus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
Aspergillus niger
Aspergillus ochraceus
Aspergt7lus parasiticus
Aspergt7lus penicilloides
Aspergt7lus restrictus
Aspergt7lus sydowii
Aspergillus tamarii
Aspergillus terreus
Aspergt7lus ustus
Aspergt7lus versicolor
Aspergt7lus wentii
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1) Pitt and Hocking (1985)
REFERENCES
BRIDGE, P.A., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.5., PATERSON, R.R.M. and
SACKlN, M.J. 1985. An integrated approach to Penicillium systematics. In Advances in Penicillium and
COWAN, S.T. 1974. Cowan and Steel's Manual for the Identification of Medical Bacteria". Cambridge:
Cambridge University Press.
FRISVAD, J.e. 1981. Physiological creiteria and mycotoxin production as aids in classificatiln of common
asymmetric Penicillia. Applied and environmental Microbiology 41: 568- 579.
- - 1985a. Oassification of asymmetri Penicillia using expressions of differentiation. In Advances in
Penicillium and Aspergillus Systematics,eds. R.A. Samson and J.I. Pitt, pp. 327-335. New York and London:
Plenum Press.
--1985b. Profiles of primary and secondary metabolites of value in classification of Penicillium viridicatum.
In Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 311-325. New
PITT, J.I. 1979. The genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces". London:
Academic Press.
PITT, J.I. and HOCKING, A.D. 1985. Fungi and food spoilage. Sydney: Academic Press.
SAMSON R.A., STOLK, A.C. and HADLOK, R. 1976. Revision of subsection Fasciculata of Penicillium and
some allied species. Studies in Mycology, Baarn 11: 1-47.
GENERAL DISCUSSION FOLLOWING AFTER THE SESSION ON COMPUTERASSISTED KEYS

.
GAMS: I'd like to ask Mr. Williams what language he used for his program.
WILLIAMS: I wrote the program on an Apple originally, and adapted it for use on an IBM
using Williams BASIC.
GAMS: Dr. Bridge, you have told us about the software development, but not about the
data you entered. Are the data used in this system the same as developed by CMI?
295
BRIDGE: The data is selected from our numerical taxonomy project.

GAMS: How easy would it be to extend the data set?
BRIDGE: It would be very easy. The data file is a standard ASCn file and can be handled by
a word processor. The program itself uses any data file in the right format.
SAMSON: For whom are you writing your key?
Bridge: At the moment, I was interested to see what we could do with the data we had.
SAMSON: It looks like a key for ascomycetes, with words like spore, reticulations, lobate
and so on. There are no characters in Penicillium like that.
BRIDGE: Certainly these terms are used for describing the ornamentation using the SEM.
SAMSON: So you need an SEM to use your key?
BRIDGE: At the moment, this is a demonstration of mixed data types running from a
matrix. This may not be a practical identification scheme, but it is an example of what
can be done. It is a methodology for an identification strategy.
GAMS: Dr. Klich has expressed some important thoughts about international coordination
of data systems. Perhaps we can find some agreement on the basic data and the
structures to be used. Most of the keys we have seen today start with Dr. Pitt's
monograph. It would be workable, provided that the system is explanable and can
grow steadily with each addition or correction being incorporated.
SEIFERT: Perhaps we should ask Dr. Pitt how he feels about everyone using his data as if it
was public domain.
PITT: I don't have any problem with people using the data from my publications. The data
in PENNAME is, in fact, derived from my private data using data from about 2600
isolates, correctly identified and filed in dBase ill+. At the moment, this is not generally
accessible but it could be made available to selected people for particular purposes.
GAMS: So your database is much more than just one record for each species?
PITT: Certainly. The database is one record for each isolate.
GAMS: How many species or records were sorted during your demonstration?
PITT: There are 68 species, and more than 100 characters. I plan to expand this to a larger
dataset.
GAMS: If we are to set up the cooperative database, we have to chose between two
alternatives. Are we satisfied with a minimal amount of information, one record for
each taxon, and distribute this as widely as possible or should we immediately try for a
very sophisticated system incorporating all the doubtful isolates, synonyms and so on?
SEIFERT: I think that we have to decide what the database is going to be used for. Who
needs records for 10,000 isolates? This might be valuable for taxonomists. But what we
have seen today are keys designed by taxonomists for general use. Perhaps Dr. Klich
can explain what purpose this international database should serve.
296
KLICH: I was thinking of a taxonomic database. I can see this will be much more
complicated than I had imagined. I was thinking of separate files for morphology, one
for biochemistry and so on. So often we publish a paper and the information disappears
into our filing cabinets or personal computers, and this is the information that I'd like to
be able to get at, information that we have developed and finished with. The problems
are who should contribute and who should have access.
GAMS: There are only relative few who will use such an extensive database. General users
may be satisfied with a limited data set, but for the working taxonomist, we need a
centrally administered, up to date, database.
PITI: The data in the database would be meaningless if the identifications are inaccurate.
The identifications would have to be checked.
GAMS: We have the same problem with Fusarium. Two or three authorities must check the
identifications before the data is entered.
SAMSON: This is impractical. How can you ask three people to check two or three
thousand isolates?
GAMS: It may not be a question of checking thousands of isolates. If we have deviating
isolates, that may be incorrectly identified, these could be checked by the authorities.
KLICH: One of the fields in the database should be who made the identification. Then you
can select data that you trust.
GAMS: Especially when we are talking about secondary metabolites.

KLICH: We have to use the information intelligently. I don't see this as a major problem.
There could be different files for different kinds of information. We don't have to make
all of it available.
GAMS: How about the data structure? Should we be using a database system, or should
we consider using more sophisticated expert system software. These are user friendly
programs, and they can be used for generating keys, or point to discrepancies in handmade keys. For example, Super Expert or Prologue are already available.
SEIFERT: The problem with expert system software is that is all first generation, and the
database systems available are all fourth generation or more. They have standard data
formats and are often compatable with the dBase format. This is important for any
international effort based on microcomputers.
GAMS: Expert system data can also be superimposed on dBase formats.
Additions to the dialogue at a later time

PITT: There are two aspects of such a database. One would be to have a strictly
nomenclatural database when were the species described, who described them, are they
297
typified, do the specimens exist, have they been neotypified? Most of this information
is available in Pitt (1979). Perhaps this could be scanned into a word-processing file and
then into a database file, if necessary. The second aspect would deal with the properties
of isolates or strains that actually have been studied. My own database includes some
2500 isolates, and the data for many is very complete. This is a dBase III+ file with
numeric fields on colony diameters and microscopic characters that allows me to
assemble data from a single species for any character I choose.
HAWKSWORTH: It might be useful as a first step to have some standardized field names
and definitions and so on. Perhaps SPAS could look after this?
CHRISTENSEN: rd like to make an appeal for the inclusion of fields relating to ecological
data such as geographical locations, substrate or habitat, and prior names for the
isolate.
SEIFERT: It's easy to add these fields onto your own database.
ONIONS: Who would actually fund the development of a database on such a large scale?
Who would do all this work?
SAMSON: Who would actually use such a database? You can already extract a lot of
information on specific isolates from the databases of individual culture collections.
7
NEW APPROACHES FOR PENICILLIUM AND
ASPERGILLUS SYSTEMATICS: MOLECULAR
BIOLOGICAL TECHNIQUES
301
A REVIEW OF MOLECULAR BIOLOGICAL TECHNIQUES FOR

SYSTEMATIC STUDIES OF ASPERGILLUS AND PENICILLIUM
E.J. Mullaney and M.A. Klich
United States Department of Agriculture
Southern Regional Research Center
New Orleans, wuisiana 70179, USA
SUMMARY
Over the last several years various molecular taxonomic techniques have been utilized for defining
evolutionary relationships and for identifying species. A review of the different techniques, their
limitations, and their impact to date on Aspergillus and Penicillium systematics are presented.
Comparisons of results obtained from molecular techniques and conventional taxonomic methods,
present trends in each technique's application, and possible application of recently developed
molecular techniques are discussed.
INTRODUCTION
Several genetic engineering procedures have been developed in the last several years.
Following closely behind these developments have been several efforts to adapt or
develop some of these molecular biological techniques for taxonomic and phylogenetic
research. The primary impetus for this move has been the perceived promise of this
technology for greater precision and clarification of taxonomic questions not satisfactorily
addressed by current methods. In fungal taxonomy, with little or no fossil record of many
species, lack of a perfect stage in some species, and few available morphological
characteristics on which to base taxonomic decisions in others, taxonomic decisions have
been difficult to make. Molecular techniques were adopted by some scientists as a means
to overcome these difficulties. A review of the previous fungal research reveals that most
of the molecular research has been clustered in taxonomically uncertain groups and that
no single technique has been uniformly employed. It is also significant that in a few cases
when different techniques were utilized to study a single fungal group the findings were
not always in agreement. Therefore, as in conventional taxonomic methods, selection of
the appropriate molecular technique for the specific problem addressed is essential.
The scope of this paper is limited to a review of molecular biological techniques in
Aspergillus and Penicillium taxonomy, genera not widely studied by these methods.
However, Aspergillus nidule/lus Eidam [more widely known as Aspergillus nidulans;
telemorph = Emericella nidulans (Eidam) Vuillemin] has been extensively studied and this
knowledge may enhance the potential development of molecular techniques in Penicillium
and other Aspergillus species. Nucleic acid isolation techniques developed for A. nidulellus
and other fungi have been used in both of these genera (Yelton et al., 1984; Biel and
Parrish, 1986; Kurtzman et al., 1986; Klich and Mullaney, 1987; Lee et al., 1988). Some of
these techniques require a minimal amount of specialized equipment and training and
should be readily adopted by nonspecialist laboratories. Additional purification of DNA
and separation of nuclear, ribosomal and mitochondrial DNA by cesium
chloride/bisbenzimide density gradient centrifugation (Garber and Yoder, 1983) or
302
E.J. Mullaney & M.A. Klich
hydroxylapatite fractionation (Britten and Kohne, 1968) is also possible. To date most
molecular taxonomic research on these genera has been on Aspergillus species.
Four molecular methods have been used in fungal systematics: G+C molar
percentage, determining the relative frequency of the nucleotides guanine and cytosine in
DNA: DNA complementarity, measuring the rate and extent of reassociation of single
stranded DNA from two isolates; ribosomal RNA (rRNA) sequence comparison,
comparing homology of nucleotide sequences from rRNAs of different species; and
restriction fragment length polymorphism (RFLP), where DNA from different isolates is
digested by a restriction enzyme, electrophoretically separated, and the resulting DNA
fragment patterns compared. Each of these methods is discussed in more detail below.
G+CVALUES
Among the simplest molecular techiques used in taxonomy, the molar percentage of
guanine plus cytosine (G+C) is a measure of DNA base composition. As G+C values
among unrelated species may be similar, and G+C ratios provide no information on the
arrangement of the bases, its taxonomic use is primarily exclusionary (Kurtzman, 1985).
This technique is often used as a initial screening step in DNA-DNA complementarity
studies.
G+C values in fungi are principally determined by thermal denaturation (Marmur
and Doty, 1962; Mandel and Marmur, 1968) and cesium chloride density gradient
equilibrium centrifugation (Schildkraut et al., 1%2; Mandel et al., 1968). As the different
types of DNA (nuclear, mitochondrial, ribosomal) will have different G+C values, they
must be separated for precise measurements, preferably by cesium chloride/bisbenzimide density gradient centrifugation.
The G+C values for several species in Aspergillus sect. Flavi (Gams et al., 1985) have
been measured by Kurtzman et al. (1986, 1987), as follows: A. flavus, 49.1-49.9; A. oryzae,
49.1-49.6; A. parasiticus, 49.1-50.0; A. sojae, 49.1-49.4; A. Ieporis, 49.2; A. nomius, 49.7-50.4; A.
tamarii, 48.6-48.6. These values, reported with DNA complementarity measurements, were
not used independently for taxonomic purposes.
DNA COMPLEMENTARITY
DNA complementarity, the reassociation of DNA from two isolates, is used as a measure
of relatedness among species. One of the frrst molecular techniques used in biological
classification (Hoyer et al., 1964), its utility has led to the development of several different
methods. Some use radioisotopes to label DNA as a means to quantify reassociation,
others monitor reassociation spectrophotometrically during temperature reduction
(Seidler and Mandel, 1971; Ellis, 1985). Kurtzman (1985) has detailed these procedures
and reviewed their use in fungi. Repeated DNA sequences can reassociate with each
other, so their presence may obscure the true reassociation value. Therefore, the accuracy
of these methods is enhanced by using highly purified nuclear DNA consisting
exclusively of unique (non-repeated) sequences. Hydroxyapatite chromatography has
been routinely used to remove repeated DNA sequences from samples to be tested. In
most studies, the complementarity values are normalized by considering the level of
reassociation of the control DNA to itself as 100%.
Measurements of DNA relatedness in Aspergillus and Penicillium have been limited to
Aspergillus sect. Flavi. Based on reassociation at 420, Kurtzman et al. (1987) proposed a new
Systematic studies of Aspergillus and Penicillium
303
species, Aspergillus nomius, for several isolates formerly considered to be A. flaws. Based
on data from a single isolate of each species, Kurtzman et al. (1986) suggested reduction of
A. oryzae, A. sojae, and A. parasiticus to the ranks of subspecific taxa of A. flavus. The
limitation on the number of isolates which can be studied due to the labour intensive
requirements of these measurements has been one factor in limited adoption of this
technique in taxonomic research in these genera.
RNA SEQUENCE COMPARISON
Ribosomes, which translate messenger RNA into protein, consist of protein and RNA
subunits of various sizes. These subunits, referred to by their sedimentation rates (e.g. 55,
185, 255), contain some of the most highly conserved nucleic acid sequences known.
Ribosomal RNA sequences have been determined in several taxonomic or phylogenetic
studies above the species level (Kurtzman, 1985; Hori and Osawa, 1987).
The small 55 rRNA has been sequenced in many filamenteous fungi. Limited success
has been reported in taxonomic studies on Basidiomycetes. Recently Bartoszewki et al.
(1987) have reported intraspecific heterogeneity in the 55 rRNAs of A. nidulellus.
Hasegawa et al. (1985) suggested that the 55 rRNA molecule is too small (about 120
nucleotides) to be statistically reliable, and proposed that the larger subunit rRNAs are
better suited for phylogenetic analysis. Preliminary sequence and secondary structure
data from the 185 and 285 rRNA have supported this (Blanz and Unseld, 1987). The
development of a new rapid rRNA sequencing technique using crude cellular lysates
(Lane et al., 1985), has the potential to provide much valuable information, but it is not
suitable for use as a sole tool on which to base all taxonomic decisions.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
RFLP uses restriction endonucleases (restriction enzymes) to cleave double stranded

DNA. This is followed by gel electrophoresis to separate the fragments by molecular
weight and net charge. After staining the DNA with ethidium bromide, the resulting
DNA fragment pattern may be observed under UV light. The same DNA will form
different patterns when enzymes that cleave at different restriction sites are used.
Southern blot analysis (Southern, 1975), also known as RFLP with hybridization,
(RLFPH) may be used to determine if isolates have regions with similar DNA sequences.
After electrophoresis, the DNA fragments are denatured into single strands, and then
transferred to a nitrocellulose membrane. A previously prepared sample of DNA (termed
the probe) is then radiolabelled, and mixed with the membrane bound DNA under
conditions which favor hybridization between fragments with identical or very similar
DNA sequences. The membrane is then washed to remove excess probe and regions of
hybridization visualized by autoradiography.
The proportion of common bands in an RFLP or RFLPH can be used as a measure of
relatedness between isolates. This method has the advantage of being simpler than either
DNA complementarity or sequencing and it correlates well with DNA sequence analysis
(Nei, 1983). However, several factors can limit the accuracy of RFLP or RFLPH analyses
(Taylor 1986; Natvig et al., 1987; Forster & Kinscherf 1988). Mutations at a recognition site
or of length (deletions or insertions), can yield a polymorphism which has little
Significance. In RFLPs, where a probe is not used, similarly sized non-homologous DNA
fragments will be read as identical, giving a false impression of similarity. However, RFLP
304
and RFLPH have served as useful tools in distinguishing closely related species and in
phylogenetic studies.
RFLP both alone and with Southern blot analysis (RFLPH) have been used in
Aspergillus and Penicillium systematics. Kozowski and Stepien (1982) used RFLPH analysis
of mitochondrial DNA (mtDNA) to assess the morphological data which has formed the
basis of Aspergillus taxonomy. They reported that two members of Aspergillus sect. Flavi, A.
oryzae and A. tamarii, were almost identical. They questioned the apparent close
relationship between A. awamori and A. niger, but acknowledged the limitations of RFLPH
analysis. In a review of Aspergillus genetic variation, Croft (1986) reported that no
intraspecific sequence variation has been reported in Aspergillus mtDNA, unlike the case
of Neurospora. However, distinct restriction site patterns have been reported to be
associated with each different species in Aspergillus sect. Nidulantes. Length mutations, i.e.
presence or absence of introns in certain regions of the mitochondrial genome, appear to
be the principle reasons for these differences. Cloned random fragments from an A.
nidulellus genomic library, used as probes in RFLPH analysis of different species in sect.
Nidulantes also proved useful as a diagnostic tool (Croft, 1986). One interesting finding
was that an A. nidulellus probe hybridized strongly to DNA from A. terreus, suggesting
that these species may be more closely related than the present taxonomies suggest. Using
probes from A. nidulellus developmental genes, Mullaney and Klich (1987) reported a
quite high degree of hybridization between A. nidulellus probes and A. terreus DNA. The
major morphological differences between A. nidulellus and A. terreus are colony colour,
vesicle diameter, and stipe colouration, and the latter two characters are variable in sect.
Nidulantes. These data suggest an overreliance on colony colour in Aspergillus
classification (Raper and Fennell, 1965; Klich and Pitt, 1988).
Previous molecular research utilizing A. nidulellus suggests that the conidia and the
complex, multicellular conidiophores that are the characteristic features of Aspergillus and
Penicillium are coded for by several developmentally regulated genes expressed only
during conidial development. Several of these genes have been cloned, including tubC
(May et al., 1985) SpoCl (Timberlake and Barnard, 1981) or cloned and sequenced such as
brlA (Adams et al, 1988). Beta3-tubulin, the product of the tubC gene, functions in the
development of microtubules during conidial mitosis. SpoCl is a 13.3 kb gene cluster that
codes for several poly(A)+RNAs that are primarily expressed during conidiophore
development. The product of the brlA gene is apparently a nucleic acid binding protein
whose presence in vegetative cells results in the expression of several other
developmentally regulated genes essential for the production of conidiophores. The
mutant allele of the brlA locus blocks conidiation by producing abnormal sterile
conidiophores with the phenotype known as ''bristle''.
This research to identify and define the specific function of genes coding for the A.
nidulellus conidiophore has provided a new potential means of measuring phylogentic
relationships in Aspergillus and Penicillium. Clones of these three A. nidulellus genes
(SpoCl, tubC, brlA) have been used in RFLPH analysis of representative species of both
Aspergillus and Penicillium (Mullaney and Klich, 1987; 1988). Under standard conditions, a
considerable degree of hybridization was found between the A. nidulellus SpoCl gene
cluster and the tubC gene and A. fumigatus, A. terreus, A. niger, A. flavus, A. ochraceus, P.
citreonigrum, P. montanense, and P.lividum DNAs. The functions of the genes in the SpoCl
gene cluster are unknown, but there is a widespread distribution of DNA sequence with
homology to the SpoCl clone in representative Hyphomycetes (Mullaney and Klich, 1988).
Less DNA sequence conservation appears to be found in the analogous brlA genes in
other species of Aspergillus and Penicillium. When the brlA gene was used as a probe in the
305
same study, this A.nidulellus gene only hybridized strongly to A. terreus DNA. This
supports the observations of Croft (1986) noted above. As additional A. nidulellus
developmental genes are characterized, both development of a model system to study
conidiogenesis in other Hyphomycetes and a source of DNA probes coding for significiant
phenotypic characteristics of Aspergillus and Penicillium can be expected. Then the
possibility of convergent evolution of conidiophores can be investigated on the molecular
level.
In a practical application of RFLPs, a technique was developed to help distinguish the
aflatoxigenic A. flavus from the koji mould A. oryzae (Klich and Mullaney, 1987). In this
study, eleven A. flavus isolates and seven A. oryzae isolates were digested with a variety of
restriction enzymes and subjected to gel electrophoresis. One enzyme, SmaI, yielded
consistent species specific patterns. All of the A. flavus isolates showed a major 3.8 kb
band, while all but one of the A. oryzae isolates showed major bands at 2.7 and 1.0 kb. The
final A. oryzae isolate had slightly larger bands (3.1 and 1.2 kb). The observed differences
in the banding patterns were shown to result from differences in nuclear rather than
mitochondrial DNA. The combined size of the two unique A. oryzae bands (2.7 and 1.0) is
approximately equal to the size of the unique A. flavus band (3.8). This suggests that A.
oryzae contains one SmaI site not found in A. flavus. The SmaI restriction digest patterns of
total DNA provided a useful adjunct to other taxonomic criteria distinguishing these two
economically important secies. Apparently, the resolution of this method is greater than
that of the reassociation method used by Kurtzman et al (1986), where the relative
reassociation values for one isolate each of A. flavus and A. oryzae was 100%.
An RFLP analysis has recently been included in the first study by the Subcommission
of Penicillium and Aspergillus Systematics of the ICTF which is nearing completion. In this
study to clarify relationships among P. glabrum, P. spinulosum and other closely related
species, RFLP results were compared to standard morphological methods, gross
physiological methods, grouping by secondary metabolite, and classification based on
isoenzymes. Agreement was reached by the majority of participants on 13 of the 15
isolates; RFLP data agreed with the majority for 10 of the 13 isolates. In this study total
DNA was used and the major visible bands after electrophoresis were most likely mtDNA
or rDNA. It is not surprising that RFLP results were in agreement with those of the other
methods. If phenotype is indeed a reflection of genotype, one would expect results from
different approaches to agree. The fact that such a simple method was effective is
encouraging for future applications.
One factor limiting the increased use of molecular technology in many locations is the
need to use radioactive material. Effective non-radioactive methods to label nucleic acids
are being developed and have been used recently in fungal taxonomic studies (e.g. Taylor
et al., 1985). The technology for the use of non-radioactive oligonucleotide probes to
selectively detect alleles differing in only a single nucleotide is now available (Bugawan et
al., 1988) and could potentially be used to distinguish morphologically similar fungal
isolates. The commercial availability of universal primers for sequencing rRNA, the ability
to rapidly sequence rRNA from crude cellular lysates (Lane et al., 1985) and commercial
development of automated sequencing technology will also have potential value. Another
new technique that has a potential impact on fungal molecular genetics is the polymerase
chain reaction (PCR) (Saiki et aI, 1988), a procedure which allows the amplification of a
few molecules of template DNA in a crude extract to several million copies in a few hours.
This will generate enough DNA to allow sequencing without the need for cloning (Wong
et al., 1987; Stoflet et al., 1988). A procedure to use PCR in vitro amplification of rRNA
genes followed by direct sequencing is also now available (Medlin et al., 1988).
306
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309
RFLP ANALYSIS OF NUCLEAR AND MITOCHONDRIAL DNA AND ITS

USE IN ASPERGILLUS SYSTEMATICS
J.H. Croft, V. Bhattacherjee and K.E. Chapman
School of Biological Sciences
University of Birmingham
Birmingham B15 2IT, UK
SUMMARY
The analysis of restriction fragment length polymorphisms (RFLP's) in both nuclear and
mitochondrial DNA is proving useful in establishing phylogenetic relationships in the section
Nidulantes of Aspergillus. Largely because of the presence of a highly diagnostic perfect stage, this
group has a firm and well established taxonomy, and the well developed genetics (making use of
sexual, parasexual and protoplast fusion methods) has enabled the concept of the biological species to
be applied. An examination of RFLP's in the nuclear and mitochondrial DNA of these species has
allowed a comparison to be made between the extent and nature of such variation in the DNA
sequences with the group structure imposed by the various systematic studies and thus permits an
interpretation of the RFLP's in terms of that systematic classification. The highly diagnostic nature of
restriction fragment variation in the mitochondrial genomes and of certain probes for detecting
nuclear RFLP's will be described.
INTRODUCTION
The classification of species in the genus Aspergillus has been based primarily on growth,
morphological and physiological characters (e.g. Raper and Fennell, 1965). Specification is
well defined in some sections, such as sect. Nidulantes where the presence of a sexual stage
has resulted in a precise classification. However, other areas of the genus are less clearly
defined, particularly those containing only anamorph species.
The genetic bases of the characters used for taxonomic purposes are generally
unknown. Consequently little information is available concerning the phylogenetic and
evolutionary relationships between the described taxa. A species should, perhaps, best be
considered as a population which is reproductively isolated from other populations: that
is, as an ideal, we should invoke the concept of the biological species in our schemes of
classification. However, the practical necessity for simple and rapid identification of
unknown isolates is recognised and accepted. Species concepts other than that of the
biological species may be appropriate for many purposes, especially in anamorph genera
where the identity of reproductively isolated populations is unknown.
Though many biochemical and physiological techniques have provided useful
information for classification, it is possible to argue that variation in the composition,
nucleotide sequence and organisation of DNA is the most likely to give a clear and
sensitive discrimination between organisms and also to indicate clearly their evolutionary
and phylogenetic relationships. Methods such as DNA base composition and overall DNA
complementarity (Kurtzman, 1985) provide an idea of levels of relationship, but are
relatively crude and difficult to interpret. For example, high complementarity is clearly an
indicator of close relationship, but too much intrageneric variation probably exists in
310
J.H. Cran et al.
degree of complementarity to allow definition of species or precisely identify individual

isolates.
An alternative method of assessing DNA sequence divergence is that of the analysis
of restriction fragment length polymorphisms (RFLP 's). RFLP analysis of the relatively
small fungal mitochondrial (mt) genome may be achieved by direct observation, following
electrophoresis, of the number and size of the fragments produced by digestion with type
II restriction endonucleases (Kozlowski and Stepien, 1982). Small regions of the much
larger nuclear genome may be analysed by the use of cloned DNA probes in Southern blot
hybridisation experiments (Southern, 1975). This technique has been used mainly to
provide large numbers of genetic markers in eukaryotic genomes, particularly those of
human origins in order to detect markers linked to genes known to cause inherited
disorders (Botstein et al., 1980; Donis-Keller et al., 1987) and more recently in plants
(Tanksley et al., 1989) in order to provide markers linked to genes for economic traits such
as resistance to diseases. The discovery of hypervariable probes based on highly
polymorphic repeated sequences in human DNA led to the development of 'genetic
fingerprinting' techniques in which each individual in the human population has a high
probability of producing a unique pattern in a Southern blot experiment thus permitting
the identification of individuals and accurate determination of their pedigrees Geffreys et
al., 1985). Direct observation of the bright bands produced by repeated sequences in
electrophoretic smears of digested nuclear DNA have also been informative. Repeated
sequences may also be highlighted by probing with labelled total nuclear DNA in
Southern blot experiments.
The variation in all of these patterns is due to differences in the DNA sequence which
alter the size and number of the fragments obtained following digestion with restriction
endonucleases. The differences may result from single base pair substitutions, small
insertions or deletions, or gross chromosomal structural changes. The generation and
detection of RFLP's is illustrated in Fig. 1.
The amount of polymorphism observed will depend both on the amount of
polymorphism actually present in the populations under study and experimental details,
for example, in Southern blot experiments, on the nature of the probe being used. The
amount of polymorphism present in a species may be expected to vary according to its
breeding system. In outbreeding organisms, such as species of Phanaerochaete, each
individual basidiospore progeny has been shown to differ when probed with random
genomic sequences, reflecting a high level of polymorphism (Raeder and Broda, 1986).
The situation in species where dispersal is probably mainly clonal such as Aspergillus or
Penicillium may be quite different. Other factors may also be important, such as the time
elapsed since the species differentiated and the history of natural selection of the species.
The types of probe used range from known characterised cloned genes of greater or
lesser expected sequence conservation to those which reveal hypervariable sequences.
Thus a probe consisting of a highly conserved gene such as that coding for ribosomal
DNA, or for tubulin, will hybridise to DNA taken from several genera and will probably
show relatively little polymorphism within a species or even within a genus. Other cloned
genes, such as the pectin lyase D gene of A. niger may provide sufficient polymorphism to
distinguish between clearly defined taxa, (Kusters-van Someren et al., 1990). At the other
extreme, some repeated DNA sequences may be located at several positions in the
genome and be highly polymorphic, thus giving a hypervariable probe which may reveal
a great deal of intraspecific variation between quite closely related isolates.
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics
probe
A
B
C
E
F
-'--~-~-I(
- - - - - --)-JE
...... , .........
ABCDEFGH
Figure 1. Generation and detection of RFLPs. A cloned genomic sequence from strain A was
used as a probe in a Southern blot experiment in which DNA from strains A to H was
isolated and digested to completion by a particular restriction enzyme. These digested DNA
samples were then electrophoresed and transferred to a DNA binding membrane and then
hybridised to the radiolabelled probe. The bands where the probe hybridised to the
homologous fragments in the digested DNA were then revealed by autoradiography.
a] The cloned fragment used as a probe was thet sequence limited by the dotted lines.
Strains A to H were polymorphic for the sites for the particular restriction enzyme used
as indicated by the vertical bars. In addition strain F contained a deletion and strains G
and H an insertion.
bl The fragments revealed for each of the eight strains by this probe are illustrated.
311
312
J.H. Croft et al.
Thus, for purposes of taxonomy, it is important to choose a suitable probe which reveals
variability at a level which is of relevance to the study being undertaken. At the present
time the selection of probes is an empirical process in fungal genera, as information to
enable a prediction of the nature of appropriate probes remains limited.
This paper will attempt to demonstrate that RFLP analysisis a useful and perhaps
definitive approach to the determination of a phylogenetic classification of genera such as
Aspergillus or Penicillium. A very precise distinction between closely related biological
species should also be possible by this technique.
GENETIC ANALYSIS OF THE SECTION NIDULANTES
Aspergillus nidul;;ms is one of the genetically best studied of fungal species. However, the
vast majority of work on A. nidulans has been carried out on mutant derivatives of one
isolate, NRRL 194, the so-called Glasgow strains (Pontecorvo et al., 1953; Clutterbuck,
1974). The background of information available from these now classical studies has
enabled a considerable amount of genetics to be carried out on other isolates of A. nidulans
and also on other species or varieties belonging to Aspergillus sect. Nidulantes, thus
providing a framework for the interpretation of RFLP studies.
Isolates which have been examined from species or varieties in the sect. Nidulantes fall
into a number of heterokaryon compatibility groups (Grindle, 1963a, b; Croft, 1985). Such
groups also exist in other sections of Aspergillus, including both teleomorph and
anamorph species (Caten, 1971). Heterokaryon incompatibility is under heterogenic
control: an allelic difference at anyone of a series of specific het genes is sufficient for two
isolates to be incompatible. In a sample of six heterokaryon compatibility groups, eight het
loci have been demonstrated, with multiple alleles at two (Dales and Croft, 1977; Dales et
al., 1983; Dales and Croft, 1983; Croft, 1985). Most pairs of isolates from the natural
environment are likely to belong to different heterokaryon compatibility groups and to
differ at a number of het loci. Considerable genetic variation exists in a species such as A.
nidulans, but detailed examination has shown that it is mainly confined to differences
between heterokaryon compatibility groups and not between isolates within a group
(Butcher et al., 1972; Croft and Jinks, 1977). Members of the same compatibility group are
clearly clonally related. It is tempting to suggest that this division of a species such as A.
nidulans into sexually interfertile but genetically distinct heterokaryon incompatible
groups which are probably efficiently dispersed by conidia could lead to their further
divergence into genetically isolated sibling species. In reality, this is probably a simplistic
model.
Nevertheless, what appear to be sibling species do occur in sect. Nidulantes. A.
quadrilineatus, A. nidulans var. echinulatus and A. rugulosus are very closely related to each
other and to A. nidulans, but are clearly distinct biologically, because sexual crosses with
A. nidulans are not fully fertile. Hybrid cleistothecia have not been produced between A.
nidulans and A. nidulans var. echinulatus despite many attempts. Crosses of A.
quadrilineatus and A. rugulosus with A. nidulans have both given rare hybrid cleistothecia,
but these contain only a very small number of recognisable ascospores and give rise to
aneuploid and allodiploid progenies. However, when protoplast fusion is used to
overcome heterokaryon incompatibility parasexual crosses are possible between these
species and allodiploids can be isolated (Dales and Croft, 1977; Croft and Dales, 1983)
Such crosses may then be haploidised and the progeny analysed. Such analysis has shown
the free segregation of all eight linkage groups among the progeny of an A. nidulans plus
A. quadrilineatus fusion but the presence of complex translocations in A. nidulans var.
313
echinulatus involving the chromosomes equivalent to the A. nidulans linkage groups ill, V
and Vill. Other genetic differences between the latter two taxa can be revealed by this
analysis, including the presence of allelic differences at one het gene on each of the
chromosome homeologues equivalent to the A. nidulans linkage groups II, VI and VII and
at least one on the I1I-V-VIII complex. One further result from protoplast fusion
experiments between these closely related species is that selectable mitochondrial
markers, such as oligomycin resistance, can be transferred readily from one species to
another. This has been shown to involve either the transfer of the whole mitochondrial
genome or a very high frequency of mitochondrial recombination (Earl et al., 1981).
At the other extreme, sect. Nidulantes includes species, such as A. unguis, A.
heterothallicus and A. stellatus, which are clearly distantly related to A. nidulans. No genetic
interaction at all is possible between A. nidulans and these species (Kevei and Peberdy,
1984). No fusion products have ever been recovered from protoplast fusion experiments
and mitochondrial transfer experiments have never been successful.
RFLP ANALYSIS OF THE SECTION NIDULANTES
Analysis of RFLPs in the mitochondrial genome is becoming widely used for detection of
variation in natural populations of fungal species, particularly of plant and animal
pathogens. It has also been used successfully in phylogenetic and evolutionary studies of
Neurospora (Taylor and Natvig, 1989). The mitochondrial genome of A. nidulans has been
almost completely sequenced, maps of restriction sites are available and functions have
been assigned to most regions of the molecule (Brown et al., 1985). As a consequence,
detailed interpretation of the restiction patterns obtained for other species of the sect.
Nidulantes has become possible. The most surprising feature of the RFLPs of the mtDNA
of those species so far examined is that no restriction site polymorphism has been detected
within a species. This remains to be confirmed for a larger sample of isolates, species and
restriction enzymes, but the results to date are quite striking. For example, some fifteen
isolates of A. nidulans from the U.K., continental Europe, North and South America and
Southern Africa all have identical mitochondrial restriction patterns for six restriction
enzymes, and each species tested has a distinct, diagnostic restriction map.
The differences in mtDNA between the closely related species have been shown to be
due almost entirely to the presence or absence of inserted sequences. These sequences are
optional introns which tend to be located in certain regions of the genome (Fig. 2). They
may make up a considerable proportion of the molecule, but may be absent in some
species. The difference between the 30kb mitochondrial genome of A. quadrilineatus and
the almost 40kb genome of A. nidulans var. echinulatus is due almost entirely to seven extra
introns in the latter species. The exonic sequences of these species are almost identical,
there being only about 0.2% base substitution (Jadayel, 1986).
This result contrasts with comparisons of the mtDNA of A. nidulans with sect.
Nidulantes species which have no genetic interaction with A. nidulans, namely A. unguis, A.
heterothallicus and A. stellatus. While it is possible to align their restriction maps with that
of A. nidulans (Fig. 3) and sequence conservation in ribosomal RNA regions is evident,
more polymorphism exists among these species. For example, comparison of a short
(124bp) sequence located in the fourth exon of the oxiA gene of A. nidulans and of A.
heterothallicus shows fifteen base substitutions (12%). Moreover, in A. heterothallicus, part of
the mitochondrial genome has been rearranged (Fig. 4) Jadayel, 1986).
J.H. Croft et al.
314
A. nidulans var. echinulatus
.'
HaeIII ~T---~~~~n---~~~----~----------~~~-----
HhaI
HindII
XhoI
A. quadrilineatus
II
..I: III
, ,
,, :
.
.
,;
,
"
II
I.
Hind~~____L-~I~II~_-.____________-L__
Hae II I
HhaI
~II'-T~~-nll--~~--~------~-r----- L_ _ _ _
~~
XhoI
j 5 kb
Figure 2. Linearised restriction site maps of the circular mitochondrial genomes of

Aspergillus nidulans var. echinulatus, A. nidulans and A. quadrilineatus aligned around a
unique XhoI site found in a tRNA gene. The position of the small and large rRNA genes is
indicated for A. nidulans. The three species are closely related with few restriction site
differences. The differences in size and restriction fragment patterns is due to the presence
or absence of optional introns, the positions of which are indicated by solid bars conected by
vertical dotted lines.
The diagnostic nature of RFLPs in nuclear DNA was revealed by the use of random
genomic probes chosen from a library of one isolate of A. nidulans cloned in the vector
Lambda EMBL3. The inserts cloned in this vector are about 17 to 23 kb in size. Most of
these clones, when used as probes in Southern blot experiments against digests of total
DNA of the strain from which they were isolated, hybridised with from three to six bands.
When these probes were hybridised with digested DNA from a range of isolates of A.
nidulans belonging to different heterokaryon compatibility groups and from different
geographical locations, very little or no polymorphi-sm was revealed. The polymorphism
that was present was limited to differences between heterokaryon compatibility groups,
and was limited to simple single restriction site differences (Fig. 5). No polymorphism was
detected within a compatibility group.
315
A.nidulans
HaeIII
II
[I
HhaI
PvuII
II:
II
xhoI
A.heterothallicus
HaeIII
HhaI
II
IJ
PvuII
II
XhoI
A.unguis
HaeIII
HhaI
II
I
II I I
PvuII
II I
III
XhoT
5kb
Figure 3. Linearised restriction maps of the circular mitochondrial genomes of Aspergillus

nidulans, A. heterothallicus and A. unguis aligned around the unique XhoI site. These three
species are less closely related than those in figure 2 and the site polymorphism is apparent.
The intron status of A. heterothallicus and A. unguis is unknown (From Jadayel, 1986).
When these probes were used with digested DNA from other sect. Nidulantes species, A.
quadrilineatus, A. rugulosus and A. nidulans var. echinulatus, hybridisation was detected,
usually to a similar number of bands, but the patterns revealed considerable

polymorphism. A more complex genetic basis for the polymorphism was indicated than
that found between heterokaryon compatibility groups within a species (Fig. 6). These
probes appear to be highly diagnostic for each of these closely related species.
When these random genomic probes were used in Southern blot experiments with
digested DNA from A. unguis, no hybridisation could be detected at all. This result was
also produced with DNA from A. niger. However, when DNA from A. terreus was probed,
hybridisation was detected (Fig. 6), suggesting that this species has some relationship with
A. nidulans.
J.H. Croft et al.
316
A.nidulans
oxiA
XhoI
cobA
oxiB
URF1 URF4
~.-A.unguis
oxiA
XhoI
-=~~----.--'
cobA URF's 1.4
~--~~----------~!----------~
~---
A.heterothallicus
XhoI URF's 1.4
cobA
oxiA
DxiB
Figure 4. A comparison of the organisation of some genes in the mitochondrial genome of

Aspergillus nidu lans, A. unguis and A. heterothallicus showing structural rearrangements
present in A. heterothallicus. The maps are aligned around the unique XhoI site. Known
introns are cross-hatched, but the intron content of A. unguis and A. heterothallicus is not
known. The maximum possible extent of the oxiA gene in A. unguis is indicated (From
Jadayel, 1986).
DISCUSSION
The results, briefly summarised here, demonstrate the highly diagnostic nature of RFLP
analysis in Aspergillus sect. Nidulantes, both in nuclear DNA, when revealed by the use of
random genomic probes, and in mtDNA. The patterns revealed by the restriction cleavage
of mtDNA are characteristic for each species as are the patterns of hybridisation revealed
by most random genomic probes used in Southern blot experiments with nuclear DNA.
The extreme conservation of the mitochondrial genome within each species is unexpected
and perhaps still requires confirmation. Nevertheless, this intraspecific conservation,
together with the presence or absence of optional introns in the otherwise similar
mitochondrial genomes of many taxa in sect. Nidulantes allows a clear distinction between
these taxa on the basis of their restriction fragment patterns, as the presence of introns
introduces additional restriction sites. The evolution of these mitochondrial genomes is
clearly of considerable interest. More taxa from sect. Nidulantes need to be examined to
Lanes
3
317
isolates
37 26
l1kb
6kb
..................
4kb
2.2kb
1.75kb
1.7kb
Figure 5. Diagrammatic representation of the autoradiograph obtained by the hybridisation

of a 32p labelled random genomic clone from Aspergillus nidulans to Hind III digests of total
nuclear DNA of nine isolates of A. nidulans from three heterokaryon compatibility (h-c)
groups The strains used were Birmingham isolates 3, 28 and 38 (h-c group A) in lanes 1 to 3,
isolates 1, 26 and 36 (h-c group B) in lanes 4 to 6, and isolates 33,37 and 74 (h-c group C) in
lanes 7 to 9. The photograph shows this single site polymorphism for isolates 37 and 26
(Chapman and Croft, unpublished).
increase the sample size, but to date each taxon examined has shown a different restriction
fragment pattern. If the variation seen so far is a general feature of the mitochondrial
genomes of other sections in Aspergillus and also in Penicillium, then mitochondrial
restriction fragment patterns will be vey useful for the assignment of isolates to genetically
related groups. The relative simplicity of the procedure for the extraction, digestion and
electrophoresis of mtDNA is an added attraction for this approach.
The nature of the random genomic probes remains unknown, but most appear to be
single copy sequences. Some random genomic probes appear to be conserved in the
species closely related to A. nidulans. Other probes appear to be based on highly repeated
sequences and show hypervariability (O'Dell et al., 1989) but so far these have not been
isolated from Aspergillus.
Very little screening of clones for use in differentiation between species has been
necessary. The conservation of these sequences within a single species is striking, the
small amount of variation present being explicable by single site variation. The
polymorphism between the closely related species is more complex, but the genetics of
these differences has not been analysed yet. The hybridisation of random genomic probes
from A. nidulans to sequences from closely related species but not to more distantly related
taxa such as A. unguis or A. niger provides a dot blot method for the rapid grouping of
related taxa. The hybridisation of A. nidulans probes to A. terreus suggest a closer
relationship than conventional taxonomy indicates.
J.H. Croft et af.
318
--
---- -
10
lOkb
5kb
3kb
1.75kb
Figure 6. Diagrammatic representation of the autoradiograph obtained by the hybridisation

of a random genomic probe from Aspergillus nidulans to EcoRI digests of DNA from
different species of Aspergillus. The strains used were: lanes 1 to 3, A. nidulans isolates 26, 74
and 258; lanes 4 and 5, A. nidulans var. echinulatus isolates 25 and 209; lane 6, A.
quadrilineatus isolate 12; lane 7, A. unguis; lane 8, A. terreus; lanes 9 and 10, A. niger,
(Chapman and Croft, unpublished).
Despite the intraspecific conservation of both nuclear and mtDNA there is considerable
genetic variation present within a species such as A. nidulans. This variation can be
demonstrated at the molecular level by the use of two classes of probe. The first is a
transposon-like sequence (supplied by Dr. M. Hynes and Dr. A. Upshall) which was
isolated from A. nidulans. Preliminary experiments showed that hybridisation patterns
produced by this probe in Southern blot experiments with nuclear DNA from other A.
nidulans isolates are heterokaryon compatibility group specific, that is genotype specific. A
probe of labelled wild-type M13 bacteriophage has produced a similar result. This virus
has been shown to contain a short repeated sequence which hybridises to DNA of all
organisms tested and possibly provides a universal hypervariable probe (Vassart et al.,
1987; Ryskov et al., 1988). Repeated sequences, showing up as bright bands in
electrophoretic digests of nuclear DNA, have been used to distinguish between strains of
A. flavus and A. oryzae (Klich and Mullaney, 1987). Although the patterns of bands
produced by repeated sequences within a species are generally consistent variation is
sometimes observed. For example, additional or different bands of repeated DNA have
been found in some isolates of A. quadrilineatus. Thus, although the random genomic
probes have revealed strong intraspecific conservation of DNA sequences, the study of
polymorphism in various classes of repeated sequence DNA suggest that they may vary
considerably between isolates within a species and thus be of interest in terms of
evolution within the genus.
The analysis of RFLPs found in Aspergillus sect. Nidulantes has been carried out
against a good background of genetic information about the taxa used, which has enabled
319
interpretation of the polymorphisms found in terms of genetic differences. This

information will be of importance when attempting to interpret the DNA polymorphism
found in anamorphtaxa such as those in Aspergillus sect. Nigri or in Penicillium. The
methods will provide information concerning the phylogenetic relationships of the
various taxa studied and thus give the taxonomist a genetic and evolutionary
interpretation of the classification produced by systematic studies.
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321
VARIATION IN PECTINOLYTIC ENZYMES OF THE BLACK ASPERGILLI:

A BIOCHEMICAL ANO GENETIC APPROACH
M.A. Kusters- van Somerenl, H.C.M. Kesterl, R.A. Samson2 and J. Visserl
1Department of Genetics, section Molecular Genetics
Agricultura! University Wageningen, The Netherlands
Oosterstraat 1, 3740 AG Baarn, The Netherlands
SUMMARY
Severa) hlack Aspergillus isolates ha ve heen analyzed hy hiochemical and genetic tools to investigate
whether such techniques are useful for rapid, ohjective identification. Western hlots were used to
screen for pectin Iyase production and Southern hlots to look for homology in these isolates with
respect to their pectin Iyase gene(s). AII A. niger isolates can easily be identified hy these methods.
They produce a similar pectin Iyase, although differences in molecular weigth and reactivity with a
monoclonal antihody exist. Also ali A. niger isolates contain at least one conserved gene: the pelD gene.
There are other hyhridizing hands visihle, prohahly resulting from heterologous hyhridization with
another pectin Iyase gene. Classification on the hasis of presence of these hands is different from
classification on the hasis of morphological features. The other species of sect. Nigri differ in one or
more of these aspects with the A. niger aggregate, exceptA. foetidus.
INTRODUCTION
The black Aspergilli (Aspergillus sect. Nigri, Gams et al., 1985) have been frequently
studied, because of their industrial importance and significance as a plant or human
pathogen. Species in this section appear tobe closely related. Identification of individual
species, however, is difficult and the severa! attempts made at classifying the section are
debatable. For example, Mosseray (1934 a and b) recognized 35 species, while Thom and
Raper (1945), and Raper and Fennell (1965) accepted 15 and 12 species, respectively. AlMusallam (1980) reinvestigated the Aspergillus sect. Nigri and accepted 7 species, of which
A. niger represented an aggregate of 7 varieties and 2 formae (see Table 1). Although a
number of species can be readily distinguished on the basis of the conidiophore structure,
shape and ornamentation of the conidia, severa! taxa are still difficult to identify.
The black Aspergillus species are well known for their ability to produce and secrete a
large variety of pectinolytic enzymes which attack pectin either by hydrolysis (the
polygalacturonases) or by transelimination (the pectin lyases). Pectin esterase saponifies
the substrate. The variation in extracellular enzyme patterns between two different A.
niger isolates (NW756 and CBS 120.49) has prompted us to investigate the use of variation
in pectin lyase as an aid in the classification within Aspergillus sect. Nigri.
Two different experimental approaches were used. First, pectin lyase pattems were
analyzed by Western blotting using antibodies raised against purified enzymes. Second, a
previously cloned gene, pectin lyase D, was used as a probe to screen restriction enzyme
digests of isolated DNA after Southern blotting. A variety of Aspergillus isolates was
studied, including (neo)type cultures and typical representatives.
M.A. Kusters-van Someren et sl.
322
Table 1. Species concepts of the black Aspergilli according to AI-Musallam (1980) and Raper
and Thom (1965)
Al-Musllllam (1980)
Raper & Thom (1965)
A CQrbonarius
A carbonarius
A heteromorphus
A heteromorphus
A ellipticus
A helicothrix
A ellipticus
A japonicus var. japonicus

A japonicus var. aculfiltus
A japonicus
A aculfilfus
A niger var. niger
A niger
Aficuum
A tubingensis
A niger var. niger f. hennebergii
A niger var. phoenicisp
A niger var. phoenicis f. pulverulentus
A
A
A
A
niger var. awamori

niger var. nanus
niger var. usamii
niger var. intermedius
Afoetidus
A phoenicis
A pulverulentus
A awamori
A foetidus
Isolates.
The cultures used were obtained from the Centraalbureau voor Schimmelcultures (Baam,
The Netherlands) and are shown in Table 2.
Media and growth conditions.
The minimal medium (MM) used had a composition as described by Pontecorvo et al.
(1953). It consists of 70 mM NaN~, IlmM KH2P04, 2 mM MgClu 6 mM KC1 and 0.9 mg
Zns04. 7H20, 0.2 mg MnCh, 0.06 mg Coel2, 0.06 mg CUs04. 5H2 0, 0.04 mg
(NH4)6Mo~24 4H20, 0.29 mg CaCI2. 2H20, 0.2 mg Fes04. 7H20 per litre. Complete
medium (CM) had the same basal composition as MM, but was supplemented with 2 g
neopeptone, 1 g vitamin assay casamino acids, 1 g yeast extract, 0.3 g sodium ribonucleate
and 2 ml vitamin stock solution (1 mg/ml thiamine, 1 mg/ml riboflavine, 0.1 mg/ml paminobenzoic acid, 1 mg/ml nicotinamide, 0.5 mg/ml pyridoxine HCI, 0.1 mg/ml
panthothenic acid, and 2 ~g/ml biotin) per litre. All media were adjusted to pH 6.0 with
NaOH before autoclaving for 20 min at 120C. For propagation of conidia, Petri dishes
containing CM solidified with 1.2% agar supplemented with 20 roM sucrose as carbon
source were inoculated by streaking out a conidial suspension. After growth for 4-6 days
at 30C, conidia were harvested by adding 5-10 ml saline/Tween [0.8% (v/v) NaC!,
0.005% (v/v) Tween 20] per Petri dish. The conidial suspension obtained was thoroughly
shaken to break conidial chains and concentrations determined with a haemocytometer.
To study the expression of pectolytic enzymes, MM with 1% (w Iv) sugar beet slices was
inoculated at 106 conidia per ml. Incubation was for two days at 28C in a New Brunswick
Variation in pectinolytic enzymes of the black Aspergilli
323
Table 2. Isolates examined
A. carbonarius
A. e1lipticus
A. helicothrix
A. heteromorphus
A. japonicus
A. aculeatus
A. niger
A. niger
A.awamori
A. phoenicis
A. nanus
A. usami
A. intermedius
A. hennebergii
A. pulverulentus
A.foetidus
A. niger
CBS 111.26 (NT), CBS 112.80

CBS 707.79 (T)
CBS 677.79 (T)
CBS 117.55 (T)
CBS 114.51 (T), CBS 621.78
CBS 172.66, CBS 115.80
CBS 554.65 (NT)
CBS 120.49
CBS 557.65 (NT), CBS 563.65
CBS 126.49, CBS 135.48
CBS 136.52, CBS 131.52
CBS 139.52 (T), CBS 553.65
CBS 559.65, CBS 117.32
CBS 118.35 (T), CBS 125.52
CBS 558.65, CBS 425.65
CBS 121.28, CBS 618.78
NW756
m ex type; (NT) ex neotype culture

orbital shaker at 250 rpm. The medium was separated from the mycelium by filtration
over cheese cloth, dialyzed against 20 mM sodium acetate buffer (pH 5.5) and stored at
-20C.
To obtain mycelial biomass for the isolation of DNA, CM supplemented with 20 mM
sucrose was inoculated with 1()6 conidia per ml. Incubation was as described above. The
mycelia were harvested by filtration over cheese cloth, washed with cold saline, squeezed
to remove excess liquid and stored at -S0c.
Isolation of chromosomal DNA.
DNA was isolated as described by de Graaff et al. (1988). Frozen mycelium (0.5 g) was
disrupted with a microdismembrator (Braun) for 1 min and 4 ml freshly prepared,
prewarmed (55C) extraction buffer [0.2 M Tris, 0.26 M NaCI, 50 mM EGTA, 20 mg/ml triisopropyl-naphthalene sulphonic acid, 120 mg/ml p-aminosalicylic acid, pH 8.5 and
37.5% (v Iv) phenol] was added. The suspension was mixed thoroughly on a vortex mixer
for 1 min, 1 ml chloroform was added and the suspension was mixed again (1 min). After
centrifugation (104 g, 10 min) in a Sorvall high speed centrifuge the aqueous phase was
extracted once with phenol/chloroform (1:1) and once with chloroform. DNA was
precipitated from the aqueous phase with 2 vol. ethanol at room temperature by
centrifugation (104 g, 10 min) in a Sorvall high speed centrifuge. The pellet was dried
under vacuum and dissolved in 500 III sterile distilled water with 20 Ilg/ml RNase A and
stored at -20C.
Restriction enzyme analysis.
Approximately 2 Ilg chromosomal DNA was digested for 2 h in a 200 III volume using 20
units of the appropriate restriction enzymes according to the manufacturer's conditions
(BRL). The DNA was then precipitated by adding 0.1 vol. 3M NaAc (pH 5.6) and two vol.
ethanol and centrifugation in a small table centrifuge (10 min, 104 g). The pellet was dried
under vacuum and dissolved in 12 J.1l of sterile distilled water. After the addition of 5 J.1l
324
M.A. Kusters-van Someren et al.
sample buffer [0.25% (w/v) bromophenol blue, 15% (w/v) Ficoll type 400], the samples
were loaded on an agarose gel [0.6% in TBE (0.89 M Tris, 0.89 M Boric acid, 27 mM EDTA)
containing 0.5 J.1g/ml ethidium bromide; the electrophoresis buffer also consisted of
TBEI ethidium bromide]. Electrophoresis was carried out for 16 h at 30 V (gel dimensions
22 x 17 em), or until the bromophenol blue dye marker had nearly run off the gel.
Southern blotting.
After electrophoresis the DNA was denatured by gently agitating the gel in a 0.5 M
NaOH, 1.5 M NaCI solution for 30 min (solution changed after 15 min). The gel was
neutralized by agitating for 30 min (solution changed after 15 min) in 0.5 M Tris-HCl (pH
7.5),1.5 M NaCl. The DNA was blotted overnight on nitrocellulose (0.45 J.1ID, Schleicher &
Schuell) using 10 SSC (1.5 M NaCI, 0.15 M tri-sodium citrate dihydrate) as transfer
buffer. The DNA was fixed on the nitrocellulose membrane by baking for 1 h in an oven at
80C (Maniatis et al., 1982).
Isolation of the probe.
The plasmid which contains the pelD gene (pGW840) was propagated in E. coli MH1
(Goddard et al., 1983) grown in LB medium (10 g trypticase peptone,S g yeast extract, 10 g
NaCl, pH 7.5 per litre) with 50 J.1g/ml ampicillin. Plasmid DNA was isolated as described
by Maniatis et al. (1982). 1 J.1g DNA was digested with BamHI and Pst! in a final volume of
20 J.11 under the conditions described by the manufacturer (BRL). The restriction fragments
were separated on a 0.8% (w Iv) agarose gel in TBEI ethidium bromide. After
electrophoresis, the desired band was cut out and the DNA was isolated by electro-elution
using sample cups from Isco Inc. (Lincoln, USA). The DNA was precipitated by adding 0.1
vol. 3M NaAc (pH 5.6) and 2 vol. ethanol and by subsequent centrifugation in a small
table centrifuge at 104 g for 10 min. The DNA pellet was dried under vacuum, suspended
in sterile distilled water and the concentration was determined by electrophoresis using
lambda DNA as standardized concentration marker.
Preparation of the probe.
50 ng of the BamHII Pst! fragment on which the pelD gene is situated was labeled using
the random priming method: 50 ng hexamers (Pharmacia) were added as well as 1 J.11 10
priming buffer (0.1 M Tris-HCl pH 7.5, 50 mM MgCl:z) in a final volume of 10 J.1l. The
mixture was heated at 100C for 3 min and cooled on ice. dGTP, dCTP and dTTP were
added (0.15 mM each), as well as 50 J.1Ci a32P-dATP and 1 U Klenow enzyme (BRL), in a
final volume of 20 J.1l. After incubation at 37C for 15 min, the non-incorporated label was
removed by centrifugation in a spun column through Sephadex G50 in TE (10 mM TrisHCl pH 8.0, 1mM EDTA pH 8.0) (Maniatis et al., 1982).
Hybridization.
Prehybridization of the Southern blot was done for 1 h at 65C in hybridization buffer.
This buffer consists of 50 mM Tris (pH 7.5), 10 mM EDTA (pH 7.5), 1 M NaCI, 0.5% (w Iv)
SDS, 0.1% (w/v) tetra-sodium diphosphate-10-hydrate, 10. Denhardt [1% (w/v) Ficoll,
1% (w/v) polyvinylpyrolidone, 1% (w/v) BSA] and, in addition, 100 J.1g/ml heatdenatured, sheared herring sperm DNA (Boehringer, Mannheim) to minimize aspecific
hybridization. The probe was heat-denatured (5 min, 100C) and then added to the
prehybridization mix. After 18 h incubation, the membrane was washed twice for 30 min
with 2. SSC, 0.5% (w Iv) SDS at 65C and once with 0.2 SSC, 0.5% (wIv) SDS (30 min,
Variation in peclinolytic enzymes of the black Aspergilli
325
65C), air dried, and exposed to a Konica X-ray film using Kodak intensifying screens for
at least 16 h at -60c.
Immunological screening for pectin lyases.
Small samples (30 Ill) of the dialyzed culture fluids were mixed with 10 III sample buffer
[0.25 M Tris-HCl pH 6.8, 8% (w Iv) SDS, 40% (v Iv) glycerol, 20% (v Iv) 2-mercaptoethanol,
0.05 mg/ml bromophenol blue] and heated (3 min, 100C). SDS-polyacrylamide gel
electrophoresis was performed according to Laemmli (1970) in a 10% slab gel. The
electrophoresis was stopped when the internal bromophenol blue marker had reached the
bottom of the gel. Proteins were transferred to nitrocellulose (0.45 11m, Schleicher &
Schuell) by electroblotting (0.8 mAl cm2 for 1 h) using the LKB Novablot system and the
continuous buffer system [39 mM glycin, 48 mM Tris, 0.0375% (w Iv) SDS and 20% (v Iv)
methanol] as described by the manufacturer. To minimize aspecific binding of the
antibody to the nitrocellulose filter, the filter was incubated with 3% (w/v) gelatin in Tris
buffered saline (TBS: 20 mM Tris-HCl pH 7.5, 0.5 M NaCl) for 1 h. After washing the blot
(2 times 5 min) in TTBS [0.05% (v Iv) Tween 20 in TBS] it was incubated overnight in 75 ml
TTBS containing 1% (w/v) gelatin and 10 III monoclonal antiserum or 0.1% (v/v)
polyclonal antiserum. Excess antiserum was removed by washing the blot (2 times 5 min)
in TTBS. Finally, the blot was incubated for 2 h in 75 ml TTBS containing 1% (w/v) gelatin
and 10 III goat-anti-mouse y-globulin alkaline phosphatase conjugate (Biolabs) when
monoclonal antibodies were used or 10 III goat-anti-rabbit y-globulin alkaline phosphatase
conjugate when polyclonal antibodies were used. After washing in TTBS (2 times 5 min)
to remove excess conjugate, the blot was washed in TBS (5 min) to remove Tween.
Detection of alkaline phosphatase activity was accomplished by immersing the blot in 0.1
M NaHC03 buffer (pH 9.8), 1 mM MgCI2, 0.3 mg/ml nitro blue tetrazolium (NBT) and
0.15 mg/mI5-bromo-4-chloro-3-indolyl phosphate (BCIP). The staining was stopped by
washing the blot in water.
The antisera used were polyclonal antibodies raised in rabbits against PLI and PLll, two
A. niger pectin lyases isolated from the commercially available pectinolytic preparation
UltrazymR (van Houdenhoven, 1975), and monoclonal antibodies raised in mice against
PLI, which recognize both PLI and PLll (M. Flipphi, A. Schots, E. Egberts, H.C.M. Kester
and J. Visser, in preparation).
RESULTS
Western blot analysis of pectin lyase.

Proteins from the culture fluids were separated on SDS polyacrylamide gels, blotted on
nitrocellulose and incubated with a monoclonal antibody raised against PLI, but which
reacts also with PLll (Fig. 1). From Fig. 1B it is clear, that A. niger CBS 120.49 produces one
pectin lyase: PLII. Under the growth condition used the pelD gene product PLI cannot be
detected. From the A. niger isolates only A. intermedius CBS 117.32 and A. hennebergi CBS
125.52 do not produce a pectin lyase reactive with the monoclonal antobody. However,
when we use polyclonal antibodies raised against PLI and PLll,these isolates do show a
pectin lyase of approximately the same molecular weight as PLll (not shown). Therefore,
the epitope recognized by the monoclonal antibody is not present in the pectin lyase
produced by these isolates. Further, there seem to be differences in the band intensities
and in the apparent molecular weigths of the pectin lyases of A. niger. Since quantitative
M.A. Kustars-van Someran et al.
326
10
11
11
11
1.
1. A. carbonarius CBS 111.26, 2. A. carbonarius CBS 112.80, 3. A. ellipticus CBS 707.79, 4. A.

helicothrix CBS 677.79, 5. A. heteromorphus CBS 117.55, 6. A. japonicus CBS 114.51, 7. A. japonicus
CBS 621.78, 8. A. aculeatus CBS 172.66,9. A. aculeatus CBS 115.80, 10. A. niger CBS 554.65, 11. A.
awamori CBS 557.65, 12. A. awamori CBS 563.65, 13. A. phoenicis CBS 126.49, 14. A. phoenicis CBS
135.48
..
10
11
11
11
1.
11
1. A. phoenicis CBS 135.48, 2. A. nanus CBS 136.52, 3. A. nanus CBS 131.52,4. A. usami CBS 139.52,
5. A. usami CBS 553.65, 6. A. intermedius CBS 559.65, 7. A. intermedius CBS 117.32, 8. A.
hennebergii CBS 118.35, 9. A. hellnebergii CBS 125.52, 10. A. pulverulentus CBS 558.65, 11. A.
pulverulentus CBS 425.65, 12. A. foetidus CBS 121.28, 13. A. foetidus CBS 618.78, 14. A. niger CBS
120.49,15. A. niger NW756
Figure 1. Western analysis of pectin lyases of various isolates of Aspergillus sect. Nigri.
Extracellular proteins were separated by SDS-PAGE, blotted on nitrocellulose and incubated
with monoclonal antibodies raised against PLI. Markers are PLI (46.3 kD) and PLII ( 45.5
kD).
327
Table 3. Division of the A. niger strains in groups on the basis of results of Western blotting
using monoclonal antibodies reacting with PLI and PLII, and Southern blotting using the
A. niger CBS 120.49 pe/D gene as a probe.
strain
CBS number
A. niger
CBS 554.65
CBS 120.49
x
x
x
x
CBS 557.65
CBS 563.65
x
x
x
x
CBS 126.49
CBS 135.48
x
x
x
x
CBS 136.52
CBS 131.52
A.awamori
A. phoenicis
A. nanus
A. usami
CBS 139.52
CBS 553.65
A. intermedius
CBS 559.65
CBS 117.32
CBS 118.35
CBS 125.52
A. hennebergii
A. pulverulentus
A. niger NW756
CBS 558.65
CBS 425.65
Western
ii
iii
x
x
x
x
x
x
x
xl
x
xl
Southern
I
II
III
x
x
x
x
x
x
x
x
x
x
x
x
x
x
n.d.
n.d.
x
x
x
Groups i, ii and iii are made on the basis of different apparent molecular weights as mentioned in the text.
0: no pectin lyase detected when using monoclonal antibodies; 1: on the basis of Western blot patterns
using polyclonal antibodies.
Groups I, II and III are made on the basis of absence/presence of fragments a, band c as mentioned in the
text. D: containing the 0.8 kb 1"'/0 fragment; n.d. not determined
analysis by Western blotting is not very accurate, we can not draw any firm conclusions
on whether the slight differences in band intensity reflect true differences in expression
level. Also, the faint band which is sometimes present just above the PLII band seems to
correlate in intensity with the intensity of the PLII band itself. Strain differences have
therefore been correlated with the position of the major band, and not with the occurrence
of the minor band.
The A. niger isolates can thus be divided into three groups (see Table 3):
(i) those with a PL of approximately the same molecular weight as that from A. niger
CBS 120.49;
those with a PL with a slightly lower apparent molecular weight, similar to the
NW756 pectin lyase and PL II from UltrazymR;
(iii) isolates with a PL with a distinctly lower molecular weight.
(li)
328
M.A. Kusters-van Someren et a/.
Group (i) is the largest and contains besides A. niger CBS 120.49 A. niger CBS 554.65, both
A. niger (awamori) strains, both A. niger (phoenicis) strains, A. niger (nanus) CBS 136.52, and
both A. niger (pulverulentus) strains. Group (li) contains A. niger (nanus) CBS 131.52, both A.
niger (usami) strains, both A. niger (hennebergii) strains (CBS 125.52 included on the basis of
the pectin lyase reactive with the polyclonal antibody), and A. niger NW756, and group
(iii) contains both A. niger (intermedius) strains (CBS 117.32 produces a PL of the same MW
as CBS 559.65, although it is detected only with polyclonal antibodies).
Of all other strains (Fig. 1a and 1b) both A. foetidus and one A. carbonarius strain
produce a PL of approximately the same MW as PUI. A. japonicus (aculeatus) CBS 172.66
shows a PL of a lower molecular weight and both A. japonicus CBS 114.51 and A. japonicus
(aculeatus) CBS 115.80 produce a PL reactive only with the polyclonal antibodies, and of an
even lower molecular weight (not shown).
Restriction enzyme patterns and Southern blotting of chromosomal DNA.
Chromosomal DNA was isolated from all isolates listed in Table 2, except from A.
pulverulentus. The DNA was digested with the restriction enzymes Pvull and Sall which
both cleave in the coding region of the pe/D gene from A. niger CBS 120.49 (Gysler et al.). It
is important to note, that the pectin lyase shown in the Western blots is PLll, and thus not
coded by the pelD gene which was used as a probe in the Southern blot experiments.
Chromosomal DNA digests were separated on an agarose gel containing the intercalating
agent ethidium bromide. When digested DNA was photographed under UV light
illumination, the DNA was seen as a smear. Some pronounced bands are discernable,
probably caused by highly repetitive, ribosomal DNA (Fig. 3). All A.niger isolates, as well
as the two A. foetidus isolates show the same pattern. Both isolates of A. japonicus and A.
aculeatus also have similar patterns, as did A. ellipticus and A. helicothrix (Fig. 3a).
After electrophoresis, the DNA was blotted on nitrocellulose and subsequently
hybridized with the Bamill/ Pst! probe containing the whole structural part of the pelD
gene from A. niger CBS 120.49 (Fig. 2). Thus, when A. niger CBS 120.49 chromosomal DNA
digested with Pvull and Sall is hybridized with this probe, three hybridizing fragments
are to be expected: a PvuII-SalI fragment of 800 bp (D in Fig. 2), and two other fragments
of approximately 500 bp which partly contain pelD gene sequences and partly flanking,
non-coding, DNA.
Autoradiograms of these blots are shown in Fig. 4 a-b. All A.niger and A. foetidus
isolates with the exception of A. intermedius CBS 117.32 and A. hennebergii CBS 125.52,
contain fragment D. The latter two isolates do, however, show a unique hybridizing band
of a higher molecular weight, which probably contains the pelD gene.
The other hybridizing bands seen probably result from heterologous hybridization of pelD
with other pel genes. With regard to these bands, the A. niger isolates can be divided into
three groups: group I contains isolates characterized by two bands (a and b) indicated by
arrows in Fig. 4 i.e. A. niger CBS 554.65, A. awamori CBS 563.65, A. phoenicis CBS 126.49 and
both A. usami isolates; group II contains isolates which show just the upper band (a): A.
niger 120.49, A. awamori CBS 557.65, A. nanus CBS 131.52, A. intermedius CBS 117.32, and
both A. hennebergii isolates; and group ill contains isolates with neither of these bands, but
showing a band of a higher molecular weight instead (c): A. phoenicis CBS 135.48, A. nanus
CBS 136.52, A. intermedius CBS 559.65 and A. niger NW756. Classification on the basis of
Variation in pectinolylic enzymes of the black Aspergilli
329
these results is clearly not in correspondence with the generally accepted taxonomic
classification (AI-Mus allam, 1980), e.g. neither the two A niger, nor the A. awamori, the
A. phoenicis, the A. nanus, the A. intermedius, or the A. hennebergii isolates (the latter ones on
basis of the presence of the 0.8 kb pelD fragment) would be classified as they are classified
at present.
With respect to the other species of the Aspergillus niger-group, there is no clear
homology with the A.niger pelD gene, except, as mentioned before, in the case of A. foetidus
which clearly resembles Aspergillus niger CBS 554.65 and both A. usami isolates. Further,
only A. carbonarius CBS 111.26 seems to contain the 0.8 kb pelD fragment. What can be
seen, however, is that the patterns of isolates A. ellipticus and A. helicothrix are highly
homologous, as was shown by the ribosomal banding patterns as well. A. japonicus CBS
114.51 and A. aculeatus CBS 115.80 seem to be more related to each other than the isolates
of A. japonicus or A. aculeatus.
5011
Pvu II
5011
Pvu II
pelD gene
+---
Born HI
probe _ _
Pst I
Figure 2. Restriction map of the A. niger CBS 120.49 pe/D gene and the position of the probe
used in the Southern blotting experiments. Fragments 0, E and F, resulting from a PvuIIISalI
digest, hybridize with the probe.
The results from both Western and Southern blotting are summarized in Table 3. In
many cases the results obtained by Western and Southern blotting confirm each other. The
A. niger isolates can easily be identified by both methods. However, isolates which are
classified as one variety often have different Southern and Western blot patterns, e.g., the
A. nanus isolates are not similar, neither are the A. intermedius isolates nor the A.
hennebergii isolates.
On the other hand, some isolates classified as different varieties of the same species
show strong homology on the Southern blot, like A. phoenicis CBS 135.48 and A. nanus CBS
136.52 as well as A. intermedius CBS 117.32 and A. hennebergii CBS 118.35.
M.A. Kusters-van Someren et al.
330
23.2
9.4
6.6
2.3
2.0
2 34 567
910111213
14
1. lambda DNA digested with Hindm, 2. A. carbonarius CBS 11126, 3. A. carbonarius CBS 112.80,
4. A. ellipticus CBS 707.79, 5. A. helicothrix CBS 677.79, 6. A. heteromorphus CBS 117.55, 7. A.
japonicus CBS 11451,8. A. japonicus CBS 621.78, 9. A. aculeatus CBS 172.66, 10. A. aculeatus CBS
115.80, 11. A. hennebergii CBS 12552, 12. A. foetidus CBS 121.28, 13. A. foetidus CBS 618.78, 14.
lambda DNA digested with Hindm
9.4
6.6
4.4
2.3
2.0
234667
9101112131416
16
1. lambda DNA digested with Hindm, 2. A. niger CBS 554.65, 3. A. awamori CBS 557.65, 4. A.
awamori CBS 563.65, 5. A. phoenicis CBS 126.49,6. A. phoenicis CBS 135.48, 7. A. nanus CBS 136.52,
8. A. nanus CBS 13152, 9. A. usami CBS 139.52, 10. A. usami CBS 553.65, 11. A. intermedius CBS
559.65, 12. A. intermedius CBS 117.32, 13. A. hennebergii CBS 118.35, 14. A. niger CBS 120.49, 15. A.
niger NW756, 16. lambda DNA digested with HindlII
Figure 3. Ethidium bromide stained agarose gel electrophoresis patterns of chromosomal
DNA digests. The lengths of the lambda fragments are shown in kb.
2 3
9101112
lA. carbonarius CBS 111.26, 2.A. carbonarius CBS 112.80, 3.A. ellipticus CBS 7CY1.79, 4A. helicothrix
CBS 677.79, SA. heteromorphus CBS 117.55, 6A. japonicus CBS 114.51, 7A. japonicus CBS 621.78,
8A. aculeatus CBS 172.66, 9.A. aculeatus CBS 115.80, 10. A. hennebergii CBS 125.52, 11. A. foetidus
CBS 121.28, 12. A. foetidus CBS 618.78
10 11 12 13 14
1. A. niger CBS 554.65, 2. A. awamori CBS 557.65, 3. A. awamori CBS 563.65, 4. A. phoenicis CBS
126.49,5. A. phoenicis CBS 135.48,6. A. nanus CBS 136.52, 7. A. nanus CBS 131.52, 8. A. usami CBS
139.52,9. A. usami CBS 553.65, 10. A. intermedius CBS 559.65,11. A. intermedius CBS 117.32, 12. A.
hennebergii CBS 118.35, 13. A. niger CBS 120.49, 14. A. niger NW756
Figure 4. Southern analysis of genomic DNA of various isolates of Aspergillus sect Nigri.
DNA was digested with Pvull and SaIT. After separation on a 0.6% agarose gel the DNA was
transferred to nitrocellulose and hybridized with the 1.6 kb BamHIlPstI probe containing
the whole structural peID gene. V-banding is caused by trailing of non- or partially degraded
RNA. Arrows indicate heterologous hybridizing bands A, B and C as mentioned in the
text.The lengths of the lambda fragments are shown in kb.
331
332
M.A. Kuslers-van Someren et a/.
DISCUSSION
In the past, classification of the black Aspergilli has largely been based on morphological
features (Mosseray, 1934 a and b; Thorn and Raper, 1945; Raper and Fennell, 1965; AlMusallam, 1980). However, advanced methods in biochemical analysis, as well as the
availability of cloned genes as molecular tools, now provide other independent criteria. In
this paper we describe an initial attempt to use biochemical and genetic features to
address existing problems in the classification of different Aspergilli. The degradation of
pectin, a complex process shared by all the isolates examined, has been taken for further
analysis. This heteropolysaccharide is present in the middle lamella and in the primary
cell wall of higher plants. A large number of different enzymes and their corresponding
structural genes are involved in catabolizing this substrate. We have limited ourselves in
this study to an immunological analysis by Western blotting of only one particular
enzyme, a pectin lyase which appears to be the major pectin lyase expressed under the
growth conditions used. This enzyme corresponds with pectin lyase II described
previously by van Houdenhoven (1975). We have also analyzed all isolates for the
presence of a pectin lyase gene, pelD, which codes for another pectin lyase. This enzyme
has recently been called PLD (Gysler et al.), but it is the same enzyme as the one described
by van Houdenhoven (1975) as PLI. Due to the high degree of specificity which
characterizes antibody-antigen interactions, immunological techniques are a powerful
method to establish how different or how related proteins are. In this study our main
interest was only to establish a rapid screening procedure using pectin lyase as a marker,
enabling us to detect qualitative strain differences. Therefore, we have used besides
polyclonal antibodies, a monoclonal antibody which recognizes a continuous epitope in
both PLI and PLII, and reacts in Western blotting with the SDS-denatured protein bound
to nitrocellulose. This approach by itself has some limitations. Apart from the fact that the
conditions for staining are not such that they lead to quantitative data, it is also required
that the gene one looks at becomes expressed. The final amount of pectin lyase which is
detected in the culture medium is the result of a complex process which, amongst others,
involves induction (depending on e.g. substrate, temperature, pH, oxygen levels,
inoculation density, submerse or surface growth), glycosylation, secretion and stability of
the secreted protein. It was possible, however, to classify the A. niger isolates which
produce a pectin lyase into three groups on the basis of differences in molecular weight.
Analysis at the level of DNA by Southern blotting is a more direct and objective way
to classify isolates. Small differences in nucleotide sequence, if present in the restriction
enzyme sites used for digestion of the genomic DNA, lead to differences in restriction
fragment patterns. Moreover, deletions or insertions larger than 50-100 bp, as well as
differences in strength of hybridization can be detected. As.can be seen from the results
shown (Fig. 4b), the pelD gene is conserved throughout all the Aspergillus niger isolates. In
the two isolates where the 0.8 kb band is absent, another band is seen, the length of which
resembles the added lengths of fragments D and E or D and F (Fig. 2). This means that the
non-coding region surrounding the gene, containg the next Pvull or Sal! site, is conserved
as well. In the 0.8 kb Pvull/ SalI fragment three introns are present (Gysler et al.,
submitted), the total length of which seems to be conserved as well. In all the other
species, except A. foetidus and A. carbonarius CBS 111.26, the pelD band is absent; other
bands are observed, but these clearly differ in strength of hybridization from the A. niger
bands. In the A. niger isolates, two other bands were seen that hybridized strongly with
the pelD gene. On the basis of presence or absence of these bands the A. niger isolates
could be separated into three groups.
333
A number of similarities exist between the results obtained by Western and Southern
blotting. The uniseriate species A. japonicus and A. aculeatus can be distinguished from the
biseriate taxa. The examined isolates of A. japonicus and A. aculeatus could not be clearly
distinguished from each other and this supports the classification of both taxa in one
species. A distinction at varietal level as proposed by AI-Musallam (1980) based on
differences of vesicle size and conidiophore-length cannot be made on the basis of the
results of our investigation.
All taxa proposed as varieties of the A. niger by AI-Musallam (1980) show homology
with respect to the two genes and the pectin lyase examined, indicating a strong
relationship amongst these isolates. Strikingly, homology was also found between A.
el/ipticus and A. helicothrix. Al-Musallam (1980) described the latter as a new species after
she isolated it as a distinct strain from the type culture of A. ellipticus and this might
explain the homology between the two isolates. A. helicothrix differs from A. ellipticus by
the production of typical cup-shaped sclerotia, a structure not found in other Aspergilli.
Perhaps both isolates represent different stages of pleomorphic species, but such a
phenomenon has not been observed in other anamorphic Aspergillus species.
The techniques applied in this paper are limited in their use to establish phylogenetic
relationships. For that purpose, sequence determination would be necessary since
differences between isolates can then be quantitated by scoring percentages of differing
nucleotides after alignment of e.g. ribosomal RNA sequences (Logrieco et al., 1990). The
power of Southern and Western blotting, however, lies in the quick, yet reliable way by
which isolates can be identified and classified.
ACKNOWLEDGEMENT
Margo Kusters acknowledges the financing of her project as part of a joint programme
between the Biotechnology Department of Ciba-Geigy A.G., Basel, Switzerland and the
Agricultural University, Wageningen, The Netherlands.
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-1934 b. Les Aspergillus de la section niger Thorn et Church. Cellule 43: 203-285.
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PONTECORVO, G., ROPER, J.A., HEMMONS, L.J., MACDONALD, K.O. and BUFfON, A.W.J. 1953. The
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DIALOGUE FOLLOWING MRS KUSTERS PRESENTATION
PATERSON: You mentioned that three of the four isolates of A. japonicus produced a low
molecular weight pectin lyase. How about the fourth one?
KUSTERS: Pectin lyase was not detected with the monoclonal or polyclonal antibodies in
that isolate by our methods. I can't explain this. Aspergillus japonicus as it is now classified
did not give us consistent results. We had two isolates each of A. japonicus var. japonicus
and A. japonicus var. aculeatus. One of the isolates of A. japonicus and A. aculeatus gave
identical results, so the varieties as they are defined are not consistent with our results.
On the basis of the Southern blot, and based on some results not shown with the
ethidium bromide stained gel of digested chromosomal DNA, the four isolates of A.
japonicus are alike.
335
A MOLECULAR ASSESSMENT OF THE POSITION OF

STILBOTHAMNIUM IN THE GENUS ASPERGILLUS
J. Dupontl, M. Dutertre2, J.-F. Lafay2, M.-F. Roquebert1 and Y. Brygoo2
lLaboratoire de Cryptogamie
Museum National d'Histoire Naturelle
75 005 Paris, France
2Laboratoire de Cryptogamie
Universite Paris Sud
91 405 Orsay Cedex, France
SUMMARY
Several Stilbothamnium and Aspergillus isolates were analysed by rapid rRNA sequencing of a variable
region at the S' end of the 28S-like rRNA molecule. This sequence permits evaluation of the genetic
divergence between species and the construction of a phylogenetic tree computed from the number of
nucleotide differences. Based on these data, the phylogenetic value of other taxonomical criteria used
for the classification of Stilbothamnium and Aspergillus is discussed.
INTRODUCTION
Stilbothamnium Henn. is a genus of seminicolous fungi characterized by abundant, large,

branched synnemata developping from seeds in tropical rain forests. These conidiomata
are up to 50 mm high and brightly coloured, greenish or golden yellow. They are covered
with Aspergillus-like heads bearing chains of echinulate conidia. Conidiomata sometimes
develop together with grey-brown lobulate, stipitate sclerotia. These sclerotia often
proved to remain sterile. Three other synnematous genera Stilbodendron H.& P.Sydow,
Penicilliopsis Solms-Laubach, and Pseudocordiceps Haum., with the same ecological
characteristics were described and discussed by Hauman (1936). Conidial structures of
this group morphologically resemble those of Penicillium or Aspergillus.
Penicilliopsis was established by Solms-Laubach (1887) as an Ascomycete genus with a
phialidic anamorph. For that reason, Samson and Seifert (1985) related the anamorph of
Penicilliopsis sensu Hauman to the generic name Sarophorum Sydow. Examination of type
material in herbaria allowed these authors to correlate the anamorphic genera
Stilbodendron and Sarophorum with the teleomorph Penicilliopsis, but not Stilbothamnium
even though its pedicellate sclerotia resemble Penicilliopsis ascostromata. The lack of
evident correlation between Stilbothamnium and Penicilliopis, in spite of the morphological
similarity of conidial apparatus, led Samson & Seifert (1985) to consider Stilbothamnium as
a subgenus of Aspergillus. They proposed the new combination Aspergillus togoensis
(Henn.) Samson and Seifert for S. nudipes Haum.(= S. togoense Henn. = S. novoguineense
H.& P. Sydow = Aspergillus coremiiformis Bartoli & Maggi).
Gams et al. (1985) subdivided Aspergillus into six subgenera some with related
teleomorphs. The subgenera were further divided into sections of similar taxonomic range
to the "groups" established by Thorn and Church (1926), Thorn and Raper (1945) and
Raper and Fennell (1%5). Aspergillus subgenus Stilbothamnium (Henn.) Samson and Seifert
appears closely related to the sect. Circumdati and Flavi of the subgen. Circumdati.
336
J. Dupont et al.
Anamorphs of subgen. Circumdati are related to teleomorphs inPetromyces Malloch &

Cain, Chaetosartorya Subr. and Hemisartorya Rai & Chowderi. Penicilliopsis is closely related
to Petromyces Malloch & Cain and Dichlaena Mont. & Durrieu, both belonging to the family
Trichocomaceae.
Morphological and structural analysis of S. nudipes shows clear relationships with the
genus Aspergillus, and in particular with the sect. Flavi (Roquebert and Nicot, 1985). It
differs from A. tamarii by the presence of coremia, septate phialides and by the mode of
differentiation of conidial walls.
Christensen (1981) includes A. coremiiformis Bartoli & Maggi in sect. Flavi but
suggested affinities with sect. Circumdati based on the pigmented, rough walls of the
stipes and yellow conidial masses.
Wicklow et al. (1989) examined A. togoensis for the production of mycotoxins and did
not find any mycotoxins characteristics of sect. Flavi but did find sterigmatocystin
believed to be an intermediate in the biosynthesis of aflatoxins. These data support the
linking of A. togoensis to sect. Flavi but not to species of sect. Circumdati which never
produce sterigmatocystin. So the question is, where does Stilbothamnium fit, with regard to
Penicilliopsis and Aspergillus, more precisely to sects Flavi and Circumdati. To study this
problem we used the analysis of ribosomal RNA, which provides a powerful taxonomic
indicator, because they are highly conserved and are universally found in living cells. The
5S rRNA was first used for this purpose (reviewed by Hori and Osawa ,1987). However,
5S rRNA is too short and too well conserved, to be suitable for studying closely related
species. One has to look at the larger rRNA molecules, 18 S (Salim and Maden, 1981;
Woese et al., 1985) and 28S (Qu et al., 1983). The development of a technique for rapid and
simple sequencing of large stretches of 18 or 285 rRNA opened the way for systematic
exploitation of the remarkable properties of these molecules as phylogenic indicators (Qu
et al., 1988). The larger rRNA subunits contain regions perfectly conserved adjacent to less
conserved ones that are useful for phylogenetic evaluation (Hassouna et aI., 1984; Michot
et al.,(1987). At the 5' end, two variable regions called D1 and D2, particularly D2 appear
to be divergent enough to give informations on relationships between genera and possibly
species (Baroin et al., 1988; Guadet et aI., 1989).
In this paper, we present preliminary results on an evaluation of the relationships
between Stilbothamnium-Aspergillus and Penicillopsis, using rapid rRNA sequencing
methodology as a tool for classification. We show that this method is efficient for this
purpose and may provide phylogenetic information as well.
The names and origins of the isolates used in this study are listed in Table 1. Species
which may be related to the genus Stilbothamnium, such as A. flavus, A. tamarii, A. ochraceus
and A. coremiiformis were included for comparison. Less closely related species such as A.
clavatus, A. nidulellus, A. fumigatus, A. versicolor and some Penicillium species were also
examined. Paecilomyces fumosoroseus was added as an unrelated species for a relative
estimation of distances.
RNA template isolation from two days old cultures was performed according to the
method described by Maccecchini et al., 1979; the method for RNA sequencing is that
developed by Sanger et al. (1971) and modified by Qu et al., (1983). The sequences were
aligned manually (Fig.l). Divergence (or distance) between two sequences was estimated
as the number of nucleotide differences, each difference counting for one. A computer
A molecular assessment of the position of Stilbothamnium in the genus Aspergillus
337
Table 1. List of sequenced isolates.
Aspergillus clllllfltus Desrn.
A. coremiiformis Bartoli & Maggi

A. flaws Link:Fr.
A. flavus var. columnaris Raper & Fennell

A. fumigatus Fres.
A. itaconicus Kinoshita
A. nidulellus Samson & Gams
A. niger van Tieghern
A. ochraceus Wilhelm
A. oryme (Ahlburg) Cohn

A. parasiticus Speare
A. tamarii Ki ta
A. thumii G. Smith
A. versicolor (Vuill.) Tiraboschi
Paecilomyces fumosoroseus (Wize) Brown & Smith
Penicilliopsis dichotomus Hauman

PeniCl1liopsis sp.
Penicillium canescens Sopp
P. implicatum Biourge
P. roqueforti Thorn
P. spinulosum Thorn
Stilbodendron sp.
Stilbothamnium nudipes Hauman
U-Brest
LO'8433%
L0'863450
LO'793244
LO'843365
L0'51508
U-Brest
L0'753053
U-Brest
L0'611673
U-Brest
U-Brest
L0'521064
LO'853389
L0'853498
U-Brest
U-Paris-Sud
L0'531525
L0'732236
L0'863455
L0'853497
L0'561517
U-Brest
U-Paris-Sud
LO'653802
LO'653801
L0'51455
U-Paris-Sud
L0'763119
U-Paris-Sud
U-Paris-Sud
LO'653800
L0'651910
L0'673456
LCP: Laboratoire Cryptogamie Paris; U-Brest: Universite de Brest; U-Paris-Sud: Universite de Paris-Sud
comparison of all the sequences provided a distance matrix (Table 2) from which a
phenetic tree was constructed by a Fitch Margoliash procedure using the Fitch program of
Felsenstein's Philip package (version 2.9). The conversion of the distant matrix into a
phylogeny is difficult and the phylogenetic tree will be subject to improvement as
additional sequences and alternative interpretation of the data are used.
RESULTS
The technique used for our experiments provides sequences of about 130 nucleotides,
which represents half of the length of the D2 domain. However, we found enough
variability to separate representative species, keeping in mind that within an incomplete
D2 domain, divergences are underestimated. More investigation is needed.
J. Dupont et al.
338
Table 2. Matrix of distances between the 17 representative strains

B
A A. tamarii
B A. flavus
CA. oryzae
D
E
Stilbothamnium nudipes
St ilbodendron sp.
A. ochraceus
A. itaconicus
A. niger
F
G
B
I A.
J A.
K A.
LA.
M P.
N P.
o P.
p p.
nidulellus
versicolor
chrysogenum
canescens
implicatum
spinulosum
Q P.
fumosoroseus
fUmigatus
clavatus
J
11
11
11
10
13
12
3
13
11
12
12
10
12
12
12
12
11
11
11
11
10
12
13
12
13
13
11
12
10
"
11
14
14
14
13
12
11
11
11
11
15
12
13
13
13
12
13
15
15
15
14
12
18
13
12
14
14
13
12
16
15
15
15
12
17
13
11
10
11
15
J5
31
30
29
14
30
10
17
16
17
13
12
17
2'
31
31
31
7B
31
32
30
32
33
34
32
13
12
10
11
Distances are expressed in absolute value; any differences (transition, transversion, each nucleotide
deletion) counts for one, without correction for multiple changes at one given site.
Comparison of aligned sequences shows that different isolates of a single species are
identical, so the sequence is considered as species specific. A. flavus, A. flavus var.
columnaris, A. parasiticus and A. coremiiformis were very similar. Similarity can also be
observed for Stilbodendron and Penicilliopsis spp as well as for P. chrysogenum and P.
roqueforti. Only one sequence representative of each of these groups was kept for data
processing.
From the phenetic tree, we can observe a separation of the 24 species into two clusters
(1) A. fumigatus, A. clavatus and Penicillium spp., (2) all the other species. In this last
branch Aspergillus and Stilbothamnium segregate clearly from Penicilliopsis, Stilbodendron
and strikingly also from A. tamarii.
The presence of Paecilomyces in this study does not imply any phylogenetic
significance; it is just an outer reference for "scaling".
DISCUSSION
Separations evidenced by partial rRNA sequences show a good correlation with

morphological classification, particularly with infrageneric taxa of Aspergillus as
established by Raper and Fennell (1965) and reviewed by Gams et at. (1985). The
subgenera Nidulantes, Fumigati and Clavati are clearly distinct.
The position of Stilbothamnium nudipes close to A. flavus as well as to A. ochraceus
supports its accomodation in the subgenus Circumdati. This finding agrees with previous
suggestions (Christensen, 1981 and 1982; Roquebert and Nicot, 1985; Samson and Seifert,
1985 and Wicklow et al., 1989). Consequently, the subgenus Stilbothamnium as proposed by
Samson and Seifert (1985) seems to be superfluous. As suggested by Samson and Seifert
(1985) we can confirm the identity of S. nudipes with A. togoensis (Henn.) Samson and
Seifert.
------T--- ----------
CT--C----- -----T---- --O-GA---O
---------G
--A-
-------A-- ---------T
-------A-- ----------
------T--- ---------- CT--C-----
-----T----
-O--GG-GCA
--A-A--
---------c
--A-
------T--- ---------- ----A----- ----A-AG-- -OO-GA-CGA ---------T

------T--- ---------- ----A----- ----A--T-- --OAGA---O
C---C----- ----A-A--- --OAGA---O ---------T --A-
---------- ---------- CT-OO----- ----A-A--- ----GA-TGA
------T--- ------A--- GC--C----- ----AT-G-- -CG-GA---O -O-------G --A-
------T--- ----------
cc-------- ----A-A--- -OO-GAAGTG

-A-CC-------- ----A-A--- ---AG-OTG- --- ______ _ -A-----c----- -----T---- --O-TA---O
--A------T--- A--------- ----c----- -----T---- ---AGAOO-O
---------- - - - - - - - - - - CC-------- ----A-A--- ---AG-OTG-
--C-G-T-A- c----C---- --A---T--- O---T----- --A----A-- --G-CGCA-O G-T------G GAO-G----- ---CA-T--- O---GG--G- -C--G----C -AA-
--C-----A- ---------- -------A-- ---------- ---------- -----T---- T----G---- AC-T------ ----A-?--- -OO-GA-CTO ---------- A-?-
--C-----?- ---------- -------A-- ---------- ---------- --G------- -----COO-- -C-TC----- ----A----- --O-G-OCGO ---------G A---
--C-----A- ---------- A------A-- ---------- ---------- -T-------T ---------- AC-TC----- ----A----- -OOTG--CGO ----C----G A-?-
--------A- -----c---- -------A-- ---------- ---------- -T-------T ---------- -C-TC----- ----A----- -T--G--OG- ----C----G A-C-
--------A- ----------
-----G--G- - ?---C----
--------A- ----------
-------AA- c----------------A- -----0----A------A- c----c----
--c------- ---------- --------------G-AA- c---------
--c------- ---------- ----------
--c------- ---------- ----------
--------A- ---------- ----------
--------A- ---------- ---------- ---------- ---------- ------T--- ---------- CT--C----- -----T---- --O-GA---O ---------G --A-
--------A- ---------- ---------- ---------- ---------- ------T--- --T------- CT--C----- -----T---- --O-----CG ---------G --1\-
------?-A- ---------- ---------- ---------- ----------
--------A- ---------- ---------- ---------- ---------- ------T--- ---------- CT--C----- -----T---- --O-GA---O ---------G --A-
CGTTTACGCC OATTATGCCA GCGTCCGTGC CQGAAGOCGC GTTCCTCGGT CCAGGCAGGC CGCATTGCOA ??CCTGGCTA TAAGGCGCCC CGAOQOGAOC OTACATTCCA GGGG
Figure 1. Aligment of 21 sequences. The sequence of A. tamarii is choosen as a model for the aligment. Identical bases are noted as dashes H,
undetermined as ? and deletion as O. The positions indicated by a are not taken into account for the distance calculus.
A. oryzae
A. thomii
S. nUdipes
Stilbodendron sp.
Penicilliopsis sp.
P. dichotomus
A. ochraceus
A. itaconicus
A. niger
A. fumigatus
A. clavatus
A. nidulellus
A. versicolor
P. chrysogenum
P. canescens
P. implicatum
P. spinulosum
P. fumosoroseus
A. tamarii
A. coremiiformis
A. flavus
to
'"
cO
~
Q)
{:
'"2
'"l:>
:J
:T
m
co
8'
C/)
::to
2-
:J
;::;:
"'8
'"
'"o
2:T
'"a
'"
III
'"'"
C>
en
g
--/
'-
Siiil
A.fum/gotus (1)
eaneseens (1)
c1avati
Subgenus
fumigati
Subgenus
Subgenus
circumdati
Subgenus
nidulantes
P. implicalum (1)
P. roque/ortli (1)
P. ehrysogenum (3)
Stlibodelldron '1'. (1)

PenleUl/opsis sp. (1)
PenleUlIopsis dieh%mus (1)
P. .plnulosum (1)
A. oehraeeus (4)
A.iIaconi us (1)
A.niger(l)
A.j1avus(6)
A. paras/licus (3)
A. coremii/ormis (1)
SIUbolluunnium nudi..
A.lhomli(l)
A. clavalus (1)
A. lamarii (1)
L-.-
. - - - - - - A. o71me (1)
A. versicolor (1)
'us (1)
Figure 2. Aspergillus and Penicillium phylogenetic tree. Phylogenetic tree is constructed from table 2 data. Correspondence with
Aspergillus subgenera is figured on the right. Horizontal distances are proportional to the number of different bases. Number of
analysed strains is reported in brackets.
PucUomyces
fumosoroseus
r--
r-
=-
!U
sa
"8
c:
!'-
'"~
A molecular assessment of the position of Stilbothamniumin the genus Aspergillus
341
A. coremiiformis appears similar to A. flavus. Hence, it should be separated from

Stilbothamnium, at variance with the synonymy established by Samson & Seifert (1985).
Ambiguously, Christensen (1981) placed it in Aspergillus sect. Flavi and later in sect.
Circumdati(Christensen, 1982). In our view, despite its resemblance to the latter, A.
coremiiformis clearly belongs in sect.Flavi. Because some species of sect. Flavi produce
mycotoxins, extensive research has been carried out by several different methods to
differentiate species. Murakami (1971), Christensen (1981), Klich and Mullaney (1987) and
Klich and Pitt (1988) concluded that A. oryzae, considered as a domesticated species, is
distinct from A. flavus; our results confirm this distinction. In this case, it appears that
ecological adaptations are correlated with hereditary changes. The distinct position of A.
oryzae is not in agreement with the opinions of Vincent and Kulik (1970), Kulik and Brooks
(1970) and Nasuno (1972). Based on nuclear DNA complementarity, Kurtzman et al. (1986)
placed A. flavus, A. oryzae, A. parasiticus and A. sojae in a single species.
The close relationship of A. thomii with A. flavus was mentioned by Christensen (1981).
Christensen and Tuthill (1985) proposed the transfer of A. thomii to sect. Flavi . Our results
confirm this proximity.
The position of A. tamarii closer to Penicilliopsis than to Aspergillus is rather unexpected
and disagrees with the analysis of mitochondrial DNA, which support the link between A.
tamarii and A. oryzae.
The close correspondence of rRNA groupings with the morphological taxonomy
confirms the phylogenetic validity of the morphological criteria used by mycologists and
the usefulness of the molecular tool. This method can be used for a rapid determination
and phylogenetic classification of isolates and will prove especially valuable for atypical
isolates.
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343
RIBOSOMAL RNA COMPARISONS AMONG TAXA OF THE

TERVERTICILLATE PENICILLIA
A. Logrieco', S.W. Peterson and D.T. Wicklow
Northern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Peoria, minois 61604, USA
SUMMARY
Ribosomal RNA sequences were determined for terverticillate Penicillia by the dideoxy nucleotide
chain termination method and oligonucleotide primers. The sequences of individual isolates were
compared for base differences upon proper alignment. Prior experience in other fungi suggests that a
single nucleotide difference in the sequence of two Penicillium isolates may indicate that they are not
the same species. A second baseline for data interpretation was provided by comparisons involving:
Penicillium, Saccharomyces, and Urnula. These intergeneric comparisons revealed >100 base differences.
The maximum number of base differences between species classified in Penicillium subgenus
Penicillium was 33 bases. Our results indicate that Penicillium aurantiogriseum NRRL 971, P. viridicatum
NRRL 963, P. verucosum NRRL 965, P. expansum NRRL 976, P. echinulatum NRRL 1151, P. hirsutum
NRRL 2032, P. granu/atum NRRL 2036, and P. puberulum NRRL 845 are distinct species. Penicillium
c/aviforme NRRL 2031 and P. c/avigerum NRRL 1003 show a closer relationship to species in subgenus
Penicillium than to P. isariiforme NRRL 2628. Morphological classification schemes that accommodate
one or more of the above isolates into a single species are not supported by our results. Three isolates
showed no base differences (i.e., P. puberulum NRRL 845, P. resticulosum NRRL 2021, and P. camemberti
NRRL 877) and may represent variants of the same species. Ecological and physiological data, as well
as secondary metabolite profiles, may be required if one is to distinguish Penicillium species by
methods other than degree of nucleic acid relatedness.
INTRODUCTION
Considerable interest and controversy has surrounded taxonomic relationships among

Penicillium species that produce terverticillate conidiophores (Samson et ai., 1976; Pitt,
1979; Frisvad and Filtenborg, 1983). This group includes important food and feed spoilage
moulds, pathogens of mature fruits and cereal grains, and "domesticated" isolates used in
the fermentation of cheeses or meats (Raper and Thorn, 1949; Pitt, 1979; Leistner, 1984). In
addition to causing deterioration and quality losses, these moulds may contaminate
agricultural products with potent mycotoxins (Frisvad 1986). Correct identification is
therefore essential to mycotoxicologists, plant pathologists and food microbiologists.
There are considerable problems in attempting to identify terverticillate Penicillia because
isolates commonly have characters found in more than one species. In approaching the
taxonomy of this group one first must deal with the extensive variation among apparently
"healthy isolates" while at the same time recognizing variation associated with strain
deterioration in culture (Williams et ai., 1985). As with any group of organisms, the views
, Permanent address: Consiglio Nazionale delle Ricerche, Instituto Tossine e Micotossine da Parassiti
Vegetali, Via Amendola, 197IF, 70126 Bari, Italy
344
A. Logrieco et al.
of taxonomists differ as to the importance of individual characters in delimiting species

(Raper and Thorn, 1949; Samson et aI., 1976; Pitt, 1979; Frisvad and Filtenborg, 1983). For
the terverticillate Penicillia these taxonomic characters include: micromorphology (i.e.,
conidia, conidiogenous structures), macromorphology (Le., colony texture and color),
physiology (Le., growth on different substrates at different temperatures and water
activities), pathogenicity, and secondary metabolite profiles (SMPs). This research
attempts to resolve some of the controversies by comparing of the ribosomal RNA (rRNA)
sequences of selected species of terverticillate Penicillia by means of the dideoxy
nucleotide chain termination method and oligonucleotide primers. We examined species
that are the object of taxonomic disagreement as well as species accepted by all Penicillium
taxonomists. To provide a baseline for RNA contrasts we examined teleomorph genera
Eupenicillium crustaceum Ludwig and Talaromyces helicus
(Raper & Fennell) Benjamin, known to have a Penicillium anamorph state, and two
unrelated ascomycetes Saccharomyces cerevisiae Hansen and Urnula craterium (Schw.) Fr.,
which presumably are only distantly related to either Eupenicillium or Talaromyces.
The isolates we analyzed for rRNA base sequences are listed in Table 1. Isolates were
predominantly from subgenus Penicillium. Eupenicil/ium crustaceum also produces
terverticillate penicillia (anamorph state = P. gladioli McCulloch & Thorn), while
Talaromyces helicus was included as a species representing Talaromyces. It produces acerose
phialides and typically biverticillate symmetrical penicilli (anamorph state = P. spirillum
Pitt).
The isolates were grown at 25C, in 100 ml of YM medium (Wickerham, 1951), on a
rotary shaker (200 rpm) for 16-36 hours, until the cultures were in log phase growth.
Ribosomal RNA isolation was according to Chirgwin et al. (1979), with the exceptions that
cells were harvested by filtration, suspended in guanidinium thiocyanate reagent (10
ml/g), and broken in a Braun cell homogenizer with 0.5-mm glass beads. Intact
undegraded rRNA, as assessed from denaturing agarose gel electrophoresis, was obtained
by this method.
The base sequences of selected regions of the large (25S) and small (18S) subunit
rRNA were determined, with specific oligonucleotide primers, by the dideoxy nucleotide
chain termination method for RNA sequencing as described by Sanger et al. (1977) and
Lane et al. (1985). Oligonucleotide primer C was purchased from Boehringer-Mannheim
(Indianapolis, IN); the other primers were a gift from Carl Woese, University of Illinois.
The first base synthesized from the small subunit primer, in relation to the S. cerevisiae
primary structure (Rubstov et al., 1980), is C, 1627. The first bases synthesized from the
large subunit primers, based on S. cerevisiae primary structure (Georgiev et al., 1981), are E,
1841 and F, 635. Sulfur-35 labeled nucleotide fragments generated in the chain extension
reactions were separated by electrophoresis on 8% acrylamide-8 M urea gels. RNA base
sequences were read from autoradiographs of the fixed and dried gels. Sequences with
few differences or apparent insertions were rerun side by side on the same gel to verify
differences. Some of the sequences were verified by repeating all steps from the
beginning. Ribosomal RNA base sequences were aligned manually with a text editor.
Alignment was necessary to compare homologous sequences. The data were evaluated
with a set of programs that measure simple matching of aligned sequences.
Ribosomal RNA comparisons among taxa of the terverticillate Penicillia
345
Table 1. Isolates examined

P. atramentosum Thorn NRRL 795 ex type,
P. puberulum Bainier NRRL 845 ex neotype,
P. roquefortiThornNRRL 849 ex type,
P. camemberti Thorn NRRL 877 ex type,
P. viridicatum Westling NRRL 963 ex neotype,
P. verrucosum Dierckx NRRL 965 ex neotype,
P. aurantiogriseum Dierckx NRRL 971 ex neotype,
P. expansum Link NRRL 976 ex neotype,
P. italicum Wehmer NRRL 983 ex neotype,
P. clavigerum Demelius NRRL 1003,
P. echinulatum Raper & Thorn ex Fassatiova NRRL 1151 ex type,
P. brevi-compactum Dierckx NRRL 2011 ex neotype,
P. resticulosum Birkinshaw et al. NRRL 2021 ex type,
P. claviforme Bainier NRRL 2031 ex neotype,
P. hirsutum Dierckx NRRL 2032 ex neotype,
P. granu/atum Bainier NRRL 2036 ex neotype,
T. helicus (Raper & Fennell) Benjamin NRRL 2106 ex type,
P. isariiforme Stolk & Meyer NRRL 2638 ex type,
E. crustaceum Ludwig NRRL 3332 ex type,
P. arenico/a Chalabuda NRRL 3392 ex type,
P. fennelliae Stolk NRRL 3697 ex type,
P. olsonii Bainier & Sartory NRRL 13058 ex neotype,
U. craterium (Schw.) Fr. SWP-1,
S. cerevisiae Hansen NRRL Y-12632.
Technical limitations, possible artefacts, and difficulties of dideoxy sequencing in

ribosomal DNA have been thoroughly considered by Elwood et al. (1985), who estimated
that 99% sequencing accuracy can be achieved with the dideoxy method and a doublestranded DNA template. Direct ribosomal RNA sequencing with dideoxy methods yields
similar accuracy; however, a small percentage of the base positions are impossible to
determine because a single stranded template is used. Therefore, we are probably
underestimating the total genetic distance between the taxa we have examined. Even so,
our sequences are representative of the complete sequences (Lane et al., 1985). The
ribosomal RNA base sequences are presented in Figures la-c. For T. helicus and U.
craterium, we were unable to read approximately 30% of the sequences located in the
region most distal from the primer. To accommodate these species a second matrix was
generated based on the readable sequences located proximal to the primer (Fig. 2b).
A baseline for data interpretation was provided by comparisons between species of
Penicillium, Eupenicillium, or Talaromyces and two outgroup species, S. cerevisiae and U.
craterium. The relative rates of sequence change for all of the species in this study can be
determined by the distance of each strain from the outgroup species. McCarrol et al.
(1981)recorded an approximate 30% sequence difference between the 18s rRNA of the
cellular slime mould Dictyostelium discoideum Raper and the ascomycetous yeast
Saccharomyces cerevisiae. If all of the isolates have been mutating at a nearly constant rate
since their divergence from a common ancestor, each strain should be nearly equally
separated from the outgroup. Our results show that the outgroup species, S. cerevisiae and
U. craterium, have approximately the same number of base differences with each of the
isolates producing a Penicillium anamorph (Figs. 2 a-b).
346
A. Logrieco et al.
(375-470)
P. pubQrulum
P.
P.
n~$tioulo.';um
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hil'sutum
P.
gJ:anulatmn
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-CU --N N.N.G.h ..
U ...
U
Figure la. Aligned sequences obtained with the F (S8Or) primer. First base synthesized with this prime1
corresponds to position 635 of S. cerevisiae 255 rRNA. Dots indicate the same base as is found in the first
line; dashes indicate missing data (in U. craterium and T. helicum) or gaps in the sequences; and N
indicates that the correct base for that position could not be determined.
347
(1561-1680)
F. puberulwn
F. oamembertl-.Ip. l"estiC'ulo:>um
P. hirsutUIIl
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P. grBnulatum
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Figure lb. Aligned sequences obtained with the E (1611r) primer. The first base synthesized from the
primer corresponds to position 1841 of S. cerevisiae 25S rRNA. Symbols are the same as Figure la.
A. Logrieco et al.
348
1378-1470)
P. pUblilrultml
P. call1ElmNrtii
P. restiaulosum
P. hirsutum
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2011
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. . . . . . . N ..
0'
- N.N -.
- C.O . . . . N . . . . . . . . U .. 0 . . . . . . -..
UU .CA . . . . . - . . . . . . . . U1:}. A . . . . . . A ..
U1:} .CNN.A .. - . . . . NN .. OU. A . . . . . . A ..
19.
20:21
2032
1151
2036
13058
roqtJe,forti
.. N .. N .. NIll. . . . . . . . . . . . N N . . . . . . . . . . . - .NN . . . . . . . . N.N . . . . -. N . . . . UN

A AG . . . . . . . . . . . . N A . . . . . . . . . . . C NCA . . . . . . . . . . N . . . . - . . . . . . . . . . .
.. ..... V .. tIN . . . . . . . . . . . . N N . . . . . . . . . . . A.N ro ....... .

......... -
Y-12632
granulatum
olsollii
.VA . . . . . . . . . . A . . . . - . . . . . . N .. ..
--NN . . . . . . . N.N . . . . - . . . . . . 0'
.NN . . . . . . . . ONN . . . . -. N . . . . . . . . .
.UA . . . . . . . . N.I1 . . . . -. N . . . . NN .. .
G C -.
3392
2Ei38
2106
P. echinulatWD
.C . . . . . . -.
.NA . . . . . . . . N.A .... - . . . . . . N .. ..
NO........
........ AN . . . . . . . . . . . . 0' A . . . . . . . . . . . NN........
.. . . . . . . . NN . . . . . . . . . . . . O N . . . . . . . . . . . -
1003
3332
arenicola
.NN . . . . . . . . N.O' . . . . - . . . . . . N ... .

.UN . . . . . . . . N.N . . . . -. N . . . . . . . . .
.. . . . . . 0' . NN . . . . . . . . . . . . 0' . N . . . . . . . . N -
.. . . . . . u ..
. . . . . . A
.. .NN .. N.N
. . . . . . . N
N CN . . . . . -.
orustaceU/l
atrameonto.'1WD
-. NN . . . . . . -.
.NN -.
"
.. .. N .. N NN . . . . . . . . . . . . NN.A . . . . . . . . . . . .. . . . . . N .. NN . . . . . . . . . . . . N N . . . . . . . . . . . -
2011
i"a..rit'orme
Brenioola
(1471-1570)
P. pUberulwn
P. call1embert;il
P. l."i;!sticulosum
P. hJ..n;:utum
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
E.
P.
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G . N . . . . . . -.
-N .N . . . . . . -.
N . N . -.
965
var.z:ucosWII
"."'pansum
S. cerevi;l'i8e
U. cra.terium
N C . . . . . . -.
13056
italiculII
P.
G. NC . . . . . . -.
N C . . . . . . -.
...
granulatum
P. o18o:mii
P.
P.
NC CNUAGCCG-A UGGAAGtJGCG CGGCAAUNAC NNGUCUGO'GA VGCCCUlJNGA UGUUCOGGGN C-NCGCGCGC TJACNCO'GA-C AGGGCCAGCG

N CN -.
.. . . . . . A .. NG........
. ........ - .NN . . . . . . . . . . 0' .... - . . . . . . N .. ..
.'5
2021
2638
2106
Y-12632
'" .--.A .
C.AGUU.A GG .
.. A . . . . . . . . . . . . . N .. N
. . . . . . . . ..
.N .... -
.N . . . . . . ..
. . . . . . . . ..
.N . . . . . . . .
. . . . . . . . ..
. . . . . . . . G. Noo . . . . . ..
...... .. N. H ........ .
.N . . . . . . . . . . . . . A .. N NN ...... ..
. . . . . . . G. N . . . . . . . . .
..N . . . . . . . . . . . . . ANNN N . . . . . . . ..
G U.NNN N
. 0 G. GUGu ..
.VA
, A G U
A.NN NN u ...
(1571-1597)
P. puberulum
P. aamemberti~
lQsticulc.S'WII
hi.r3UtUIII
lIiIahinulatWII
grallulatulII
845
.77
.,.
13058
ol.sonii
roqueforti
verruaosum
965
3
P. italiculII
P. e.'l.'pansuJII
P. br.;ovi-canpaatWil
2011
P.
".
alav~gerum
1003
963
3697
P. f"nn"lliae
P. Ololvi.t'o.L'llle
2031
P. IItIalRentOoS'um
7.5
P. lIura:ntiogriselml
971
3332
Z. cruoS'tace-WI
3392
P. are-ni oola
isari.t'o.l1ll8
2638
P. helicUlll
2106
S. cerevisis"
Y-12632
I). araterium
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P. viridicatulII
AGO-AGCACG AGUCAUC-AG CUCGOGC
2021
2032
1151
... - .. N ...
. A-UA.O
... NNNN-N 0 ....
NNN
-.CG
... -.CG . . . . . . . . . . -NN ..... ..
. 11.- ...
A ... G.A . . . . . . . - . . . . 0' ....
. 11. G.A - O.C.N
Figure le. Aligned sequences obtained with the C primer. The first base synthesized corresponds to
position 1627 of the S. cerevisiae 18S rRNA. Symbols are the same as in Figure la.
349
6"
2021
2032
1151
2036 1305lJ
97'
2011
1003
963
3697
14
14
16
15
16
16
14
15
19
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971
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3332
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983
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24
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15
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13
16
11
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13
13
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11
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20
20
23
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24
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31
117
118
116
25
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27
114
1"
"
119
29
35
,.
30
US
125
120
109
116
Figure 2a. Matrix of base differences between the Penicillium species and S. cerevisiae. Gaps in sequences
are counted as mismatches to any base present Base positions for which the correct base could not be
determined for one or more strains were excluded from the calculation. Total sequence length analysed is
708 positions.
845
3332
3697
2638
2106
3332
3697
14
2638
16
22
19
2106
27
31
26
27
URNULA
85
88
81
85
75
Y-12632
80
84
83
79
72
URNULA
75
Figure 2b. Matrix of base differences between Eupenicillium, Talaromyces, Urnula, and Saccharomyces.
Results were calculated as in figure 2a. Total length of sequence examined, 555 bases.
350
A. Logrieco et al.
It was recently proposed that the teleomorph genera Eupenicillium and Talaromyces with
Penicillium anamorphs, represent separate lines of evolution involving cleistothecial

Ascomycetes (Malloch, 1985). Malloch theorized that species in subgenus Biverticillium are
more closely related to Talaromyces since they can degrade cellulose and were probably
derived from species colonizing decayed wood such as Trichoma in the subfamily
Trichocomoideae. Malloch (1985) classified Penicillium anamorphs with a marked affinity
for starchy or oily substrates in the subfamily Dichlaenoideae. The latter would
encompass those species in subgenus Penicillium that are commonly isolated from
agricultural products (Pitt, 1979). Eupenicillium crustaceum and T. helicum differed by 31
bases in the abbreviated sequence length. Because the non-readable portion of the
sequence contained numerous base differences in other species, we suggest that these
genera could have as many as 40-45 different bases over the entire sequence length.
Urnula craterium and S. cerevisiae differed from E. crustaceum and T. helicum by 72-88 bases
in the abbreviated sequence length (Fig. 2b). At the same time, U. craterium and S.
cerevisiae differ from each other by 75 bases (Fig. 2b). These results suggest that the two
major teleomorph genera having Penicillium anamorphs can be traced to the same branch
in Ascomycete evolution. If the two genera had entirely independent origins we would
have expected a number of base differences equivalent to that recorded in contrasts
involving Urnula and/or Saccharomyces.
All but three of the isolates we examined differed by one or more bases and may
represent distinct species (Fig. 2a). Strains having no base differences (Le., P. puberulum
NRRL 845, P. resticulosum NRRL 2021, and P. camemberti NRRL 877) may represent
variants of the same species. In heterothallic yeasts, isolates of a sexually reproducing
species have an identical ribosomal RNA sequence, but isolates identified as Siblings, on
the basis of mating reactions and DNA complementarity, differ by as few as 2 and up to 7
base substitutions. Six isolates of S. cerevisiae representing isolates from different sources
had identical base sequences (5. Peterson and C. P. Kurtzman, unpublished). If these data
are representative of other fungi, a single nucleotide difference in the sequence of two
Penicillium isolates suggests that they are not the same species. This information will aid in
the resolution of several questions about taxonomic and evolutionary relationships among
the isolates of terverticillate Penicillia that we sequenced. Our results indicate that P.
Verrucosum NRRL 965, P. viridicatum NRRL 963, P. aurantiogriseum NRRL 971, P. hirsutum
NRRL 2032, and P. puberulum NRRL 845 (all ex neotype cultures) represent distinct
species. Samson et al. (1976) accommodated these and several other species in P.
verrucosum Dierckx. At that time, this was justified primarily on the basis of
morphological characteristics of the conidiogenous structures (e.g., fasciculate Penicillia
with two-staged, sometimes three-staged branched, rough-walled conidiophores and
globose to subglobose, smooth to slightly rough-walled conidia). Samson et al. (1976)
recognized strain NRRL 965 as the neotype culture of P. verrucosum and included this
strain in P. verrucosum var. verrucosum Samson et al., along with strain NRRL 963 (= P.
viridicatum Westling). Pitt (1979) retained P. verrucosum as a species and distinguished it
from P. viridicatum. Frisvad and Filtenborg (1983) used SMPs to place these and other
isolates of terverticillate Penicillia into species and provisional nonbotanical subgroups.
The authors proposed that SMPs, combined with recognizable microscopic and simple
physiological criteria, should be one of the bases for the establishment of a new
classification system of the terverticillate Penicillia. It was the authors' intent to allow
mycologists time to consider these experimental groupings before formally erecting new
varieties or species. Stolk and Samson (1985), citing "practical reasons" and the SMPs of
Frisvad and Filtenborg (1983), decided to reverse their earlier classification scheme
351
(Samson et al., 1976) and list these Penicillia as species. Our results provide evidence that
these distinct Penicillium chemotypes represent distinct species.
P. puberulum NRRL 845 and P. camemberti NRRL 877, ex type, showed identical base
sequences. At the same time, P. camemberti NRRL 877 and P. aurantiogriseum NRRL 971, ex
neotype, differed by 15 bases. This result does not support the hypothesis that P.
aurantiogriseum is the wild-type ancestor of the domesticated cheese mould P. camemberti
as suggested by Samson (1985). Cruickshank and Pitt (1987) reported that P. puberulum
(NRRL 2040, ex neotype) produced zymograms, suggesting synonomy with P.
aurantiogriseum, but our data indicating 13 base substitutions argues strongly against this
(Table 2a). The authors considered P. commune Thom NRRL 890a ex neotype to be
incorrectly placed in P. puberulum by Pitt (1979). The rRNA base sequences of this P.
commune strain were not examined and, therefore, cannot address the question of whether
P. commune, like P. puberulum, should also be recognized as a synonym of P. camemberti.
"Domesticated" Penicillia used in food fermentations were derived from naturally
occurring "wild" species (Samson, 1985) but Penicillium taxonomists may disagree as to
which species represent the "wild" progenitor (Polonelli et al., 1987). Frisvad and
Filtenborg (1983) established the chemotype P. camemberti II to include species formerly
classified in P. commune Thom. Penicillium camemberti was recognized as a domesticated
form of P. commune, the wild form occurring in nature (Polonelli et al., 1987). The search
for a wild-type strain of P. camemberti is now answered with the type strain of P.
puberulum isolated from corn. Because P. camemberti was described in 1906, while P.
puberulum was described in 1907, the combination P. puberulum var. camemberti would be
unacceptable according to the rules of nomenclature. Our data do not support placement
of P. puberulum in synonomy with P. aurantiogriseum (Samson et al., 1976) because the
neotype isolates differed by 15 bases. Raper and Thom (1949) noted that P. puberulum
NRRL 1889 and P. puberulum NRRL 845 came from the same original source, Thom No.
4876.20, a strain isolated from Zea mays L. and the basis of a classic paper on penicillic acid
formation by Alsberg and Black (1913). Strain NRRL 845, received by C. Thom in 1935,
had changed in cultural appearance, becoming more loose in texture and lighter sporing,
and resembled P. commune. Thom and Raper (1949) were not certain of the taxonomic
position of P. puberulum. The production of velvety colonies led Thom (1930) to place P.
puberulum in the Asymmetrica-velutina section, but Thom and Raper (1949) noted the
development of limited fasiculate structures in older colonies, and other characters
suggested a relationship to Penicillium cyclopium series in the Asymmetrica-fasciculata
section.
P. resticulosum was originally isolated as a culture contaminant in Birkinshaw's
laboratory (Raper and Thom, 1949). P. puberulum NRRL 845 and P. resticulosum NRRL
2021 have identical base sequences. P. puberulum NRRL 845 is a loose-textured, lightly
sporulating, cultural variant of isolate NRRL 1889. Both NRRL 845 and NRRL 1889 were
extensively investigated in Birkinshaw's laboratory and it is interesting to speculate that
NRRL 2021 represents another cultural variant of P. puberulum NRRL 1889. P. puberulum is
reported to form limited fasiculate structures suggesting a relationship to the P. cyclopium
series in the Asymmetrica-fasciculata section (Raper and Thom, 1949).
Samson et al. (1976) considered P. resticulosum to be a floccose variant of P. expansum.
This is not supported by our results, which show that P. expansum and P. resticulosum
differed by 9 bases. Pitt (1979) suggested that P. resticulosum was a distinct, rare species,
but reduced it to synonymy with P. expansum (Cruickshank and Pitt, 1987). It is important
to recognize that the three isolates with identical base sequences (i.e., P. puberulum NRRL
845, P. resticulosum NRRL 2021, P. camemberti NRRL 877) show identical numbers of
352
A. Logrieco et al.
different bases in contrast with other Penicillium isolates (Fig. 2a). The observation that
some species assigned by Raper and Thom (1949) to the sections Asymmetrica subsect.
Funiculosa and subsect. Lanata represent cultural variants of isolates classified in
subsections Velutina or Fasiculata (Samson et al., 1976; Pitt, 1979) is consistent with our
findings.
Frisvad and Filtenborg (1983) proposed that P. arenicola, P. fennelliae and P. olson ii,
species that Pitt (1979) included in subgen. Penicillium, were taxonomically distinct from
"true species" of tervertici1late Penicillia. We could not separate P. fennelliae or P. olsonii
from the more typical species belonging to subgen. Penicillium on the basis of substantial
differences in rRNA base sequences. P. arenicola showed a consistent pattern of higher
numbers of base differences when contrasted with the other terverticillate Penicillia. Stolk
and Samson (1985) noted that P. arenicola is not a typical Penicillium, but retained it in
Penicillium in agreement with Pitt (1979). Pitt (1979) included P. fennelliae in subgen.
Penicillium on the basis of the orginal illustrations, but noted that the isolates he examined
produced predominantly biverticillate Penicillia. Our results indicate a closer relationship
to species in subgen. Penicillium than to P. isariiforme in subgenus Biverticillium.
Raper and Thom (1949) classified P. olsonii in sect. Biverticillata-Symmetrica. Our results
suggest that P. olsonii NRRL 13058 (ex neotype) is more closely aligned with species
classified in subgen. Penicillium (Pitt, 1979).
P. claviforme NRRL 2031 (= P. vulpinum Cooke & Massee) Seifert & Samson) and P.
clavigerum NRRL 1003 share more bases in common (14 base differences) than either taxon
does with P. isariiforme NRRL 2628 (28 and 24 differences, respectively). Raper and Thom
(1949) placed P. claviforme and P. clavigerum in subsection Fasciculata because they form
coremia, but Pitt (1979) classified these species in subgen. Biverticillium with P. clavigerum
being placed in synonymy with P. duclauxii. Frisvad and Filtenborg (1983) distinguished
P. isariiforme on the basis of SMPs and strongly yellow-colored mycelium, agreeing with
its placement in subgen. Biverticil/ium, with P. claviforme and P. clavigerum remaining in
subgen. Penicillium. Our base se quence data supports their classification.
P. eyclopium var. echinulatum Raper & Thom was not validly published and Fassatiova
(1977) validated and raised it to species status. Our results confirm that P. echinulatum
NRRL 1151 and P. aurantiogriseum NRRL 971 (= P. eye/opium) are distinct species. P.
granulatum Bainier (= P. glandicola (Dud.) Seifert & Samson) is recognized as sharing
characteristics in common with P. verrucosum and P. brevicompactum (Pitt, 1979) but our
results indicate that P. granulatum NRRL 2036 shares more bases with P. puberulum, P.
olson ii, and P. hirsutum.
Classification schemes which rely on physiological characters (e.g., growth rates, toxin
production) as well as morphological characters are supported by our results. Ecological
and physiological data as well as SMPs are required if one is to distinguish Penicillium
species by methods other than degree of nucleic acid relatedness. Wicklow (1985)
observed that physiological attributes (Pitt, 1979), and SMPs (Frisvad et al., 1983) are
ecologically relevant characters that define the fungal niche. The fundamental niche of a
fungus can be defined in the laboratory by careful control of climate, substrate chemistry,
and interacting organisms (McNaughton, 1981). IT the niche parameters of two isolates are
distinct, it is likely they occupy different niches and would represent different species.
Williams et al. (1985) suggest that the considerable variation we find in subgen.
Penicillium may result from the "rapid adaptation of a relatively few ancient species to take
advantage of the many new nutritional niches provided by man during the few millennia
of his agricultural activity." An example of this is demonstrated by our results showing
that the domesticated white cheese mould P. camemberti has no base differences with the
353
naturally occurring wild species P. puberulum. At the same time, those terverticillate
Penicillia whose sequences differ by one or more bases represent species that predate
human agriculture.
REFERENCES
ALSBERG, C. L. and BLACK, O.F. 1913. Contributions to the study of maize deterioration; biochemical and
toxicological investigations of Penicillium puberulum and Penicillium stoloniferum. U.S. Department of
Agriculture Bureau of Plant Industry, Bulletin 270, pp. 1-47.
CHIRGWIN, J. M., PRZYBYLA, A.E., MACDONALD, R.J. and RUTIER, W.J. 1979. Isolation of biologically
active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299.
CIEGLER, A., FENNELL, D.l., SANSING, G.A. , DETROY, R.W. and
BENNETI, J.A.1973. Mycotoxin-producing isolates of Penicillium viridicatum: classification into subgroups.
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CRUICKSHANK, R. H. and PITI, J.I. 1987. Identification of species in Penicillium subgenus Penicillium by
enzyme electrophoresis. Mycologia 79:614-620.
ELWOOD, H. J., OLSEN, G.L. and SOGIN. M.L. 1985. The small subunit ribosomal RNA gene sequences
from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata. Molecular Biological Evolution 2:
399-410.
RAMIREZ, O. 1977. A taxonomic study of Penicillium series Expansa Thorn emend. Fassatiova. Acta
Universitatis Carolinae Biologica 12: 283-335.
FRISVAD, J. C. 1986. Taxonomic approaches to mycotoxin identification (taxonomic indication of mycotoxin
content in foods). In Modern Methods in the Analysis and Structural Elucidation of Mycotoxins, ed. R. J.
Cole, pp. 415-457. New York: Academic Press.
FRISVAD, J. C. and F1LTENBORG, O. 1983. Classification of terverticillate Penicillia based on profiles of
GEORGIEV, O. I., NIKOLAEV, N., HADJIOLOV, A.A., SKRYABIN, K.G., ZAKHARYEV, V.M. and BAYEV,
A.A. 1981. The structure of the yeast ribosomal RNA genes. 4. Complete sequence of the 25S rRNA gene
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LANE, D. J., PACE, B., OLSEN, G.J., STAHL, D.A., SOGIN, M.L. and PACE, N.R. 1985. Rapid determination
of 16s ribosomal RNA sequences for phylogenetic analyses. Proceedings of the National Academy of Science,
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LEISTNER, L. 1984. Toxigenic Penicillia occurring in feeds and foods. In Toxigenic Fungi-Their Toxins and
Health Hazard. eds. H. Kurata and Y. Uenopp. pp. 162-171. Tokyo: Kodansha.
MALLOCH, D. 1985. The Trichocomaceae: relationships with other Ascomycetes. In Advances in Penicillium
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MCCARROLL, R., OLSEN, G.J., STAHL, Y.D., WOESE, C.R and SOGIN, M.L. 1983. Nucleotide sequence of
the Dictyostelium discoideum small-subunit ribosomal ribonucleic acid inferred from the gene sequence;
evolutionary implications. Biochemistry 22: 5858-5968.
MCNAUGHTON, S. J. 1981. Niche: Definition and generalizations. InThe Fungal Community: Its
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Marcel Dekker.
PITI, J. L. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London:
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POLONELLI, L., MORACE, G., ROSA, R, CASTAGNOLA, M. and FRISVAD, J.e. 1987. Antigenic
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BAYEV, A.A. 1980. The complete structure of yeast ribosomal RNA genes. I. The complete nucleotide
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DIALOGUE FOLLOWING DR. PETERSON'S PRESENTATION

GAMS: I would like to ask Dr. Taylor and Dr. Peterson where they would draw the line to
distinguish species using your techniques. Are your techniques sensitive enough to
really distinguish species?
TAYLOR: This figure gives an indication of what molecular techniques can and cannot tell
us. Molecular techniques can give us this whole story if we do enough work. In this
diagram, we see species diverging and becoming extinct, diverging and becoming
extinct, as we pass through time. Finally, at the bottom, we see the species on the left is
355
quite distinct and no one has any problem recognizing it. The three species on the right,
however, remain close together, and are difficult to distinguish, no matter what methods
are used. With anamorphic genera there will always be the problem that closely related
species are going to be difficult to distinguish. If these taxa are important, such as being
mycotoxin producers, then they will be distinguished for practical reasons. If not, if
nobody cares about them, they will be lumped together.
PETERSON: I agree with Dr. Taylor. Ribosomal RNA shows us the phylogeny but doesn't
give us ability to assign a taxonomic level to a taxon. So, we're seeing a pattern of descent
and it's still a philosophical decision whether something is a species or a variety.
CAMS: You said you could not distinguish some of the terverticillate Penicillia at all, but in
your diagrams you show differences of two or three base changes. Is this not sufficient?
PETERSON: Our work with heterothallic species of yeasts, in which we do have a biological
species concept, is the only way we have of calibrating what these base changes mean
taxonomically. In sexually reproducing species, up to two base changes may exist in a
single species. If there were fifteen bases differences, the case for considering these
distinct species is overwhelming.
PITT: The work that is done with yeasts is fascinating, but it is irrelevant to the kind of
fungi we are considering here. It's impossible to relate a yeast species to a Penicillium or
Aspergillus species. The genome sizes are so different. We don't know anything about the
mating patterns in these moulds, of course. I think you should ask quite a different
question. Can you take ten isolates of P. aurantiogriseum and ten of P. commune, which
people in this area consider to be separate species, and make the distinction between
intraspecific variation in the parameter you are measuring, and the variation between
species?
PETERSON: The point is well taken. We have been planning to take this approach in our
laboratory. We had planned to use P. chrysogenum rather than P. commune. This needs to
be done.
PITT: When you do this work, please have your isolates checked by at least one other
taxonomist.
357
RIBOSOMAL DNA RESTRICTION STUDIES OF TALAROMYCES

SPECIES WITH PAECILOMYCES AND PENICILLIUM ANAMORPHS
J.W. Taylor1, J.I. Pitt2 and A.D. Hocking2
1Department of Plant Biology
University of California
Berkeley, California 94720, USA
2CSIRO, Division of Food Processing
North Ryde 2113, Australia
SUMMARY
Ribosomal DNA (rDNA) in species of Talaromyces and related genera were examined in an initial
attempt to understand their phylogenetic relationships. The variability in the nuclear rDNA repeat
unit was studied by the restriction fragments of total DNA that hybridized to the rDNA repeat unit of
Neurospora crassa (pMF2). Each fragment was treated as a taxonomic character with two states, present
or absent. Pairwise comparisons of all taxa were used to produce a matrix of similarity coefficients
which were subjected to UPGMA cluster analysis.
A comparison of 29 species with 21 having multiple isolates showed 14 with a majority of the
isolates as closest neighbours. Talaromyces species with Paecilomyces anamorphic states cluster with
Byssochlamys and Thermoascus species having Paecilomyces anamorphic states and not with Talaromyces
species having Penicillium anamorphs. The results also indicate that the strictly anamorphic Penicillium
species are not mixed in with the holomorphic species, but with one exception are clustered in a
group that is well separated from most of the Talaromyces species.
INTRODUCTION
The evolutionary relationships of Talaromyces species are interesting because the

morphology of both teleomorphs and anamorphs of these fungi indicate a close
relationship to other genera of Trichocomaceae (Fig. 1). We are applying molecular
methods to evolutionary studies of these fungi to add new data to the already welldescribed morphological features. At the present time, morphology is used to determine
both the evolutionary history of these fungi and the evolution of their morphology.
Morphology can be freed of this dual role by using molecular data to determine
phylogenetic relationships and then studying the evolution of morphology against the
background of genetic relatedness.
Talaromyces shares ascomata characters or anamorphic characters with the
holomorphic fungi Hamigera, Byssochlamys and Thermoascus (Stolk and Samson, 1971, 1972;
Subramanian, 1979; Malloch, 1981, 1985). Anamorphs of Talaromyces are assignable to
Penicillium subgenus Biverticillium, Paecilomyces and Geosmithia (Pitt, 1979a, 1979b; Samson,
1974). Obviously, there are many possible evolutionary questions involving these taxa. For
our initial study, we chose two topics: the first concerned the relative importance to
systematics of teleomorphic and anamorphic reproductive morphology, and the second
concerned the relationships of strictly anamorphic fungi to their presumed holomorphic
relatives.
The first topic concerns Talaromyces species with Paecilomyces anamorphs: are their
closest affinities with Talaromyces species with Penicillium anamorphs, as current
J.w. Taylor et al.
358
taxonomies suggest, or are they more closely related to Byssochlamys or Thermoascus

species which have Paecilomyces anamorphs? Stolk and Samson (1971) separated Hamigera
from Talaromyces on a difference in teleomorph morphology (asci formed in chains vs. asci
formed singly), but included species with Penicillium and Paecilomyces anamorphs. Pitt
(1979a) retained Hamigera in Talaromyces but did not treat the species with Paecilomyces
anamorphs. The main distinction between Talaromyces and Byssochlamys, the presence of
hyphae surrounding the asci in Talaromyces and its absence in Byssochlamys, is not
absolute. Stolk and Samson (1971) noted that some Byssochlamys cultures demonstrate a
covering over the asci, while Pitt (1979a) emphasized the difficulty in distinguishing single
asci from short chains, and of detecting croziers in the absence of detailed studies of
nuclear behavior. Talaromyces remains heterogeneous and ill-defined: on the one hand, it
includes species with asci formed both in chains and singly; on the other the separation
from Byssochlamys is not clear.
Merimbla
Hamigera
Eupenicillium
Penicillium
Talaromyces
Geosmlthia
Byssochlamys
Thennoascus
Paecilomyces
Figure 1. Chart of Talaromyces and relatives. Teleomorphic genera are inside the box,
anamorphic genera outside. The links between teleomorphs and anamorphs are shown by
the broad lines.
The second question concerns Talaromyces species with Penicillium anamorphs: are they
closely interrelated with strictly anamorphic species in Penicillium subgen. Biverticillium?
Penicillium subgen. Biverticillium species are morphologically similar to some Penicillium
anamorphs of Talaromyces species, suggesting a close relationship. This similarity may be
superficial, however, as Pitt (1988) has noted that, "... anamorphs of Talaromyces and
species in subgenus Biverticillium appear to be quite distinct." Molecular evidence may
help mycologists realize the long held but perhaps unrealistic goal of aligning strictly
anamorphic species with the holomorphic taxa from which they presumably arose.
To address these questions, variability in ribosomal DNA repeat regions was assessed
by comparing restriction fragments of miniprep DNA showing sequence identity with a
cloned rDNA repeat unit from Neurospora crassa (pMF2, Free et al., 1979). Although rRNAs
are evolutionarily conservative, the non-coding regions between the genes are known to
be much more variable (Jorgenson and Cluster, 1988); our approach sampled variability in
Ribosomal RNA restriction studies of Talaromyces species
359
both the coding and non-coding regions. Distinct fragments were treated as characters
with two states, present or absent, and subjected to UPGMA cluster analysis. This
approach has been used with mitochondrial DNA (mtDNA) and rDNA in Agaricus
(Anderson et al., 1986), with random nuclear DNA fragments in Neurospora (Natvig et al.,
1987), and with mtDNA in Phytophthora (Forster et al., 1988).
Fungi and cloned DNA.

The names, classification and culture collection accession numbers of the fungi studied are
given in Table 1. Plasmid pMF2, which contains the nuclear rDNA repeat unit of
Neurospora crassa (Free et al., 1979), was obtained from Dr. Peter Russell of Reed College,
Oregon through Dr. Michael Milgroom of Cornell University, New York.
Culture methods.
The fungi were maintained on Czapek Yeast Extract Agar (CYA, Pitt, 1988) or Malt Extract
Agar (MEA, Pitt, 1979a) at 25C or 30C. To produce mycelium for DNA extraction, the
fungi were grown in 250ml flasks containing 100 ml of broth (MEA or CYA without agar)
on a rotary shaker at 25C or 30C for from two to seven days.
DNA extraction.
Mycelium was harvested from broth through Miracloth (Calbiochem, La Jolla, California)
by vacuum filtration on a Buchner funnel, folded in the Miracloth, pressed briefly between
pads of paper towelling, frozen in liquid N2, and lyophilized. Lyophilized mycelium was
ground in a mortar and pestle and stored in microcentrifuge tubes at -20C. DNA was
extracted from the ground mycelium by the miniprep methods of Lee et al. (1988) or 20lan
and Pukkila (1986).
Digestion and electrophoresis.
From 5 J.1l to 10 III of DNA isolated by miniprep was digested by restriction enzymes using
the manufacturer's suggested conditions (New England Biolabs, Beverly, Massachusetts;
Bethesda Research Labs, Gaithersburg, Maryland; Pharmacia, North Ryde, N.S.W.).
Miniprep DNA that did not digest to completion was further purified by spermine
precipitation (5 J.1l of miniprep DNA was incubated with 1 J.1l of 100mM spermine on ice
for 15 minutes, microcentrifuged for 2 minutes, and the pellet resuspended in restriction
buffer; Alan Brownlee, CSIRO Division of Animal Production, Prospect, N.S.W., personal
communication; Hoopes and McClure, 1981). Digestion fragments were separated
electrophoretically adjacent to a molecular size marker (1.0kb marker, Bethesda Research
Labs, Bethesda, Maryland) in 1.0% agarose gels in O.IM Tris, 125 mMNaAcetate, and 1.0
mM EDTA. DNA in gels was stained with ethidium bromide (0.5 Ilg per ml) and
photographed with Polaroid film.
Southern hybridization.
Target DNA was transferred in alkali from agarose to a nylon membrane (ZetaProbe,
BioRad) in alkali by the general method of Reed and Mann (1985). Probe DNA (pMF2)
was labeled with 32p dATP by the general method of Rigby et al. (1977) using a nick
translation kit (BRESA, Adelaide, S. Australia). Hybridization and washing were carried
out at 65C by the general method of Reed and Mann (1985) using 7.0% lauryl sulphate,
J.w. Taylor et al.
360
Table 1. Taxa and isolates studied.
Isolates
Taxon
Genus TalllTomyces
Section TalllTumyces
Series Flavi
+T.f1avus
- T. striatus
Series Lutei
+T.luteus
Series Trachyspermi
T. trachyspermus
T. mimosinus
T. intermedius
Section Purpureus
T. purpureus
Section Thermophilus
+T. thermophilus
Section Paecilumyces
+T. byssochlamyoides
+T.leycettanus
Section Emersonii
+T. bacillisporus
+T. emersonii
Genus Byssochlamys
B.fulva
+B. nivea
Genus Hamigera
H.avellanea
Genus Penicillium
Subgenus Bivertidllium
Section Simplicium
Series Miniolutea
+P. minioluteum
+P. funiculosum
+P. purpurogenum
Series Islandica
P. islandicum
+P. variable
Genus Geosmithia
G. putterilli
G. cylindrospora
Genus Thermoascus
Th. crustaceus
Genus Paedlomyces
Section Paecilomyces
Pa. variotii
Section Isarioidea
Pa. farinosus
+Pa. marquandii
+Pa.liladnus
~1265,404,629,
1019,2098,3380,1976,2268,
2386,2417
~717,2080
~1941,1010,2235,1727
~1792,
1026
~1875
~ 3526 (CBS
152.65)
~1731
~2155,
1791
FRR 3523 (CBS 413.71), ~ 3524 (CBS 533.71)

FRR 1655, ~ 3525 (CBS 398.68)
FRR 947, 1025
~1324,1326,3221
~1125,2785,3493
~2205,2231
~1938
1741
1823, 1630
FRR 1061, 1147
~1095,
~ 833,
~1036,
~1048,
1399
1055
FRR 2037, 2024

FRR 1366, 2673
~1328,
1563
~1658,3054
FRR2670
FRR 3583, 2015
FRR 895, 1079
+ Majority of isolates clustered together in UPGMA analysis of all isolates

- MajOrity of isolates not clustered together.
FRR: CSIRO Division of Food Processing, N. Ryde, AustraIia.
CBS: Centraalbureau voor Schimmelcultures, Baarn.
361
Table 2. Number and molecular weight of restriction fragments hybridizing to NeurosporR

cloned nuclear rDNA (pMF2) from all isolates.
EcoRl
BRmHI
BglII
HindIII
Drill
kbp
kbp
kbp
kbp
kpb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
9.0
8.6
8.0
7.6
7.0
6.7
6.4
5.7
5.4
5.2
5.0
4.7
4.4
4.2
4.0
3.7
3.4
3.2
3.0
2.8
2.2
1.8
1.2
0.8
0.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
15
12
10.5
9.25
8.25
7.3
6.5
5.9
5.2
4.5
4.0
3.5
3.0
2.7
2.0
1.8
1.5
1.3
1
2
3
4
5
6
7
8
9
10
11
12
13
11.3
10.0
9.3
8.7
8.0
7.6
7.1
6.0
5.1
4.0
2.9
2.3
1.6
1
2
3
4
5
6
7
8
9
10
11
12
13
17
13
10
9.5
9.1
8.5
8.0
7.5
6.2
5.5
4.7
4.3
2.8
1
2
3
4
5
6
7
8
9
10
20
18
15
14
12
11
9.3
8
7.5
7.0
0.5% powdered milk and 1% polyethylene glycol (20M) in place of Denhardt's solution (cf.
Maniatis et aI., 1982). Hybridization of probe DNA to target DNA was visualized by
autoradiography (Fuji X-Ray film) over 3 to 24 hours.
Data analysis.
Molecular lengths of restriction fragments hybridizing to plasmid DNA were determined
by superimposing the autoradiographs over enlargements of photographs showing the
digested DNAs and the molecular length markers. For each enzyme, the molecular weight
was determined for each hybridizing fragment; each fragment was assigned a number so
that fragments of the same size from different isolates have the same number (Table 2).
Numbered fragments were treated as characters with two states, present or absent, and
their distribution was tabulated for all the fungi studied (Table 3). These discrete character
state data were converted to Jaccard similarity coefficients (Sokal and Sneath, 1963;
Simqual in Rholf, 1988) which emphasizes the positive matches over the negative matches
as is appropriate for restriction fragment data. The similarity coefficients were subjected to
UPGMA (unweighted pair-group method, arithmetic avearage clustering) with the "find"
option to search for trees of equally good fit (SAHN in NTSYS; Rholf, 1988). How well the
trees represent the similarity values was assessed by comparing the similarity coefficient
362
J.W. Taylor et a/.
matrix with a matrix of cophenetic values synthesised from the similarities taken from
each tree, using COPH in NTSYS, Sokal and Sneath (1963). If matrix correlations between
the original similarity coefficients and the cophenetic values, asessed by MXCOMP in
NTSYS, are greater than 0.9, the fit of tree to the similarity coefficient matrix is considered
very good (Rholf, 1988). Numerical methods were carried out using an IBM PCI AT
computer (registered) or its equivalent.
RESULTS
Selection of Restriction Enzymes.

Patterns of DNA hybridization of pMF2 to miniprep DNA from T. flavus 1265 were tested
for digestions made with 16 enzymes: SspI, Seal, HinfI, Bcn, DraI, Rsal, Clal, HindIII, Bgill,
EcoRV, Xbal, Taql, Sail, NsiI, BamHI and EcoRI. Six enzymes were selected for further
study: EcoRI, BamHI, Bgill, DraI, HindIII, and TaqI.
Hybridizations of pMF2 to miniprep DNAs of all isolates showed that five of these
enzymes gave from 10 to 25 distinct fragments in all isolates (Table 2) and from one to
three distinct bands per isolate (Table 3). Digestions with TaqI, which recognizes only four
nucleotides, produced too many small fragments hybridizing to pMF2 to be analyzed in
1.0% agarose.
Multiple isolates from a single species.
EcoR! fragments hybridizing to pMF2 were examined from at least two isolates in 21 of the
29 species studied (Table 1). UPGMA cluster analysis of all the isolates showed that 14 of
the species with multiple isolates had a majority of the isolates as closest neighbors
(marked "+" in Table 1), but 7 did not (marked "-" in Table 1). We consider this molecular
evidence generally supportive of the current species concepts. In subsequent comparisons,
often only one isolate was analysed from species where patterns were identical.
Talaromyces species with Paecilomyces anamorphs.

To assess relationships of Talaromyces species having Paecilomyces anamorphs with those
having the more usual Penicillium anamorphs and with other taxa having Paecilomyces
anamorphs, isolates of T. byssochlamyoides and T. leycettanus, both of which have
Paecilomyces anamorphs, were compared with: (i) Talaromyces species having typical
Penicillium anamorphs (T. flavus, T. luteus, and T. trachyspermus); (ii) Talaromyces species
having Penicillium anamorphs that also resemble Paecilomyces (T. intermedius and T.
thermophilus); (iii) Talaromyces species with the Hamigera type of ascus development having
Penicillium anamorphs (T. striatus and T. mimosinus); and (iv) Thermoascus and
Byssochlamys species having Paecilomyces anamorphs.
UPGMA cluster analysis based on restriction fragments hybridizing to pMF2 gave one
tree (Fig. 2) which showed that Talaromyces species with Paecilomyces anamorphs clustered
closest to Byssochlamys nivea and Thermoascus crustaceus, fungi which also have
Paecilomyces anamorphs. Of the three Talaromyces species with Penicillium anamorphs that
show some resemblance to Paecilomyces, none clustered with the species having
Paecilomyces anamorphs. The two B. fulva isolates did not cluster with the other
Byssochlamys or Thermoascus species, nor did they cluster together.
0629Tlla
Talaromyces/Penicillium
Talaromyces/Paecilomyces
Byssochlamys/Paecilomyces
Thermoascus/Paecilomyces
363
"
1.00
1265Tlla
0.56
3380Tlla
0.27
1976 Tlla
0.24
2098 Tlla
0.67
0404 Tlla
0.36
I
II
2268 TIIa
I
1
0.15
1019 Tlia
0.55
2386 Tlla
1.00
2417T11a
0.10
1941 nUl
0.88
1010 nUl
1.00
2235 TlUl
0.23
1875 Tmlm 0.14

3493 Btul
0.08 "
1792 Ttra 0.14

3526 TInt
0.57
1731 Tpur 0.06

1727 nUl
0.04
0717 Tstr 0.13
2080 Tstr
0.04
3523 Tbys 0.60

'655 Tley 0.33
2205 Bnlv 0.22"
'328lHcr 0.40 A
'563 THer 0.02 A

'026 Ttra 0.07
'125 Btul 0.03 "
2155Tthr 1.00
1791 Tthr
0.000
0.200
0.400
0.600
0.800
1.000
Figure 2. Relationships of TalaromyceslPenicillium, TalaromyceslPaecilomyces,

ThermoascuslByssochlamys, and ByssochlamyslPaecilomyces using UPGMA cluster analysis
of restriction fragments hybridizing to cloned Neurospora nuclear rDNA (pMF2). The
correlation between the similarity matrix supporting this tree and the matrix of cophenetic
values extracted from the tree is 0.927 (see Data Analysis in Material and Methods).
J.w. Taylor et a/.
364
Table 3. lCharacters and 2character states based on restriction fragments hybridizing

Neurosporll cloned nuclear rDNA (pMF2)
3Isoillte
0629
1019
1265
2098
3380
0404
1976
2268
2386
2417
1727
1941
1010
2235
1026
1792
3526
1731
2155
1791
0717
2080
1875
3523
1655
1125
3493
2205
1328
1563
1095
0833
1630
1823
1061
1147
1036
1399
1048
Tfla
Tfla
Tfla
Tfla
Tfla
Tfla
Tfla
Tf1a
Tfla
Tfla
T1ut
Tlut
Tlut
Tlut
Ttra
Ttra
Tint
Tpur
Tthr
Tthr
Tstr
Tstr
Tmim
Tbys
Tley
Bful
Bful
Bniv
THcr
THcr
Pmin
Pfun
Pfun
Pfun
Ppur
Ppur
pis1
Pisl
Pvar
EcoRI Chllrllders
BllmHl Chllr/lCters
0000000001111111111222222
1234567890123456789012345
0000000001111111111
1234567890123456789
0000000000000001100000001
0000001000000000100000000
0000001000000000100000000
0000000000000000101000001
0000001000000000100000000
0000000000000000101000001
0000001000000000100000000
0000000000000000101000001
0000000000000000110000001
0000000000000000110000001
0000100000000001000000000
0000000010000000001000000
0000000010000000001000000
0000000010000000001000000
0000000000000000010000010
0001000000000100000001000
0000000100000000100000000
0000000100000000100000000
0000000000000000010010000
0000000000000000010010000
0100000000100000000001100
0000000000010010000000000
1000010100000000001010000
0000000000010000001000000
0000100000010000001000000
0000000001000000010000000
1001000100001010000001000
0000100000010000000100000
0000000010000000001000000
0000000000100000001000000
0100000001000000010000000
0000000000000001010000000
0100000100000000010000000
0000000100000000010000000
0000000000000000010010000
0000000000000000010010000
0100000000100000010000000
0000000000100000010000000
0100000000000100000000000
0001000000000000000
0000000100000000100
0000000100000000100
0000100000000000000
0000000100000000100
0000000010001000000
0001000000000000000
0000000010110000000
0000000010001000000
0000000010001000000
0001000000000000000
0000100000000000000
0000100000000000000
0000100000000000000
xxxxxxxxxxxxxxxxxxx
0000010100000000000
0000010000000000100
0001000000000000000
0000001000000100000
0000001000000100000
0000010000000000000
xxxxxxxxxxxxxxxxxxx
0000100100000010000
0000001001000100000
0000000000100100000
xxxxxxxxxxxxx~xxxxx
0000100000000000000
0010001000000000000
0000000010000000100
0000000010000000100
0000001000000000000
0000000010000001000
0000001000000000000
0000000010000001000
0000010000000000000
0000010000000000000
0000001000000000000
0000001000000000000
0000000101000000000
3Isolate
0629
1019
1265
2098
3380
0404
1976
2268
2386
2417
1727
1941
1010
2235
1026
1792
3526
1731
2155
1791
0717
2080
1875
3523
1655
1125
3493
2205
1328
1563
1095
0833
1630
1823
1061
1147
1036
1399
1048
Tf1a
Tf1a
Tf1a
Tf1a
Tf1a
Tf1a
Tf1a
Tf1a
Tf1a
Tfla
T1ut
T1ut
T1ut
T1ut
Ttra
Ttra
Tint
Tpur
Tthr
Tthr
Tstr
Tstr
Tmim
Tbys
T1ey
Bful
Bful
Bniv
THcr
THcr
Pmin
Pfun
Pfun
Pfun
Ppur
Ppur
pisl
pis1
Pvar
BglII Characters
DraI Characters
HindIII Char.
0000000001111
1234567890123
0000000001111
1234567890123
0000000001
1234567890
0000001000101
0001000000000
0001000000000
0001000000000
0010000000000
0001000000000
0010000000000
0010000000000
0010000000000
0010000000000
0100000000000
0001000000000
0001000000000
0001000000000
1000000000000
0001000000000
0010000000000
0010000000000
0010000000000
0010000000000
0000000010010
0000100000000
0001000000000
0000001000000
0000001000000
0000100000000
0000001000100
0000001000000
0000010000000
0000001000000
0000001000000
0000010000000
0000000010100
0000010000000
0000010000000
0000010000000
0000001000000
0000001000000
0100000000000
0000100000000
0000100000000
0000100000000
0000100000000
0001000000000
0000100000000
0001000000000
0001000000000
0001000000000
0001000000000
0010000000000
0000100000001
0000100010001
0000100010001
xxxxxxxxxxxxx
xxxxxxxxxxxxx
xxxxxxxxxxxxx
xxxxxxxxxxxxx
xxxxxxxxxxxxx
xxxxxxxxxxxxx
0000001001001
0000001001001
0000010001001
0000001000000
0000001000000
xxxxxxxxxxxxx
0010000010010
0000000100000
0000001000000
0000000100000
0000010000000
0000010000000
0000001000000
0000100000000
0000100000000
0100000100000
1100000100000
1100000100000
xxxxxxxxxxxxx
0010001000
1000000000
1000000000
1000000000
1000000000
1000000000
0000001000
1000000000
1000000000
1000000000
1000000000
0000001000
0000001000
0000001000
xxxxxxxxxx
1000000000
1000000000
1000000000
0000000100
0000000100
1000000000
1000000000
1000010000
0100000000
0100000000
0100000000
0001001000
0100000000
0100000000
0100000000
0000010001
0000100010
0000010001
0000010010
0000010010
0000010010
0000010001
0000010001
0000001000
1 Characters are based on restriction fragments are given in Table 2.

2 Character states: O=absent, 1=present, x=no data.
3 Isolate names and classification are given in Table 1.
365
366
J.W. Taylor et al.
Penicillium subgenus Biverticillium.

To assess relationships between species of Penicillium subgen. Biverticillium and
Talaromyces, isolates of the strictly anamorphic Penicillium subgen. Biverticillium were
compared with: (i) Talaromyces species having typical Penicillium anamorphs; (ii)
Talaromyces species having anamorphs resembling Paecilomyces (Le. T. intermedius and T.
thermophilus); and (iii) Talaromyces species having the Hamigera type of ascus development
(Le. T. striatus and T. mimosinus).
UPGMA cluster analysis based on restriction fragments hybridizing to pMF2 gave one
tree (Fig. 3) which showed that Penicillium subgen. Biverticillium isolates group together
and, with the exception of P. variabile 1048, did not closely cluster with Talaromyces species
having typical biverticillate Penicillium anamorphs.
DISCUSSION
The size of the rDNA repeat unit of N. crassa, which we used as our probe (pMF2), is ca
lOkbp. The sum of the sizes of the fragments hybridizing to pMF2 in particular digests
was found to be ca 9 to 10 kbp (Tables 2 and 3). However, the sums for single isolates were
not equal for different enzymes and for many isolates the sizes of the HindIII fragments
were much larger (ca 20kbp). The inequality of sums for different enzymes was probably
due to our inability to resolve restriction fragments smaller than 3-400 bp in 1% agarose.
The very large HindIII fragments did not appear to be due to incomplete digestion and
their presence is difficult to explain.
The variability in the sum of the sizes of the hybridizing fragments indicates that the
approach used is relatively crude. Fragments of similar size are being compared, and
some regions are not accounted for in each isolate because small fragments could not be
analyzed. Also, the cluster analyses combined changes in fragment size due to restriction
site changes (caused by nucleotide substitutions or small length mutations) with fragment
size changes due to large length mutations (cf. Taylor, 1986). In spite of the simplicity of
this technique, it has provided interesting information on the evolutionary relationships of
Talaromyces and suggests questions that deserve intensive study by more laborious
techniques such as DNA sequencing.
In 14 of the 21 cases where more than one isolate from a species was examined,
conspecific isolates were closest neighbors in cluster analysis (Table 1). We consider this
molecular evidence generally supportive of species concepts in these fungL However,
cases where isolates did not cluster should be studied further. Perhaps convergent
evolution or unsuspected misidentifications have occurred.
The comparison (Fig. 2) of Talaromyces species having Paecilomyces anamorphs with
Talaromyces species having Penicillium anamorphs and with Byssochlamys and Thermoascus
species having Paecilomyces anamorphs shows that Talaromyces species with Paecilomyces
anamorphic states cluster with Byssochlamys and Thermoascus and not with Talaromyces
species having Penicillium anamorphs. Perhaps in these cases the anamorphs may be a
better indicator of relatedness than the ascomata of the teleomorphs. This result, if
confirmed by sequence analYSiS, will be important in Ascomycete systematics.
In the comparison of species in Penicillium subgen. Biverticillium with Talaromyces
species having Penicillium anamorphs (Fig. 3), the strictly anamorphic Penicillium species,
with one exception, have clustered in a group that is well separated from the Talaromyces
species. It may be inferred that anamorphic Penicillium species in subgen. Biverticillium
arose from holomorphs on rare occasions and that most of their interspecific diversity has
367
0629 Tlia 0.15
Talaromyces/Penicillium
Penicillium
subgenus Biverticillium
1019 Tlla 1.00
I 1265 Tlia
1976 Tlia 0.24

2098 Tfla 0.67
0404 Tlla 0.36

2268 Tlla 0.55
2386 Tlia 1.00
J2417 Tlia
[J
0.56
3380 Tlia 0.27
0.12
1941 Tlut 0.88

lOla Tlut
1.00
2235 Tlut 0.23

1875 Tmim 0.07
1727 Tlut 0.05
3526 Tint 0.57
1731 Tpur 0.10

2155 llhr 1.00
I 1791
Tthr 0.04
1792 llra 0.18
1048 PIlar 0.05

0717 Tstr 0.13
2080 Tstr 0.03
1630 Pfun 0.35
1095 Pmin 0.46
I
0.000
1036 Pisl 0.90.

1399 Pisl 0.15.
0833 Pfun 0.45
1823 Pfun 0.33

1061 ppur 0.67
1147 Ppur 0.01

1026 Ttra
0.200
0.400
0.600
O.BOO
1.000
Figure 3. Relationships of TalaromyceslPenicillium and anamorphic Penicillium subgenus

Penicillium using UPGMA duster analysis of restriction fragments hybridizing to cloned
Neurospora nuclear rDNA (pMF2). The matrix correlation between the similarity matrix
supporting this tree and the matrix of cophenetic values extracted from the tree is 0.931 (see
Data Analysis in Material and Methods).
368
J.w. Taylor et al.
developed since their divergence from the holomorph. If supported by further

investigation, this result has application throughout the Deuteromycotina, where
mycologists have hoped to link strict anamorphs with their nearest teleomorphic relative.
This result indicates that in many cases a one-to-one relationship does not exist.
The questions addressed here are representative of the many that will be important to
understanding the relationships of Penicillium and its anamorphic and teleomorphic
relatives. Our results, although preliminary, indicate that currently accepted species are
generally well defined, but that relationships at higher taxonomic levels, or between
holomorphs and strict anamorphs, are less clear. This survey has shown that questions of
interest to students of Trichocomaceae in particular and Ascomycotina in general can be
approached by molecular methods: needed now is a thorough investigation with a more
powerful technique. We are beginning to use DNA polymerase chain reaction
amplification (PCR, Saiki et al. 1985, Mullis and Faloona 1987) of rDNA and direct
sequencing of the product (Wrischnik et al. 1987, Gyllensten and Erlich 1988) to obtain
better data on fungal evolution.
ACKNOWLEDGEMENTS
We thank N. Charley of CSIRO Division of Food Processing for assistance with fungal
cultures, and J. Mattick, K. Finney, M. Bills, and S. Livingstone of CSIRO Division of
Biotechnology for making the molecular work possible at North Ryde, and W. Stone of the
Jepson Herbarium, University of California, Berkeley for helping to format the figures.
Support for this research was provided by CSIRO and NSF (!NT 8702240, BSR 8516513).
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JORGENSON, R. A. and CLUSTER, P. D. 1988. Modes and tempos in the evolution of nuclear ribosomal
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MANIATIS, T., FRITSCH, E. F. and SAMBRooK, J. 1982. Molecular Cloning: A Laboratory Manual. Cold
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MULLIS, K. B. and FALooNA, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase catalysed
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NATVIG, D.O., JACKSON, D. A. and TAYLOR, J. W. 1987. A random fragment hybridization analysis of
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PITT, J.I. 1979a. The Genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic
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PITT, J. I. 1979b. Geosmithia gen. nov. for Penicillium lavendulum and related species. Canadian Journal of
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PITT, J.I. 1988. A laboratory guide to common Penicillium species. 2nd ed. North Ryde, N.5.W.: CSIRO
REED, K. R. and MANN, D. A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic
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RHOLF, F. J. 1988. NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 1.4. Setauket,
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SAMSON, R. A. 1974. Paecilomyces and some allied Hyphomycetes. Studies in Mycology, Baarn 6: 1-119.
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STOLK, A. C. and SAMSON, R. A. 1971. Studies on Talaromyces and related genera. I. Hamigera gen. nov.
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TAYLOR, J. W. 1986. Fungal evolutionary biology and mitochondrial DNA. Experimental Mycology 10: 259269.
WRISCHNIK, L. A., HIGUCHI, R. G., STONEKING, M., ERLICH, H. A., ARNHEIM, N. and WILSON, A. C.
1987. Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified
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Cellular Biology 6: 195-200.
DIALOGUE FOLLOWING DR. TAYLOR'S PRESENTATION

SAMSON: Dr. Frisvad and I recently completed a chemotaxonomic study looking for a
connection between the biverticillate Penicillia and Talaromyces. We also found no
connection. There was some similarity between P. variabile and some Talaromyces species
just as you found. T. flavus divided into two distinct groups, which correspond to the
two varieties Dr. Stolk and I described in 1972. These varieties are chemically quite
distinct. Strains of T. flavus var. macrosporus are now becoming a problem during food
processing because of their heat resistant ascospores.
TAYLOR: We also have two general groups within Talaromyces flavus that more or less
correspond with the two varieties. But there were a couple of isolates which, based on
the ribosomal repeat region, fall into the wrong variety. It would be interesting to look at
the secondary metabolites of those particular isolates. It may be that the intergenic region
is variable and the same mutation has occurred in different isolates. This lack of
correlation is not due to misidentification.
370
J.w. Taylor et al.
Talaromyces byssochlamydoides is very close to

Byssochlamys. That's why it has that name, of course. The ascomata of this species are
more typical for Talaromyces, which is why we placed it there.
SAMSON: I'm not surprised to see
We don't know a priori which of the characters we are looking at is evolutionarily

more stable. By comparing morphology with the evolutionary story that we get from a
completely different kind of character, we may be able to select the characters that are
most stable.
TAYLOR:
SAMSON: What strikes me is that the Paecilomyces teieomorphs,
Talaromyces, Thermoascus
and Byssochlamys, are a relatively homogeneous group: they are thermophilous and have
similar ascospores. Only the ascomata are different.
8
NEW APPROACHES FOR PENICILLIUM AND
ASPERGILLUS SYSTEMATICS: BIOCHEMICAL AND
IMMUNOLOGICAL TECHNIQUES
373
SECONDARY METABOLITES AS CONSISTENT CRITERIA IN

PENICILLIUM TAXONOMY AND A SYNOPTIC KEY TO PENICILLIUM
SUBGENUS PENICILLIUM
J.e. Frisvad and O. Filtenborg

SUMMARY
As taxonomic characters in Penicillium, morphology and profiles of secondary metabolites have
proved to be consistent and reproducible, provided they are recorded in a standardized and
systematic way. Techniques for standardising profiles of secondary metabolites include the use of
griseofulvin and preferably other commercially available secondary metabolites, and the use of
standardised media, inoculation and incubation conditions and extraction techniques. Confirmation
of results obtained using thin layer chromatography is strongly recommended, for example by high
performance liquid chromatography and diode array detection, especially when new or usual results
are found. Examples of consistent production of secondary metabolites in known culture collection
strains of important Penicillium species are provided in this paper. A synoptic key to Penicillium
subgen. PeniCl1lium, based on secondary metabolites, is also given.
INTRODUCTION
Biochemical products such as citric acid (Citromyces Wehmer), oxalic acid (Penicillium
oxalicum Currie et Thom) and citrinin (P. citrinum Thom), mycelial colours and diffusible
pigments have been used in Penicillium taxonomy for a long time (Thom, 1930). Despite
the monumental works of Raistrick and coworkers on the identification of a great number
of Penicillium metabolites (Turner and Aldridge, 1983), individual secondary metabolites
did not influence the systematics of Penicillium much before 1980. Ciegler et al. (1973) used
penicillic acid, ochratoxin A and citrinin as criteria in their subdividing P. viridicatum
Westling into subgroups, but did not draw any taxonomic conclusions. However, Frisvad
(1981,1983), emphasized secondary metabolites, backed up by physiological characters,
when he proposed "P. viridicatum o-c" (= P. verrucosum Dierckx), "P. crustosum pA" (= P.
crustosum Thom), "P. cyclopium p" (= P. aurantiogriseum Dierckx), "P. melanochlorum" and
"P. caseiphilum" as species concepts. Later work (Frisvad, 1985; Cruickshank and Pitt, 1987)
has validated these concepts, which are now recognised as P. verrucosum Dierckx, P.
crustosum Thom, P. aurantiogriseum Dierckx, P. solitum Westling and P. commune Thom
respectively. In introducing profiles of secondary metabolites into Penicillium taxonomy,
Frisvad and Filtenborg (1983) provisionally used the subgroup concept of Ciegler et al.
(1973), but these were later transferred species, varieties or chemotypes to correct names
(Frisvad, 1985; Frisvad and Filtenborg, 1989).
Profiles of secondary metabolites has been proposed as objective and consistent
taxonomic characters by Frisvad and coworkers (Filtenborg et al., 1983; Frisvad and
Filtenborg, 1983; Frisvad and Thrane, 1987; Frisvad, 1988, 1989; Frisvad et a/., 1989).
Although some isolates in Penicillium may in time lose the ability to produce one or two
secondary metabolites, the remaining profile of can be sufficient to identify an unknown
374
J.e Frisvad &O. Filtenborg
(Frisvad, 1989), provided a standardized method is used for the analysis. An effective,
standardized system with diode arrays detection high performance liquid
chromatography (HPLC-DAD) method for analysis of secondary metabolites in fungi has
been introduced by Frisvad and Thrane (1987). However, advanced liquid
chromatographs are available to few mycologists, so thin layer chromatography (TLC) is
the method of choice in the average mycological laboratory. Filtenborg and Frisvad (1980)
and Filtenborg et al. (1983) introduced a simple TLC method for the analysis of
mycotoxins and other secondary metabolites from growing fungi. The method,
application of small agar plugs to TLC plates with or without extraction) results in a quite
low sensitivity, so optimial inoculation techniques, media and incubation conditions, etc.,
are necessary.
Some authors have reported good results with the agar plug method (Blaser and
Schmidt-Lorenz, 1981; Abarca et al., 1988; Klich and Pitt, 1988). Other authors have in
reported inconsistent or irreproducible results with Penicillium species (Paterson et al.,
1987; Bridge et al., 1986, 1987; Land and Hult, 1987; Stenwig, 1988). For example, Stenwig
(1988) reported that clearest and most intense spots on the TLC plates were produced by
all isolates in a species, but that less distinct spots showed poor reproducibility, caused by
metabolite concentrations varying from above to below their detection limit in different
chromatographic runs. He also stated that detection of the weak spots improved if several
isolates of a species were compared on the same TLC plate, or if standards were used.
In this paper, we report on ways to consistently and reproducily detect secondary
metabolites of Penicillium species by the agar plug method. A synoptic key for Penicillium
subgen. Penicillium, based on secondary metabolites and other characters is also provided.
Ex type and authentic cultures of Penicillium subgen. Penicillium species were examined
for production of secondary metabolites using the TLC agar plug method (Filtenborg and
Frisvad, 1980; Filtenborg et al., 1983, Frisvad et al., 1989). For TLC analysis agar plugs were
taken from Czapek yeast extract agar (CYA), malt extract agar (MEA), yeast extract
sucrose agar with Difco yeast extract (YES) and yeast extract sucrose agar with Sigma (Y4000) yeast extract (SYES) (Frisvad and Filtenborg, 1983). Trace metals were added to all
media (Frisvad and Filtenborg, 1983). Cultures were incubated for 5 to 21 days in the dark
at an upright position in 9 em plastic Petri dishes in polyethylene bags, with holes to avoid
carbon dioxide accumulation.
TLC plates were eluted in CAP (chloroform/acetone/2-propanol, 85:15:20) and TEF
(toluene/ethylacetate/90% formic acid, 5:4:1) with griseofulvin as external standard.
Plates eluted in CAP were treated with 1% Cerium sulphate in 50% sulphuric acid. Plates
eluted in TEF were treated in two ways: with cold 50% sulphuric acid for intracellular
metabolites; and with anisaldehyde spray for extracellular metabolites. Anisaldehyde
spray contains 0.5% anisaldehyde in methanol/acetic acid/sulphuric acid, 17:2:1 and
visualisation is by heating for 8 min at 1300 (Frisvad et al., 1989).
TLC plates were also scanned with a CAMAG TLC scanner and UV reflectance
spectra recorded. High performance liquid chromatography (HPLC) with diode array
detection (DAD) was also employed using the method of Frisvad and Thrane (1987). In
that way UV spectra could be compared from HPLC and TLC to assure a good
confirmation of the identity of the secondary metabolites by comparison of fungal
metabolites with standards.
Secondary metabolites as consisitenl criteria in Penicillium taxonomy
375
Different ways of improving the consistency of detection of secondary metabolites in the

terverticillate Penicillia were tested (Table 1).
Table 1. Methods for increasing production and/or reproducibility of detection of secondary
metabolites in agar cultures.
Use of fresh cultures or cultures directly from freeze dried tubes or from silica gel, i.e.
avoidance of repeatedly transferred cultures.
Avoidance of carbon dioxide accumulation during incubation.
Combined point and streak inoculation.
Use of agar plugs from different places in the fungal colony.
Repeated application of several plugs on the same spot.
Use of several effective media, such as CYA, YES, SYES, MEA). Special media may be necessary
for particular secondary metabolites.
Use of more than one incubation temperature: 25 is generally best, but 20 or 30 may be much
better in some cases.
Optimization of extraction time and extraction technique: usually chloroform/methanol, 2:1 is
excellent, but chloroform/acetone or other mixtures may be significantly better in some cases.
Use of known standards (see Table 2).
Use of known cultures with good secondary metabolite production as standards (Frisvad,
1985).
Optimization of all the above conditions on good, authentic cultures from each taxonomic
group.
Usually the standard conditions recommended by Filtenborg et al. (1983) were sufficient
for reproducible recovery of important secondary metabolites, but each laboratory should
optimise the methods, using fresh isolates with known profiles of secondary metabolites
(Frisvad, 1985). Medium composition is of particular importance (Filtenborg et al., 1990) In
some cases a particular combination of medium and spray reagent is 10 to 100 times as
sensitive as the standard method. For example, patulin is best produced on potato
dextrose agar and visualized by MBTH spray (Frisvad et al., 1989) and verrucosidin is
produced optimally on Merck malt extract agar and visualized as a intracellular
metabolite by anisaldehyde spray (El-Banna and Leistner, 1987). The use of commercially
available standards (Table 2) greatly improves the TLC identification procedure and use is
strongly recommended.
The intracellular alkaloids roquefortine C, meleagrin, oxaline and penitrem A are
produced very consistently on CYA by all isolates in the relevant species, indicated in (the
synoptic key). The production of these alkaloids on YES and SYES is usually limited,
though better in strongly sporulating cultures. Analysis for intracellular mycotoxins
produced on CYA and eluted in CAP using Cerium sulphate spray is therefore
recommended as the first step in the identification of the terverticillate Penicillia by
profiles of secondary metabolites.
J.e Frisvad & O. Filtenborg
376
Table 2. Commercially available standards of secondary metabolites produced by
Penicillium.
Mycotoxin
Optimal production medium
Alternariol monomethylether
Chaetoglobosin C
Citreoviridin
Citrinin
Cyclopiazonic acid
Dipicolinic acid
Gliotoxin
Griseofulvin
Kojic acid
Mycophenolic acid
3-nitropropionic acid
Ochratoxin A
Patulin
Penicillin
Penicillic acid
PR-toxin
Rubratoxin B
Secalonic acid D
CYA
CYA,SYES
YES,SYES
YES,SYES
CYA
YES
TGY (Frisvad et al., 1987)
YES,SYES
YES
YES
YES
YES,SYES
YES, SYES, Potato dextrose agar
TGY (Frisvad et al., 1987)
YES, Raulin-Thom (Betina, 1989)
YES
YES
YES,CYA
Four important indol alkaloids detected consistently as intracellular metabolites on TLC plates
eluated in CAP after visualization with Cerium sulphate spray:
Roquefortine C: Orange spot, Rf 0.46 (a) in P. crustosum (not fluorescing)
Penitrem A:. Blue spot, Rf 1.16 in P. crustosum (not fluorescing)
Meleagrin: Yellow spot, Rf 0.65 in P. chrysogenum (a) (olive yellow fluorescence)
Oxaline: Olive yellow, Rf 0.71 in P. atramentosum (olive green flourescence)
(a) Rf value relative to
griseofulvin; (b) P. chrysogenum also produce roquefortine C
The next recommended step is analysis for intracellular metabolites produced on CYA
and YES, eluated in TEF. By this procedure a variety of mycotoxins can be detected:
xanthomegnin, and brevianamide A, yellow and viomellein, yellow brown, in daylight;
griseofulvin, a blue flourescence under UV light; citrinin, a yellow green fluorescing tail;
ochratoxin A, a turquoise fluorescing spot, mycophenolic acid, a violet blue fluorescing
spot, and cyc1opiazonic acid, a brown fluorescing tail. Spraying with cold sulphuric acid
visualizes cylopenin and viridicatin, which produce a violet blue fluorescence) and the
characteristic series of 6 bluish flourescing metabolites in P. echinulatum, the penechins.
Further spraying with anisaldehyde and heating visualizes chaetoglobosin C,
verrucosidin and compactin.
The third recommended step in the identification procedure is analysis for
extracellular metabolites produced on YES and SYES agar, eluted in TEF and visualized
by anisaldehyde spray and heating. Predominantly, extracellular mycotoxins such as
penicillic acid, patulin and terrestric acid, a yellow spot in daylight, are detected in this
system.
Secondary metabolites as consisitent criteria in PenicilUum taxonomy
377
Table 3. Consistent production of known secondary metabolites by Penicillium coprophilum
Isolate
CBS 477.75
CBS 473.75
FRR1403
NRRL 13627
IMI285526
IMI321500
IMI293196
IMI321501
ATCC64629
Griseofulvin
Roquefortine C
Meleagrin
Oxaline
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table 4. Consistent production of known secondary metabolites by Penicillium griseofulvum
Isolate
NRRL2300
NRRL993
NRRL2159A
NRRL A-23324
NRRL A-26914
IMI296933
ATCC9260
IMI293195
IMI285525
lF07010
CSIR 1399
CSIR 1082
FRRI232
Griseofulvin
Cyclopia'Zonic
acid
Patulin
Roquefortine C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table 5. Consistent production of secondary metabolites by Penicillium brevicompactum
Isolate
IMI40225
IMI92044
IMI 94149
IMI92219
IMI17456
IMII25546
IMI285520
IMI293191
NRRL859
NRRL A-23329
CBS 317.59
CBS 256.74
CBS 210.28
NRRL886
Mycophenolic acid
Raistrick phenols
Brevianamide A
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
J.e Frisvad & O.
378
Rltenborg
Many secondary metabolites are produced very consistently in Penicillium species as

shown in Tables 3, 4 and 5, and Fig. 1. Fig. 1 shows HPLC traces of two P. echinulatum
isolates. The traces are strikingly similar, and they are also similar to traces produced by
the culture ex type (NRRL 1151) and several other isolates tested by this method.
Cyclopenin, cyclopenol, viridicatin, viridicatol and the penechins were all consistently
produced. UV spectra as determined by either HPLC DAD or UV reflectance spectra on
TLC plates were valuable in designating the chromophore families in the different species
(Fig. 2.).
4121121
'iic
11.
3121121
2-
.
11.
0
>.
2121121
c ii
..
>.
...
L
")
P.
echfnulatum ex bakers yeast
.
~
a
...
-;:
")
1121121
:J
II:
E
121
-1121121
-2121121
-3121121
-4 121
P.
10
20
Tl me
echfnulatum ex cucumber
(mf n. )
30
4121
Figure 1. HPLC traces of two isolates of P. echinulatum. Note the very high qualitative
similarity and the quantitative differences in individual compounds. All major peaks, except
those marked, are members of the penechin chromophore family.
For example, P. coprophilum always produces griseofulvin, dechlorogriseofulvin,

roquefortine C, meleagrin and oxaline irrespective of geographic source or habitat,
including soil, dung, feedstuffs or foods). Study of P. coprophilum metabolites by HPLC
DAD revealed a consistently produced series of unknown specific secondary metabolites
as well. Similarily P. griseofulvum always produce griseofulvin, dechlorogriseofulvin,
cyclopiazonic acid, roquefortine C and patulin (Table 4).
Except for brevianamide A, all known alkaloids are produced very consist ently by
all isolates in each relevant terverticillate Penicillium species (Frisvad and Filtenborg,
1989). The insecticidal toxin brevianamide A (Paterson et at., 1987) is produced by two
species, P. brevicompactum and P. viridicatum, but only by a minor proportion of isolates.
Other metabolites are consistent produced by P. brevicompactum (Table 5) and P.
viridicatum, however, so this is not a major problem. Consistent production of
mycophenolic acid and the Raistrick phenols clearly identify P. brevicompactum.
Secondary metabolites as consisitent criteria in Penicillium taxonomy
UV 24.619
140
379
(6) OT TERVA08A.D
A PENECHIN FROM
P.
ECHINULATUM
120
100
80
II:
E
60
40
20
250
300
Wavelength
350
(nm)
400
450
Fig. 2. UV spectrum of a major penechin obtained by diode array detection in a HPLC run of
an extract of P. echinulatum NRRL 1151.
100
220nm
450nm
Fig. 3. UV reflectance spectrum of the major penechin (Rf value relative to griseofulvin in
TEF: 1.0) obtained by a CAMAG TLC scanner. Note the similarity to the UV spectrum
obtained by HPLC-DAD (see Fig. 2).
380
J.e Frisvad & O. FiHenborg
Some extracellular mycotoxins such as patulin, penicillic acid and citrinin are not
consistently produced by some isolates of P. expansum or P. aurantiogriseum on the
standard media, but are other media often assist toxins production. Land and Hult (1987),
for example, could not detect patulin in some isolates of P. expansum. When some of those
isolates were tested on oatmeal agar, MEA or potato dextrose agar, we found large
amounts of patulin. The other Penicillium isolates from wood investigated by Land and
Hult (1987), which were P. roqueforti and P. commune, also produced their typical
mycotoxins in our hands (Frisvad and Filtenborg, 1989). Bridge et al. (1986, 1987) also
reported inconsistent secondary metabolite production by some isolates of P. viridicatum,
P. glandicola, P. crustosum and P. commune.
These fungi were all very efficient producers of their usual mycotoxins in our hands
(Frisvad and Filtenborg, 1989).
SYNOPTIC KEY TO PENICILLIUM SUBGENUS PENICILLIUM
As secondary metabolite production by species in Penicillium subgen. Penicillium is very

consistent when the recommended methods of analysis listed in Table 1 are followed, we
offer the following synoptic key. It is based on individual secondary metabolites,
physiological characters and some morphological characters.
List of included species and varieties.
Species bracketted (26-28) are not classified in subgen. Penicillium, but are included because of similarities in
metabolites.
1.
2.
3a.
3b.
3c.
3d.
3e.
4.
Sa.
6a.
6b.
7.
5b.
5c.
8.
9.
10.
11.
12.
13a.
13b.
14.
15.
16a.
16b.
P. aethiopicum Frisvad 1989

P. atramentosum Thorn 1910
P. aurantiogriseum Dierckx 1901 var. aurantiogriseum
P. aurantiogriseum var. polonicum (Zaleski) Frisvad 1989
P. aurantiogriseum var. melanoconidium Frisvad 1989
P. aurantiogriseum var. neoechinulatum Frisvad et al. 1987
P. aurantiogriseum var. viridicatum (Westling) Frisvad et Filtenborg 1989
P. brevicompactum Dierckx 1901
P. camemberti Thorn 1906
P. chrysogenum Thorn 1910 var. chrysogenum
P. chrysogenum var. dipodomyis Frisvad et al. 1987
P. clavigerum Demelius 1922
P. commune Thorn 1910 chemotype I
P. commune chemotype II
P. confertum (Frisvad et al.) Frisvad 1989
P. coprophilum (Berk. et Curt.) Seifert et Samson 1985
P. coprobium Frisvad 1989
P. crustosum Thorn 1930
P. digitatum (Pers.:Fr.) Sacco 1832
P. echinulatum (Raper et Thorn) Fassatiova1977 chemotype I
P. echinulatum chemotype II
P. expansum Link 1809
P. fennelliae Stolk 1969
P. glandicola (Oud.) Seifert et Samson 1985 var. glandicola
P. glandicola var. glaucovenetum Frisvad 1989
17a.
17b.
18a.
18b.
18c.
18d.
18e.
19.
20.
21.
22.
23a.
23b.
24a.
24b.
25.
(26.
(27.
(28.
381
P. griseofulvum Dierckx 1901 var. griseofulvum

P. griseofulvum var. dipodomyicola Frisvad et al. 1987
P. hirsutum Dierkx 1901 var. hirsutum
P. hirsutum var. albocoremium Frisvad 1989
P. hirsutum var. allii Frisvad 1989
P. hirsutum var. hordei (Stolk) Frisvad 1989
P. hirsutum var. venetum Frisvad 1989
P. italicum Wehmer 1894
P. solitum Westling 1911
P. mononematosum (Frisvad et al.) Frisvad 1989
P. olsonii Bain. et Sart. 1912
P. roqueforti Thorn 1906 var. roqueforti
P. roqueforti var. carneum Frisvad 1989
P. verrucosum Dierckx 1901 chemotype I
P. verrucosum chemotype II
P. vulpinum (Cooke et Massee) Seifert et Samson 1985
P. sclerotigenum Yamamoto 1955)
P. oxalicum Currie et Thorn 1915)
P. lanosum Westling 1911)
Synoptic key.
Production of griseofulvin: 1,9, 17a, 17b, 26, 28
Production of mycophenolic acid: 4, 23a, 23b
Production of ochratoxin A: 24a, 24b
Production of citrinin: 14, 18b, 24b
Production of cyclopiazonic acid: 5a, 5b, 5c, 17a, 17b
Production of chaetoglobosin C: 13b, 14
Production of kojic acid: 28
Production of secalonic acid D: 27
Production of PR-toxin: 23a
Production of patulin: 10,14, 16a, 16b, 17a, 17b, 23b, 25
Production of penicillic acid: 3a, 3b, 3c, 3d, 3e15, 18b, (23b)
Production of penicillin: 6a, 6b
Production of terrestric acid: (3a), 11, 18a, 18b, 18d, 18e
Production of roquefortine C: 2, 6a, 8, 9, 11, 14, 16a, 16b, 17a, 18a, 18b, 18c, 18d, 18e, 23a,
23b, 25, 26, 27
Production of meleagrin: (2), 6a, 8, 9, 16a, 16b, 18b, 18c
Production of oxaline: 2, 3c, 9, 16a, 25, 27
Production of cyclopenin and viridicatin: 3a, 3b, 3c, 3d, 3e, (5c), 11, 13a, 13b, 18b, 18c, 18e,
20,25
Production of palitantin: 5b, 5c, 13b, (20)
Production of rugulovasine A: 2, 5b
Production of viridicatumtoxin: 1
Production of tryptoquivalins: 1,12
Production of aurantiamine: 3a, 3d
Production of viridamine: 3e
Production of verrucofortine: 3a, 3b, 3e
Production of verrucosidin: 3a, 3b, 3c
Production of penitrem A: (3a), 3c, 7, 11, 16a, (16b)
382
J.C Frisvad & O. Filtenborg
Production of xanthomegnin and viomellein: 3a, 3e

Production of brevianamide A: (3e), (4)
Production of botryodiploidin: (4), (23b)
Production of asperphenamate: 4
Production of Raistrick phenols: 4, 7, 14
Production of pebrolides: 4
Production of xanthocillin X: (6a)
Production of emodic acid: (6a)
Production of isofumigaclavine A: 7, (5b), (5c), 23a
Production of penechins: 13a
Production of mycelianamide: (17a)
Production of cyclopiamine: (17a)
Production of compactin: 18a, 20, 28
Production of carolic acid: 18d
Production of cyclopaldic acid: 5b, (5c), 21
Production of isochromantoxin: 21
Production of verrucolone: 24a, 24b
Production of marcfortin: 23a
Production of deoxybrevianamide E: 19
Production of 5,6-dihydro-4-methoxy-2H-pyran-2-one: 19
Production of puberulic acid: (3a)
Production ofrot in apples: 11, 14,20
Production of rot in citrus fruits: 12, 19
Production of rot in sweet potato: 26
Production of rot in garlic: 18c
Production of rot in onions and bulbs: 18a, 18b, 18e
Growth at 37C: I, (6a), (6b), 8, 21, 27
Good growth on creatine sucrose agar (and good base production): 2, (3b), 5a, 5b, 5c, 9,10,
II, 13a, 13b, 14, 16a, 16b,20,23a,23b,25
No acid production on creatine sucrose agar: 2, (4), (6a), (6b), 7, (9), 10, 12, 15, (17a), 18c,
18e, 19, (20), 22, 23a, 23b, 24a, 24b, 26, 28
Good growth on nitrite sucrose agar: 2, 4, (5a), (5b), (5c), 7, 9, 10, (11), (16a), (16b), 18c, 18e,
(19), (20), 22, 23a, 23b, 24a, 24b, 26, 27, 28
Growth on 0.5% acetic acid: 23a, 23b
Conidia very dark green: 2, 3c, 6b, (5c), 10, (II), 13a, 13b, 20, (27)
Conidia blue green: 3a, 3b, 3d, (6a), 16b, 18a, 18b, (18d), 18e, (20)
Conidia olive green: 12
Conidia grey: 17a
Conidia white or grey green late in the growth phase: Sa, (mutants)
Conidia cylindrical with rounded ends: 12, IS, 19
Conidia ellipsoidal: I, (3), (4), (6a), (6b), 7, (5b), (5c), 8, 9, 10, 11, 12, (13b), 14, 15, 16a, 16b,
19, (20),21,22,25,26,27
Conidia echinulate: 3d, 13a, 13b
Conidia finely roughened: 4, IS, 18d, (20), 22,28
Conidia> 3.5 ~m long: 12, 19, 26, 27
383
Short phialides 6.5 ~): 17a, 17b

Quite short phialides with a thick-walled neck: 6a, 6b, 21
Rami divergent: 2, 6a, 6b, (8), (12), (13b), 17a, 17b, (18d), 21, 28
Penicilli multiramulate: 2, (4), 6a & b, (21),22
Very few rami: 12, 15,26,27, (28)
Stipes rough on MEA: 1, (3a), (3b), (3c), 3d, 3e, (Sa), 5b, 5c, (7), 11, 13a, 13b, 14, 16a, 16b,
18 a,b,c,d,e, 20, 23a, 23b, 28
Production of sclerotia on MEA: 10, 23a, 26
Formation of conidial crusts: (3a), (5b), 11, 19, 27
Production of synnemata with distinct capitula: 9, 10, (13b), (14), 16a, 16b, (17a), (18a), 18b,
18d, (18e), (19),25
Synnemata yellow: 18a, 18d
Synnemata without sterile stalks: 7
Colonies floccose: Sa (repeatedly transferred strains)
Weak sporulation on YES agar: 3a, 3d, 3e, Sa, 5b, 7, 11, (14), 18b, 18d, (19, 20, 24a,
24b, (25), 28
Violet brown reverse on YES: 19, 24b
Dark blackish green reverse on CYA: 23a
Colonies < 20mm MEA, 1 week, 25 C: 2, 3a,c, (4), Sa, (6b), (7), (8), (9), (10), 16a,
16b, (17a), 21, 22, 23a, 24a, 24b, (25)
Colonies> 40 mm MEA, 1 week, 25 C: (3b), (6a), (11), (12), (14), (15), (18a,b,c,e), (19),
(23a,b), 26, 27
Colonies> 40 mm YES, 1 week, 25 C: 1, (2), 3a, (3b), (3e), (5 b,c), 6a, 9, 11, 12, (13a,b),
14, 16a, (18a,b,c,d,e), 19, (20),22, (23a), 23b, (25), 26, 27
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FRISVAD, J.C 1981. Physiological criteria and mycotoxin production as aids in identification of common
asymmetric Penicillia. Applied and Environmetal Microbiology 41: 568-579.
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FRISVAD, J.C 1989. The connection between the Penicillia and Aspergilli and mycotoxins with special
emphasis on misidentified isolates. Archives of Environmental Contamination and Toxicology 18: 452-467.
FRISVAD, J.C and FILTENBORG, O. 1983. Classification of terverticillate Penicillia based on profiles of
FRISVAD, J.C and FILTENBORG, O. 1989. Chemotaxonomy of and mycotoxin production by the
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secondary metabolites in fungal cultures by thin-layer chromatography and high-performance liquid
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DIALOGUE FOLLOWING DR FRISVAD'S PRESENTATION
Pm: SPAS (Subcommission on Penicillium and Aspergillus Systematics) is now considering

circulating morphologically "typical" isolates of selected Penicillium species, to allow
people to calibrate their medium and incubation systems. Perhaps we could also
consider doing the same thing with a selected set of producers of secondary metabolites.
385
ELECTROPHORETIC COMPARISON OF ENZYMES AS A

CHEMOTAXONOMIC AID AMONG ASPERGILLUS TAXA:
(1) ASPERGILLUS SECTS. ORHATI AND CREMEI
J. Sugiyama and K. Yamatoya

Institute of Applied Microbiology
University of Tokyo
Bunky~ku,Tokyol13,Japan
SUMMARY
Polyacrylamide slab gel electrophoresis with specific staining for five enzymes was used as a
chemotaxonomic criterion for comparing 35 isolates assigned to 12 species in Aspergillus subgen.
Ornati sect. Ornati and three species in subgen. Circumdati sect. Cremei. These were glucose-6phosphate dehydrogenase, malate dehydrogenase, fumarase, alcohol dehydrogenase, and glutamate
dehydrogenase. A numerical analysis was performed on the basis of the similarity values in patterns
of five enzymes. The results are presented as a dendrogram. Isolates from different geographical and
ecological habitats within the same species were identical or very similar in the enzyme patterns. In
the dendrogram, isolates were divided into five major clusters. Isolates within the same species were
linked to each other with a high similarity value of 73% or more, whereas each species was linked to
one another with a similarity value of 15 to 70 %. The three dusters were a heterogeneous assemblage
in teleomorphs or ubiquinone systems, or both. Comparisons of enzyme patterns are considered to be
useful tools at the species level in the classification of these Aspergillus taxa.
INTRODUCTION
Traditionally, circumscription and classification among Aspergillus taxa have been based
on differences in cultural and morphological characteristics (Raper and Fennell, 1965), and
a need for newer approaches has become apparent. Gams et al. (1985) classified Aspergillus
into six subgenera and 18 sections: the complexity of the genus is emphasised by the fact
that this single anamorph genus is associated with eleven teleomorph genera in the
Trichocomaceae. In addition to the diversity of teleomorphs, the heterogeneity of
Aspergillus ubiquinone systems has been reported by Kuraishi et al. (1985, 1990). It is time
that the taxonomic systems in Aspergillus and its associated teleomorphs be revised on the
basis of modern taxonomic criteria.
One of the six Aspergillus subgenera, Aspergillus subgen. Ornati sect. Ornati is
associated with four different teleomorph genera, Dichlaena, Hemicarpenteles, Sderocleista,
and Warcupiella, and includes some strictly anamorphic species as well (Table 1). This
subgenus is heterogeneous with respect to ubiquinone systems, as also shown in Table l.
Three major ubiquinone systems, Q-9, Q-lO, and Q-I0(H2), occur in subgen. Ornati
(Kuraishi et al., 1985,1990).
Enzyme electrophoresis has been made for the classification of Aspergillus isolates by
Nealson and Garber (1%7), Nasuno (1971, 1972a-b, 1974), and Kurzeja and Garber (1973).
However, this technique has not yet been fully evaluated as an aid in Aspergillus
taxonomy. In this paper, the electrophoretic comparison of enzymes has been studied as a
chemotaxonomic tool for the classification of Aspergillus subgen. Ornati.
J. Sugiyama & K. Yamatoya
386
Table 1. Genera, species and ubiquinone systems in the Aspergillus omatus group and in
Aspergillus subgen. Oman.
A. omatus group:
Raper & Fennell (1965)
Aspergillus subgen. Oman:

Gams et al. (1985)
Teleomorph genus
Ubiquinone
systemb
A. paradaxus
A. citrisporus
A. arnatus
A. spinulasus
A. acanthasporus
A. paradoxus
A. citrisporus
A. ornatulus
A. warcupii
Hemicarpenteles
Hemicarpenteles
Sc/erocleista
Sc/erocleista
Q-I0
Q-9
Q-9
Q-9
Q-I0
A. brunneo-uniseriatus
A. raperi
Warcupiella
Aspergillus sp.
Dichlaena
A. brunneo-uniseriatus
aA. raperi
A. apicalis
A. brunneo-uniseriatus var. nanus
a A. ivoriensis
Q-9
Q-I0(H2)
Q-I0
Q-I0(H2)
Q-IO(H2)
a Aspergillus spp. with Hiille cells are excluded from subgenus Ornati.
From Kuraishi eI al. (1985, 1990).
Isolates examined.
Thirty isolates assigned to 12 species, both teleomorph and anamorph, in Aspergillus
subgen. Ornati sect. Ornati (= the A. ornatus group of Raper and Fennell, 1965) were
examined. Five isolates of three Chaetosartorya species in subgen. Circunuiati sect. Cremei (=
the A. cremeus group) were also included for comparison. Names, culture collection
numbers and other relevant information are listed in Table 2. The species names shown in
this table are as received from culture collections, but the appropriate teleomorph names
have been used.
Cultivation and harvest of mycelia.
The isolates were cultivated in 500 ml flasks containing 200 ml of YM broth supplemented
by 1 % glucose (pH 5.8) for 48 to 60 h at 27C on a reciprocal shaker. The mycelia were
harvested by filtration and were washed twice with 0.05 M Tris-HO buffer, pH 7.8. The
harvested mycelia were stored in a freezer at -20C until used.
Preparation of cell-free extracts for electrophoresis.
Mycelia were distrupted by sonication for 6 to 10 min at 200 W (Tomy Seiko, Model UR200P, Tokyo, Japan) and at 5C in the same buffer solution. Usually 10 to 15 g of wet
packed mycelia were employed. The homogenates were then centrifuged twice, for 30 min
at 10,000 g and 5C, and for 90 min at 100,000 g and 5C. The resulting supernatant was
used as an enzyme source for electrophoresis. It was stored in a freezer at -20C until just
before use.
Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillus taxa: 1

Table 2. Isolates used
Species
Isolate"
Source
Hemicarpente/es acanthosporus
IF0949O(T)
IF096CY7
JCM2268
JCM2269
Soil, Papua New Guinea

Soil, Papua new Guinea
Sclerae/eistJz ornata
NRRL 2256(1')
= JCM 2354
JCM2263
CBS 425.68
Soil, Wisconsin, U.S.A.
NRRL 2162(1')
=IF08172
= JCM 2355
IF09127
Opossum dung, New Zealand
Sc/eraeleistJz thaxteri
JCM2264(1')
JCM2350
Caterpillar dung, U.S.A.

Soil, Massachusetts, U.S.A.
Warcupie/la spinu/osa
IFO 31800(1')
= JCM 2358
JCM2270
Jungle soil, Borneo
Chaetosartorya chrysella
(Kwon & Fennell) Subram.
NRRL 5084(1')
NRRL5085
Forest soil, Costa Rica

Chaetosartorya CTemea
NRRL 5081(1')
Chaetosartorya stromatoides
NRRL4519(1')
= IFO 9652
Soil,Panama
Wiley & Simmons
Aspergillus apica/is
CBS 236.81(1')
Wheat bean, India
Aspergillus brunneo-uniseriatus
IF06993(1')
=JCM2348
Soil, India
Singh & Bakshi
Aspergillus brunneo-uniseriatus var. nanus
JCM2349(1')
Garden soil, India
Aspergillus ivoriensis
JCM2353(1')
Aspergil/us raperi Stolk
IF06416(1')
=JCM2356
JCM2266
Garden soil, Belgian Congo
Udagawa & Takada
(Raper et a/.) Subram.
Hemicarpenteles paradoxus
Sarbhoy & Elphick
Subram.
Subram.

Forest soil, U.S.S.R.
Dog dung, U.K.
Soil, Singapore
(Kwon & Fennell) Subram.
B.s.Mehrotra & Basu
Sankaran & Zachariah

Bartoli & Maggi
Garden soil, Belgian Congo
>(1'): Strain derived from the type isolate. Culture collections: IFO, Insitute for Fermentation, Osaka; JCM,
Japan Collection of Microorganisms, Wako; NRRL, USDA, Northern Regional Research Center Peoria,
Illinois; CBS, Centraalbureau vaor Schimmelcultures, Baam.
387
388
Electrophoresis and staining of enzymes.

Electrophoresis and staining of enzymes were carried out as described by Yamazaki and
Komagata (1981) with minor modifications. An apparatus (Rapidus Slab-gel
Electrophoresis AE-6220, Atto Co. Ltd., Tokyo, Japan) was used for vertical gel
electrophoresis. A 3.75 % polyacrylamide slab gel, 120 x 120 x 2 mm, with 12 slots, was
prepared by the method of Yamazaki (1982). Extracts (0.06 to 0.1 ml) were applied to the
slots. Enzymes were separated using Tris-HCl buffer, pH 8.9 in the gel and Tris-glycine
buffer, pH 8.3 in the electrotrode vessels with 15 to 25 rnA current for ca 5 h at 5C.
Bromophenol blue was used as the tracking dye. After electrophoresis, the gel was
removed from the mould and stained to visualize the enzymes.
Enzymes.
The following five enzymes were examined: glucose-6-phosphate dehydrogenase
(G6PDH; NADP-dependent, EC 1.1.1.49), malate dehydrogenase (MDH; NAD-dependent,
EC 1.1.1.37), fumarase (Fmase; NAD-dependent, EC 4.2.1.2), alcohol dehydrogenase
(ADH; NAD-dependent, EC 1.1.1.1), and glutamate dehydrogenase (GDH; NADPdependent, EC 1.4.1.4). These enzymes were chosen because they play important roles in
the metabolism of glucose by moulds.
Relative mobilities of enzyme bands and numerical analysis.
The relative mobilities (Rm) of the enzyme bands were calculated as the ratio of the
distance that the enzyme moved from the origin to the distance that the tracking dye
moved. The similarity values in enzymatic patterns were calculated by the following
formula: S (%) = 2NAB / (NA + NB) x 100 (S, similarity value; NAB, the number of
enzyme bands with identical Rms; NA, the number of enzyme bands of isolate A; NB, the
number of enzyme bands of isolate B). These similarity values were averaged to find the
overall similarity between isolates. Clustering was achieved by means of the unweightedaverage linkage technique (Sneath and Sokal, 1973). Computations were performed on a
FACOM M-380 computer in the Institute of Physical and Chemical Research (RIKEN),
Wako-shi, Saitama Pref. using a FORTRAN program written by Dr. T. Kaneko. The results
of the numerical analysis are presented as a dendrogram.
RESUL1S AND DISCUSSION
Figure 1 shows representative examples of G6PDH and GDH electrophoretic patterns for
the isolates of Hemicarpente/es acanthosporus, Sclerocleista thaxteri, S. ornata, Chaetosartorya
chrysella and Aspergillus apicalis. Isolates from different geographical and ecological
habitats within the same species showed identical or very similar enzyme patterns. GDH
produced several enzyme bands which were characteristic in each species. Table 3 shows
Rm's of five enzymes in 25 isolates of Aspergillus and three associated teleomorphs,
Hemicarpenteies, Sclerocleista, and Warcupiella in subgen. Ornati sect. Ornati, and five
isolates of three Chaetosartorya species with Aspergillus anamorphs in subgen. Circumdati
sect. Cremei.
The G6PDH, MDH, Fmase, ADH, and GDH patterns were characteristic for each
species, and clear differences were detected bewteen species. G6PDH and MDH produced
one, two or three bands. Fmase and ADH produced one or two bands. GDH produced a
single band. No enzyme band for Fmase and ADH was present in the isolates of
Hemicarpenteles acanthosporus, Warcupiella spinulosa, Chaetosartorya chrysella, C. cremea,
Electrophoretic comparison of enzymes as a chemotaxonornic aid among Aspergillus taxa: 1
G6PDH
IFO 9490
IFO 9607
H. acanthosporus
JCM 2269
JCM 2268
IFO 6416
A. raperi
-{
JCM 2266
JCM 2356
GDH
IFO 9490
IFO 9607
H. acanthosporus
JCM 2269
JCM 2268
s.
thaxteri - {
JCM 2264
JCM 2356
C. cht'ysella
NRRL 5084
-{
NRRL 5085
A. apicalis--CBS 236.81
S. ornata---CBS 425.68
Figure L G6PDH and GDH electrophoretic patterns for 13 strains in Aspergillus sects. OmaH
andCremei.
389
390
Table 3. Comparison of electrophoretic Rm values of enzymes from 30 strains in Aspergillus

sects. Ornati and Cremei.
G6PDH
MDH
Enzymes (Rm x 1(0)

Fmase ADH
GDH
Hemicarpenteles acanthosporus
IF09490
IF09607
JCM 2268
JCM2269
52,55,59
52,55,59
52,55,59
52,55,59
54,56,58
54,56,58
54,56,58
54,56,58
49
49
49
49
-"
54
54
54
54
Hemicarpente/es paradoxus
NRRL2162
IFO 8172
IF09127
JCM2355
49,51
49,51
49,51
49,51
64,77
64,77
64,77
64,77
81
81
81
81
50,53
50,53
50,53
50,53
70
70
74
70
Sc/eroc/eista ornata
NRRL2256
JCM2263
JCM2354
CBS 425.68
57,61
57,61
57,61
57
72,76,79
72,76,79
72,76,79
72,76,79
75,80
75,80
75,80
75,80
78
78
78
74
87
87
87
87
Sc/eroc/eista thaxteri
JCM2264
JCM 2350
52,56
52,56
66,71,74
66,71,74
67,74
67,74
75
75
84
84
Warcupiella spinulosa
IF031800
JCM 2270
JCM2358
64,68
64,68
64,68
43,49
43,49
43,49
50,58
61,66
50,58
Chaetosartorya chrysella
NRRL5084
NRRL5085
64
64
56,60
56,60
57
57
Chaetosartorya cremea
NRRL 5180
63
55,58
61
Chaetosartorya stromatoides
NRRL4519
IFO 9652
68,72
68,72
60,63
60,63
50
50
Aspergillus apicalis
CBS 236.81
69,71
53,57
54
61,63
55,57
66
66
53
53
72
72
Aspergillus brunneo-uniseriatus JCM2349

var. nanus
54,57
48,52
58
84
JCM2353
55,59
56,69
Aspergillus raperi
IF06416
JCM 2266
JCM2356
52,55,58
52,55,58
52,55,58
49,51
49,51
49,51
Aspergillus brunneo-uniseriatus IF06993

JCM2348
a Not detected.
67
67
67
55
55
31,33
69
65
64
64
64
68
68
68
Aspergillus apicalis
9490
2348
6993
CBS 236.81
JCM
IFO
JCM 2270
IFO 31800
JCM 2358
JCM 2266
JCM 2356
IFO 6416
JCM 2349
9607
2268
2269
Similarity value
50
Figure 2. Dendrogram based on the similarity values of the electrophoretic mobilities of enzymes from the 30 strains examined in
Aspergillus sects. Ornati and Cremei.
s[
brunneo-uni seri atus
A. brunneo-um sen at us
var. nanus
Aspergi 11 us raperi
Warcupiella spinulosa
{A.
3[
21
5085
2353
4519
9652
5081
5084
IFO 8172
JCM 2355
NRRL 2162
IFO 9127
NRRL 2256
JCM 2354
JCM 2263
CBS 425.68
JCM 2264
JCM 2350
NRRL
NRRL
NRRL
JCM
Chaetosartorya s tromatoi des NRRL
IFO
Hemicarpenteles acanthosporus IFO
IFO
JCM
JCM
Chaetosartorya cremea
Chaetosartorya chryse 11 a
Sclerocleista thaxteri
Sclerocleista ornata
Hemi carpente les paradoxus
100:1:
m
Ci)
Co>
~
cQ
:t.
:::l
co
II>
91.
a.
(>
2.
~
~
(\)
g.
II>
en
~
(\)
(\)
!3:::l
.a.
5"
Cil
g8
"C
(3
0-
392
C. stromatoidea, Aspergillus brunneo-uniseriatus var. nanus, and A. raperi. The isolates within
the same species produced identical or very similar patterns for each enzyme. Sclerocleista
ornata CBS 425.68 had a single band for G6PDH and a different band (Rm 0.74) for ADH.
Three other isolates of S. ornata showed identical patterns for all enzymes. Warcupiella
spinulosa JCM 2270 showed a different Fmase pattern. The two isolates IFO 6993 and JCM
2348 of A. brunneo-uniseriatus produced different patterns for G6PDH.
The relative mobilities for five enzymes from the isolates tested are shown as a
dendrogram to emphasize the similarities in enzyme patterns (Fig. 2). The isolates tested
separated into five major clusters. Isolates in the same species were linked to each other
with similarity values of 80 % or more except for S. ornata isolates (73% similarity). Species
were linked with similarity values of 15 to 70%.
Cluster 1 included Hemicarpenteles paradoxus, Sclerocleista ornata and S. thaxteri. These
showed little relationship in enzyme patterns, with similarity coefficients of only 18% and
32.5%. H. acanthosporus, the second Hemicarpenteles species examined, clustered quite
separately from H. paradoxus. As H. paradoxus has the Q-9 ubiquinone system, and H.
acanthosporus the Q-I0 (Kuraishi et al., 1985, 1990), a strong case exists for exclusion of H.
acanthosporus from Hemicarpenteles. It showed a much higher relationship in enzyme
similarity profiles with S. stromatoides, which, however, also has the Q-9 system.
Cluster 2 included isolates of three Chaetosartorya species, Aspergillus ivoriensis and
Hemicarpenteles acanthosporus. Chaetosartorya cremea and C. chrysella were linked with a
similarity value of 70%, while C. stromatoides was only distantly related, with a similarity
value of 32.5%. A. ivoriensis, tentatively placed in sect. Ornati by Samson (1979) despite its
production of Hiille cells, showed 50% similarity to C. cremea and C. chrysella, but less to
other species. Although Gams et al. (1985) excluded species with Hiille cells from sect.
Ornati, our data on this point are equivocal.
Cluster 3 included the isolates examined from Warcupiella spinulosa, Aspergillus raperi
and A. brunneo-uniseriatus var. nanus. Intraspecific variation within this cluster was small,
but the different species showed only a low level of similarity: 56% for A. raperi and A.
brunneo-uniseriatus var. nanus, and 48% for A. raperi and A. spinulosa. A. brunneo-uniseriatus
clustered alone (Cluster 4), and the two isolates examined, IFO 6693 and JCM 2348, were
linked with a similarity value of only 80%. The two isolates were culturally and
microscopically quite different, particularly in the shape and size of phialides and conida.
The identity of JCM 2348 is therefore questionable. A. brunneo-uniseriatus clustered quite
separately from A. brunneo-uniseriatus var. nanus, with a similarity value of only 25%. As
these taxa also have different ubiquinone systems, Q-9 and Q-I0 (H2) respectively
(Kuraishi et al., 1990), their relationship is very doubtful.
The final cluster, 5, with A. apicalis, showed a very low level of relationship to any
other species. Placement in sect. Ornati appears questionable.
Several conclusions may be drawn from the results shown here. Taxa in subgen.
Ornati are heterogeneous in their enzyme patterns as well as their teleomorph-anamorph
connections and ubiquinone systems. However, the enzyme patterns were characteristic
for each species. Therefore, electrophoretic comparisons of enzymes will be of value in
distinguishing species, and may provide valuable information for subgeneric taxonomy of
Aspergillus and associated teleomorphs also.
ACKNOWLEDGEMENTS
We thank the curators of culture collections from which cultures were examined: CBS,
393
IFO, JCM, and NRRL. We also thank Dr. T. Kaneko, Tokyo University of Agriculture,
Tokyo and Dr. K. Suzuki, Japan Collection of Microorganisms, The Institute of Physical
and Chemical Research, Wako for the computing and Dr. M. Yamazaki for helpful
comments on the electrophoresis. A Grant-in-Aid for Co-operative Research (A) (No.
61300005) from the Ministry of Education, Science and Culture, Japan to J.S., which partly
supported this research is gratefully acknowledged.
REFERENCES
GAMS, W., CHRISTENSEN, M., ONIONS, A. H., PITT, J. I. and SAMSON, R. A. 1985. Infrageneric taxa of
Aspergillus. In Advances in Penicillium and Aspergillus Systematics, eels. R. A. Samson and J.1. Pitt, pp. 5562. New York and London: Plenum Press.
KURAISHI, H., KATAYAMA-FUJIMURA, Y., SUGIYAMA, J. and YOKOYAMA, T. 1985. Ubiquinone
systems in fungi. I. Distribution of ubiquinones in the major families of Ascomycetes, Basidiomycetes,
and Deuteromycetes, and their taxonomic implications. Transactions of the Mycological Sodety of Japan 26:
383-395.
KURAISHI, H., ITOH, M., TSUZAKI, N., KATAYAMA, Y. YOKOYAMA, T. and SUGIYAMA, J. 1990.
Ubiquinone systems as a taxonomic tool in Aspergillus and its teleomorphs. In Modern Concepts in
Penidllium and Aspergillus Oassification, eds. R. A. Samson and J. I. Pitt, pp. 407-420. New York and
KURZEJA, K.C. and GARBER, E. D. 1973. A genetic study of electrophoretically variant extracellular
amylolytic enzymes of wild-type strains of Aspergillus nidulans. Canadian Journal of Genetics and Cytology
15: 275-287.
NASUNO, S. 1971. Polyacrylamide gel disc electrophoresis of alkaline proteinases from Aspergillus species.
Agricultural and Biological Chemistry 35: 1147-1150.
NASUNO, S. 1972a. Differentiation of Aspergillus sojae from Aspergillus oryzae by polyacrylamide gel disc
electrophoresis. Journal of General Microbiology 71: 29-33.
NASUNO, S. 1972b. Electrophoretic studies of alkaline proteinases from strains of Aspergillus flavus group.
Agricultural and Biological Chemistry 36: 684-689.
NASUNO, S. 1974. Further evidence on differentiations of Aspergillus sojae from Aspergillus oryzae by
electrophoretic patterns of cellulase, pectin-lyase, and acid proteinase. Canadian Journal Microbiology 20:
413-416.
NEALSON, K. H. and GARBER, E. D. 1967. An electrophoretic survey of esterases, phosphatases, and
leucine amino-peptidases in mycelial extracts of species of Aspergillus. Mycologia 59: 330-336.
RAPER, K. B. and FENNELL, D. I. 1965. The Genus Aspergillus. Baltimore: Williams & Wilkins.
SAMSON, R. A. 1979. A compilation of the Aspergilli described since 1965. Studies in Mycology, Baarn 18: 138.
SNEATH, P. H. A. and SOKAL, R. R. 1973. Numerical Taxonomy. 2nd edition. San Francisco: W. H.
Freeman.
YAMAZAKI, M. 1982. Electrophoretic patterns. In Biseibutu no Kagaku Bunrui Jikkenho (Chemotaxonomic
Methods for Microorganisms), eel. K. Komagata, pp. 184-209. Tokyo: Gakkai Shuppan Center.
YAMAZAKI, M. and KOMAGATA, K. 1981. Taxonomic significance of electrophoretic comparison of
enzymes in the genera Rhodotorula and Rhodosporidium.lnternational Journal of Systematic Bacteriology 31:
361-381.
395
ELECTROPHORETIC COMPARISON OF ENZYMES AS A

CHEMOTAXONOMIC AID AMONG ASPERGILLUS TAXA:
(2) ASPERGILLUS SECT. FLA VI
K. Yamatoya1, J. Sugiyama1 and H. Kuraishi2
1Institute of Applied Microbiology
University of Tokyo
Bunkyo-ku, Tokyo 113, Japan
2Faculty of Agriculture
University of Agriculture and Technology
Fuchu, Tokyo 183, Japan
SUMMARY
Polyacrylamide slab gel electrophoresis with specific staining for eight enzymes was used as a
chemotaxonomic aid in comparing 41 isolates in Aspergillus subgen. Circum dati sect. Flavi. The eight
enzymes were 6-phosphogluconate dehydrogenase, malate dehydrogenase, phosphoglucomutase,
glucose-6-phosphate dehydrogenase, fumarase, alcohol dehydrogenase, lactate dehydrogenase, and
glutamate dehydrogenase. Numerical analysis was applied to similarity values of elctrophoretic
relative mobilities of the eight enzymes. Five major clusters were obtained. A. flavus, A. kambarensis, A.
toxicarius, and other very closely related Aspergillus taxa formed one major cluster at a 63% similarity
level. This cluster could be divided into two subclusters, corresponding to A. flavus and A. parasiticus.
A. tarnarii, A. leporis, A. nomius, and A. avenaceus formed the separate major clusters at a similarity level
of 38% or less; each cluster corresponded to a single species. The ubiquinone systems of 27 isolates
were also determined. A. avenaceus, A. flavus var. asper and A. leporis possessed the Q-lO ubiquinone
system, while all others had Q-10(H2)' The heterogeneity in the ubiquinone systems suggests that
Aspergillus sect. Flavi is in need of revision.
INTRODUCTION
Aspergillus subgen. Circumdati sect. Flavi (Gams et ai., 1985), previously known as the 'A.
flavus group' (Raper and Fennell, 1965), includes industrially, agronomically and
medically important species. Hence a great deal of interest persists in the classification of
these species.
Recently Kurtzman et al. (1986) studied the DNA base composition and DNA-DNA
homology of 17 isolates of A. flavus, A. parasiticus, A. oryzae and A. sojae. All four species
had high (69-100 %) nuclear DNA complementarity and a similar genome size. They
interpreted their data as indicating that these taxa represented a single species. They
proposed the following taxonomic designations, taking into account morphological
differences: A. flavus subsp. flavus var. flavus, A. flavus subsp. flavus var. oryzae, A. flavus
subsp. parasiticus var. parasiticus and A. flavus subsp. parasiticus var. sojae. Klich and
Mullaney (1987) and Klich and Pitt (1988) have disagreed with this taxonomic approach.
In this paper, we present data on electrophoretic relative mobility values of eight
enzymes in 41 isolates in Aspergillus sect. Flavi, and attempt to evaluate these in relation to
the conflicting concepts of speciation mentioned above, and our zymogram studies in
Aspergillus subgen. OrnaH (Sugiyama and Yamatoya, 1990). We also wished to compare
our data to modern chemotaxonomic approaches such as DNA base composition, DNADNA homology, and ubiquinone systems.
396
K. Yamatoya et a/.
Table 1. Isolates studied.
Species
Isolate
SOUTce
A. avenaceus
lAM 13120
lAM 13121 (1')
NRRL482
NRRL 1957(NT)
NRRL3251
NRRL 13135
NRRL 13136
lAM 2670
lAM 13119(1')
NRRL 447(N1)
NRRL451
lAM 2630
lAM 2735
lAM 12166
lAM 13833
lAM 2660
lAM 2719
lAM 2683
lAM 2699
lAM 2955
lAM 2956
lAM 2957
lAM 2958
lAM 2750
NRRL465
NRRL502(1')
lAM 2150
NRRL 5595(1')
NRRL5594
lAM 2631
lAM 2669
lAM 12768(1')
NRRL 3216(1')
NRRL 3161
NRRL5919
NRRL 13137(1')
NRRL429
NRRL 13139
lAM 2138
JCM2252
JCM2253(1')
Seed peas, London, u.K.

Seed peas, London, U.K.
Wehmer's A. flavus isolate
Mouldy cellophane, South Pacific
Walnuts, U.s.A.
Mouldy peanuts, U.S.A.
Kangaroo rat cheek pouch, U.S.A.
Shoyu-koji, Chiba, Japan
Butter, Hokkaido, Japan
Sake-koji, Japan
Soy Sauce, China
Miso-koji, Korea
Sake-koji, Kyoto, Japan
Okinawa, Japan
Cassava, South Africa
Amazake-koji, Tokyo, Japan
Shoyu-koji, Tokyo, Japan
Shoyu-koji, Tokyo, Japan
Tarnari-koji, Japan
Shoyu-koji, Kumamoto, Japan
K. Saito (IFO 4083)
Miso-koji, Nagoya, Japan
T. Takahasi (IFO 4134)
Shoyu-koji, Tottori, Japan
J. Takamine's Lab., Japan
Sugarcane, Hawaii
ATU A-8-3 = CLMR
Koji, Japan?
Koji, Manchuria?
P Shoyu-koji, Tokyo, Japan
Shoyu-koji, Chiba, Japan
Core sample, Niugata, Japan
Dung of rabbit, U.S.A.
Cycas circinalis, Polynesia
Diseased alkalibes, U.S.A.
Mouldy wheat, U.S.A.
Soy sauce, China
Kangaroo rat cheek pouch, U.S.A.
Amazake-koji, Japan
ATCC 15517 (as A. parasiticus)
Groundnuts, Uganda
A. flavus var. flavus
A. flavus var. asper

A. flavus var. oryme
A. oryme
A. oryme var. magnasporus

A. oryme var. microsporus
A. oryme var. micro-vesiculosus
A. oryme var. pseudoflavus
A. oryme var. sporofallus
A. oryme var. tenuis
A. oryme var. viridis
A. flavus var. parasiticus
A. parasiticus
A. flavus var. sojae
A. sojae
A. kambarensis
A.leporis
A. nomius
A. tamarii
A. toxicarius
Strain derived drom the type; (NT) strain derived from neotype.
Culture collections: lAM, Institute for Applied Microbiology, University of Tokyo, NRRL, USDA,
Northern Regional Research Center, Peoria, Illinois; JCM, Japan Collection of Microorganisms, Wako.
(T)
397
Isolates examined.
Forty-one isolates assigned to Aspergillus sect. Flavi were examined, including 17 NRRL
isolates used by Kurtzman et al. (1986, 1987) to obtain data on DNA base composition and
DNA relatedness values. Names, culture collection numbers, and ecological and
geographical sources are listed in Table 1. Most species names were used according to
their designation when they were received from culture collections.
Electrophoretic comparison of enzymes.
The cultivation, harvest of mycelia, electrophoresis and staining procedures of eight
enzymes used in this study were as described previously (Sugiyama and Yamatoya, 1990).
The numerical analysis based on the enzyme patterns was also made as described
previously (Sugiyama and Yamatoya, 1990).
In addition to the five enzymes examined previously in isolates of Aspergillus subgen.
Ornati (Sugiyama and Yamatoya, 1990), three further enzymes were studied. These were
6-phosphogluconate dehydrogenase (6PGDH; NADP-dependent, EC 2.7.5.1),
phosphoglucomutase (PGm; NADP-dependent, EC 1.1.1.41) and lactate dehydrogenase
(LDH; NAD-dependent, EC 1.1.1.27).
Extraction and analysis of ubiquinones.
Ubiquinone systems for 27 Aspergillus isolates were determined as described previously
(Kuraishi et al., 1985). The abbreviations used for ubiquinones were indicated as described
previously (Sugiyama and Yamatoya, 1990).
Summarized in Table 2 are the ubiquinone systems in 27 isolates assigned to of Aspergillus

subgen. Circumdati sect. Flavi obtained in this study together with some data reported by
Kuraishi et al. (1990). The four isolates of A. avenaceus, A. flavus var. asper and A. leporis
examined possessed the Q-lO ubiquinone system. All others examined possessed the Q10(H2) system.
Figure 1 shows representative examples of G6PDH and 6PGDH patterns for seven
Aspergillus isolates from the lAM, JCM and NRRL culture collections. The isolates of five
taxa produced identical enzyme patterns for G6PDH, whereas these isolates were clearly
divided into two groups for 6PGDH.
Table 3 presents data on the Rm values for eight enzymes from 41 isolates tested in
Aspergillus sect. Flavi. On the whole, isolates within the same taxon were identical or
showed very similar patterns for all enzymes. G6PDH produced three enzyme bands for
each isolate. MDH produced one, two or three bands. Fmase produced two bands in most
isolates; A. nom ius produced a single band, while Fmase was not detected in A. leporis.
ADH and LDH produced one or two bands, except that LDH was present in A. leporis.
6PGDH, PGm and GDH produced a single band in most isolates; however, no 6PGDH
bands were seen in A. avenaceus or A. leporis.
A dendrogram based on calculated similarity values of eight enzymes in 41 isolates,
examined by the unweighted-average linkage method, is shown in Fig. 2. This
dendrogram was drawn from all enzyme bands detected. Figure 3 shows a dendrogram
K. Yamatoya et al.
398
Table 2. Ubiquinone in Aspergillus isolates examined.

Species
Isolate
Ubiquinone system
A avenaceus
lAM 13120
lAM 13121m
NRRL482
NRRL 1957(NT)
NRRL3251
NRRL 13135
NRRL 13136
lAM 13119m
NRRL 447(NT)
NRRL451
lAM 2660
lAM 2719
lAM 2683
lAM 2699
lAM 2955
lAM 2956
lAM 2957
lAM 2958
lAM 2750
NRRL465
NRRL502m
NRRL5594
NRRL559Sm
lAM 12768m
NRRL3216m
NRRL3161
NRRL5919
NRRL 13137m
NRRL429
NRRL 13139
JCM2252
Q-IO*
Q-IO*
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO*
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-I0
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)"
A flavus var. flavus
A flavus var. asper

A flavus var. oryzae
A oryzae var. magnasporus
A oryzae var. microsporus
A
A
A
A
A
A
oryzae var. micro-vesiculosus

oryzae var. pseudoflavus
oryzae var. sporofallus
oryzae var. tenuis
oryzae var. viridis
flavus var. parasiticus
A flavus var. sojae

A kambarensis
Aleporis
A nomius
A tamarii
A toxicarius
Footnotes: see Table 1.
": Data from Kuraishi et al. (1990).
based on only the deeply stained bands for eight enzymes; those with underlines in Table
3 were omitted as weakly staining or unstable. No significant differences were found
between the two dendrograms.
The isolates tested separated into five major clusters, as shown in Figs. 2 and 3. Thirtytwo isolates assigned to A. flavus and other closely related taxa formed one major cluster
(Cluster 1) at a 63% similarity level (Fig. 2). This cluster was further divided into two
subclusters, I-a and I-b. Subcluster I-a contained twenty-two isolates of A. flavus, A. flavus
var. asper, A. flavus var. flavus, A. flavus var. oryzae, A. oryzae and most of its varieties, and
A. kambarensis. These isolates in this sub-cluster were linked to each other with a high
similarity values of 73 % or more. Also, Subcluster I-a was composed of the isolates
having only the Q-I0(H2) system except A. flavus var. asper lAM 13119 (Table 2).
Subcluster I-b contained 10 isolates of A. flavus var. parasiticus, A. flavus var. sojae, A.
oryzae var. magnasporus lAM 2719, A. parasiticus, A. sojae, and A. toxicarius. These isolates
were linked to each other with a similarity value of 86% or more. All isolates in this
399
G6PDH
A. oryzae v. magnasporus
lAM 2260
lAM 2719
A. oryzae
lAM 2735
A. toxicari us
JCM 2252
JCM 2253
A. flavus v. parasiticus
NRRL 502
A. flavus v. flavus
NRRL 1957
6PGDH
lAM 2260
lAM 2719
A. oryzae
lAM 2735
A. toxicarius
JCM 2252
JCM 2253
A. fl avus v. parasiticus
NRRL 502
A. flavus v. flavus
NRRL 1957
Figure 1. G6PDH and 6PGDH electrophoretic pattern for seven isolates in Aspergillus
subgen. Circum dati sect. Flavi.
subcluster were of the same Q-lO(Hv system as in Subcluster I-a (Table 2). Therefore, the
isolates in these subclusters can be regarded as an electrophoretically homogeneous
taxonomic group which corresponds to a single species. However, further taxonomic
investigations will be required to determine the taxonomic position of A. flavus var. asper
having the Q-I0(H2) system.
Cluster 2 contained three isolates of A. tamarii having the Q-I0(H2) system. Enzyme
patterns for these isolates showed more than more than 94% similarity. This cluster was
linked to Ouster 1 with a relatively low similarity value of 37%.
Cluster 3 contained only A. leporis NRRL 3216, which was characterized by us as
having the Q-I0 system (Table 2). This cluster was linked to Cluster 2 with a low similarity
value of 27%.
Cluster 4 contained three isolates of A. nomius, of which two isolates (NRRL 13137 and
NRRL 3161) had identical enzyme patterns (100% similarity). Isolate NRRL 5919 was
linked to these two isolates with a similarity value of 73%. The three isolates of A. nomius
were characterized by the same Q-I0(H2) system as Clusters 1 and 2. A. nomius appears to
be a distinct species.
400
K. Yamatoya et al.
Table 3. Comparison of electrophoretic Rm values of enzymes from 41 isolates in Aspergillus

subgen. Circum dati sect. Flavi
Enzymes (Rm xlOO)
Speci es
A. avenaceus
lAM
lAM
NRRL
NRRL
NRRL
NRRL
NRRL
fl avus
lAM
fl avus v. asper
lAM
f1 avus v. oryzae
NRRL
NRRL
oryzae
lAM
lAM
lAM
lAM
oryzae v. magnasporus
lAM
lAM
oryzae v. mi eros porus
lAM
lAM
oryzae v. mi era-yes i culosus lAM
oryzae v. pseudofl avus
lAM
oryzae v. soprofa 11 us
lAM
C,Jryzae v. tenuis
lAM
oryzae Y. vi ridis
lAM
flavus v. parasiticus
NRRL
NRRL
parasiticus
lAM
flavus v. sojae
NRRL
NRRL
sojae
lAM
lAM
kambarens i s
lAM
leporis
NRRL
A. flavus v. flavus
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
Strai ns
A. nom; us
A. tamar; i
A. taxi car; us
-: Not detected.
13120
13121
482
1957
3251
13135
13136
2670
13119
447
451
2630
2735
12166
13883
2660
2719
2683
2699
2955
2956
2957
2958
2750
465
502
2150
5595
5594
2631
2669
12768
3216
NRRL 3161
NRRL 5919
NRRL 13137
NRRL
429
NRRL 13139
lAM 2138
JCM 2252
JCM 2253
G6PDH
MDH
Fmase
ADH
LDH
65,67
65,67
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
76,79
77 ,81
77 ,81
80,83
80,83
~,72
.u,83
80
83
72
83
72
72
83
83
72
,83
73
.I ,72 1l,83
.I ,72
83
83
72
40,45,~
83
72
40,45,.48.
40,44,~
f!1,72 .u,83
fil.,72 n,80
40,44,.48.
57,72 .u,83
40,44,.48.
2,72 n,S3
40,44
40,44,.48. 76,79 .'l,72 .u,S3
72 n,83
40,44,.48. 76,79
79
83
40,44
76,79
72
83
40,45,'!ll 76,79
83
76,79
72
40,45,~
83
72
40,45,.48.. 76,79
83
76,79
72
40,45
76,79
84
38,42
72
76,79
38,42
72
84
84
76,79
38,42
72
76,79 ~,72 !If ,84
39,42
76,79
72 !If.,84
39,42
40,44
76,79
72 .82.,84
76,79
72 .8Z.,84
39,42
83
73
40,45,~ 76,79
80
80
80
80
80
80
82
80
80
80
91
55,59,62
55,59,62
61,65,68
61,65,68
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
40,45,~
62,65,67
62,65,67
62,65,67
40,45,.48.
40,45,~
40,45,.48.
40,45,.48.
40,45,.48.
40,43
40,45,.48.
40,45,'!ll
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
62,65,67
59,61,65 59
56,59,61 40,49,.I
56,59,61 40,49
56,59,61 40,49,fil.
62,65,67 50,54,63
62,65,67 50,54,63
62,65,67 50,54,63
62,65,67 40,44
62,65,67 40,44
6PGDH
2.2,72 .u,83 80
ru.
72
70
56
70
78
91
80
80
80
80
80
78
80
91
91
91
91
91
91
91
80
69
73
57
73
73
69
76,78 ..,65 l..,86
86
76,78
64
76,78
64 L5,86
76,79
83
72
83
76,79
72
87
87
87
72
72
72
91
91
PGm
78
78
74
74
74
74
74
74
74
74
74
74
74
74
74
74
69
74
74
74
74
74
74
74
69
69
69
69
69
69
69
74
83
62,66
62,66
62,66
72
72
72
69
69
GDH
80
80
71
71
71
71
71
71
7'
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
71
70,74
70
70
70
71
71
71
71
71
3251
13135
13136
2670
12768
2735
2630
2957
2956
2750
447
12166
2683
2699
1957
482
13119
2955
451
2660
2958
13883
465
502
2150
5594
5595
2669
2252
2253
2631
2719
NRRL
NRRL
lAM
NRRL
NRRL
lAM
JCM
JCM
lAM
lAM
lAM 13120
lAM 13121
NRRL 13137
NRRL 3161
NRRL 5919
Similarity value
50
Figure 2. Dendrogram based on the similarity values of the electrophoretic mobilities of enzymes from 41 isolates examined in
Aspergillus subg. Circumdati sem Flavi; this dendrogramn was drawn from all enzyme bands detected.
5 [
A. avenaceuS
A. nomius
3216
NRRL
A. lepari s
3 [
2 [
A. tamari i
A. sojae
~: ~~;~~ari us
lAM 2138
NRRL 13139
NRRL
429
l-b I
A. paras i ti cus
A. fl avus soj ae
A. fl a.us paras it i cus
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
l-al A.
A.
A.
A.
A.
A.
A. flavus v. flavus
NRRL
NRRL
NRRL
lAM
fl avus
lAM
kambarensis
lAM
aryzae
lAM
lAM
aryzae v. sporofallus
lAM
aryzae v. pseudaflavus
lAM
aryzae v. viridis
NRRL
flavus v. aryzae
lAM
oryzae
lAM
oryzae v. mi crosporus
lAM
NRRL
flavus v. flavus
NRRL
lAM
fla.us v. asper
oryzae v. micro-vesiculosus lAM
NRRL
flavus v. oryzae
lAM
oryzae v. magnasporus
lAM
oryzae v. tenui s
lAM
aryzae
100%
I~
I\)
!'!
IQ.
CQ
"
D>
Co
;;.
o
e!.
i
CD
g.
D>
'<
j;l
CD
D>
".
"S-
.a
l""
Sl.
iii
lAM 13120 I
lAM 13121
NRRL 13137
NRRL 3161
NRRL 5919
A. nom; us
3216
NRRL
A. avenaceus
f-----
429 I
NRRL
NRRL 13139
lAM 2138 1
A. leporis
A. tamari i
502
NRRL
465
NRRL
lAM 2150
NRRL 5594
NRRL 5595
lAM 2669
lAM 2631
JCM 2252
JCM 2253
lAM 2719
Similarity value
50
f--
f--
Figure 3. Dendrogram based on the similarity values of the electrophoretic mobilities of enzymes from 41 isolates examined in
Aspergillus subgen. Circumdati sect. Flavi; this dendrogram was drawn from deeply stained bands for the respective enzyme.
5 [
4 [
3 [
Z [
A. aryzae v. magnasporus
A. taxi car; us
A. flavus v. sojae
I-b A. saJae
'
['A. nO'.
'. "''';'''"'
paras iti cus
A.
A.
A.
l-al A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
NRRL
NRRL
NRRL
NRRL
NRRL
lAM
lAM
NRRL
lAM
lAM
lAM
lAM
lAM
lAM
lAM
lAM
lAM
3251
13135
1957
482
13136
2670
flavus
12768
kambarens i s
447
fl avus v. oryzae
2735
oryzae
2630
2699
oryzae v. microsporus
2956
oryzae v. pseudoflavus
2957
oryzae v. sparofa11us
2750
aryzae v. viridis
2683
aryzae v. microsporus
12166
aryzae
13119
flavus v. asper
oryzae v. micro-vesiculasus 1M 2955
451
NRRL
fl avus v. aryzae
1M 2660
oryzae v. magnasporu5
lAM 2958
oryzae v. tenui s
lAM 13883
oryzae
'A. fl avus. fl avus
100%
III
:-
!a
~
~
?'
~
Ig
..
",,~
502
5595
429
NRRL
NRRL
NRRL
I------.J
Similarity value
I
50
L
Jf-
1-------------.
NRRL 13137
NRRL 3216 - - - - - - - - - - - - - - - - -_ _ _ _ _ _ _ _ _ _ _ _
447
1957~
NRRL
NRRL
Figure 4. Dendrogram based on the data of DNA-DNA homology between A. flavus and other phenotypically similary species
reported by Kurtzman et al. (1987).
flavus v. oryzae
flavus v. parasiticus
flavus v. sojae
tamarii
nomius
leporis
~~. ",,~
1 a
6[A.
A.
2 [ A.
5 I: A.
3 I: A.
100';
I\)
!'!
tc9.
co
:s
D>
c-
o
!!!.
;;l.
i
CD
g.
D>
III
rn
CD
CD
9-
~
:s
~:s.
"C
404
K.
Yamatoya et al.
Cluster 5 contained two isolates of A avenaceus, with identical enzyme patterns and the
same Q-10 system as in A flavus var. asper, and Aleporis (Table 2). This cluster was linked
to Cluster 4 with a low similarity value of 12% (Fig. 2).
As already mentioned, recently Kurtzman et al. (1986) studied taxonomic relationship
between the isolates of A. flavus and six phenotypically similar species in the A flavus
group by DNA base composition and DNA-DNA homology. G+C ratios in the DNAs of
these isolates ranged from 48.6 to 50.4 mol%. Their data showed no significant difference
in the DNA base composition. However, DNA relatedness values reported by Kurtzman
et al. (1986, 1987), divided the seven Aspergillus taxa into four major clusters, and we
present this data as a dendrogram here (Fig. 4). A good cOlTelation was found between the
two dendrograms (Figs. 2 and 3) based on the similarity values in enzyme patterns
obtained in this study and the dendrogram (Fig. 4) based on the DNA-DNA homology
values reported by Kurtzman et al. (1986,1987). These cOlTelations support a conclusion
that the respective major cluster in Figs. 2 and 3 colTeponds to a single species. We suggest
that A flavus, A oryzae, A parasiticus, A sojae, and other closely related taxa in Cluster 1
are accommodated in a single species, A flavus and A parasiticus. From our data, the three
taxa A fiavus var. asper, A leporis and A avenaceus, characterized by the Q-10 system,
should be excluded from Aspergillus sect. Flavi.
Combinations of chemotaxonomic criteria such as electrophoretic comparison of
enzymes, ubiquinone systems, DNA base composition, and DNA-DNA homology can
support or question taxonomic decisions in recent years based mainly on morphological
species concepts (e.g., Murakami, 1971; Christensen, 1981; Murakami et al., 1982; Klich and
Pitt, 1985, 1988). Such chemotaxonomic methods will be a valuable tool for clarification of
interrelation among Aspergillus taxa lacking teleomorphs.
In conclusion, we emphasise the usefulness of electrophoretic comparisons of
enzymes and the ubiquinone system for classifying taxa of Aspergillus and associated
teleomorph genera.
ACKNOWLEDGEMENTS
We thank Dr. T. Nakase (JCM) and Dr. S. W. Peterson (NRRL) for supplying the cultures.
We also thank Dr. T. Kaneko, Tokyo University of Agriculture, Tokyo and Dr. K. Suzuki,
Japan Collection of Microorganisms, The Institute of Physical and Chemical Research,
Wako for their helpful suggestions with respect to the computation of data and Dr. M.
Yamazaki for helpful comments on the electrophoresis. Grateful acknowledgement is
made of a Grant-in-Aid for Co-operative Research (A)(No. 61300005) from the Ministry of
Education, Science and Culture, Japan to J. S., which partly supported this research.
REFERENCES
CHRISTENSEN, M. 1981. A synoptic key and evaluation of species in the Aspergillus jlavus group. Mycologia
73: 1056-1084.
GAMS, W., CHRISTENSEN, M., ONIONS, A. H., pm, J. I. and SAMSON, R. A. 1985. Infrageneric taxa of
Aspergillus. In Advances in Penicillium and Aspergillus Systematics, eds. R. A. Samson and J. I. Pitt, pp. 5562. New York and London: Plenum Press.
rapid differentiation of Aspergiil/us f1avus from Aspergillus oryzae. Experimental Mycology 11: 170-175.
KLICH, M. A. and pm, J. I. 1985. The theory and practice of distinguishing species of the Aspergillus flavus
group. In Advances in Penicillium and Aspergillus Systematics, eds. R. A. Samson and J. I. Pitt, pp. 211220. New York and London: Plenum Press.
405
KLICH, M. A. and PITI, J. I. 1988. Differentiation of Aspergillus fllWUS from A. parasiticus and other closely
systems in fungi. I. Distribution of ubiquinones in the major families of Ascomycetes, Basidiomycetes
and Deuteromycetes and their taxonomic implications. Transactions of the Mycological Society of Japan 26:
383-395.
KURAISHI, H., ITOH, M., TSUZAKI, N., KATAYAMA, Y., YOKOYAMA, T. and SUGIYAMA, J. 1990.
Ubiquinone systems as a taxonomic tool in Aspergillus and its teIeomorphs. In Modern Concepts in
Penicillium and Aspergillus Classification, eds R. A. Samson and J. I. Pitt, pp. 407-420. New York and
KURTZMAN, C. P., SMILEY, M. J., ROBNETI, C. J. and WICKLOW, D. T. 1986. DNA relatedness among
wild and domesticated species in the Aspergillus flavus group. Mycologia 78: 955-959.
KURTZMAN, C. P., HORN, B. W., and HESSELTINE, C. W. 1987. Aspergillus nomius, a new aflatoxinproducing species related to Aspergillus flavus and Aspergillus tamarii. Antonie van Leeuwenhoek 53: 147-158.
MURAKAMI, H. 1971. Classification of the koji mold. Journal of General and Applied Microbiology 17: 281-309.
MURAKAMI, H. HAYASHI, K. and USHIJIMA, S. 1982. Useful key characters separating three Aspergillus
taxa: A. sojae, A. parasiticus, and A. toxicarius. Journal of General and Applied Microbiology 28: 55-{;0.
RAPER, K. B. and FENNELL, D. I. 1965. The Genus Aspergillus. Baltimore: Williams & Wilkins.
SUGIYAMA, J. and YAMATOYA, K. 1990. Electrophoretic comparison of enzymes as a taxonomic aid
among Aspergillus taxa. I. Aspergillus sects. Ornati and Cremei. In Modern Concepts in Penicillium and
Aspergillus Classification, eds. R. A. Samson and J. I. Pitt, pp. 385-393. New York and London: Plenum
Press.
DIALOGUE FOLLOWING DR. SUGIYAMA'S PRESENTATION

TAYLOR: It looks to me as if Aspergillus flavus and A. oryzae, identified on morphological
characters, are very close and perhaps do not split completely. Maybe these really should
be considered a single species, the only differences being the ability to produce certain
toxins or enzymes.
SUGIYAMA: Taxonomically, these comprise one single species, I think, based on the
enzyme pattern.
Pm: On the other hand, one could argue that there is a split between your groups 1A and
lB. This would still allow us to distinguish A. flavus and A. parasiticus.
SUGIYAMA: At the moment, I think that Cluster 1 consists of two species, A. flavus and A.
parasiticus.
PITT: Comparing your two papers, it is interesting to see that the divergence in section
Ornati is so much greater than you have shown in section Flavi. It's nice to see a modem
technique that correlates so well with what earlier observers have seen in studying
morphology.
407
THE UBIQUINONE SYSTEM AS A TAXONOMIC AID IN ASPERGILLUS

AND ITS TELEOMORPHS*
H. Kuraishi l , M. Itohl, N. Tsuzald l , Y. Katayama l , T. Yokoyama2
and J. Sugiyama3
1Faculty of Agriculture
Tokyo University of Agriculture and Technology

Fuchu, Tokyo 183, Japan
2Institute for Fermentation

Osaka Yodogawa-ku Osaka 532, Japan
3Institute of Applied Microbiology
University of Tokyo
Bunkyo-ku, Tokyo 113, Japan
SUMMARY
Ubiquinones (Q) are important carriers in the electron transport chain of respiratory systems. It has
been shown that the number of isoprene units of the ubiquinone side chain is an excellent aid in
identification of genera or subgeneric taxa in microbial taxonomy.
In Aspergillus, 9 or 10 isoprene units (Q-9 and Q-10) occur, together with a hydrogenated form Q1O(H2). We have studied the ubiquinone systems in Aspergillus in relation to the taxonomy of Raper
and Fennell, who subdivided Aspergillus into species without metulae, species with or without
metulae and species with metulae. In species without metulae, Q-9 occurs in sects Aspergillus and
Restricti and most species in sect. Cervini; and Q-lO in sects Fumigati and Clavati. Subgen. Ornati was
heterogeneous in ubiquinone systems, in agreement with the observations of morphological
heterogeneity. In the sections which mayor may not produce metulae, belonging to sects Nigri and
Cremei possessed Q-9 ubiquinone, as did A. wentii. Nearly all species in sects Wentii, Flavi, Circumdati,
Candidi and Sparsi possessed the Q-10(H2) system. Species in sects Nidulantes, Versicolores, Usti, Terrei
and Flavipedes, which include species always producing metulae, all possessed Q-lO(H2) ubiquinone
without exception.
Xerophilic species had Q-9 or Q-10, while nearly all species having HiiUe cells possessed only the
Q-10(H2) system. The isoprene units of ubiquinone were highly correlated with morphological and
physiological characters in the infrageneric taxa of Aspergillus.
INTRODUCTION
The isoprene side chains of ubiquinone molecules are easily characterised from mycelia
and fruiting structures of fungi by high performance liquid chromatography after
extraction. Previously, Kuraishi et al. (1985) determined the general distribution of
ubiquinone systems in representative species in the major families of the Ascomycotina,
Basidiomycotina and Deuteromycotina. We demonstrated the usefulness of the
ubiquinone system as an aid in the classification of fungal taxa in the major families of
these subkingdoms, and for the elucidation of their phylogeny. Sugiyama et al. (1988)
This paper constitutes Part III of a series entitled "Ubiquinone systems in fungi"
408
H. Kuraishi et al.
showed that rust and smut fungi possessed 0.9 and 0.10, respectively, and the value of
the ubiquinone system in their systematics was evaluated.
This paper describes the distribution of ubiquinone systems in Aspergillus and its
teleomorphs, in relation to the 18 group organisation of Raper and Fennell (1965) or the six
subgenera and 18 sections presented by Gams et al. (1985). The relationship between
morphology and ubiquinone systems is also discussed.
Isolates studied.
The isoprene side chains of ubiquinones were analysed on 397 isolates from 131 species of
Aspergillus, representing both teleomorphs and anamorphs. These isolates are listed in
Table 1.
Table 1. Principle ubiquinone systems in Aspergillus and its teleomorphs
Subgenus Aspergillus
Section Aspergillus
Eurotium
E. amstelodami Mangin
lAM 2035, lAM 13826(FRR 548), lAM 13827(FRR 2792), IFO 5721, lFO 6667,
JCM 1565(NT), JCM 2605
E. chevalieri Mangin
lAM 2001, lAM 13859(FRR 2773), lAM 1386O(FRR 2795), IFO 5883, lFO 30570,
JCM 1568(NT)
E. chevalieri Mangin var. intermeilium (Thorn et Raper) Malloch et Cain
lF05322
E. echinulatum Delacroix
lFO 5862, JCM 157O(NT)
E. haluphilicum Christensen et al.
lFO 7054, IFO 8156
E. herbariorum (Wiggers) Link : Fries
JCM 2602, JCM 2603
E. leucocarpum Hadlok et Stolk
JCM 1574(NT)
E. pseudoglaucum Blochwitz
lAM 2578, JCM 1579
E. repens de Bary
lFO 4332, IFO 7463, JCM 2600
E. rubrum Konig et al.
lAM 138%(FRR 1968), lAM 13897(FRR 2887), lAM 13898(FRR 321)
lAM 13899(FRR 2970), IFO 4089, lFO 7712
E. tonuphilum Ohtsuki
lFO 4089, IFO 7712
E. umbrosum (Bainier et Sartory) Malloch et Cain
lF08207
E. reruphilum Samson et Moucchacca
JCM 1583(1')
Edyuillia
E. athecia (Raper et Fennell) Subramanian
JCM 185O(T)
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
The ubiquinone system as a taxonomic aid in Aspergillus and its teleomorphs
409
Aspergillus
A. proliferans Smith
JCM 1729
Q-9
Section Restricti
A. caesiellus Saito
lPO 4882, JCM 1852
Q-9
A. conicus Blochwitz
Q-9
IFO 4047, IFO 6399
A. gracilis Bainier
JCM 1726
Q-9
A. penicilloides Spegazzini
lAM 1389O(FRR 2177), lAM 13891(FRR 2178), lPO 30615, NRRL 4550
Q-9
lPO 8155
Q-(8(49%)+9(51 %
A. restrictus Smith
lAM 13844(FRR 2973), lAM 13845(FRR 2176), IFO 7101, IFO 7683, JCM 1727(T) Q-9
Subgenus Fumigati
Section Fumigati
Neosartorya
N. aurata Malloch et Cain

IFO 8783, IFO 9817
N. aureola (FennelI et Raper) Malloch et Cain
IF08105
N. fennelliae Kwon-Chung
JCM 1946 mate A, JCM 1947 mate a
N. fischeri (Wehmer) MalIoch et Cain
lAM 2024, lAM 13863(NRRL 4161), lAM 13864(FRR 181),
JCM 1740(T), lPO 5866, lPO 8790
N. fischeri var. glabra (FennelI et Raper) MalIoch et Cain
IFO 8789, IFO 9857, IFO 30571, lPO 30572
N. fischeri var. spinosa (FennelI et Raper) Malloch et Cain
lPO 5955, IFO 8007, lPO 8780, lPO 8782, IFO 30573
N. fischeri var. verrucosa (Udagawa et Kawasaki) Malloch et Cain
IF08779
N. quadricincta (Yuill) MalIoch et Cain
lPO 8778, IFO 9842, JCM 1855(T)
N. stramenia (Novak et Raper) MalIoch et Cain
IFO 9611, IFO 31358, JCM 1856(T)
Aspergillus
A. brevipes Smith
lPO 5821, JCM 1734(T)
A. fumigatus Fresenius
lAM 3006, lAM 13869(FRR 163), lAM 13870(SRRC 43), lPO 8868,
IF09733
A. unilateralis Thrower
lPO 8136, JCM 1737(T)
Section Cervini
A. cervinus (Massee) Neill
lAM 2752, lAM 13857 (FRR 3531), lAM 13858(FRR 3532), JCM 1722
A. kanagawaensis Nehira
lAM 13874 (FRR 3520), lAM 13875(FRR 3528), JCM 1723(T)
A. nutans McLennan et Ducker
IFO 7869, IFO 8134, JCM 1730(T)
A. bisporus Kwon-Chung et Fennell
JCM 1721(T)
Q-10
Q-10
Q-10
Q-lO
Q-lO
Q-lO
Q-lO
Q-10
Q-10
Q-9
Q-9
Q-9
Q-IO(H2)
(cont.)
410
H. Kuraishi et al.
Table 1 (cant)
Subgenus Ornati
Hemicarpenteles
H. aeanthosporus Udagawa et Takada

IFD 9490(T), IFD 9607
H. paradoxus Sarbhou et Elphick
lAM 13886(FRR 2162), lAM 13887(NRRL 5162), IFO 8172(T), IFD 9127,
JCM 2355(T)
Sclerocleista
S. arnatus Raper et al.
lAM 13880(FRR 2291), IFD 4Ot2, IFO 8130, JCM 2263, JCM 2354(T)
S. thaxteri (Subramanian) von Arx
JCM 2264(T), JGM 2350(T)
Warcupiella
W. spinulosa (Warcup) Subramanian
IFO 31800(T), IFO 32023, JCM 2358(T), NHL 2824
Aspergillus
A. brunneo-uniseriatus Singh et Bakshi
!FO 6993, JCM 2348(T)
A. brunneo-uniseriatus var. nanus Singh et Bakshi
IFD 32018, JCM 2349
A. apicalis Mehrotra et Basu
CBS 236.81
A. iuoriensis Bartoli et Maggi
Q-lO
Q-9
Q-9
Q-9
Q-10
Q-9
Q-10(H2)
Q-10
JCM 2353(T)
Q-IO(H2)
JCM 2265, JCM 2266, JCM 2356(T)
Q-10(H2)
A. raperi Stolk
Subgenus Clavati
Section Clavati
A. cIavatus Desmazieres
lAM 2002, lAM 13832(FRR 3029), lAM 13833(SRRC 19), rAM 13834(SRRC 52),
IFD 5837, rFO 8605, lFO 8606, IFO 8865
Q-10
A. e/avato-naniea Batista et al.
JCM 1858(T)
Q-lO
A. giganteus Wehrner
rFD 5818, JCM 1719
Q-IO
A. longivesica Huang et Raper
JCM 172O(T)
Q-(9(49%)+10(46%
Subgenus Nidulantes
Section Nidulantes
Emericella
E. aurantiobrunnea (Atkins et al.) Malloch et Cain
rFD30837
Q-(10(64%)+1O(H2)(36%
rFD30830
Q-IO(H2)
IF030839
Q-IO(H2)
rFD30840
Q-IO(H2)
rFD 30559, rFD 30560
Q-IO(H2)
E. hieolor Christensen et States

E. c/eistominuta Mehrotra et Prasad
E. desertorum Samson et Mouchacca
E. foveolata Horle
E, frutieulosa (Raper et Fennell) Malloch et Cain
IFD30841
Q-(10(52%)+ 10(H2)(48%
411
E. heterothallica (Kwon et al.) Malloch et Cain

IFO 30842, lFO 30843
Q-l()(H2)
E. na'Ollhoensis Christensen et States
IF030836
Q-l()(H2)
E. nidulans (Eidam) Vuillernin
lAM 2006, lAM 13876(NRRL 189), lAM 13877(NRRL 1092),
IFO 9852, JCM 2728, IFO 30872
Q-l()(H2)
E. nidulans var. acristata (Fennell et Raper) Subramanian
IF030063,IF030844
Q-l()(H2)
E. nidulans var. dentata (Sandhu et Sandhu) Subramanian
Q-l()(H2)
IF030845, IFO 30907
E. nidulans var. echinulata (Fennell et Raper) Godeas
lFO 30248, lFO 30412, lFO 30896
Q-l()(H2)
E. nidulans var.lata (Thorn et Raper) Subramanian
IFO 30847
Q-l()(H2)
E. nivea Wiley et Simmons
IFO 4112, IFO 9653('1), IFO 9654, IFO 32020
Q-l()(H2)
E. ptITVIIthecia (Raper et Fennell) Malloch et Cain
IFO 30848
Q-(10(85%)+1()(H2)(15%
E. purpurea Samson et Mouchacca
IF030849
Q-l()(H2)
E. quadrilineata (Thorn et Raper) Benjamin
lAM 13895(FRR 3539),
Q-(10(34%)+10(H2)(66%
lFO 5859, IFO 30850, IFO 30911, JCM 2730
Q-l()(H2)
E. rugulosa (Thorn et Raper) Benjamin
lAM 13900(FRR 1587), lAM 13901(SRRC 93), IFO 5863,
Q-l()(H2)
IF08626, IFO 8629, IF030853, lFO 30913,JCM 2729
IFO 30852
Q-(10(12%)+ 1()(H2)(88%
E. spectabilis Christensen
IFO 30854
Q-l()(H2)
E. striata (Rai et al.) Malloch et Cain
IF030856
Q-l()(H2)
E. sublata Horie
IFO 30906
Q-(10(12%)+ I()(H2)(88%
E. unguis Malloch et Cain
lAM 13911(NRRL 216), lAM 13912(FRR 220), IFO 8087, IFO 30857, JCM 2726,
JCM 2727
Q-l()(H2)
E. variecolor Berkeley et Broome
CBS 119.37, CBS 134.55, CBS 135.55, CBS 136.55, CBS 597.65, IFO 30855,
IFO 31666, IFO 32051, lFO 32052
Q-l()(H2)
E. violacea (Fennell et Raper) Malloch et Cain
lFO 8106, JCM 2725('1)
Q-l()(H2)
Aspergillus
A. multicolor Sappa
lFO 8133
Section Versicolores
A. asperescens Stolk
IF05996
A. caespitosus Raper et Thorn
lAM 13853(FRR 1929), lAM 13854(FRR 3521), IFO 8086
A. janus Raper et Thorn
IF07627
A. silvaticus Fennell et Raper
IF08173
Q-l()(H2)
Q-l()(H2)
Q-l()(H2)
Q-l()(H2)
Q-l()(H2)
(cont.)
412
H. Kuraishi
et a/.
Table 1 (cont)
A. sydowi (Bainier et Sartory) Thorn et Church
lAM 13905(FRR 546), lAM 13906(FRR 2991)
Q-IO(H2)
IF04096
Q-IO(H2)
lF04097
Q-IO(H2)
lF04114
Q-IO(H2)
A. syduwi var. achlamidosporus Nakazawa et al.

A. sydowi var. inaequalis Nakazawa et al.
A. varians (Wehrner) Raper et Fennell
A. versicolor (Vuillemin) Tiraboschi
lAM 12156, lAM 13848(FRR 658), lAM 13849(SRRC 109), IFO 4105, IFO 4411,
lFO 8004, IFO 30338, lFO 31223, JCM 2608, JCM 2609
Q-IO(H2)
Section Usti
A. deflectus Fennell et Raper
IFO 6357
Q-I0(H2)
lAM 13892(FRR 1852), lAM 13893(FRR 3537)
Q-IO(H2)
A. puniceus Kwon et Fennell
A. ustus (Bainier) Thorn et Church
lAM 2057, lAM 13913(NRRL 279), lAM 13914(FRR 664), IFO 7013, IFO 8878
Q-IO(H2)
Section Terrei
A. terreus Thorn
lAM 3004, lAM 13909(FRR 1910), lAM 13910(SRRC 100), lFO 6123, IFO 7078,
IF030537
Q-I0(H2)
A. terreus var. african us Fennell et Raper
Q-I0(H2)
lF08835
A. terreus var. aureus Fennell et Raper
lFO 30536(T), lFO 31217
Q-I0(H2)
Section F/avipedes
Fennellia
F. flavipes Wiley et Simmons

lAM 2523, lAM 13865(SRRC 22), lAM 13866(FRR295), IFO 9655(T)
F. nivea (Wileyet Simmons) Samson
JCM 2731, JCM 2732<n
Aspergillus
A. carneus (van Tieghem) Blochwitz
Q-IO(H2)
Q-IO(H2)
lAM 13855(NRRL 527), lAM 13856(FRR 2977), IFO 5861, IFO 30898, lFO 30899
Q-I0(H2)
A. iizukiJe Sugiyama
IF08869
Q-IO(H2)
A. niveus Blochwitz
lAM 13878(NRRL5505), lAM 13879(FRR3516)
Q-IO(H2)
Subgenus Circumdati
Section Wentii
A. terrieola Marchal
IF05867
A. terrieola var. americana Marchal
IFO 5446
Q-IO(H2)
lAM 2759<n
Q-IO(H2)
A. thomii Smith
A. wentii Wehmer
Q-IO(H2)
lAM 13915(FRR 1778), lAM 13916(FRR 2627), IFO 4108, IFO 6578, lAM 7126,
lFO 8864, IFO 8879, JCM 2724
A. wentii var. minimus Nakazawa et al.
lF04110
Q-9
Q-9
The ubiquinone system as a taxonomic aid in Aspergillus and its teJeomorphs
413
Section Flavi
A. avenaceus Smith
007539,008129
Q-1O
A. fIavus Link : Fries
lAM 13835(SRRC l00A), lAM 13836(SRRC 167), 00 7600, lFO 30107, 00 30180,
JCM 2604
Q-IO(H2)
A. flavus var. asper Sasaki
AHUB-8,IF05324
Q-I0
A. flavus var. columnaris Raper et Fennell
JCM 2605
Q-IO(H2)
A.~(~burg)COhn
lAM 2609, lAM 1388l(SRRC 266), lAM 13882(SRRC 2103),

lAM 13883(SRRC 2104), JCM 2606
A. parasiticus Speare
lAM 13888(SRRC 1039), lAM 13889(IMI 26862), 00 30179, JCM 2720
A. sojae Sakaguchi et Yamada
lAM 13902(SRRC 299), lAM 13903(SRRC 1124), 00 5241
A. tamani Kita
lAM 13907(ATCC 26950), lAM 13908(VDR 33), 00 7465
A. toxicarius Murakami
lFO 30108
A. zonatus Kwon et Fennell
00 8817,JCM2254
Section Nigri
A. aculeatus Iizuka
lAM 2907(T), 00 31348
A. awamori Nakazawa
lAM 2387, 00 4033
A. awamori var. fumeus Nakazawa et al.
004122
A. awamori var. fuscus Nakazawa et al.
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-IO(H2)
Q-9
Q-9
Q-9
OOOU
OOO~
A. awamori var. minimus Nakazawa et al.

A. awamori var. piceus Nakazawa et al.
00 OM
A. carbonarius (Bainier) Thorn
lAM 13830(SRRC 16), lAM 1383l(FRR 369), 00 4039, IFO 5864
A. ficuum (Reichardt) Hennings
lFO 4280, IFO 4320
A. foetidus (Nakazawa) Thorn et Raper
lAM 13867(NRRL 337), lAM 13868(NRRL 341), 00 4312
A. helicothrix AI-Musallan
JCM 1861(T)
A. heteromorphus Batista et Maia
JCM 1862(T)
A. japonicus Saito
00 4060, IFO 4337, 00 4403
A. japonicus var. aculeatus
lAM 13871(FRR 5118), lAM 13872(SRRC 168), lAM 13873(NRRL 1782)
A. niger van Tieghem
lAM 3001, JCM 1864(NT), lAM 1384O(SRRC 60), lAM 1384l(SRRC 61)
A. niger var. awamori (Nakazawa) Al-Musallam
lAM 13838(FRR 4795), lAM 13839(FRR 3534)
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
(cent.)
414
H. Kuraishi et al.
Table 1 (cont.)
A. niger var. plwenicis (Corda) AI-Musallam

JCM2610
A. plwenicis (Corda) Thorn
IFO 8873, IFO 8874
A. pulverulentus (McAlp.) Thorn
lFO 4282, IFO 4353
A. saitoi Sakaguchi e/ al.
lAM 2209(T)
A. saitoi var. kagoshimaensis Sakaguchi et al.
lFO 2190
A. usamii Sakaguchi et al.
lAM 2185(T), IFO 4388
Section Circumdati
Q-9
Q-9
Q-9
Q-9
Q-9
Q-9
Petromyces
P. alliaceus Malloch et Cain
lAM 13824(SRRC 8), lAM 13825(FRR 3518), IFO 5320, IFO 7538, JCM 1948(T) Q-I0
Aspergillus
A. dimorphicus Mehrotra e/ Prasad
JCM 1952(T)
Q-9
A. /anosus Kamal et Bhargava
JCM 1955(T)
Q-I0
A. auricomus (Gueguen) Saito
lAM 13851(NRRL 391), lAM 13852(SRRC 1031), JCM 1949(T)
Q-IO(H2)
A. bridgeri Christensen
Q-1O(H2)
JCM 1950(T)
A. campestris Christensen
JCM 1951(T)
Q-I0(H2)
A. e/egans Gasperini
Q-IO(H2)
JCM 1953
A. insulicola Montemayor et Santiago
JCM 1954(T)
Q-IO(H2)
A. melleus Yukawa
lAM 2066, JCM 1956, JCM 2607
Q-IO(H2)
A. ochraceus Wilhelm
ATCC 1008, lAM 2022, lAM 13842(SRRC 65), lAM 13843(FRR 400), IFO 4344,
Q-IO(H2)
IFO 4345, IFO 4346, lFO 8559, JCM 1958
A. ochraceus var. microsporus Tiraboschi
IF04073, IFO 4409, IF04410
Q-HJ(H2)
A. ochraceoroseus Bartoli et Maggi
JCM 1957(T)
Q-IO(H2)
A. os/ianus Wehmer
lAM 13884(FRR 2115), lAM 13885(FRR 3504), JCM 1959
Q-IO(H2)
A. petrakii Voros
JCM 196O(T)
Q-IO(H2)
A. robustus Christensen e/ Raper
JGM 1961(TI
Q-IO(H2)
A. sclerotiorum Huber
lAM 13846(FRR 3505), lAM 13847(FRR 3507), JCM 1962(T)
Q-IO(H2)
A. sulphureus (Fresenius) Thorn et Church
JCM 1963
Q-IO(H2)
Section Candidi
A. candidus Link : Fries
lAM 2018, lAM 13828(NRRL 303), lAM 13829(NRRL 2297),
lAM 13850(NRRL 312), lFO 4322, JCM 1867
Q-IO(H2)
415
Section Cremei
Chaetosartorya
C. chryseUa Kwon et Fennell

IFO 32022
C. cremea Kwon et Fennell
IAM 13861(NRRL 5081), lAM 13862(FRR 3534), IFO 32021
C. stromatoides Wileyet Simmons
IFO 9652(T), IFO 9656, IFO 9657, IFO 9658, IFO 9659
A. itaconicus Kinoshita
lF04419
Section Sparsi
Aspergillus
A. goralchpurensis Kamal et Bhargava
JCM 2267(T)
A. hiplanus Raper et Fennell
JCM 2347(ST)
A. divers us Raper et Fennell
JCM 2351(ST)
A. funiculosus Smith
JCM 2352(T)
A. sparsus Raper et Thorn
lAM 13904(FRR 1933), JeM 2357(T)
Q-9
Q-9
Q-9
Q-9
Q-9
Q-10(H2)
Q-10(H2)
Q-10(H2)
Q-10(H2)
Extraction and analysis of ubiquinones.

Mycelia grown on potato sucrose agar, or malt yeast extract agar were used for the
extraction of ubiquinones. Xerophilic species were grown on potato sucrose agar
containing 20% sucrose. Extraction, purification and characterisation of ubiquinones were
carried out as described previously (Kuraishi et al., 1985). Where one type of ubiquinone
molecule constituted more than 90% of the types found in a particular species, it was
considered to be the major quinone type.
Influence of growth conditions.

Emericella fruticulosa IFO 30841 and E. parvathecia IFO 30848, which have two molecular
species, Q-10 and Q-10(H2) as major ubiquinones, were grown on potato sucrose agar
adjusted to two pH values, incubated for two weeks, harvested and ubiquinones
analysed. Under these conditions, Q-1O and Q-1O(H2) were equally produced by E.
fruticulosa, while in E. parvathecia most of the ubiquinone molecules were Q-10 with a
small proportion of Q-10(H2). As shown in Table 2, pH had no effect on ubiquinone
composition.
A. longivesica JCM 1720 produces both short and long conidiophores, the latter only
under the light, and it normally produces equal levels of Q-9 and Q-I0. As shown in Table
3, no differences were seen in the ubiquinone composition of hyphae cultivated under
lights, and harvested at various ages. It is concluded that the molecular composition of the
ubiquinone system is a stable character in fungal mycelia.
Ubiquinone systems in Aspergillus and its teleomorphs.
Major ubiquinone systems in the isolates studied are shown in Table 1.
H. Kuraishi et af.
416
Table 2. Ubiquinone compositions of Emericella fruticulosa IFO 30841 and Emericella

parvathecia IFO 30848 grown on the different pH media.
pH
E. fructiculosa IFO 30841

Q-10
Q-10(H2)
E. parvathecia IFO 30848

Q-10
Q-10(H2)
5.6
52%
48%
88%
12%
7.0
46%
54%
89%
11%
Table 3. Ubiquinone compositions of Aspergillus longivesica JCM 1120 obtained at various

ages and cultivated under the light.
Dark
Light
1 week
2 weeks
4 weeks
Q-9
Q-I0
8%
59%
34%
8%
57%
35%
8%
57%
36%
Q-8
Q-9
Q-I0
8%
59%
34%
7%
59%
34%
7%
59%
34%
Subgenus Aspergillus
The group includes species which are xerophilic and have no metulae. It is divided into
two sections, Aspergillus and Restricti.
In sect. Aspergillus, Q-9 was the major ubiquinone system found in 38 isolates. Species
in sect. Restricti also predominantly possessed the Q-9 system. Four isolates of A.
penicillioides had the Q-9 system, but A. penicil/ioides IFO 8155 produced equal amounts of
Q-8 and Q-9. The occurrence of Q-8 in this isolate was very unusual.
A large number of Ascomycetes produced ubiquinones with hydrogenated isoprene
side chains such as Q-10(H2) (Kuraishi et al., 1985), but hydrogenated ubiquinones have
not been found in xerophilic fungi.
Subgenus Fumigati
Subgen. Fumigati includes sects Fumigati and Cervini; metulae are not produced. In sect.
Fumigati, all 36 isolates examined from Neosartorya (six species and three varieties) and
Aspergillus (three species) produced Q-10. In sect. Cervini, four species were examined:
three species produced the Q-9 system, but A. bisporus JCM 1721 was exceptional, having
the Q-10(H2) system.
Subgenus Ornati
Subgen. Ornati, corresponding to the A. ornatus group of Raper and Fennell (1965) is an
admittedly artificial grouping of species without metulae (Raper and Fennell, 1965;
Christensen and Tuthill, 1985). The subgenus is clearly heterogeneous, as it includes four
teleomorph genera (Fennell, 1977; Samson, 1979; Gams et al., 1985; Christensen and
Tuthill,1985).
417
In the teleomorph genus Hemiearpenteles, H. aeanthosporus (two isolates) possessed the

Q-10 system, while H. paradoxus (five isolates) had Q-9. Different ubiquinone systems in
one teleomorph genus are most unusual. Sclerocleista ornata and S. thaxteri (seven isolates)
possessed the Q-9 system, while four isolates of Wareupiella spinulosa had Q-lO. The
Aspergillus species were also heterogeneous, with Q-9, Q-1O and Q-10(H2) all being
produced. Both Q-9 and Q-10 were produced by A. brunneo-uniseriatus and A. apiealis.
In bacteria, ubiquinone systems are species specific, so it appears to be unacceptable
for varieties in one species to have different systems. As A. brunneo-uniseratus had the Q-9
system, while A. brunneo-uniseriatus var. nanus had Q-1O(H2), further taxonomic study
seems indicated.
A relationship may exist between the production of the Q-lO(H2) system and the
production of Hillle cells. A. ivoriensis produces Q-lO(H2) and has Hillle cells, and appears
unrelated to other species of this subgenus (Samson, 1979). A. raperi, also a Q-10(H2)
species, was described as producing abundant Hillle cells, and A. bisporus in sect. Cervini,
has Hillle cells and the Q-10(H2) system. Species which have uniseriate conidial heads,
Hiille cells and the Q-10(H2) system should probably be excluded from sect. Cervini and
subgen. Ornati.
Here, we tentatively propose to divide subgen. Ornati into two groups with Q-9 or
Q-lO.
Subgenus Clavati
Subgen. Clavati sect. Clavati is morphologically well defined and produces phialides only.
Species examined (11 isolates) possessed Q-1O except A. longivesiea, the single isolate of
which produced about 50% of Q-9 in addition to Q-10.
Subgenus Nidulantes
This subgenus has five sections, sects Nidulantes, Versieolores, Usti, Terrei and Flavipedes:
metulae are always present and Hiille cells are often produced. In sect. Nidulantes, which
includes a number of Emerieella species, most isolates possessed the Q-lO(H2) system, but
five isolates had Q-1O as well. In sects Versieolores, Usti, Terrei and Flavipedes, only the Q10(H2) system was found.
Subgenus Circumdati
In subgen. Cireumdati, metulae mayor may not be present, and this large subgenus is
divided into seven sections. In sect. Wentii, A. wentii (nine isolates) had the Q-9 system, but
A. terrieola and A. thomii had Q-10(H2). The result for A. wentii was surprising, as
Kozlowski and Stepien (1982) reported that mitochondrial DNA fragments of A. wentii
linked to those of A. tamarii and A. oryzae, which belong to sect. Flavi and have Q-10(H2).
Christensen and Tuthill (1985) described that the A. wentii group is heterogenous like the
A. ornatusgroup. Our results suggest that A. wentii (subgroup I) is clearly distinguished
from the group which includes A. thomii and A. terrieola (subgroup II) based on the
ubiquinone isoprenologue.
In sect. Fiavi, the Q-10(H2) system was almost universally found, but the Q-10 system
was found in A. avenaeeus and A. flavus var. asper. It appears likely that such Q-I0 species
are incorrectly classified in sect. Flavi and that detailed characterization of Q-lO strains are
necessary for their correct taxonomic placement.
All forty isolates examined from sect. Nigri, comprising 13 species, have Q-9
ubiquinones. In sect. Cireumdati, 13 anamorphic species had the Q-10(H2), while A.
dimorphieus and A. lanosus were exceptional. A. dimorphieus had Q-9 system, which was
418
H. Kuraishi et at.
morphologically characterized by Mehrotra and Prasad (1%9) by branched conidiophores.

According to Samson (1979) this species might be close to A. petrakii, but this taxon has Q10(H2). A. lanosus had the Q-10 system, and shows some similarity with A. fresenii (= A.
sulphureus), which has Q-10(H2), on the basis of the long conidiophores. Since these two
species were analyzed on the ubiquinone system using one single isolate, their
infrageneric position can be elucidated after the investigation of further isolates.
Petromyces is the only teleomorph in this section; Q-10 ubiquinones are produced.
Therefore, subsect. Circumdati can be divided into two subgroups I and IT consisting of
species with the teleomorph Petromycetes (Q-10) and other taxa (Q-I0(H2.
In sect. Candidi, with a single species, A. candidus, six isolates tested produced Q10(H2). In sect. Cremei, where we examined three species of Chaetosartorya and one
Aspergillus, all isolates were of Q-9. Four species in sect. Sparsi produced Q-10(H2), but
only one isolate of A. gorakhpurensis (group A. sparsus) had a Q-9 system which have
usually metulae, whereas other species in this group had Q-10(H2). According to Samson
(1979), A. gorakhpurensis is biseriate, and considered as related to A. pulvinus of the A.
versicolor group. Unfortunately, the quinone system of A. pulvinus was not analysed and
detailed re-examination on the morphological characteristics might be worthwhile for this
species.
In summary, species belonging in subgenera Aspergillus, Fumigati, Ornati and Clavati,
where species do not form metulae, mainly produced Q-9 or Q-10, although a few species
had Q-I0(H2). Species belonging to subgen. Nidulantes, which always produce metulae,
and often have Hiille cells, produce the Q-10(H2) system without exception. In subgen.
Circumdati, a large and undoubtedly taxonomically heterogeneous subgenus, the
ubiquinone systems produced were rather more complex than in the other subgenera.
Molecular species of ubiquinone differed from section to section, or sometimes, as in sects
Wentii and Circumdati, within sections as well.
The species of Aspergillus and its teleomorphs fall into 18 groups, which have been
widely accepted. Figure 1 shows the main ubiquinone molecules of groups in the
schematic presentation according to the order of the groups described by Raper and
Fennell (1%5). Among the groups, each of A. ornatus, A. ochraceus and A. wentii groups
was further divided into two subgroups. In Fig. 2, the intrageneric subgenera and sections
as proposed by Gams et al.(1985) are presented with their ubiquinone species. Like Fig. 1,
the two sections and a subgenus were separated into two distinct clusters on the basis of
ubiquinone isoprenologue. The schematic presentation of groups or sections with
subgenera shown in Figs. 1 and 2 is modified and rearranged as shown in Fig. 3 on the
basis of ubiquinone system, metulae and Hillie cells.
Morphological characters are considered to originate in the chromosomal DNA. On
the other hand, ubiquinone would be distributed in the membrane of mitochondria to act
as a carrier in electron transport. Although it is not clear whether biosynthesis of
ubiquinone molecules is coded only on chromosomal DNA, mitochondrial DNA or both,
it was found that there is some correlation between the presence or absence of metulae
and ubiquinone isoprenologue. In the species belonging to subgenera which have no
metulae, Q-9 or Q-I0 was distributed as a major ubiquinone except for a few species such
as A. bisporusand a member of subgenera Ornati, which have a dihydrogenated
ubiquinone.
Further examinations including other biochemical approaches are required for the
strains which have ubiquinone systems contradictious in the infrageneric taxa.

A. terreus group
r-------------------A.
--------------------~
A. flavipes group --------------....._"'1:

A.
~~-----------A.
ustus group
419
glaucus group
cervinus group
A. ornatu8 group
A. clavatus group
A. fumigatuB group
A. wentii group
A. rsstrictu8 group
A.
A. niger group
A. candidus group ----------__~~'_
\~~.L-------------
A. cremeUB group
-.....:::::~C:::;::;:::;.>"
A. ochraceus group----______________~
A. spars us group
Metulae strictly absent
Metulae absent or present

Metulae strictly present
Figure 1. Schematic presentation of the main ubiquinone systems of the groups in the genus
Aspergillus and its teleomorphs according to Raper and Fennell (1965).
Sec t. Spars i ------------------______~
r------------------- Sec t.
Sect. Cremei -----------------,~;
~,__-----------
As pe rgi II us
Sect. Restricti
Sect. Fumigati
Sect.
Sec t. Ni 9 ri
-----------<
Subgen. Ornati
Sect. Flavi
Sect. Wentii
....-.--
-.,
Sect. Flavipede8------------~~. .1
Sect.
Terrei----------------------~
Cervini
Sect.
Clavati
Sect.
Nidulantes
~~~-------------Sect.
Sect.
Versicolores
Usti
Metulae strictly absent
!fetulae absent or present

Figure 2. Schematic presentation of the main ubiquinone systems of the subgenera and
sections in the genus Aspergillus and its teleomorphs according to Gams et al. (1985).
420
H. Kuraishi et al.
. - - - - - - - - - Sect. Aspergillus
Sect.
Ftavipeds
Sect.
Terrei
Sect.
Usti
Sect.
Versicolores
Sect.
Cervini
Sect.
Nidu l.antes
Sect.
Fumigati
Se ct.
Sparsi
Subgen. Ornati I I
Se ct.
Candidi
Sect.
Cravati
Se ct.
Circumdati
Sect.
Nigri
Se ct.
Flavi
Sect.
Cl'emei
Sec t.
Wen t i i I I
Sec t.
Wen tii I
,.~------ Sect.
Restricti
"---L- Subgen.
---------""-...::::::::::;::=t:::::-"""--------
Meculae strictly absent
' - - - - - - - - - - - - Sect.
Ornati I
Circumdati I
M"etulae absent or present

Figure 3. Rearrangment of the subgenera and section of Aspergillus and its teleomorphs
based on the ubiquinone systems.
REFERENCES
CHRISTENSEN, M. and TUTHILL, D.E. 1985. Aspergillus: an overview. In Advances in Penicillium and
FENNELL, D.T. 1977. Aspergillus taxonomy. In Genetics and Physiology of Aspergillus, eds. J.E. Smith and
J.A. Pateman, pp. 1-21. London: Academic Press.
GAMS, W., CHRISTENSEN, M., ONIONS, A.H., PITT, J.I. and SAMSON, R.A. 1985. Infrageneric Taxa of
Aspergillus. In Advances in Penicillium and Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 5562. New York and London: Plenum Press.
KOZLOWSKI, M. and STEPIEN, P.P. 1982. Restriction enzyme analysis of mitochondrial DNA of members
of the genus Aspergillus as an aid in taxonomy. Journal of General Microbiology 128: 471-476.
systems in fungi. I. Distribution of ubiquinones in the major families of Ascomycetes, Basidiomycetes
and Deuteromycetes, and their taxonomic implications. Transactions of the Mycological Society of Japan 26:
383-395.
MEHROTRA, B.S. and PRASAD, R. 1969. Aspergillus dimorphicus and Emericella cleisto-minuta spp. nov. from
Indian soils. Transactions of the British Mycological Society 52: 331-336.
RAPER, K.B. and FENNELL, D.I. 1965. The Genus Aspergillus. Baltimore: Williams and Wilkins.
SAMSON, R.A. 1979. A compilation of the Aspergilli described since 1965. Studies in Mycology, Baarn 18: 138.
SUGIYAMA, J., ITOH, M., KA TAYAMA, Y., YAMAOKA, Y., ANDO, K., KAKISHIMA, M. and KURAISHI,
H. 1988. Ubiquinone systems in fungi. II. Distribution of ubiquinones in smut and rust fungi. Mycologia
80: 115-120.
The ubiquinone system as a taxonomic aid in
Aspergillus and its teleomorphs
421
DIALOGUE FOLLOWING DR. KURAISHI'S PRESENTATION
SAMSON: This is a very impressive study. None of the isolates of Hemicarpenteles paradoxus
that I have seen produced ascospores. So, I tend to agree that the placement of this
species is doubtful.
CHRISTENSEN: As I recall, A. thomii is in the A. wentii group, but has been suggested to be a
member of section Flavi. Do you have an opinion on this?
KURAISHI: Although A. thomii and nearly all isolates in sect. Flavi have the Q-IO(H2)
system, we need to study this problem more carefully.
423
IMMUNOLOGICAL DIFFERENTIATION BETWEEN PENICILLIUM AND

ASPERGILLUS TAXA
B. Fuhrmann1, M.F. Roquebert2, V. Lebreton1 and M. van Hoegaerden1
IChemunex S.A.
94700 Maisons Alfort
2l.aboratoire de Cryptogamie
Museum National d'HistoireNaturelle
75005 Paris, France
SUMMARY
Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence have been used to detect
species of Penicillium, Aspergillus and other moulds. Thirteen species of Penicillium subgenera
Aspergilloides, Furcatum and Penicillium and five species of Aspergillus were tested for their
immunological reaction with monoclonal antibodies directed to P.glabrum (Wehmer) Westling,
A.versicolor (yuill.) Tiraboschi, Geotrichum candidum link and Mucor racemosus Fres. Cross reactions
showed that monoclonal antibodies directed to M. racemosus , G. candidum and A.versicolor are specific.
Similar reactions with monoclonal antibodies from P. glabrum or A.versicolor show that one antigenic
determinant is shared by all species of moulds tested. Other monoclonal antibodies show that at least
one epitope is shared by Penicillium and Aspergillus but not other genera listed above. At least one
epitope is shared by all Penicillium subgen. Aspergilloides and Aspergillus, and at least one by all tested
species of subgen. Aspergilloides. With the monoclonals tested, no distinction was found between
subgen. Furcatum and subgen. Penicillium. Antigenicity appears to correlate well with morphological
data. ELISA tests are faster than microscopical or biological examination. The method seems
appropriate for defining precise relationships between taxa.
INTRODUCTION
Mould identification is based primarily on colony characteristics and micromorphology.

Morphological and biochemical variations in cultures are frequent, even in single spores
(Bridge et al., 1986). Thus these criteria are not always reliable. The problem is particularly
acute for Penicillium and Aspergillus where the large number of species and varieties make
identifications difficult even for an experienced mycologist. Moreover, standard methods
require examination of at least one week old cultures. For medical or industrial purposes,
this length of time is often too long. Mycologists are attempting to propose other criteria
for identifying moulds rapidly and reliably. Immunological methods may be the most
efficient of all for this purpose. They were first investigated for medically important fungi
(Pepys and Longbottom, 1978; Polonelli and Morace, 1985, 1986; Morace et al., 1984;
Sekhon et al., 1982; Standard and Kaufman, 1977; Kaufman and Standard, 1987). The
specificity of the tests showed them to be of value not only for medical identifications but
also for resolving taxonomic problems in other fields of mycology CHingand et al., 1983;
Morace et al., 1984; Polonelli et al., 1984, 1985, 1987; Lin et al., 1986; Notermans et al., 1986;
van der Heide and Kauffman, 1987) and for characterization of an atypical isolate among
typical ones (Dupont et al., 1988). Our objective was to identify reliably fungal
contaminants present in dairy products, in the fastest way possible. The principle of the
method was to detect antigenic sites of the cell wall by using enzyme linked
424
B. Fuhrmann et al.
immunosorbent assays (EUSA) and immunofluorescent methods, with specific mould

antibodies. A first experiment used Penicillium viridicatum Westling antiserum (Fuhrmann
et al., 1989). Results show that this antiserum contained antibodies reacting with all tested
isolates from Penicillium subgen. Penicillium and Furcatum but not with the subgen.
Aspergilloides, nor other tested moulds. This discrimination between subgenera permitted
a first step in identification. On the other hand, adsorption experiments provided evidence
similar biochemical structures between P. glabrum (Wehmer) Westling, Aspergillus
versicolor (Vuill.) Tiraboschi and A. fumigatus Fres. In this case, antigenicity seemed to
correlate with morphology,i.e. apical enlargment of the stipes, a common feature in
Aspergillus and Penicillium subgen. Aspergilloides. More specific and precise recognition
with monoclonal antibodies (MAB) was next attempted. This is reported here. Reactions
with MAB have the advantage of reacting with one and only one antigenic site on cell
walls. Each taxon being characterized by a set of antigenic determinants, the knowledge of
the composition of this set may help in identification.
Antigen production.
Fungal strains (Table 1) were cultured and prepared by the protocol described by
Fuhrmann et al. (1989). The mycelium of P. glabrum (LCP 88.2494), A. versicolor (LCP
88.2500), Mucor racemosus (LCP 88.3095), Geotrichum candidum (LCP 88.2502) and P.
viridicatum (LCP 88.2485) was used to immunize mice and rabbits. The other strains were
tested for cross reaction with antibodies obtained from immunization.
Antibodies production.
Rabbit immunization protocol with P. viridicatum was described by Fuhrmann et al. (1989).
For monoclonal antibody production, Balb/c mice were injected intravenously or
intraperitoneally with 200 or 400 g of mycelium. Three days after the last boost injection,
the mouse spleens were removed for fusions. Splenocytes were pooled with azaguanine
resistant murine myeloma P3-X63-Ag8-65-3 or NS1 cells 3 and fused according to the
procedure of KOler et Milstein (1975). Positive hybridomas were cloned by limiting
dilution in presence of macrophages or thymocytes and were amplified as ascitic fluids in
mice primed with pristane. The different antibodies obtained by these protocols and
proposed by Chemunex, are grouped in Table 2.
EUSA assays.
In ELISA tests, antigens (in this work, from mycelium) are bound to the plastic surface of a
microtiter plate. Incubation with polyclonal or monoclonal antibodies then allows binding
with those specific for the immobilized antigens from the mycelium. The fixed antibodies
are detected when incubated with a second specific antigen labeled with an enzyme. After
adding the enzyme substrate, the optical density of the stained product is measured (van
der Heide and Kauffman,1987). The protocol used here was described by Fuhrmann et al.
(1989). The percentage of cross reaction was expressed as: 0.0. (test fungal strain with
antibody)- 0.0. (test fungal strain with the preimune/ O.D.(test immunogen with
antibody)- O.D.(test immunogen with preimune). This percentage indicates binding of the
antibodies to the antigen of the mycelium. Cross reaction is considered not significant if
under 12%.
Immunological differentiation between Penicillium and Aspergillus taxa
425
Table 1. Fungal mycelium investigated by ELISA and immunofluorescence.

Penicillium subgen. Aspergilloides
P. glabrum
882494,8930, MUCL 11337, MUCL 158571, 51162, 282, 8931,
MUCL 134661, 8932
783183,53798,722169
P. spinulosum
581377
P. decumbens
803265,763119
P. implicatum
P. montanense
49459
P. thomii
51417,488
P. purpurescens
65412
Penicillium subgen. Furcatum
P. raistricldi
76803,853436
P. jensenii
1389
P. dtrinum
682011,793276
P. waksmanii
692046
P. janthinellum
54268,53105
571533,51782
P.oxalicum
P. melinii
773173
Penicillium subgen. Penicillium
P. aurantiogriseum
882485,50212,611628,753045,863439,873488,882486,
LMUB 8736, LMUB 8737, LMUB 8738, LMUB 8739
P. roqueforti
882492,75146,75148,55352
P. camemberti
882493
P. chrysogenum
561177,72284
P. breuicompactum:
611633, 52699
873492,843384
P.expansum
Aspergillus
A. versicolor
882500, IP 1187-79, 62141, 642270, 763140,833361,863444
A. fumigatus
882499, IP 864, 65516, 722155
A. parasiticus
IP 1142-76, 531525
A. niger
853502,853501,742280
A.flavus
IP 855,753053,863450,853495
A. nidulans
681992
Eurotium
E. repens
882498
E. rubrum
50115
E. amstelodami
50142
E. chevalieri
51991
Mucorales
M. racemosus
60427,863459,631816,763095
M.plumbeus
843350, 49435
M. drdnelloides
601609
M. lamprosporus
72151
M. dispersus
61629
Mfuscus
833343
M.ambiguus
732209
M. genevensis
783178
M. hiemalis
637
M.mucedo
305
Other genera
Absidia corymbifera
Geotrichum candidum
Geotrichum capitatum
U/oc/adium chartarum
IP 1129
882502,51590, IP 287, IP 285, IP 1447-83, 2288, 53577
IP 1647, IP 1640-86
882504
(cont.)
426
B. Fuhrmann et al.
Table 1 (cont.)
Trichoderma longibrachiatum
Trichoderma harzianum
Trichoderma reesei
Fusarium solani
Fusarium decemcellulare
Fusarium sp.
813296
8717
49119
882507
Alternaria tenuissima
Alternaria alternata
882503,681988,681989
843390,853387,863465
Phialophora mustea
Phialophora malorum
793219,833332
882506
Botrytis cinerea
831835,83555,823392
882505, 823394
823408, 803308
All isolates are from Laboratoire de Cryptogamie (LCP), except those indicated with IP: Institut Pasteur,
Paris, LMUB: Laboratoire de Microbiologie de I'Universite Libre de Bruxelles and MUCL: Mycotheque
Universite Catholique de Louvain, Belgium.
Immunofluorescence tests.
In the immunofluorescence tests, the antigen-antibody reaction is used for confirmation of
binding. The principle is similar to ELISA, but the second antibody (specific for the first) is
labelled with a fluorochrome and detected with an epifluorescence microscope. The
protocol was described by Fuhrmann et al. (1989).
Table 2. Antibody production
Immunogen
Antigen
Monoclonal antibody
Penicillium g/abrum
8824941
Aspergillus versicolor
8825001
Mucor racemosus
883095
Geotrichum candidum
8825021
PFI-46/5
PF3-93
PF3-139
AVI-15/37
AVI-17/58
AVl-32/70
MR3-20/12
MR3-3/10
Gel-17/1
Immunogen
Antigen
Polyclonal antibody
Penicillium viridicatum
8824851
B3
RESULTS
A fungus or group of fungi may be characterized by the presence or absence of a specific

antigenic site, in comparison with other antigenic profiles (Table 3). All Hyphomycetes
and Mucorales tested (94 strains) have a common antigenic determinant (1) not shared
427
Immunological differentiation between Penicillium and Aspergillus taxa
Table 3. Cross reaction results

FUNGAL MYCEUUM
NUMBER
Al
A2
A3
A4
a
882494
a
+ 85
Penicillium gl.brum (AJ
8930
5
0
+ 55
a
11337
+ 40
3 0
158571
51182
0 0
5
0
282
0
8821
0
134881
0
8832
2 0
783183
+ 50
P. aplnu/".um (A)
0
3
53788
0
2
722188
60
581377
+
P. de"UlllbaM (A)
48458
+ 80
P. mon"naMa (A)
0
0
51417
P. 'homll (A)
488
0
0
15412
P. purpunecaM (AJ
0
103215
+ 93
P. Impllc.'um (A)
713118
+ 77
71803
75
p. r.I.'r/ckll (F)
+
853431
+ 43
0
1389
+ 66 5
P. /anHnl (F)
882011
p. cJt,lnum (F)
+ 46
7'3278
+ 89
692046
+ 66
P ...t.manll (FJ
0
2
54268
p. 'en,hlne/lum (F)
53105
5 0
571533
P. ox.lleum (FJ
51782
8
0
773173
P. me/ln# (FJ
0
882485
+ 128
P. v."uca.um (P)
0
50212
39
+
811628
+ 21
P. 8urantJogri um(PJ
753045
+ 46
863438
+ 85
873488
+ 56
882488
0
0
8738
0
8737
8738
8738
5 4
882482
+ 65
P. roque/Drtll (P)
75146
0 0
+ 21
0
75148
2
55352
0 0
1 0
882493
+ 106
P. camembe,tli (P)
581177
+ 71
P. chry.ogenum (P)
72284
+ 84
611633
+ 65
P. brevicompactum (P)
52891
+ 72
873482
+ 66
P. ."peneum (P)
843384
+ 47
882500
3 0
+ 100
Aeperl/lllu. ver.Jcolor
0 0
118778 + 93
82141
+ 100
842 270
+ 100
763140
+ 100
833361
+ 100
863444
+ 100
882489
+ 96 0 0
A. lumig.tu.
2 0
884
+ 84
6551&
+ 100
722155
+ 67
(A,. Subgenue Aapergilloidee, (F).. Subgenua Furcatum, (P) Subgenua
+. Antigen present; ... no Antigen; Following numbers c:orreapond to the
AS
a
0
0
0
0
0
0
0
0
0
0
0
ANTIGENES
A8
laC
93
82
89
93
58
85
89
55
10:
78
+
+
+
+
+
+
+
+
++
+
+ 10~
+ 24
+ 63
+ 10
0 + 96
0 + 39
+
+
0 +
+
37
46
58
67
+
0 +
+
a +
65
51
60
36
0
0 +
+
+
+
+
+
+
0 +
0 +
+
+
0 +
0
0 +
0 +
0 +
+
+
+
+
+
+
0 +
+
+
10C
21
83
53
73
78
31
38
30
90
75
93
21
41
87
82
88
43
51
45
51
47
92
66
A8
A7
3
0
0
0
0
+ 32
+ 94
1
1
0
0
+ 95
+ 88
2
0
2
0
10
0
0
a
0
11
0
10
5
3
2
2
+
+
+
+
+ 44 +
+ 73 +
+ 89 +
0 + 42 +
0 + 13: +
+ 92 +
+..58 +
11
10
1
5
0
10
5
0
0
0
0
025
100 +
216
laC + 100
100
100
10C + 63
10C
38 + 84
210
10C + 88
100 + 67
11
14
10
13
+ 110
+ 51
+ 75
+ 81
+ 100
+ 72
+ 93
+ 38
+ 91
0
8
+ 24
+ 100
0
0
0
0
0
0
a
0
0
0
0
0
0
+ 67
+ 100
+ 73
+ 88
+ 96
+ 72
76
100
100
100
72
100
53
17
+ 103
+
+
+
+
+
+
+
14
15
5
6
11
12
8
7
8
1
2
2
2
3
0
0
0
0
0
0
a
4
11
0
0
0
0
0
0
0
0
0
a
2
0
7
a
0
0
0
0
0
0
Al0
A8
+ 100 7
12
13
+ 58
8
68
+
12
8
9
8
9
8
+ 100
+ 100 5
100
9
+
8
1
2
1
0
0
0
0
5
+ 100
+ 100
0
0
Penicillium
percentage of ero.. reaction.
(cont.)
B. Fuhrmann et a/.
428
Table 3. Continued.
FUNGAL MYCEUUM
NUMBER
AI
114216+
531525 +
853502 +
A. nlge,
853501 +
742280 +
855
+
A. n.....
753053 +
813450 +
853485 +
881 2 +
A. nldull.'ua
8824.8 +
Eurolium ,.~".
50115
E. ,ubrum
+
50142
E. ._'e/odemf
+
51
1
+
E. ch.vM/erl
Mucor ,.c.mtMua
183015 +
80427
+
813451 +
831811 +
843350 +
"- plum"'"
48435
+
101801 +
M. c/t'c/n.llold
722151 +
M. 'amp,oaporua
111821 +
"- dl.".,.ua
833343 +
"- lac".
II.amblguua
73220' +
783178 +
M.gan .,,,naia
837
M. hl"malla
+
305
AL mut:MIo
1121
Abeldle corymblf.,.
+
882502
Geotrlchum candidum
51580
287
285
1447-83
2288
53577
1647
G. capit.tum
1640-86
882504 +
Ulocl.dlum chart.rum
Trichoderma long/brach/atum 882505 +
823394 +
803308 +
T. harz/anum
823408 +
813296 +
T. IUHI
17
+
Fus.rium lIolanl
49119
+
F. dllcamc.elJul.,.
882507 +
F. .p
882503 +
AII.rnarla lenui ima
681988 +
&1119118 +
843390 +
A.
853387 +
863465 +
783219
Phl.'oph"'8 mu.I.II.
833332
811250&
p. ma/arum
11135
Botrytle ciner
555
3392
A. pIIriUcua
1111.,,,.,.
A2
56
100
100
72
45
100
42
66
74
54
75
72
25
100
31
47
42
84
100
51
44
100
100
87
68
84
92
107
9
0
0
6
0
0
0
70
70
100
100
69
89
108
68
87
37
96
78
55
34
88
AS
A4
A3
-0
- 11 - 0
-4
+
+
+
+
+
+
+
+
+
6
10C
82
79
75
78
80
78
75
73
0
11
+
+
+
+
+
+
+
+
+
+
+
+
100
100
88
87
91
98
100
100
94
100
44
92
11
0
-- a - 0
-0
+ 10C
+ 75
--
5
2
+ 80
17
+ 24
+ 30
+ 30
+ 96
7
0
ANTIGENES
A8
A7
21
39
+ 48
+ 89
0
0
0
0
0
0
0
0
0
0
0
0
0
0
--------
0
4
0
0
- 02
10
11
1
0
-0
0
0
0
0
0
0
0
8
0
-- 6 -
A8
+ 94 + 44
+ 76 + 10~ + 100
70
100
+ 3;
+ 24
+ 50
+ 59
+ 151 + 227
+ 100
--
5
0
8
2
8
0
0
+ 100
+ 100
0
+ 100 - 0
+ 100 - 0
+ 10C
3
0
0
0
0
0
11
0
0
0
0
0
0
0
0
0
--
-----
--
A8
- 12
-
- 12 -
0
0
+ 28
+ 100 - 9
+ 100 - 10
+ 100 - 11
+ 100
5
+ 211
0
10
+ 53
8
0
10
8
2
0
-8
-6
3
0
0
1
8
0
0
1
0
0
0
3
0
3
0
0
- 11 -- 012 - 00
-- 00 - 00
0
0
-- 00 0
-- 0 0
- 00 - 00
-- 0 0
10
- 40
-
-0
-- 00
- 00
-- 0
- 00
-- 00
0
6
11
0
0
0
- 07 - 0 - 5
0
-- 1011
-- 70
-0
-4
Al0
0
2
0
2
0
7
0
- 011 - 0
0
0
0
0
0
0
0
11
- 11
- 01
-
11
0
0
- 45 00
- 70 - 00 0
- - -
+. Antigen pre_nt . no Antigen, FollOWing number. correspond to the percentage of ero.. reaction.
Imrrunological differentiation between PenicilHum and Aspergillus taxa
429
with Geotrichum. This genus is characterized not only by the absence of any common
antigen with other fungi, but also by the presence of a specific one (2). These results were
obtained with nine isolates of G. candidum and G. capita tum, and are in agreement with the
distance of this genus with Endomycetous affinities from the other genera studied here.
The 14 strains of Mucor have one antigen different from Hyphomycetes; cross reaction
with the corresponding MAB (3) is 0%.
Aspergillus versicolor, A. fumigatus, A. flavus and A. parasiticus and Eurotium rubrum, E.
amstelodami and E. chevalieri have in common three antigenic determinants (1,5 and 6).
The conjugation of the three is representative of Aspergillus, but not specific to it. Antigen
6 is produced in common with Fusarium and Trichoderma, 5 with Penicillium and 1 with
each of these genera. Eurotium species are distinct from anamorphic Aspergilli by the
absence of one antigen (7).
In our experiments, Aspergilli are the best characterized by four antigenic
determinants: 1,5,6, 7. In this genus, A. versicolor species can be recognized by one species
specific antigen (10).
For comparison, we tested representatives of Penicillium subgen. Aspergilloides,
Furcatum and Penicillium. It appears that tested species in subgen. Aspergilloides have three
antigens in common with Aspergillus and only two with subgen. Penicillium: 1 and 5. In
these experiments, no differences were detected between subgen. Furcatum and
Penicillium, which have two antigens in common with Aspergillus (11 and 5) and a specific
one (8, polyclonal) .
...
Geot:l'i.ehwn
0
0
Mucor
*
*
l\
Aspergi Hus
AspergilZoides
Bubgen.
l\
o Penicilliwn subgen.
t:,. AS
AI
A3
A6
A7
A8
Eurotiwn
A A2
{!
1\
*l\
T:l'i.choderma
Fusariwn
*...
Purcatwn subgen.
Q*
t;;;.
t;;;.
AspergiHus versicolor
P.gZabrwn
.::t
p.spinuZosUJn
P.purpurescens
AIO
Figure 1. Schematic representation of antigenic sites on the cell wall.
430
B. Fuhrmann et al.
DISCUSSION
Antibodies produced against specific antigenic structures of fungal cells have been shown
to be important tools in classification and identification tests (Van der Heide and
Kauffman, 1987). Production of species specific MAB is needed to use the corresponding
species as an immunogen. Heterologous crossing with a given MAB permits
establishment of clusters based on the presence or absence of one antigenic site (Fig.l).
This study showed that the method allows distinctions at different levels of classification:
generic, subgeneric and specific, even if the number of tested species is limited. Aspergillus
and Penicillium have two antigens in common both genera may be separated from other
tested fungi by a specific antigen (5).
Penicillium subgen. Aspergilloides appears to be closer to Aspergillus (3 antigenic sites in
common) than to subgen.Penici/lium (2 antigenic sites in common). These results confirm
those obtained with antisera against P. verrucosum (Fuhrmann et al., 1989). In this case,
morphological features like apical enlargment of the stipes are correlated with antigenic
similarities. This suggests a relative coherence of Aspergillus and Penicillium subgen.
Aspergilloides and the intermediate position of subgen. Aspergilloides between Penicillium
and Aspergillus. Aspergillus is well characterized with four antigens, of which three are
common with Eurotium and three with Penicillium subgen. Aspergilloides. Subgenus
Aspergilloides does not share antigen 6 with Aspergillus and Eurotium and, by this fact, and
of course many other features, is not completely included in Aspergillus.
P. raistrickii G. Smith has antigen (7) in common with all tested species of subgen.
Aspergilloides. This species is included by Pitt (1979) in subgen. Furcatum, who considered
that it resembled P. thomii in subgen. Aspergilloides by production of sclerotia and rough
walled vesiculate stipes, giving the appearance of being a "metulate P. thomii". Raper and
Thorn (1949) also previously noted a similarity between the two species. Our results
provide further evidence of proximity between P. raistrickii and P. thomii, as both species
reacted very strongly with MAB PF3.93 (88 and 94%).
P. glabrum (= P.frequentans), P. purpurescens and P. spinulosum possess a common
antigen, A9, absent in other species of subgen.Aspergilloides. Morphologically these species
are very close (Raper and Thorn, 1949; Pitt, 1979), and immunogenic reactions confirm this
relationship. Species tested of subgen. Penicillium and subgen. Furcatum do not react with
the common Aspergillus subgen. Aspergilloides antigen and show a common reaction with
antiserum A8. Differences in species reactions with this antiserum have been discussed
earlier (Fuhrmann et al., 1989). These reactions, however, are not so specific as MAB, as
they detect more than one antigenic determinant in the same experiment. The highest
level of identification in these experiments was obtained with A. versicolor who has four
Aspergillus antigens and one species specific antigen. Trichoderma and Fusarium, both of
which have teleomorphs in the family Nectriaceae, have a common antigenic determinant,
shared with Eurotium and Aspergillus which are both in the family Trichocomaceae. These
two families are grouped in the order Hypocreales by Malloch (1979). A6 antigen may be
representative of this community but more strains need to be tested. Mucor
(Zygomycotina) has an antigen in common with the Ascomycetes (AI) and
Hyphomycetes with ascomycetous affinities. Surprisingly, this antigen does not react with
Geotrichum, whose teleomorph Dipodascus is an Ascomycete. Geotrichum is antigenically
segregated from other filamentous fungi with which it does not share any antigens. It is
characterized by A2 antigen, which is specific to the genus.
The MAB EliSA method has shown to be very efficient for systematic purposes and
to identify species, genera and groups. Results are in accordance overall with accepted
Imrrunological differentiation between PenicilHum and Aspergillus taxa
431
Table 4. Specificity of antibodies

MotwClolUd antibody cross reaction with antigen specificity
AVl-15/37
GCl-17/11
MR3-20/12
MR3-3/10
PFl-46/5
AVl-17/58
PF3-93
91 strains
48 strains
55 strains
so strains
101 strains
69 strains
77 strains
Al
A2
A3
A4
AS
A6
A7
PF3-139
83 strains
A9
AVI-32/70
62 strains
AI0
Hyphomycetes and Mucorales

Genus Geotrichum
GenusMuCOT
Specific for Mucor species
Genera Penicillium and Aspergillus
Genera Aspergillus, Trichoderma and Fusarium
Genus Aspergillus (except Eurotium) and
Penicillium subgenus Aspergilloides
Specific for some Penicillium spp of subgenus
Aspergilloides
A. versicolor
Polyclontll antibody cross reaction with antigen specificity
B3
73 strains
A8
Penicillium subgenus Furcatum
classifications. They have proved to be useful to pinpoint the intermediate position of

some taxa. MAB has the advantage of using standard reagents produced indefinitely in
vivo or in vitro. They bind to one, and only one, epitope and are quite specific. Using
ELISA, many isolates may be tested and identified rapidly.
REFERENCES
BRIDGE, P.D., HAWKSWORTI'I, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S and PATERSON, R.R.M. 1986.
Morphological and biochemical variations in single isolates of Penicillium. Transactions of the British
DUPONT, J., POLONELU, L. and MORACE, G. 1988. Application de I'immunologie (anticorps
monoclonaux) a la caracterisation d'une souche de P. camemberti Thorn. I.e fAit 68: 435-442.
FUHRMANN, B., ROQUEBERT, M.F., van HOEGAERDEN, M. and STROSBERG, A.D. 1989.
Immunological differentiation of Penicillium species. Canadian Journal of Microbiology 35 (in press).
HEIDE, van der S. and KAUFFMAN, F. 1987. Serological methods for taxonomic diagnostic research of
yeasts. Studies in Mycology, Baarn 30: 351-360.
HINGAND L., LE COZ, S., KERLAN, C. and JOUAN, B. 1983. Application de I'immunofluorescence la
detection de Phoma exigua var. foveata, agent de la gangrene de la pomme de terre. Agronomie 3: 51-56.
KAUFMAN L. and STANDARD P.G. 1987. Specific and rapid identification of medically important fungi by
exoantigen detection. Annual Review of Microbiology 41: 209-225.
KOHLER, G. and MILSTEIN,C. 1975. Continuous cultures of fused cells secreting antibody of predefined
specificity. Nature 256: 495.
LIN, H.H., LISTER R.M. and COUSIN, M.A. 1986. Enzyme linked immunoabsorbent assay for detection of
mold in tomato puree. Journal of Food Science 51: 180.
MALLOCH, D. 1979. P1ectomycetes and their anamorphs. In The Whole Fungus, Vol.1, ed. W.B. Kendrick,
pp. 153-165. Ottawa: National Museums of Canada.
MORACE, G., ORSINI, D., CASTAGNOLA, M. and POLONELU, L. 1984. Exoantigen studies of
Phanerochaete chrysosporium and Sporotrichum pruinosum cultures. 19iene Moderna 81: 314-321.
NOTERMANS, S., HEUVELMAN, CJ., van EGMOND, H.P., PAULSH, W.E. and BESLING, J.R. 1986.
Detection of mold in food by enzyme-linked immunosorbent assay. Journal of Food Protection 49: 135-142.
PEPYS, J. and LONGBOTIOM, J.L. 1978. Immunological methods in mycology. In Handbook of
Experimental Immunology. Third edition, ed. D. Weir, pp. 1-27. Oxford: Blackwell Scientific Publications.
B. Fuhnnann et al.
432
PITT J.I. 1979 The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic
Press, London.
POLONELLI, L., ORSINI D., CASTAGNOLA, M. and MORACE, G. 1984. Serological identification of
aflatoxin potential producing Aspergillus species. Igiene Moderna 81: 1128-1136.
POLONELLI, L. and MORACE, G. 1985. Antigenic characterization of Micrsoporum CIl7Iis, M. distortum, M.
equinum, M. ferrugineum and Trichophyton soudanense cultures. MycopathologiJl92: 7-10.
POLONELLI, L., CASTAGNOLA, M., D'URSO, C. and MORACE, G. 1985. Serological approaches for
identification of Aspergillus and Penicillium species. In Advances in Penicillium and Aspergillus
Systematics, eds. R.A. Samson and }.1. Pitt, pp. 267-280 New York and London: Plenum Press.
POLONELLI, L. and MORACE, G. 1986. Specific and common antigenic determinants of C.albicans isolates
detected by monoclonal antibodies. Journal of Clinical Microbiology 8: 366-368.
POLONELLI, L., MORACE, G., ROSA, R., CASTAGNOLA, M. and FRISVAD, J.e. 1987. Antigenic
RAPER, K.B. and mOM, e. 1949. A Manual of the Penicillia. Baltimore: Williams and Wilkins.
SEKHON, AS., LI, J.S.K. and GARG, A.K. 1982. Penicillium marneffei: serological and exoantigen studies.
MycopathologiJl77: 51-54.
STANDARD, P.G. and KAUFMAN, L. 1977. Immunological procedure for the rapid and specific
identification of Coccidioides immitis cultures. Journal of Clinical Microbiology 5: 149-153.
DIALOGUE FOLLOWING DR. ROQUEBERTS PRESENTATION
Do you have any idea of the class of immunoglobulins or the chemical nature of
your wall antigens?
LATGE:
ROQUEBERT:
saccharides.
The immunoglobulins are IgM. We believe that the antigens are poly-
PITT: I'd be intrigued to see how Paecilomyces fits in with Aspergillus and Penicillium using
the techniques that you have described today.
433
THE SIGNIFICANCE OF YEAST EXTRACT COMPOSITION ON

METABOLITE PRODUCTION IN PENICILLIUM
O. Filtenborg, J.e. Frisvad and U. Thrane
SUMMARY
In the literature and in our own experience, significant variations occur in morphological
characteristics and production of secondary metabolites by cultures grown on YES (2% yeast extract
and 15% sucrose) agar, a substrate which is often used in screening for mycotoxins in moulds. In this
investigation we have demonstrated a very Significant influence of yeast extract brand (Difco, Sigma
Y4000 and Y0325, Oxoid, Merck, Lab M and Gibco) on the production of mycotoxins in YES by some
important Penicillia. Using a TLC screening method the variation in mycotoxin production due to the
use of different brands of yeast extract ranged between detection in 5 days and none detected in 4
weeks. The difference in mycotoxin production was often accompanied by differences in several other
characteristics like pH changes of the substrate, sporulation, colony diameter and reverse colour. We
have been unable to find which components in the yeast extracts were responsible for the observed
changes, but the addition of MgS04 appeared to be a satisfactory compensation in most respects. So it
is suggested that this compound in general is added to the YES formula, along with previously
suggested compounds like ZnS04 and CuSO(, thus making this substrate a very valuable and reliable
tool in screening for production of secondary metabolites and in mould taxonomy. Further it is
suggested to use pH registration monitoring in the cultures parallel to screening for secondary
metabolites, since pH differences proved to be a useful indication of significant changes in the
detected profiles.
INTRODUCTION
It is generally recognized that substrate composition has a major impact on morphology
and production of secondary metabolites of mould cultures (Aharonowitz, 1980; Betina,

1984; Orvehed et ai., 1988; Constantinescu, 1990). The importance of trace metals e.g. Zn,
Cu, and magnesium on mycotoxin production and pigmentation has been the subject of
several investigations (Weinberg, 1970; Coupland and Niehaus, 1987a, b; Mashaly et ai.,
1988; Jackson et ai., 1989) and so has the influence of C and N sources (Krumphanzl et ai.,
1982; Haggblom and Ghosh, 1985; Coupland and Niehaus, 1987b; Orvehed et ai., 1988).
During our taxonomic studies of moulds we have now and then experienced
surprisingly large morphological differences between cultures of the same isolate on
substrates differing only in the origin of the ingredients. The substrates which exhibit
these differences are CYA, made with Czapek Dox Broth (Difco) and yeast extract 5 gil,
and YES (15% sucrose + 2% yeast extract) agar, which have a worldwide use in studies of
morphology and production of secondary metabolites in moulds (Pitt, 1979; Frisvad and
Filtenborg, 1983; Betina, 1984; Samson and Pitt, 1985). Yeast extract and peptones are
important components in substrates used for mycotoxin production and it is known that
considerable variations occurs in its composition due to variations in raw materials and
production conditions (Odds et ai., 1978; Bird et ai., 1985; Bridge et at., 1985). We have
tested (unpublished data) the influence of different brands of yeast extract on morphology
434
O. FiHenborg et s/.
and secondary metabolite profiles of mould cultures on CYA and YES. We found that
cultures on CYA were only slightly influenced by the choise of yeast extract brand,
whereas cultures on YES were very much so. Compared to Difco, the yeast extract from
Oxoid, Sigma Y-0375, Lab M, Gibco and Merck in general supported a significantly lower
production of secondary metabolites, if any, as well as a reduced sporulation and less
colourful reverses. On the other hand, the yeast extract Sigma Y-4000 supported increased
production of certain secondary metabolites compared to Difco, while other metabolites
were produced in equal or slightly smaller amounts. Similarly Sigma Y-4000 yeast extract
supported greater sporulation and more colourful reverses than Difco. All tested brands
of yeast extract produced a significant variation in colony diameters of cultures on YES
(Filtenborg, Frisvad and Thrane, unpublished results).
This investigation aimed to establish the differences in yeast extract composition
which were responsible for the significant variations in morphology and production of
secondary metabolites of cultures on YES. It is important to clarify these differences as
they may be the cause of disagreements concerning specific secondary metabolite
profiles.
As obtaining the exact composition of all brands of yeast extract from the
manufacturers has been impossible, this investigation is based on the lack of certain salts
and trace metals in YES, inspired by the above mentioned results with CYA which implies
the importance of adding Czapek Dox Broth.
Fungi.
Isolates used were Penicillium expansum 11nk (Frank 597 = IBT 3034), Penicillium roqueforti
Thorn IBT 3035, Penicillium verrucosum Dierckx chemotype II IBT 3038 and Penicillium
crustosum Thorn IBT 3036.
Substrates.
A number of YES substrates were used all with 2% yeast extract, 15% sucrose and 1.5%
agar, differing only in the brand of yeast extract: Difco (DYES), Oxoid (aYES), Sigma Y4000 (SlYES), Sigma Y-0375 (S2YES), Lab M (LYES), Merck (MYES) and Gibco (GYES).
The substrates were prepared with and without the addition of Mg504' 7H20 (0.5 gil) or
Czapek Dox Broth (Bacto) (35 gil). The pH of SlYES was adjusted to 6.5, as it was pH 4.5
without adjustment. Other media were close to pH 6.5 unadjusted.
Inoculation.
Inoculation was in triplicate. Observed variations in triplicate colony diameters did not
exceed 3 mm. The agar plug TLC method (Filtenborg et al., 1983) was used in metabolite
screening, with samples taken from all three colonies. Variation in metabolite
concentrations between triplicates was never significant. Measurement of pH was
performed by applying the agar plugs, used for the metabolite screening, onto pH
indicator strips (Acilit pH 0-6, Merck art. 9531 and Neutralit pH 5-10, Merck art. 9533).
Measurements were made on all three colonies, and variation did not exceed 0.5 pH
units.
The significance of yeast extract composition on metabolite production in Penicillium
435
Preliminary investigations performed with Penicillium verrucosum demonstrated that the

addition of Czapek Dox Broth to YES substrates containing different brands of yeast
extract almost completely eliminated the observed differences in morphological
characteristics and ability to produce secondary metabolites. On the other hand, addition
of trace metals, Cu and Zn, did not change the culture characteristics. Each component in
the Czapek Dox Broth was then added separately to the YES substrates. Only the addition
of MgS04 appeared to have an effect, and the effect was very much the same as observed
with Czapek solution. Further investigations then aimed to see if other important moulds
responded to the addition of MgS04like Penicillium verrucosum. The importance of MgS04
concentration was also checked.
When Penicillium expansum was grown all expected secondary metabolites were detected
after 7 days in YES with Difco or Sigma Y-4000 yeast extract, but none were detected when
Oxoid, Sigma-0375, Gibco, Lab M or Merck were used (Table 1).
Table 1. Influence of MgS04 addition on growth and metabolite production by Penicillium
expansum on various YES formations after 7 days incubation.

Medium
DYES (a)
OYES
SIYES
S2YES
LYES
MYES
GYES
DYES
OYES
SIYES
GYES
MYES
LYES
S2YES
DYES
OYES
SIYES
MGS04
added
Colony'"
(mm)
Sporulation
pH
Roquefortine C
Citrinin
Patulin
64
60
60
weak
weak
some
NT
NT
NT
NT
weak
weak
some
NT
NT
NT
NT
some
some
some
5.5
3.5
3.5
3.0
3.5
3.0
3.0
4.0
3.5
3.0
5.5
5.5
3.5
3.0
7.0
5.0
4.0
+3(b)
+4
+2
(+)
+1
+4
+4
+3
(+)
+4
+3
+2
+5
+5
+2
+1
+4
+4
+2
+4
+4
+6
+3
+3
+3
+5
+3
+5
+5
NT(c)
NT
+
+
+
+
+
+
+
Cz(d)
Cz
Cz
NT
NT
62
59
61
NT
NT
NT
NT
61
62
60
+4
+6
+3
(a) for formulae see text; (b) - no metabolite was detected, +1: detection limit, +2, +3 etc., indicates arbitraly
chosen values for increasing metabolite-concentrations; (e) NT, not tested; (d) addition of Czapek Dox
broth (35g/\).
By adding MgS04, alone or as a component of Czapek Dox Broth, citrinin and patulin
could be detected in every YES substrate tested, with citrinin dominating at pH values
above 5.5 and patulin at low pH. As YES is not the optimal substrate for roquefortine C
production, detection can be variable, as seen in Table 1. Addition of Czapek Dox Broth,
o. Filtenborg et al.
436
which stabilises the final pH at a slightly higher level than does MgS04 alone, appeared to
increase detection of roquefortine C significantly.
Increasing or decreasing the MgS04 concentration to 2.5 or 0.1 gIl produced only a
minor effect, with metabolite production tending to increase with increasing
concentration. Increasing concentration of Czapek Dox Broth to 90 gIl Significantly
decreased patulin and citrinin production, but increased the roquefortine C concentration.
This effect may be due to nitrate inhibition of production of polyketides (Ward and
Packter, 1974; Grootwassink et al., 1980; Orvehed et al., 1988).
After 14 days incubation, citrinin concentration was higher or remained constant and
detection was possible on every subtrate, although very weak on OYES. The level of
roquefortine C did not change significantly, wheras the level of patulin significantly
decreased and detection was only possible in Sl YES, with and without added MgS04. The
pH values in general increased to 6-7 between 7 and 14 days incubation, but the pH of
Sl YES rose only to about 4, and this may account for the detection of patulin in that
medium. This is in agreement with results obtained for canescin production of P. canescens
Sopp by Brian et al., (1953), who stated that "a variety of media were suitable for its
production, the main requirements being that the pH shall not rise above 6.5". It was of
particular interest to note that media which did not support production of citrinin
supported accumulation of one of its suggested precursors, 2-carboxy-3,5-dihydroxybenzylmethylketone (Turner, 1971).
Variations in colony diameter were only of minor importance. Differences in
sporulation, reverse colour and other morphological characteristics were observed, but
reduced significantly by the addition of MgS04.
Table 2. Influence of MgS04 addition on growth and metabolite production by Penicillium
TOquefOrti on various YES formations after 7 days incubation.
Medium
GS04
added
DYES(a)
OYES
SIYES
S2YES
LYES
MYES
GYES
DYES
OYES
SIYES
GYES
MYES
LYES
S2YES
DYES
OYES
SIYES
Footnotes: see Table 1.
+
+
+
+
+
+
+
Cz(d)
Cz
Cz
Colony f2J
(mm)
Sporulation
pH
PR-toxin
RoquefortineC
60
heavy
some
heavy
NT
NT
NT
NT
heavy
heavy
heavy
NT
NT
NT
NT
heavy
heavy
heavy
4.5
6.0
4.5
6.5
6.0
5.5
6.0
4.0
4.5
4.0
5.0
5.0
5.0
5.0
5.0
5.5
4.5
+3(b)
+4
+1
+5
+5
46
57
NT(c)
NT
NT
NT
63
58
60
NT
NT
NT
NT
53
49
53
+4
+4
+1
+4
+1
+1
+1
+3
+4
+2
+2
+2
+1
+2
+1
+1
+2
+2
+3
437
Penicillium roqueforti
PR-toxin was only detected when P. roqueforti was grown on DYES and S1 YES. PR-toxin is
more frequently, but not invariably, detected when MgS04 was added alone, but not as
component of Czapek Dox Broth (Table 2). However, increasing the MgS04 concentration
to 2.5 gil (data not shown) significantly improved the detection frequency. When Cz
broth was added PR-toxin was not produced. Again, this may be due to nitrate inhibition,
or it may be an effect of pH, or both. After 14 days of incubation PR-toxin concentration
decreased in substrates without MgS04, but increased in substrates with MgS04 added.
Only minor pH changes were observed during prolonged incubation (14 days), except in
DYES with added Czapek Dox Broth, where the pH rose to 7, and here it was not possible
to detect PR-toxin.
Roquefortine was detected in every case, at higher concentrations in substrates
without added MgS04. This differs from the detection of this toxin in P. expansum (see
Table 1), but the pH values were higher in the P.roqueforti cultures.
Differences were observed in colony diameters, sporulation and reverse colours on
the various media, especially OYES, but again addition of MgS04 greatly reduced these.
Penicillium crustosum
Terrestric acid was detected when P. crustosum was grown in each of the substrates, but
little was found in OYES without MgS04 (Table 3). CYA is a much better substrate than
YES for production of penitrem A and roquefortine C (Filtenborg et al., 1983), but MgS04
and Czapek Dox Broth increased production of both alkaloids quite significantly.
Table 3. Influence of MgSD4 addition on growth and metabolite production by Penicillium
crustosum on various YES formations after 7 days incubation.
Medium
DYES(a)
OYES
SIYES
S2YES
LYES
MYES
GYES
DYES
OYES
SIYES
GYFS
MYES
LYES
S2YES
DYES
DYES
SIYES
MGS04
added
Colony 0
(mm)
Sporulation
pH
51
50
55
NT(c)
heavy
weak
heavy
NT
NT
NT
NT
heavy
heavy
heavy
NT
NT
NT
NT
weak
weak
heavy
3.5
4.0
3.0
3.5
4.0
7.0
4.0
3.5
3.5
3.0
4.0
3.5
3.0
3.0
4.5
5.0
3.5
NT
NT
NT
+
+
+
+
+
+
+
Cz(d)
Cz
Cz
Footnotes: see Table 1
53
50
53
NT
NT
NT
NT
52
53
54
Terrestric Penitrem
acid
A
+7(b)
(+)
+4
+3
+3
+3
+3
+5
+6
+2
+3
+5
+4
+3
+7
+6
+5
Roqueforline
+3
+2
+5
+2
NT
+1
+1
+4
+4
+2
+2
+3
+1
+6
+1
+6
NT
NT
NT
+3
+3
+2
NT
NT
NT
NT
+5
+4
+6
O. Filtenborg et a/.
438
Table 4. Influence of MgSO, addition on growth and metabolite production by Penicillium

verrucosum on various YES formations after 7 days incubation.
Medium
DYES(a)
OYES
SIYES
S2YES
LYES
MYES
GYES
DYES
OYES
SIYES
DYES
OYES
SIYES
GYES
MYES
LYES
S2YES
MGS04
added
Cz(d)
Cz
Cz
+
+
+
+
+
+
+
Colony
(mm)
34
28
34
NT(c)
NT
NT
NT
34
30
34
32
32
32
NT
NT
NT
NT
Sporulation
NT
NT
NT
NT
weak
NT
NT
NT
NT
Reverse
colour
pH
Citrinin
Ochratoxin A
VB (e)
4.5
5.0
4.5
5.0
4.0
4.5
5.0
5.5
6.0
5.5
5.0
4.0
4.5
4.5
3.5
5.0
3.5
+2(b)
+3
+3
+2
+1
+1
+1
C
VB
NT
NT
NT
NT
C
C
LVB
VB
VB
VB
NT
NT
NT
NT
+5
+6
+6
+4
+5
+6
+5
+5
+5
+3
+1
+5
+6
+6
+3
+2
+3
+4
+2
+3
+3
Footnotes (a) to (d) see Table 1; (e) VB, violet brown; C, cream-coulored, LVB, light violet brown.
Penicillium verrucosum
After 7 days incubation, P. verrucosum produced citrinin and ochratoxin A in significant
amounts only on DYES and SlYES (Table 4). The addition of MgS04 resulted in good
detection of both toxins on every substrate tested. After 14 days incubation, P. verrucosum
produced citrinin on every substrate, but ochratoxin A production was still variable. The
violet brown reverse colour which is diagnostic for P.verrucosum chemotype II when
grown on YES (Frisvad, 1983), did not appear on DYES. However, the characteristic colour
appeared on every substrate when MgS04 was added.
CONCLUSIONS
The detection of the mycotoxins citrinin, patulin, roquefortine C, PR-toxin, terrestric acid,
penitrem A and ochratoxin A on YES, using a simple TLC screening procedure (Filtenborg
et al., 1983, Frisvad et al., 1989) has been shown here to be dependent on the brand of yeast
extract used in the substrate. In several cases detection was not possible at all, even after
14 days of incubation. Only Difco and Sigma Y-4000 yeast extracts provided reasonably
reliable secondary metabolite production by different Penicillia.
This is of course a very serious and unacceptable problem, as YES is a very valuable
substrate for taxonomy based on secondary metabolites. This may explain reports in the
literature (Bridge et al., 1986, 1987; Land and Hult, 1987; Stenwig, 1988) that the significant
and consistent profile of secondary metabolites observed by us for at great number of
Penicillium species (Frisvad and Filtenborg, 1983, Frisvad and Filtenborg, 1989), may be
difficult to reproduce everywhere. Perhaps it was pure luck that there was Difco yeast
439
extract on our shelves when we started to work on mycotoxin producing moulds using
the agar plug method some 13 years ago.
However, according to the results in this investigation, the problem can be overcome
by including MgS04 (0.5 gil) in YES, in addition to the ZnS04 and CUS04 recommended
previously (Smith, 1949; Frisvad and Filtenborg, 1983). MgS04 obviously compensates for
differences in yeast extract composition between brands, and perhaps between batches,
which we have shown significantly affects production of secondary metabolites, and gross
morphology and colours of mould cultures on YES. However, clarifying the exact nature
of these differences has not yet been possible. Further investigations will be carried out to
solve these problems.
Sigma Y-4000 yeast extract appears to be unique. The final pH of S1YES, around 4.5
makes pH regulation necessary to avoid softening of the agar gel. During growth of
cultures on SlYES, pH initially decreases significantly, as in other substrates, but the usual
subsequent pH increase is minimal in S1YES compared to the other media. Sporulation
was often significantly heavier on S1YES, at the expense of mycelium production, and
often the secondary metabolite profile was increased both qualitatively and quantitatively
on Sl YES compared to the other media.
Based on the results reported here it is suggested that culture pH always be measured
at the time of screening for secondary metabolite profiles. In our experience knowledge of
pH level may assist in detection of culture changes or problems with the screening
procedure. Significant pH deviations mean that the screening must be carried out after a
different incubation time, on a different substrate or perhaps that the culture is
contaminated.
REFERENCES
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BETINA, V., ed. 1984. Mycotoxins: production, isolation, separation and purification. Amsterdam: Elsevier.
BIRD, N.P., CHAMBERS, J.G., LEECH, R.W. and CUMMINS, 0.1985. A note on the use of metal species in
microbiological tests involving growth media. Journal of Applied Bacteriology 59: 353-355.
BRIAN, P.V., HEMMING, H.G., MOFFATT, J.5. and UNWIN, e.H. 1953. Canescin, an antibiotic produced
by Penia1lium canescens. Transactions of the British Mycological Society 36: 243-247.
BRIDGE, P.O., HAWKSWORTH, Z., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, R.R.M. and
SACKIN, M.J. 1985. An integrated approach to Penicillium systematics. In Advances in Penicillium and
Aspergillus Systematics, eds. R.A. Samson and J.1. Pitt, pp. 281-309. New York and I.ondon: Plenum Press.
BRIDGE, P.O., Hawksworth, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S. and PATERSON, R.R.M. 1986.
Mycological Sodety 87: 389-396.
BRIDGE, P.O., HUDSON, L., KOZAKIEWICZ, Z., ONIONS, A.H.5. and PATERSON, R.R.M. 1987.
Investigation of variation in phenotype and DNA content between single conidium isolates of single
Penicillium strains. Journal of General Microbiology 133: 995-1004.
CONSTANTINESCU, O. 1990. Standardisation of methods in Penicillium identification. In Modem Concepts
in Penicillium and Aspergillus Classification, eds. R.A. Samson and J.I. Pitt, pp. 17-25 New York and
I.ondon: Plenum Press.
COUPLAND, K. and NIEHAUS, W.G. 1987a. Stimulation of altemariol biosynthesis by zinc and magnesium
ions. Experimental Mycology 11: 60-64.
COUPLAND, K. and NIEHAUS, W.G. 1987b. Effect of nitrogen supply, Zn++, and salt concentration on
kojic acid and versicolorin biosynthesis by Aspergillus parasiticus. Experimental Mycology 11: 206-213.
FILTENBORG, O. and FRISVAD, J.e. 1980. A simple screening method for toxinogenic moulds in pure
culture. Lebensmittel Wissenschaft und Technologie 13: 128-130.
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FILTENBORG, 0., FRISVAD, J.C and SVENDSEN, J.A. 1983. Simple screening method for molds producing
FRISVAD, J.C 1983. A selective and indicative medium for groups of Penicillium viridicatum producing
FRISVAD, J.C and FILTENBORG, O. 1983. Classification of terverticillate Penicillia based on profiles of
FRISVAD, J.C and FILTENBORG, O. 1989. Terverticillate Penicillia: Chemotaxonomy and mycotoxin
production. Mycologia 81 (in press).
FRISVAD, J.C FILTENBORG, O. and THRANE, U. 1989. Analysis and screening for mycotoxins and other
secondary metabolites in fungal cultures by thin-layer chromatography and high-performance liquid
GROOTWASSINK, J.W.D. and GAUCHER, G.M. 1980. De novo biosynthesis of secondary metabolism
enzymes in homogenous cultures of Penicillium urticae. Journal of Bacteriology 141: 443-455.
HAGGBLOM, P.E. and GHOSH, J. 1985. Postharvest production of ochratoxin A by Aspergillus ochraceus and
Penicillium viridicatum in barley with different pH levels. Applied and Environmental Microbiology 49: 787790.
JACKSON, M.A., SLINNINGER, P.J. and BOTHAST, R.J. 1989. Effect of zinc, iron, cobalt and manganese on
Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures. Applied and
KRUMPHANZL, V., SIKYTA, B. and VANEK, Z. eds. 1982. Overproduction of microbial products. London:
Academic Press.
LAND, CI. and HULT, K. 1987. Mycotoxin production by some wood-associated Penicillium spp. Letters in
MASHALY, R.I., HABIB, S.L., EL-DEEB, S.H., SALEM, M.H. and SAFWAT, M.M. 1988. Isolation,
purification and characterization of enzyme(s) responsible for conversion of sterigmatocystin to aflatoxin
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237-246.
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relation to polyketide synthesis in the fungus Alternaria alternata. Experimental Mycology 11: 187-1%.
PITT, J.I. 1979. The genus Penicillium and its teleomorphic states Eupenicilium and Talaromyces. London:
Academic Press.
SAMSON, R.A. and PITT, 1.1., eds. 1985. Advances in Penicillium and Aspergillus Systematics. New York en
SMITH, G. 1949. The effect of adding trace elements to Czapek-Dox medium. Transactions of the British
STENWIG, H. 1988. Thin-layer chromatography of plugs from agar cultures as an aid for identification of
moulds in routine mycological examination of animal feeds. Acta Agriculture Scandinavia 38: 215-222.
TURNER, W.B. 1971. Fungal metabolites. London: Academic Press.
WARD, A.C. and PACKTER, N.M. 1974. Relationships between fatty acid and phenol synthesis in
Aspergillus fumigatus. European Journal of Biochemistry 46: 323-333.
WEINBERG, E.D. 1970. Biosynthesis of secondary metabolites: roles of trace elements. Advances in Microbial
Physiology 4: 1-44.
DIALOGUE FOLLOWING DR. FILTENBORG'S PRESENTATION
We've recently been noticing problems with yeast extracts. I devised CYA as a
successor to Raper's com steep liquor because yeast extract is much more widely
available. The yeast extract that was available at the time had the advantage of
containing all the trace elements and accessory nutrients that the fungi might require for
growth. It compensated for differences in water supplies, grades of chemicals, including
agar, and so on. The addition of Smith's trace elements, at that time, was either
unnecessary or counterproductive. Later, it became apparent in some European labs that
PITT:
441
CYA was no longer producing good sporulation. It is now clear that copper is the
problem. Too little copper inhibits sporulation because it is a cofactor for the enzymes
that produce the pigmentation of the Penicillium conidia. Too much copper is toxic. The
problem is that the medium makers are now using a more purified yeast extract that is
less suitable for fungal work. The cure for this is to add 0.05% CuS04.5H20 and 0.1 %
FeS04.5H20 to CYA.
FRISVAD: The problem may not be as simple as the absolute concentration of copper.
Metals might be chelated by some yeast extracts.
Pm: There is a fair amount of flexibility in copper concentration.
FRISVAD: You may get sporulation, but poor colour production also. The conidia may be
brown if insufficient copper is present. Magnesium sulfate, as we mention in the paper,
helps get the proper colouration with YES. It is already present in CYA.
ONIONS: Is there any problem with malt extracts? Malt extracts are highly variable.
Pm: This is true, but it doesn't seem to cause any problems.
FRISVAD: We have considered this in the Subcommission on Penicillium and Aspergillus

Systematics, and we have a lot of data on colony diameters and so on using different
malt extracts in different laboratories. Eventually, this data should be analyzed.
9
TAXONOMIC STUDIES ON THE TELEOMORPHS OF
PENICILLIUM AND ASPERGILLUS
445
CHEMOTAXONOMY OF EUPENICILLIUM JAVANICUM

AND RELATED SPECIES
J.e. Frisvad1, R.A. Samson2 and A.e. Stolk2



3740 AG &am, The Netherlands
SUMMARY
The secondary metabolites of isolates of Eupenicillium javanicum and related species were examined by
thin layer chromatography and high-performance liquid chromatography. E. javanicum produces
xanthomegnin and viomellein, while E. ehrlichii differs by the production of brefeldin A, palitantin
and penicillic acid. E. zonatum produce janthitrems, xanthomegnin and brefeldin A, indicating an
intermediary position between the two former taxa. Isolates of P. janthinellum also produces
xanthomegnin and related compounds and this support its identity as the anamorph of E. javanicum.
Chemically P. cremeogriseum fits very well with E. ehrlichii and it could be considered as the anamorph
of this ascomycete. Production of janthitrems and brefeldin A indicates that P. piscarium is the first
available name for the anamorph of E. zonatum. The slow growing species E. meloforme, E. lineolatum,
E. angustiporcatum and E. cryptum all differ from E.javanicum and should be recognized as distinct
species.
INTRODUCTION
In their monograph of the ascomycetes genus Eupenicillium and related Penicillium

anamorphs, Stolk and Samson (1983) accommodated several species of Eupenicillium as
synonyms of E. javanicum. They were regarded as synonyms on the basis of the
production of single asci, broadly lenticular ascopores with spinulose valves and identical
phialide shape. E. Ievitum (Raper and Fennell) Stolk and Scott, E. lineolatum Udagawa and
Horie and E. meloforme Udagawa and Horie also proved to be closely related, but differed
by the ascospore ornamentation. These three taxa were therefore recognized as varieties.
Stolk and Samson (1983) also found, that P. janthinellum as the anamorph of E. javanicum.
Based on an original drawing of in Oudemans' herbarium, they also considered that the
neotypification of P. simplicissimum (Oud.) Thorn by Pitt (1979) was incorrect and that P.
janthinellum was a synonym of P. simplicissimum.
Only a few connections are known to exist between anamorphic nonsclerotial
Penicillia and Eupenicillium species. A connection between P. janthinellum and E. javanicum
and related species would be of great theoretical and practical importance, especially
because strains of the teleomorphic state are also important in biotechnology (Hamlyn et
al., 1987).
Chemotaxonomy, especially based on profiles of secondary metabolites and
extracellular enzymes, has been a succesfull independent taxonomic criterion in
Penicillium and Aspergillus taxonomy (Frisvad, 1989a). In this paper we evaluate the
species related to E. javanicum based on chemotaxonomic data.
J.e. Frisvad 6t a/.
446
Table 1. Production of mycotoxins by isolates in species related to Eupenicillium jRvRnicum.
Species
Number
E. javanicum
NRRL707
FonnernRme
CBS 341.48
CBS 251.66
NRRL2078
NRRL2079
CCMF-374
CBS 349.51
NRRL2016
NRRL2674
CBS 191.67
CBS 346.68
IMII08033
NRRL904
ATCC42743
IBTNIPBI0
IBT]MRS2
E.zonatum
P. oligosporum
P. janthinellum
P. raperi
P. janthinellum
P. janthinellum
P. janthinellum
P. janthinellum
P. citreuviride
CBS 992.72
79-4L-61
P. janthinellum
NRRL2022
P. piscarium
IBTLO}03
E. ehrlichii
E. ehrlichii
NRRL708
NRRL2083
E. /Jrefeldianum
NRRL710
CBS 235.81
CBS 233.81
CBS 234.81
E. brefeldianum
E. brefeldianum
E. /Jrefeldianum
E. brefeldianum
NRRL2093
CBS 577.70
E. /Jrefeldianum
E. javanicum
Mycotoxins produced
xanthomegnin
palitantin (weak)
xanthomegnin
palitantin (weak)
xanthomegnin
palitantin (weak)
xanthomegnin
palitantin (weak)
xanthomegnin
xanthomegnin
palitantin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
xanthomegnin
brefeldin A (weak)
janthitrems
brefeldinA
janthitrems
brefeldinA
paspaline
xanthomegnin
brefeldinA
janthitrems
brefeldinA
palitantin
penicillic acid
brefeldinA
penicillic acid
palitantin
fulvic acid
brefeldinA
brefeldinA
brefeldinA
brefeldinA
paspaline
paspalinine
brefeldinA
brefeldinA
palitantin
fulvic acid
Chemotaxonomy of Eupenicllium javanicum and related species
Species
Number
Former name
Mycotoxins produced
CBS 682.77
E. brefeldianum
brefeldinA
CBS 421.66
CBS 291.62
NRRL3389
CBS 277.83
P.onobense
IMI253737
P. pulVl1lorum
NRRL2026
IMI177905
IMI19OO29
P.simplicissimum
sensu Raper & Thorn
CBS 372.48
P. species
FRR 1893
E. brefeldianum
E. brefeldianum
P. cremeogriseum
P. sajarrwii
447
penicillic acid
paIitantin
brefeldinA
brefeldinA
brefeldinA
brefeldinA
brefeldinA
P. simplicissimum
P. simplicissimum
P. janthinel/um
penicillic acid
penicillic acid
penicillic acid
verrucologen
penicillic acid
E.leoitum
CBS 345.48
CBS 228.81
E.ludwigii
CBS 417.63
E. lineolatum
CBS 188.77
E. angustiporcatum
CBS 202.84
E. meloforme
CBS 445.74
CBS 446.74
CBS 447.74
E.cryptum
ATCC60138
Note: Several secondary, but specific metabolites were found in all isolates but they could not be
identified.
Isolates of E. javanicum and related species (Table 1) were grown on CZ, CYA, MEA, YES,
and OAT agars at 25 C and on CYA at 37 C and examined after one, two and three weeks
for morphological, physiological and chemical characters. For secondary metabolite
production all isolates were examined using the agar plug method (Filtenborg et al., 1983),
while ex type cultures and some other representative isolates of all species were examined
by high performance liquid chromatography (HPLC) with diode array detection (Frisvad
and Thrane, 1987).
448
J.C. Frisvad et al.
All isolates of E. javanicum and all strongly coloured isolates of P. janthinellum produced
xanthomegnin and related metabolites (Table 1) supporting the viewpoint of Samson and
Stolk (1983) that this species is the anamorph of E. javanicum. Similar growth rates and
other physiological features, including growth at 37C and weak growth on creatinesucrose agar also supported that conclusion. Production of palitantin in some isolates
indicated relationship to E. brefeIdianum and E. ehrlichii, however. E. zonatum produced
xanthomegnin, brefeldin A and janthitrems, so from a chemotaxonomic point of view this
species is intermediate between E. javanicum and E. ehrlichii. The anamorph of E. zonatum
differs from P. janthinellum by the very rough conidia, but the size and shape of the
ascospores resemble those of E. javanicum (Fig. 1). E. zonatum shares secondary metabolites
with E. javanicum and E. ehrlich ii, but the production of janthitrems seems to be unique to
the former species. The production of janthitrems and very rough conidia by P. piscarium
suggests that the latter is the anamorph of E. zona tum.
The less strongly coloured isolates in E. ehrlichii and E. brefeldianum were very good
producers of brefeldin A and palitantin and some of them produced penicillic acid and
fulvic acid. Pitt (1979) and Stolk and Samson (1983) emphasized the intermediate position
of E. ehrlichii between E. javanicum and E. brefeldianum. The strong micromorphological
resemblance between E. ehrlichii and E. brefeIdianum was fully supported by their identical
profiles of secondary metabolites. The connection between E. ehrlichii and weakly
coloured strains of P. janthine/lum is also substantiated by their common production of
palitantin and brefeldin A.
P. cremeogriseum Chalabuda (Fig. 2) would fit the weakly coloured isolates and this
name appear to be the first available for the anamorph of E. ehrlichii.
Among the anamorph species examined P. piscarium and P. onobense both produce
brefeldin A suggesting relationship to P. cremeogriseum. However, P. piscarium has
distinctly rough conidia and stipes and the conidia are globose to subglobose and clearly
related to E. zonatum. P. onobense seems to be closely related to P. brasilianum because of
its rough stipes and ellipsoidal spirally roughened conidia, but the profiles of secondary
metabolites are very different: P. brasilianum produces penicillic acid, verrucologen,
viridicatumtoxin and verruculotoxin (Frisvad, 1989b).
Because P. simplicissimum has been used for several taxa, the name should maybe
restricted to isolates related to CBS 372.48 = NRRL 902, the isolate on which Raper and
Thorn (1949) based their concept of that species. Chemotaxonomically this isolate
produced unique secondary metabolites different from those of P. pulvillorum, P.
brasilianum, P. piscarium, P. cremeogriseum, P. ochrochloron, P. onobense or P. janthine/lum.
The situation is further complicated by strains such as P. janthinellum FRR 1893 and "P.
solitum" CBS 288.36, both producers of verrucologen and penicillic acid. These strains may
be related to P. brasilianum, but they do not produce verruculotoxin, viridicatumtoxin or
other metabolites unique for P. brasilianum.
We did not find any known secondary metabolites in E. levitum, E. lineolatum and E.
meloforme and since we have not observed similarities in the chemical profiles of these taxa
with E. javanicum, we tentatively place them as separate species (Fig. 3).
Several attempts to induce the teleomorph in the ex type culture of E. cryptum
Gochenaur & Cochrane (1986) failed, but the description, as well as the micrographs and
drawings strongly suggest a close relationship with the other ascosporic species of
Eupenicillium series Javanica. The type culture of E. angustiporcatum Takada & Udagawa
(1983) produces only a few, reduced conidiophores (Fig. 3m-n), agreeing with those of the
series Javanica. No teleomorph was observed. According to Takada and Udagawa's
449
'
"
110 pm
.:.
'.
:.0 j
GO' ,0 0
0 0 .' 0 Q
Figure 1. P. simplicissimum - teleomorph: E. javanicum CBS 310.48, a. ascus-development; b.

ascospores; c. conidiophores; d. conidia CBS 283.36 e. conidiophores; f. conidia, CBS 992.72 g.
conidiophores; h. conidia, CBS 340.48 i. conidiophores; j. conidia
450
J.C. Frisvad et al.
00
CJ
o
b
O~
a
00
o
10 pm
Figure 2. P. cremeogriseum teleomorph: E. ehrlichii CBS 235.81 a. conidiophOles; b. conidia;
c. ascospores CBS 417.16 g. conidiophores; h. conidia; i. ascospores CBS 324.68;
j. conidiophores; k. conidia; 1. ascus-development; m. ascospores
.'.
..
..
0
o
0
0
()
o abO
00 0
o Of8
o~
10 ILm
Figure 3. P. meloforme - teleomorph: E. meloforme CBS 443.75 a. conidiophores; b. conidia
CBS 447.74 Co ascus-development; d. ascospores P. Tilsile - telemorph: E. leviium, CBS 345.48
e. conidiophores; f. conidia; g. ascus-development; h. ascospores P. lineolilium - teleomorph:
E. lineoliltum, CBS 187.77 i. conidiophores; j. conidia; k. ascus-development; I. ascospores P.
IlngustispoTcllium, CBS 202.84 m. conidiophores; n. conidia
451
J.C. Frisvad et al.
452
Table 2. Production of secondary metabolites in fast growing species related to Eupenicillium

javanicum.
Species
E. javanicum
P. janthinellum
++
++
E.zonatum
++
P. piscarium
E. ehrlichii
P. cremeogriseum
++
++
w
w
++
++
++
++
++
++
++
++
w
w
++
++
P. simplicissimum
sensu Raper & Thorn
pulvillorum
++
P. brasilianum
++
P.
P.onobense
++
P.ochrochloron
E.levitum
E.ludwigii
1: Xanthomegnin
2: Penicillic acid
3: Brefeldin A
4: ]anthitrem
5: PaIitantin
6: Verrucologen
7: Viricatumtoxin
w: weak production
++: strong production
description the ascospores were ornamented with two prominent well-separated

equatorial ridges and the valves show several low ribs. The species is probably related to
E. lineo/atum.
The production of secondary metabolites in E. javanicum, E. ehrlichii and other species
was apparently not dependent on the production of ascomata. Cultures grown on OA or
MEA, where ascomata production was usually prolific, did not contain extra secondary
metabolites compared to those produced on other media where ascomata were absent.
The profiles of secondary metabolites of ascomatal isolates were identical to those
produced by conidial isolates obtained from sectors in some isolates of E. javanicum or E.
ehrlichii. Furthermore the profiles of secondary metabolites in E. javanicum isolates were
alike those produced by strictly conidial strains of P. janthinellum and the same was the
case for E. ehrlichii isolates compared to P. cremeogriseum, and E. zonatum as compared to
P. piscarium. An overview of known mycotoxins produced by species previously regarded
as closely related to E. javanicum, P. janthinellum or P. simplicissimum (Table 2) shows that
several closely related taxa could be recognized
This means that comparison of profiles of secondary metabolites could assist to
elucidate relationships between anamorphs and teleomorphs. However it is still possible
that some secondary metabolites are morph related, as Wicklow and Shotwell (1983) and
Chemotaxonomyof Eupenicllium javanicum and related species
453
Wicklow and Cole (1982) found that some fungal tremorgens were only found in the
sclerotia (and not in the conidia) of Aspergillus flavus.
ACKNOWLEDGEMENTS
REFERENCES
DOMSCH, K.H., W. GAMS and T.-H. ANDERSON. 1980. Compendium of soil fungi. Academic press,
London.
FILTENBORG, 0., FRISVAD, J.e. and SVENDSEN, J.A. 1983. Simple screening method for molds producing
FRlSVAD, J.e. 1989a. The use of high-performance liquid chromatography and diode array detection in
fungal chemotaxonomy based on profiles of secondary metabolites. Botanical Journal of the Linnerm Society
99: 81-95.
--1989b. The connection between the Penicillia and Aspergilli and mycotoxins with special emphasis on
FRISVAD, J.e. and THRANE, U. 1987. Standardized high performance liquid chromatography of 182
GOCHENAUR, S.E. and E. COCHRANE. 1986. Eupenicillium cryptum sp. nov., a fungus with self-limiting
growth and restricted carbon nutrition. Mycotaxon 26: 345-360.
HAMLYN, P.F., D.S. WALES and B.F. SAGAR. 1987. Extracellular enzymes of Penicillium. In J.F. PEBERDY
(ed.): Penicillium and Acremonium, pp. 245-286. New York and London: Plenum Press.
pm, J.1. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London and New
York: Academic Press.
RAPER, K.B. and e. THOM. 1949. Manual of the Penicillia. Baltimore: Williams and Wilkins.
STOLK, A.e. and SAMSON, R.A. 1983. The ascomycete genus Eupenicillium and related Penicillium
TAKADA, M. and S.-I. UDAGAWA. 1983. Two new species of Eupenicillium from Nepalese soil. Transactions
of the Mycological Society of Japan 24: 143-150.
WICKLOW, D.T. and COLE, R.J. 1982. Tremorgenic indol metabolites and aflatoxins in sclerotia of
Aspergillus flavus: an evolutionary perspective. Canadian Journal of Botany 60: 525-528.
WICKLOW, D.T. and SHOTWELL,O.L. 1983. Intrafungal distribution of aflatoxins among conidia and
sclerotia of Aspergillus flavus and Aspergillus parasiticus. Canadian Journal of Microbiology 29: 1-5.
DIALOGUE FOLLOWING DR. FRISVAD'S PRESENTATION
PITT: In Penicillium, especially subgen. Penicillium, your approach to taxonomy using

secondary metabolites tends to split species more than my morphological concepts.
However, secondary metabolites appear to be more conservative in what I would
consider to be older species and genera, such as the soil Penicillia and species of
Aspergillus. Here, you tend to be seeing fewer species using your techniques than some of
the morphologists do. In the species you have just discussed, I would tend to be more
conservative and maintain species that you are tending to lump together. I have tended
to maintain Eupenicillium javanicum as a monoverticillate species and am rather surprised
to see the well developed metulate forms you showed included in that species. Dr.
454
J.e. Frisvad et al.
Samson and Stolk's concept of this species is undoubtedly broader than mine, and we
must do more work before we will come to a consensus.
TAYLOR: Is there any variability in the wall of the ascocarp of any of the Eupenicillium
species you have looked at?
SAMSON: Yes. The wall can be either pseudoparenchymatous or sclerenchymatous.
PITT: How many other species have you actually looked at, and how many possible
connections have you seen? When Dr. Taylor looked at Ta/aromyces, there were very few
connections. I would expect to see more in Eupenicil/ium but still not very many.
FRISVAD: Perhaps P. decumbens and Eupenicillium meridianum might be very closely related.
Maybe there are a few other connections. In my opinion, the Eupenicillium euglaucum
complex may have a lot of species in it.
PATERSON: In some of the HPLC traces, some of the peaks have quite similar retention
times. Is the diode ray detector completely satisfactory for distinguishing between peaks
with similar retention times?
FRISVAD: You sometimes see that the peaks aren't very well separated at all or maybe
completely together. So then we can take ultraviolet spectra from the up slope and the
down slope and distinguish two compounds that way. If they are really close, then you
get a mystical ultraviolet spectrum that you cannot interpret.
455
THE GENUS NEOSARTORYA: DIFFERENTIATION BY SCANNING

ELECTRON MICROSCOPY AND MYCOTOXIN PROFILES
RA. Samsonl, P.V. Nielsen2 and J.C. Frisvad2
lCentraalbureau voor Schimmelcultures
Technieal University of Denmark
SUMMARY
Isolates of Neosartorya including extype cultures were examined morphologically and
chemotaxonomicallyand allocated to nine taxa (N. aurata, N. aureola, N. fennelliae, N. fischeri, N. fischeri
var. glabra, N. fischeri var. spinosa, N. quadricincta, N. spathulata, and N. stramenia), three chemotypes
and three undescribed species. All the species were clearly distinguished by their ascospore
morphology and their characteristic profile of secondary metabolites. Tremorgens were produced by
many species including N. fischeri, N. aureola and N. fischeri var. spinosa. N. fennelliae produced
vidiricatumtoxin, found for the first time outside the genus Penicillium. Trypacidin, was found in N.
aureola, N. fennel/iae and N. fischeri var. glabra.
INTRODUCTION
The genus Neosartorya was established by Malloch and Cain (1972) for members of "the
Aspergillus fischeri" series, a series in the "Aspergillus fumigatus group" of Raper and Fennell
(1965) (= subgen. Fumigati sect. Fumigati; Gams et ai., 1985). Raper and Fennell accepted 5
species and 3 varieties in the N. fischeri series, separated by ascospore ornamentation and
colour of the ascomata. Malloch and Cain (1972) listed 10 taxa, including three
teleomorphs in Aspergillus, later re classified by Samson (1979) and von Arx (1981) in the
teleomorphic genera Hemicarpenteles and Chaetosartorya. In additon of the taxa accepted by
Raper and Fennell (1965), Kwon-Chung and Kim (1974) and Takada and Udagawa (1985)
described two heterothallic species, N. fenneiliae and N. spathulata.
Table 1. Reported production of mycotoxins by Neosartorya species
Taxa
Mycotoxins and antibiotics
N. aurata
Helvolic acid (Turner and Aldridge, 1983)

Fumitremorgin A, B, C, verruculogen (Horie and Yamazaki,
1981; Patterson et al., 1981; Nielsen et aI., 1988), Avenaciolide
(Turner and Aldridge, 1983), Terrein (Mizawa et al., 1%2)
Canescin (Turner and Aldridge, 1983)
Cyclopaldic acid (Turner and Aldridge, 1983)
Canadensolide (Turner, 1971)

N. fischeri var glabra
N. quadricincta
N. stramenia
456
R.A. Samson et al.
Table 2. Examined isolates of Neosarlorya

N. aurata Warcup:
CBS 466.65 WB 4378, T, ex soil, Brunei
N. aureola Fennell & Raper:

CBS 105.55 = NRRL 2244, ex soil, Gold Coast; CBS 106.55 = NRRL 2391, ex soil Liberia;
CBS 651.73 A and B, ex soil, Surinam
N. fennelliae Kwon-Chung & Kim:
CBS 598.74 = NRRL 5534, mating a and CBS 599.74 = NRRL 5535, mating A, T ex rabbit eye ball,
USA; CBS 410.89 = NHL 2951 ("N) and CBS 411.89 = NHL 2952 ("a"), ex marine sludge, Japan
N. fischeri var. fischeri :
CDS 111.51 ex Camellia sinensis; CBS 113.64 ex man; CBS 525.65 = WB 4161; CBS 544.65 = WB
181, T, original C. Wehmer ex Dahlia tuber which were preserved in alcohol, Switserland
isolatL'd by E. Fischer; CBS 404.67 T, Aspergillus fischeri var. thermomutatus, ex mouldy
cardboard, Canada; CBS 681.77, ex soil, Baam; CBS 832.88 = IBT 3023, ex soil Lyngby, Denmark;
IBT 3003, 3012 and 3013, ex soil Lyngby, Denmark; IBT 3005, ex soil Urmston, UK; IBT 3007,
3008,3009, ex soil Holte, Denmark; WB 4075 = 1M! 16143 ex garden Soil, UK
N. fischeri var. glabra :
CBS 111.55 = WB 2163, T. ex old tires, USA; CBS 112.55 = NRRL 2392, ex garden soil, South
Australia; CBS 165.63, ex soil, the Netherlands; IBT 3004, 3006, ex soil Urmston, UK; IBT 3010,
3011 ex soil Lyngby; IBT 3014 and 3015 ex soil, Sweden; WB 4179, soil Australia;WB 2392 = CBS
112.55, garden soil, Australia; IMI 144207, ex canned strawberries; RO 343; RO 27-3
N. fischeri var. spinosa:
CBS 483.65 = WB 5034, T, ex soil Nicaragua; CBS 297.67 ex soil, Pakistan; CBS 865.70 from
unrecorded source, India; CBS 448.75 = NHL 5083, T, Aspergillus fischeri var. verrucosus ex
Japan; CBS 161.88 = TRTC 50994, ex soil Sudan; IBT 3001, Anatto seeds, Brazil; IBT 3002, ex soil,
Lyngby, Denmark; WB 4076 IMI 16061, ex hay, UK; WB 5034 = CBS 483.65, T, ex soil,
Nicaragua
N. quadricincta Yuill
CBS 135.52 WB 2154, T. ex cardboard, UK; CBS 941.73, ex soil, Surinam; IMI 58374 ex soil, NT,
Australia; WB 4175 ex soil Australia; WB 2221 ex fabric, Florida, USA; WB 2154 = CBS 135.52, T,
ex cardbord, UK
N. spathulata Takada & Udagawa

CBS 408.89 = NHL 2948 ("A") and CBS 409.89 = NHL 2949 ("a"), T ex soil, Formosa
Neosartorya spp.
CBS 652.73 A and 652.73 B, ex soil, Surinam; CBS 290.74 ex Acer pseudoplatanus, The
Netherlands; IBT 3016, ex soil, Lyngby, Denmark; RO 342; IBT 3017
N eosartorya stramenia
CBS 498.65 WB 4652, T. ex forest soil, Wisconsin
Isolates of Neosartorya fischeri have frequently been reported as heat resistant spoilage
fungi in foods and beverages (Kavanagh et aI., Beuchat, 1986; 1963; Scott and Bernard,
1987; Conner and Beuchat, 1987; Baggerman and Samson, 1988; Nielsen et aI., 1988;
Samson, 1989). In addition, several mycotoxins have been reported from Neosartorya
species (Table 1).
457
Correct identification of Neosartorya taxa is therefore important. This paper reassesses the
taxonomy of this genus, primarily on the basis of ascospore morphology and profiles of
secondary metabolites.
The isolates examined are listed in Table 2. They were grown on CYA, MEA, YES, and OA
(Samson and van Reenen-Hoekstra, 1988) at 25C and on CYA and MEA at 37C.
Examination was made after one and two weeks for morphological, physiological and
chemical characters.
For scanning electron micropscopy (SEM), mature cleistothecia were transferred to
aluminium stubs which were covered with double sided adhesive tape. A small drop of
water containing some Tween-SO has added and the cleistothecia crushed. The suspension
was air dried, coated with gold and examined by SEM JEOL. For comparison of
ornamentation patterns, cleisthothecia were also fixed in glutaraldehyde and chemically
dehydrated prior to critical point drying as described by Samson et al. (1979).
For secondary metabolite production, all isolates were examined using the agar plug
method (Filtenborg et al., 1983), Representatives of all species, including extype cultures,
were examined by high performance liquid chromatography (HPLC) with diode array
detection (Frisvad and Thrane, 1987). For this purpose, isolates were grown on 3 plates
each of CYA, YES and Sigma Y-4000 Yeast extract sucrose agar (SYES) for two weeks.
After extraction with 150 ml chloroform/methanol (2:1) for 2 min in a Colworth
Stomacher, filtration and evaporation of the solvent the procedure was as described by
Frisvad and Thrane (1987).
Ascospore morphology.
Light microscopical examination of the taxa accepted by Raper and Fennell (1965) showed
that the species can be readily recognized, on the basis of the colour of ascomata and
ascsopore morphology. However, for the varieties of N. fischeri where the differentation is
only based on the ornamentation of the ascospores, light microscopy proved to be more
difficult. SEM, however, provided a much clearer picture of ascospore ornamentation, and
proved to be consistent and typical for each taxon. Specimens fixed in glutaraldehyde and
critical point dried showed similar surface structures to ascospores which were unfixed
and airdried. The latter simple preparation technique was therefore chosen to examine all
species.
Raper and Fennell (1965) described the ascospores of N. fischeri var. fischeri as having
convex surfaces bearing anastomising ridges. Under SEM the ascospores are typically
reticulate (Fig. 1 A-B). Ascospores of N. fischeri var. glabra observed by Fennell and Raper
(1955) to be smooth or nearly so, were finely roughened under the SEM (Fig. 1 C-D).
Ascospores N. fischer of var. spinosa are spinulose, although some gradation exists from
rough to distinctly spinulose (Fig. 1 C-F, 2 A-B). Ascospores of N. fischeri var. verrucosa
(Fig. 2 B), are identical with N. fischeri var. spinosa, and so the two taxon are synonymous.
Spinulose ascospores were also observed in N. aureola (fig. 2 E-F), but this species can be
separated from N. jischeri var spinosa by yellow rather than white ascomata. Ascospores of
N. stramenia (Fig. 4 A-B) and N. aurata (Fig. 2 C-D) resemble those of N. fischeri var. glabra,
but again the ascomata are yellow. The type culture of N. quadricincta produces only very
limited and small ascomata, but the ascospores have the four crests characteristic of
458
A.A. Samson et al.
Figure 1. Scanning electronmicrographs of ascospores. A-B. N. fischeri A. CBS 544.65 =WB

181, B. CBS 832.88 =IBT 3023, CoD. N. fischeri var. glahra C. CBS 112.55 =NRRL 2392, D. IBT
3004, E-F. N. fischeri var.spinosa, E. CBS 483.65 =Wb 5034, F. WB 4076 =IMI 16061 (all
3500 x).
Figure 2. Scanning electronmicrographs of ascospores. A-B. N. fischeri var. spinosa, A. mT

3001. B. CBS 448.75 (T of A. fischeri var. verrucosus) CoD. N. "ur"ta CBS 466.65 = WB 4378, EF. N. aureola, E. CBS 105.55 NRRL 2244, F. CBS 651.73 A (all 3500 x).
459
460
A.A. Samson
et at.
Figure 3. Scanning electronmicrographs of ascospores. A-B. N. quadricincta CBS 135.52 = WB

2154, B. WB 4175, CoD. N. fennelliae, C.CBS 598.74 x CBS 599.74, D. CBS 410.89 x CBS 411.89,
E-F. N. spathulata CBS 408.89 x CBS 409.89 (all 3500 x).
Figure 4. Scanning electronmicrographs of ascospores. A-B. Neosartorya stramenia, CBS

498.65 =WB 4652, C-F. Neosartorya spp. C. mT 3002, D. mT 3016, E. CBS 652.73 A, F. 652.73 B
(all 3500 x).
461
462
A.A. Samson et al.
this species, while the convex surface is slightly reticulate (Fig. 3 A-B). Ascospores of the
heterothallic N. fennelliae, produced after are mated in pairs, have a distinct cerebriform
surface structure (Fig. 3 C-D). N. spathulata isolates produce almost smooth-walled
ascospores with distinct crests (Fig. 3 E-F). This species also produces a quite different
Aspergillus anamorph as described by Takada and Udagawa (1985).
Amongst the material examined, several isolates were found with ascospores different
from any described species (Fig. 4 C-F). Their secondary metabolites were also distinct, so
these isolates may represent three undescribed species.
Secondary metabolites profiles.
Secondary metabolite data correlated well with the morphological data. All species
studied produced a characteristic profile of secondary metabolites as evaluated by HPLCDAD. Two examples of consistent secondary metabolite production by isolates of the
same taxon are depicted: in Fig. 5 and in Fig.6. Nearly all isolates of N. fischeri var. fischeri
produced fumitremorgin A, B, C and verrucologen, Horie and Yamazaki (1981), found
fumitremorgin production in only a small proportion of isolates they examinated, but the
differences in results may be explained by differences in media used for mycotoxin
production. We have found CYA, YES and Sigma Y-4000 YES are a very effective
combination of media for mycotoxin production.
Isolates identified as N. fischeri var. glabra and N. fischeri var. spinosa produced on
morphological criteria different profiles of secondary metabolites (Table 3). However,
morphologically similar isolates of N. fischeri var. glabra produced two quite distinct
secondary metabolite profiles. Two of them produced very large amounts of mevinolins
(CBS 112.55 and RO 27-3) while three others, including the ex type culture of N. fischeri
var. thermomutatus, produced a somewhat heterogeneous range of metabolites.
The potentially tremorgenic tryptoquivalins were found for the first time in N. aureola,
N. fischeri var. fischeri, N. fischeri var. glabra chemotype III and N. fischeri var. spinosa (Table
I, 3). Tryptoquivalins have previously earlier been found in A. fumigatus and A. clavatus
(Buchi et al., 1977). Two tryptoquivalins were produced in very high amounts by N. aureola
CBS 651.73 A and B.
N. fennelliae isolates were found to produce viridicatumtoxin which was previously
only found in Penicillium (Frisvad, 1989). Viriditoxin was found in N. aureola (CBS 106.55
and 671.73B) and Neosartorya sp. (CBS 652.73A & B). Xanthocillin derivatives were
produced by N. spathulata and Neosartorya sp. (IBT 3002). Trypacidin, another metabolite
characteristic of Aspergillus fumigatus was also produced by N. fennelliae and the ex type
culture of N. fischeri var. thermomutatus. Two families of secondary metabolites with
characteristic UV spectra (FUA and FUB), found in A. fumigatus, were also produced by
several Neosartorya species (Table 1). However, although some secondary metabolites
were common to several species in Neosartorya and Aspergillus sect. Fumigati, each
Neosartorya taxon we investigated produced a specific distinct profile of secondary
metabolites.
CONCLUSIONS
On the basis of differences in ascospore ornamentation and profiles of secondary

metabolites the described taxa in Neosartorya can be well separated. The two varieties
glabra and spinosa of N. fischeri differ from var. fischeri that they could be raised to species
rank (also compare Kozakiewicz, 1989). Since there are some biochemical and
morphological similarties between isolates asigned to N. fischeri var. spinosa and N. fischeri
213'11
Neosartorya fischerivar.1I1abra CBS 111.55 (ex type)
lee
:J
a:
-lee
IMJ 144207
113
2'11
Ti me
eml n. )
313
4'11
Figure 5. Comparison of HPLC traces of two isolates of N. fischeri var. glabra, CBS 111.55 (ex
type) and IMI 144207. The prominent peak at 29.3 min. was unique to var. glabra
chemotype I.
Neosartorya fischeri IBT 3013
1'11'11'11
I~
-1'11'11'11
I
I'll
I
2'11
T I me
3'11
It
Neosartoryafi,cheri IMI16143
4'11
eml n. )
Figure 6. Comparison of HPLC traces of two strains of N. fischeri, IBT 3013 (soil, Denmark)
and IMI 16143 (soil, Great Britain). Both strains produce fumitremorgins A, B, and C and
verrucologen two Iryptoquivalins (T) and the unknown metabolite PUB.
463
464
R.A. Samson et al.

Table 3. Secondary meta bolites determined in the Neosartorya isolates investigation
N. aurata
CBS 466.65
N. aureola
CBS 10555
CBS 10655
CBS 651.73
CBS 651.73
N. fennelliae
CBS 598.74
CBS 599.74
NHL2951
NHL2952
CBS 544.65
WB4075
IMI16143
IBT 3005
IBT 3007 (NT)
IBT 3008 (NT)
IBT3009
IBT 3012 (NT)
IBT 3013
IBT 3023
CBS 448.75 (NT)
N. fischeri var. glabra chemotype I
CBS 111.55
IMII44207
IMII02173
IBT3004
IBT 3006 (NT)
IBT3010
IBT 3011 (NT)
IBT 3014 (NT)
IBT 3015 (NT)
N. fischeri var. g/abra chemotype II
WB2392
CBS 112.55
R027-3
N. fischeri var. glabra chemotype III
CBS 404.67
CBS 941.73
IMII6061
R0343
WB4076
WB4179
N. fischer var. spinosa
CBS 483.65
CBS 297.67
IBT 3001
CBS 681.77 (NT)
WB5034
+
+
+
+
+
+
+
+
+
Mycotoxins produced
4
5
7
6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
465
hfycotoxinsproduced
2
N. quadricincta
CBS 135.52
WB2154
IM158374
WB4175
WB2221
N. spathulata
NHL2948
NHL2949
N. stramenia
CBS 498.65
N. species 1
CBS 652.73
CBS 652.73
CBS 290.74
N. species 2
IBT3016
N. species 3
R0342
N. species 4
I
BT3002
1 ;FUA
2 ; tryptoquivaline
3 ; trypacidin
4 ; virid icatumtoxin
5 ; Fumitremorgins A,B,C and verrucologen
+
+
+
7 ; mevinolins
8; terrein
9; kotanin
NT ; Not tested by HPLC-DAD
var. glabra chemotype III a more detailed investigation is required to solve the taxonomic
classification of these taxa.
It is important to note that both varieties glabra and spinosa are the only two species
found on food products. IMI 144207 and IMI 102173 were both found in canned
strawberries from UK and IBT 3001 was found on anatto seeds from Brazil. The two
isolates asigned to var. glabra examined did not produce any known mycotoxins, but var.
spinosa IBT 3001 produced tryptoquivalins. Kozakiewicz (1989) incorrectly stated that the
type of Neosartorya fischeri CBS 544.65 = WB181 = 1M! 211391 was isolated from canned
apples. This culture was received by Wehmer (1907) from Prof. E. Fischer and isolated
from tubers of Dahlia flowers, which were preserved in alcohol. In our analysis we did not
find any known mycotoxins in this isolate.
The significance of the food-borne taxa will be further examined. The isolates
representing the new species will be described elsewhere.
ACKNOWLEDGEMENTS
The authors thank the NATO Scientific Affairs Division (Brusser, Belgium) for the research
466
R.A. Samson et al.
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DIALOGUE FOLLOWING DR. SAMSON'S PRESENTATION
TAYLOR: Does anyone know how these ascospores are dispersed in nature? Or is the
whole cleistothecium dispersed?
SAMSON: This is only speculation, but I think that the whole ascoma is dispersed. Insects
might eat them and then excrete the ascospores.
TAYLOR: It is possible that the ascospore ornamentation in these fungi is involved in
dispersal, and so the ascospore morphology may be evolving quite rapidly compared to
other characters.
PITT: Regarding the dispersal of the teleomorph in nature, perhaps the real answer is that
these things don't occur in nature! Neosartorya species are found in processed foods
because of their heat resistance. Have you looked at other Aspergillus teleomorph genera
that appear to be closely related, such as Sclerocleista. The number of known isolates of
these genera is very low. They all have the same ascocarp wall and appear to me to be
very closely related.
SAMSON: I would like to combine some of these genera. But we have to consider the quite
distinct anamorphs. I'm very interested in the studies that Dr. Taylor and Dr. Kuraishi
have done, which might help us understand the relationships among these teleomorph
genera. Perhaps we should consider erecting a teleomorph genus that has different
anamorphs in section Ornati.
TAYLOR: Has anyone crossed isolates of Neosartorya fennelliae from different parts of the
world to see if it is really all one species, or if there might be vegetative compatability
groups?
SAMSON: Takada and Udagawa published a study in Japanese. Judging from their tables,
they saw lots of isolates and tried to establish the mating pattern.
CHRISTENSEN: We should be looking in the tropics a lot more for Aspergillus species and
their teleomorphs.
SAMSON: I agree. The heat shock technique, in which you heat the sample at 6Q-70C, or
microwave a sample in a petri dish for one minute, is very effective.
TAYLOR: Bob Metzenburg at Wisconsin has developed a chemical method that simulates
heat shock. Using it, he has found a number of homothallic Neurospora species from
tropical Mexican and Central American soils.
469
PARTICIPANTS
Australia
Dr. ].1. Pitt
e.S.I.R.O.
Division of Food Research
P.O. Box 52,
NORlli RYDE, N.S.W. 2113
Czechoslovakia
Dr. O. Fassatiova
Dept. of Cryptogamic Botany
Benatska2,
128 01 PRAHA 2
Belgium
Prof. Dr. G.L. Hennebert
Laboratoire de Mycologie Systematique et
Appliquee
MUCL, Universire Catholique de Louvain
Place Croix du Sud 3,
1348 LOUVAIN-LA-NEUVE
Dr. J.F. Berny
Laboratoire de Mycologie Systematique et
Appliquee
MUCL, Universire Catholique de Louvain
Place Croix du Sud 3,
1348 LOUVAIN-LA-NEUVE
Dr. D. Stynen
ECO/BIO, Diagnostics Pasteur
Woudstraat 24
3600GENK
Canada
Dr. K.A. Seifert
Forintek Canada Corp.
800 chemin Montreal Road,
OTTAWA, Ontario KIG 3Z5
Denmark
Dr J.e. Frisvad
Food Technology Lab., Building 221
2800LYNGBY
Dr O. Filtenborg
Food Technology Lab., Building 221
2800LYNGBY
Federal Republic of Germany
Dr. Helgard I. Nirenberg
Institut rur Mikrobiologie
Biologische Bundesanstalt fiir Land- und
Forstwirtschaft
Konigin Louise Str. 19,
1000 BERLIN 33
France
Dr. J.-P. Latge
Institut Pasteur
28, Rue du Dr. Roux
75724 PARIS
Dr. J.-P. Debeaupuis
Institut Pasteur
28, Rue du Dr. Roux
75724 PARIS
Dr. Eveline Gueho
Institut Pasteur
28, Rue du Dr. Roux
75724 PARIS
Dr. Marie-France Roquebert

Laboratoire de Cryptogamie
M.N.H.N.
12, Rue de Buffon,
75005 PARIS
Mad. Joele Dupont
Laboratoire de Cryptogamie
MN.H.N.
12, Rue de Buffon,

75005 PARIS
470
Italy
Prof.L. Polonelli
Istituto di Microbiologia
Facolta di Medicina e Chirurgia
Universita degli Studi di Parma
Via A. Gramsci 14,
1-43100 PARMA
Japan
Dr. Junta Sugiyama
Institute of Applied Microbiology
Univ. of Tokyo
Bunkyo-ku, TOKYO
Prof. Hiroshi Kuraishi
Faculty of Agriculture
Tokyo Univ. of Agriculture and Technology
5-8 Saiwaicho 3-chome
Fuchu, TOKYO 183 Japan
Netherlands
Prof. Dr. K.W. Garns
P.O. Box 273,
3740 AG BAARN
United Kingdom
Dr. p.o. Bridge
C.A.B. Mycological Institute
Ferry Lane,
KEW, Surrey TW9 3AF
Dr. Jim Croft
Department of Genetics
University of Birmingham
P. O. Box 363,
BIRMINGHAM BIS 2TT
Prof. Dr. D.L. Hawksworth
Ferry Lane,
KEW, Surrey TW9 3AF
Dr. Z. Lawrence
Ferry Lane,
KEW, Surrey TW9 3AF
Dr. Agnes H. S. Onions
The nineteenth, North Road
Dunbar,
EASTLOTHIANEH421 AY
Drs. Ellen S. van Reenen-Hoekstra

P.O. Box 273,
3740 AG BAARN
Dr. R.R. Paterson

Ferry Lane,
KEW, Surrey TW9 3AF
Dr. R.A. Samson

Centraalbureau voor Schimme1cultures
P.O. Box 273,
3740 AG BAARN
Mr. A.P. Williams

Leatherhead Food Research Association
Randalls Road,
LEATHERHEAD, Surrey KT 22 7RY
Drs. Amelia C. Stolk

Wittelaan 19,
3743CPBAARN
Dr Veronia Hearn
Public Health Laboratory Serivce
Mycological Reference Laboratory
61 Colindale Avenue,
WNDON NW9 SHT
Dr. J. Visser
Section Molecular Genetics
Dept. of Genetics, Agricultural University
Dreyenlaan 2,
6703 HA WAGENINGEN
Turkey
Dr. N. Aran
Dept. Food Science, Tubitak
Marmara Scientific and Industrial
Research Institute
GEBZE/ISTANBUL
United States of America

Prof. Dr. M. Christensen
Dept. of Botany
Aven Nelson Building, University of Wyoming
LARAMIE, Wyoming 82071
Dr. Maren Klich
USDA Southern Regional Research Center
P.O. Box 19687,
NEW ORLEANS, LA 70179
471
United States of America

Dr. S. Peterson
United States Department of Agriculture,
NRRL
1815 North University Street,
PEORIA, Illinois 61604
Dr. Ed. Mullaney
USDA Southern Regional Research Center
p.o. Box 19687,
NEW ORLEANS, LA 70179
Dr. John Taylor
Dept. of Botany, University of California
2017 Life Sciences Building,
BERKELEY, California 94720
Sweden
Dr. O. Constantinescu
The Herbarium, University of Uppsala,
P.O. Box 541,
5-75121 UPPSALA
Switserland
Mrs Nadine Braedlin
NESTEC Ltd, Quality Assurance Dept.
55 Avenue Nestle,
1800VEVEY
473
INDEX
Arachis
hypogaea 179
Aspergillus
acolumnaris 202, 203
aculeatus 328, 329, 333, 334
alutaceus 28, 84
amstelodami 6
anomalus205
apicalis 388, 392, 417
aurata221
avenaceus397,404,417
awamori 304,329
bisporus 416, 417, 418
brevipes201,204,206,221,241
brunneo-uniseriatus 392, 417
var. nanus 392,417
caesiellus 250, 251, 252, 253, 255
candidus 69, 418
carbonarlus 328, 329, 332
chrysogenums 338
clavatus 336, 338, 462
conicus 249, 250, 251, 255
coremiiformis 335, 336, 338, 341
cremeus386
dimorphicus 417
duricaulis 201, 204, 206, 219, 221, 239, 241
ellipticus 328, 329, 333
fennelliae 235
ficuum 84, 85
fischeri 235
flavescens 77
flavus9,27,28,39,70, 221,235,259,260,262,
263,264,265,275,302,303,304,305,318,336,
338,341,395,398,404,405,429,453
subsp. flavus var. flavus 395
subsp. flavus var. oryzae 9, 395
subsp. parasiticus 9
subsp. parasiticus var. parasiticus 395
subsp. parasiticus var. sojae 9, 395
var. asper 397, 398, 399, 417
var. columnaris 338
var. flavus 398
var. oryzae 398
var. parasiticus 398
var. sojae 398
foetidus 274, 328, 329,332
fresenii 418
fumigatus 70, 77,201, 202, 203, 204, 205, 206,
210,212,219,221,225,226,229,232,235,237,
239,241,242,243,244,245,251,304,336,338,
424,429,462
var. acolumnaris 202, 205, 242
var. albus 205
var. ellipticus 205, 229, 241
var. fulvoruber 205

var. griseobrunneus 205
var. sclerotiorum 204, 205
glaucus 249
var. tonophilus 255
gorakhpurensis 418
gracilis 249, 250, 251, 255
var. sartoryi 252, 253
griseus 77
helicothrix 328, 329, 333
hennebergii 328, 329
heterothallicus 313
hollandicus 6
intermedius 328, 329
itaconicus 249, 250
ivoriensis 392,417
japonicus 328, 329, 333, 334
var. aculeatus 334
var. japonicus 334
kambarensis 398
lanosus 417, 418
leporis 265, 302, 397, 399
longivesica 415, 417
nanus 328, 329
neo-ellipticus 205
nidulans 77,84,86,87,94,96,221,301,312,
313,314,317,318
var. echinulatus 312, 313, 315
nidulellus 84, 86, 87, 94, 301, 303, 304, 305, 336
niger 39,70, 77,84,85,94,98,210, 221,274,304,
315,317,328,329,332,333
var. niger 85
var. phoenicis 84
nomius260,263,264,265,302,303,397,399
ochraceus70,84,304,336,338,418
ornatus 386, 416, 417, 418
oryzae9,108,259,262,263,264,275,302,303,
304,305,318,341,395,398,404,405,417
var. magnasporus 398
parasiticus 9,27,70, 259,260, 262,263,264,265,
275,302,303,338,341,395,398,404,405,429
penicillioides 249, 250, 251, 253, 255, 256, 416
petrakii 418
phialiseptus 205
phoenicis 39, 84, 85,328,329
pulverulentus85,328
pulvinus 418
quadrilineatus 312, 313, 315, 318
raperi392,417
restrictus 70,249,250, 251,253,255,256
rugulosus 312, 315
sojae 9, 259, 262, 263, 264, 275, 302, 303, 341,
395,398,404
spinulosa 392
474
stellatus 313
sulphureus 418
tamarii 39, 70, 260, 263, 275, 302, 304, 336, 338,
341,399,417
terreus 39,43, 70, 304, 305, 315, 317
terricola 417
thorrdi341,417,421
togoensis 336, 338
toxicarius 275, 398
unguis 313, 315, 317
unilateralis 201, 204, 206, 220, 221, 239, 241
usanU 328, 329
ustus 70
versicolor 28, 70, 336, 418, 424, 429, 430
virens 77
viricola 255
viridinutans 201, 204, 206, 221, 241
vitricola 257
lVentii70,417,418,421
Beta
vulgaris 190
Brassica
campestris
var. toria 186
Byssochlamys
fulva 362
nivea362
Chaetosartorya
chrysella 388, 392
cremea 388, 392
stromatoidea 392
Corerrdum
coprophilum 131
glandicola 131
silvaticum 133
vulpinum 132
Dictyostelium
discoideum 345
Emericella
fruticulosa 415
nidulans 69, 86, 87, 94, 96, 301
parvathecia 415
Eupenicillium
angustiporcatum 448
brefeldianum 448
cinnamopurpureum 69
crustaceum 128, 344, 350
cryptum448
ehrlichii 168,448,452
euglaucum 454
javanicum 6,7,91,157, 169, 171,445,447,448,
452,453
var. javanicum 151
levitum 445, 448
lineolatum 445, 448, 452
meloforrne 445, 448
meridianum 454
sheari 166
zonatum448
Eurotium
amstelodarrd 6, 69, 274, 429
chevalieri 69, 429
herbariorum 69
manginii69
medius 69
repens 69, 70
rubrum 69, 429
Fusarium
oxysporum 65
Geotrichum
candidum 424, 429
capitatum 429
Globodera
rostochienses 156
rostochiensis 157
Herrdcarpenteles
acanthosporus388,392,417
paradoxus 392, 417, 421
Hormodendrum
pedrosi210
Lagenaria
vulgaris 179
Mucor
racemosus 424
Neosartorya
aurata 219, 239, 457
aureola 219, 221, 241, 245, 457
fennelliae 205, 239, 241, 455, 462, 467
fischer
var. glabra 204
fischeri 69, 70, 98, 204, 212,221,239,241,242,
245,456,457,465
var. fischeri 201, 203, 229, 457, 462
var. glabra 229, 241, 242, 457, 462
var. glabrata 212
var. spinosa 212, 229, 457, 462
var. thermomutatus 462
var. verrucosa 457
quadricincta 201, 206, 219, 241, 457
spathulata 219, 229, 455, 462
stramenia 219, 221, 241, 457
Neurospora
crassa 86, 358, 359, 366
Paecilomyces
fumosoroseus 336
li1acinus 198
Penicillium 350
aeruginosum 127, 131
aethiopicum 105, 106, 205
albidum 168
album 129, 130
allahabadense 176, 186, 192
allii 106, 131
aragonense 128
475
arenicola 106,352
aromaticum 130
forma microsporum 108, 126
aromaticum-<:asei 130
asperosporum 159
asturianum 128
atramentosum68, 106, 126, 142
atrovenetum 159, 169
atroviride 130
aurantiacum 179, 192
aurantioalbidum 129
aurantiocandidum49, 106, 129
aurantiogriseum 8, 28, 31, 35, 39, 41, 43, 68, 70,
83,106,107,109,110,111,114,115,129,133,
140,142,146,291,350,351,352,355,373,380
var. album 142
var. aurantiogriseum 129
var. poznaniense 109, 110
var. viridicatum 129
aurantiovirens 111, 129
australicuml09,110,130
baculatum 126
bialowiezense 132
biforme 108, 129
biourgeanum 132
biourgei 130
blakeslei 129
brasilianum 166, 169, 205, 448
brevicompactum 5, 28, 35, 68, 70, 107, 108, 112,
113,131,132,142,144,147,194,352,378
var. magnum 132
brunneorubrum 87, 126
brunneostoloniferum 107, 132
brunneoviolaceum 106, 129
camemberti 22, 68, 94,108, 109,128,129, 142,
350,351,352
var. rogeri 129
camerunense 108, 126
canadense 106
candidum 94, 108, 129
canescens 149, 153, 156, 168, 171, 172, 194, 195,
198,436
cameolutescens 106, 129
casei 130
var. compactum 114, 130
caseicola 108, 129
caseiperdens 130
caseiphilum 373
chlorophaeum 108, 126
chrysogenum 22, 28, 31, 68, 77, 83, 84, 87, 88,
94,98,108, 109, 125, 126, 140, 146, 165, 173,355
citreonigrum 169, 304
citreoroseum 87, 126
citrinum 69, 169, 373
c1aviforme 9, 83, 132, 133, 142,352
clavigerum 132, 133, 142, 352
commune 68, 70, 107, 109, 110, 115, 125, 128,

129,140,351,355,373,380
concentricum9,83,109,110,113,131
conditaneum 129
conservandi 114, 130
coprophilum 9, 68, 83, 109, 110, 113, 126, 131,
378
coralligerumI59,169,171
cordubense 129
corylophilum43, 69, 70, 169, 171
corymbiferum 112, 131
corynephorum 169
cremeogriseum 168, 197,448,452
crustosum 22, 49,62,63,68,70, 110, 114, 130,
131,140,146,373,380,434,437
var. spinulosporum 110, 130
cyaneofulvum 87, 125, 126
cyc10pium 62, 68, 70, 83, 106, 107, 128, 129, 351,
373
var. album 130
var. aurantiovirens 107, 129
var. echinulatum 111, 130, 352
daleae 149, 153, 157
decumbens 454
dierckxii 169
digitatoides 127
digitatum22, 39, 43, 68, 70, 110, 111, 127
var. latum 127
divergens 131
diversum 189, 191
var. aureum 192
duc1auxii 132, 352
duninii 127
echinulatum 39, 43,68,111, 130,352,376,378
echinulonalgiovense 169
ehrlichii 169
elongatum 131
epsteinii 129
estinogenum 159, 169
expansum22,28,39,49,50,57,62,63,64,68,
70,103, 111, 130, 131, 140, 194, 351, 380, 434,
435,437
var. crustosum 62
farinosum 110, 130
fasciculatum 77
fellutanum 69
fennelliae 111, 128,352
flavidomarginatum 108, 126
flavoglaucum 109, 129
flexuosum 112, 126
frequentans 83, 194,430
funiculosum 142, 144, 173, 175, 176, 179, 182,
189,192
fusco-flavum 253, 255
fuscoglaucum 129
gaditanum 186, 189
glabrum 69, 83, 97, 128, 194, 275, 305, 424, 430
476
gladioli 128, 344
glandicola 9, 68, 83, 112, 126, 131, 147,352,380
glaucum 77, 131
gorgonzolae 113, 130
granatense 151, 153, 157
granulatum 9, 83, 112, 131, 142, 147,285,352
var. globosum 142, 146
griseobrunneum 107, 132, 147
griseofulvum 39, 68,112,126,132,194,195,378
var. dipodomyicola 112
griseopurpureum 168, 171
griseoroseum 83, 87, 109,125, 126, 140, 165
hagemii 107, 132
harmonensel08,l26
herquei 159
hirsutum 43, 68, 112, 113, 131,350,352
hordei 28, 112, 113, 131, 194, 195
indonesiae 6
isariiforme 352
islandicum69, 176, 191
italicum 22, 39, 68, 70, 110, 113, 127, 128, 146
var. album 127
var. avellaneum 127
janczewskii 149, 151, 153, 157, 165, 166, 168,
169,171,172,194,195
janthinellum 91, 149, 151, 156, 165, 166, 171,
193,197,198,445,448,452
janthogenum 131
japonicum 113, 127
javanicum 6, 7
jensenii 22, 69, 125, 168, 171
johanniolii 129
kabunicum 165
kap-laboratorium 131
kapuscinskii 168, 172
kojigenum 125
korosum 186
lanogriseum 130
lanoso-coeruleum 106,109
lanosogrisellum 127
lanosogriseum 109
lanosoviride 109, 115, 130
lanosum 125, 169
leucopus 131
lividum304
luteum 182
majusculum 130, 136, 137
mali 114, 130
maltum 127
manginii 169, 172
mariaecrucis 169
marneffei 192
marquandii 198
martensii 106,107, 129
mediolanense 130
megasporum 39, 43
melanochlorum 130, 373
meleagrinum 108, 125, 126, 142

var. viridiflavum 165
melinii 149, 156, 157,169,194,195
miczynskii 69, 169, 170, 172
minioluteum 69,173,176,179,186,189,192
monstrosum 132
montanense 275, 304
murcianum 168
musae 131
nalgiovense 125
nigrescens 77
nigricans 151, 168
nordicum 130
notatum 49,87,88,108,125,126
novae-zeelandiae 159, 169
ochraceum 115, 128, 129, 140
var. macrosporum 109, 129
ochrochloron 195, 196,197,198,448
olivaceum 127
var. italicum 127
var. norvegicum 127
olivicolor 115, 129
olivinoviride 115, 129
olsonii 68, 70, 113, 122, 132, 142, 144, 147, 352
onobense168,169,448
oxalicum 39, 127, 128, 136, 165, 169,373
paecilomyceforme 129
palitans 109, 115, 129, 140
var. echinoconidium 111, 130
paraherquei 159
patris-mei 107, 132
patulum 112, 126
paxilli 166, 169
pedemontanum 171
phialosporum 165
pinophylum 69,173,176,179,182,183,186
piscarium 166, 168, 169,448,452
plumiferum 131
polonicum 129
porraceum 106, 129
primulinum 191, 192
proteolyticum 179
pseudocasei 110, 130
psittacinum 114, 130
puberulum 106, 107, 109, 125, 129, 140, 146,
350, 351-353
var. camemberti 351
pulvillorum 169, 448
purpurescens275,43O
purpurogenum 69
val. rubrisclerotium 182
raciborskii 169
rademirici 189, 190, 191
radiatolobatum 168
radulatum 149, 156, 157, 169
rais trickii 169, 430
resticulosum 111, 127, 131
477
restrictum 22,194,195
rogeri 129
roqueforti 22,28,68,70,77, 113, 114, 128, 130,
144,291,338,380,434,437
var. punctatum 109, 130
var. viride 114, 130
var. weidemannii 113, 130
roseocitreum 126
rubens 108, 126
rubicundum 176, 179,192
rubrum 189
rugulosum 69
sajarovii 168
samsonii 186, 189
schneggii 131
sclerotigenum 128, 169
sclerotiorum 22
simplicissimum69, 149, 151, 156, 157, 169, 172,
198,445,448,452
siwvae 169
smithii 169
solitum 33,68,107, llO, ll4, 130, 131, 136, 137,
142, 166, 192,373
var. crustosum 140
soppii 169, 170
spinulosum 22, 69, 97, 275, 305, 430
steckii 169
stephaniae 129
stilton 130
stoloniferum 107, 108, 132
sua volens 130
szaferi 132
tabescens 132
tardum 165
terraconense 127
terrestre 110, 128, 130
thomii430
trimorphum 195, 196, 197
urticae 112, 126
variabile 39, 69, 131, 176, 179, 366, 369
varians 179, 181, 182
ventrusosum 127
verrucosum 27, 28, 31, 68, 83, 107, 114, 115, 128,
129,130,350,352,373,430,434,435,438
var. album 130
var. corymbiferum 131
var. cyclopium 62, 107, 129
var. cyclopium strain ananas-olens 126
var. melanochlorum 110, 114, 130
var.ochraceum129
vesiculosum 130
victoriae 165
virescens 130
viridicatum 8, 49, 69, 83, 105,107, 114, 115, 128,
129,350,373,378,380,424
viridicyclopium 106, 129
volgaense 107, 132
vulpinum 9, 22, 83, 126, 132, 352

weidemannii 130
var. fuscum 130
westlingii 165, 169, 170, 171
zacinthae 186
wnatum452
Phoenix
dactylifera 84
Pichia
guiIliermondii 229
Pritzeliella
caerulea 131
Psedotsuga
menziesii 171
Ramalina
siliquosa 79
Rana
macrodon 190
Saccharomyces
cerevisiae 344,345,350
Sclerocleista
ornata 388, 392, 417
stromatoides 392
thaxteri 388, 392, 417
Sordaria
humana 39, 43
Sterigmatocystis
nidulans 86
Stilbothamnium
novoguineense 335
nudipes 335, 336, 338
togoense 335
Stilbum
humanum131
Talaromyces
byssochlamyoides 362, 370
f1avus 69, 70, 362, 369
var. macrosporus 369
helicus 344, 345, 350
intermedius 362, 366
leycettanus 362
luteus 362
mimosinus 362, 366
striatus 362, 366
thermophilus 362, 366
trachyspermus 362
Thermoascus
crustaceus 362
Trichophyton
rubrum210
Urnula
craterium 344, 345, 350
Uromyces
viciae39
Ustilago
ficuum84
phoenicius 84
478
Warcupiella
spinulosa 388, 392, 417
Yersinia
enterocolitica 212
Zacintha
verrucosa 186
Zea
mays 179, 182,351

Modern Concepts in Penicillium and Aspergillus Classification

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Modern Concepts in

Penicillium and Aspergillus

NATO ASI Series

The series is published by an international board of publishers in conjunction with the

Plenum Publishing Corporation

Kluwer Academic Publishers

Computer and Systems Sciences

Recent Volumes in this Series

Volume 179-lmmunological Adjuvants and Vaccines

Series A: Life Sciences

Springer Science+Business Media, LLC

Proceedings of the Second International Pnicillium

Library of Congress Catalog1ng-1n-Publ1cat1on Data

I n t e r n a t i o n a l Pnicillium and Aspergillus NATO Advanced Research

ISBN 978-1-4899-3579-3 (eBook)

1990 Springer Science+Business Media New York

SYSTEMATICS OF PENICILLIUM AND ASPERGILLUS

J.I. Pittl and R.A. Samson2

Division of Food Processing

'lCentraalbureau voor Schimmelcultures

J.I. Pitt & R.A. Samson

Systematics of Penicillium and Aspergillus

isolates, cannot be overemphasized. Their significance in the effective utilisation of fungi

Shortcomings of Raper and Thom's work.

J.I. Pitt & R.A. Samson

(b) Failure to typify.

(c) The problem of naming sexual states.

Systematics of Penicillium and Aspergillus

J.I. Pitt & R.A. Samson

Systematics of Penicillium and Aspergillus

J.I. Pitt & RA Samson

Systematics of Penicillium and Aspergillus

as far as possible, to those in Bangkok or Bogota, Berlize or Bhutan, as well as those

J.I. Pitt & R.A. Samson

Systematics of Penicillium and Aspergillus

DIALOGUE FOLLOWING DR. PITT'S PRESENTATION

How about identification of Penicillium and Aspergillus by serological

STANDARDIZATION IN PENICILLIUM IDENTIFICATION

MATERIAL AND METHODS

Values affected by interference of the colonies

Standardization in Penicillium identification

Standardization in Penicillium identification

RESULTS AND DISCUSSION

Preparation of inoculum. and inoculation.

Figure 3. Variation in colony diameters of 36 species of Penicillium grown on G25N in

Glass versus plastic Petri dishes.

Standardization in Penicillium identification

Figure 4. Histogram showing the differences in colony diameters of 36 species of Penicillium

DIALOGUE FOLLOWING DR. CONSTANTINESCU'S PAPER

Standardization in Penicillium identification

IDENTIFICATION OF PENICILLIUM AND ASPERGILLUS SPECIES IN

o Filtenborg & J.e.

MATERIAL AND METHODS

Fungi. The investigation of metabolite production in pure culture was performed on

Table 1. Influence of media on secondary metabolite production of selected Penicillium and

Aspergillus species after incubation for seven days.

(a) ec = extracellular detection, ic = intracellular detection; better than intracellular detection.

= no detection, ec>ic = extracellular detection

Identification of Penicillium and Aspergillus species in mixed cultures

85:15:20 (CAP) followed by Ce(S04)2-spray for DRBC and elution in toluene/

Production of secondary metabolites on isolation media by pure cultures.

Figure 1. TLC-metabolite profiles of pooled pure cultures of several Penicillium species on

o FiHenborg & J.e. Frisvad