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o
E
F
G
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
ISBN 978-1-4899-3581-6
DOI 10.1007/978-1-4899-3579-3
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
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
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
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.
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.
"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
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
12
GREUTER, W. et al. 1988. International Code of Botanical Nomenclature adopted by the Fourteenth
International Botanical Congress, Berlin, July-August, 1987. Konigstein, W. Germany: Koeltz Scientific
Books.
HAWKSWORTH, D.L. 1990. Problems and prospects for improving the stability of names in Aspergillus and
Penicillium. In Modem Concepts in Penicillium and Aspergillus Classification, eds. R.A. Samson and J.I.
Pitt, pp. 75-82. New York and London: Plenum Press.
HAWKSWORTH, D.L. and SUTTON, B.C 1974. Article 59 and names of perfect state taxa in imperfect
genera. Taxon 23: 563-568.
KLICH, M.A. and MULLANEY, E.J. 1987. DNA restriction enzyme fragment polymorphism as a tool for
rapid differentiation of Aspergillus flavus from Aspergillus oryzae. Experimental Mycology 11: 170-175.
KLICH, M.A. and PIIT, J.1. 1985. The theory and practice of distinguishing species of the Aspergillus flavus
group. In Advances in Penicillium and Aspergillus Systematics, eds. RA. Samson and T.I. Pitt, pp. 211-220.
New York and London: Plenum Press.
- - 1988. Differentiation of Aspergillus f1avus from A. parasiticus and other closely related species.
Transactions of the British Mycological Society 91: 99-108.
KULIK, M.M. and VINCENT, P.G. 1973. Pyrolysis gas-liquid chromatography of fungi: observations on
variability among nine Penicillium species of the section Asymmetrica, subsection Fasciculata.
Mycopathologia et Mycologia Applicata 51: 1-18.
KURTZMAN, CP., SMILEY, M.T., ROBNEIT, C]. and WICKLOW, D.T. 1986. DNA relatedness among wild
and domesticated species in the Aspergillus flavus group. Mycologia 78: 955-959.
ONIONS, A.H.S., BRIDGE, P.D. and PATERSON, R.R 1984. Problems and prospects for the taxonomy of
Penicillium. Microbiological Sciences 1: 185-189.
PATERSON, R.RM. 1986. Standardized one- and two-dimensional chromatographic methods for the
identification of secondary metabolites in Penicillium and other fungi. Journal of Chromatography 368: 249264.
PIIT, T.I. 1973. An appraisal of identification methods for Penicillium species: novel taxonomic criteria based
on temperature and water relations. Mycologia 65: 1135-1157.
- - 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
- - 1984. The value of physiolOgical characters in the taxonomy of Penicillium. In Toxigenic Fungi - Their
Toxins and Health Hazard, eds. H. Kurata and Y. Ueno, pp. 107-118. Amsterdam: Elsevier.
- - 1988. A Laboratory Guide to Common Penicillium Species. 2nd ed. North Ryde, N.S.W.: CSIRO
Division of Food Processing.
PIIT, J.I. and CRUICKSHANK, R.H. 1990. Speciation and synonymy in Penicillium subgenus Penicillium towards a definitive taxonomy. In Modern Concepts in Penicillium and Aspergillus Classification, eds.
RA. Samson and J.1. Pitt, pp. 103-119. New York and London: Plenum Press.
RAPER, K.B. 1957. Nomenclature in Aspergillus and Penicillium. Mycologia 49: 644-662.
RAPER, K.B. and FENNELL, D.1. 1965. The Genus Aspergillus. Baltimore, Maryland: Williams and Wilkins.
RAPER, K.B. and THOM, C 1949. A Manual of the Penicillia. Baltimore, Maryland: Williams and Wilkins.
SAMSON, RA. 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
and London: Plenum Press.
SAMSON, RA. and pm, T.I. 1985. Check list of common Penicillium species. In Advances in Penicillium and
Aspergillus Systematics, eds. RA. Samson and J.1. Pitt, pp. 461-463. New York and London: Plenum Press.
SAMSON, R.A., STOLK, A.C and HADLOK, R. 1976. Revision of the Subsection Fasciculata of Penicillium
and some allied species. Studies in Mycology, Baarn 11: 1-47.
SEIFERT, K.A. and SAMSON, RA. 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
London: Plenum Press.
S0DERSTR0M, B. and FRISVAD, J.C 1984. Separation of closely related asymmetric Penicillia by pyrolysis
gas chromatography and mycotoxin production. Mycologia 76: 408-419.
SOPP,O.J.-O. 1912. Monographie der Pilzgruppe Penicillium mit besonder erucksichtigung der in Norwegen
gefungdenen Arten. Skrifter udgivna af Videnskabsselskabet i Christiana 11: 1-208.
STOLK, A.C and SCOIT, D.B. 1967. Studies on the genus Eupenici/lium Ludwig 1. Taxonomy and
nomenclature of Penicillia in relation to their sclerotioid ascocarpic states. Persoonia 4: 391-405.
13
STOLK, Ae., SAMSON, R.A., FRSIVAD, J.e. and FlLTENBORG, O. 1990. The systematics of the
terverticillate Penicillia. In Modem Concepts in Penicillium and Aspergillus Classification, eds. R.A.
Samson and J.I. Pitt, pp. 121-137. New York and London: Plenum Press.
THOM, e. 1906. Fungi in cheese ripening: Camembert and Roquefort. Bulletin of the Bureau of Animal
Industry, U.S. Department of Agriculture 82: 1-39.
- - 1910. Cultural studies of species of Penicillium. Bulletin of the Bureau of Animal Industry, U.S. Department
of Agriculture 118: 1-109.
--1930. The Penicillia. Baltimore, Maryland: Williams and Wilkins.
--1954. The evolution of species concepts in Aspergillus and Penicillium. Annals of the New York Academy of
Sciences 60: 24-34.
THOM, e. and CHURCH, M.B. 1926. The Aspergilli. Baltimore, Maryland: Williams and Wilkins.
THOM, e. and RAPER, K.B. 1945. A Manual of the Aspergilli. Baltimore, Maryland: Williams and Wilkins.
--1946. Aspergillus or what? Science, N. Y. 103: 735.
VOSS, E.G. et al., eds. 1983. International Code of Botanical Nomenclature adopted by the Thirteenth
International Botanical Congress, Sydney, August, 1981. Utrecht: Bohn, Scheltema and Holkema.
WESTLING, R. 1911. Uber die griinen Spezies der Gattung Penicillium. Arkiv fUr Botanik 11: 1-156.
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:
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
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
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
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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
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
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
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
mycotoxins and other secondary metabolites. Applied and Environmental Microbiology 46: 1301-1310.
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
London: Plenum Press.
- - 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
and London: Plenum Press.
- - 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
London: Plenum Press.
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
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.
28
Frisvad
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
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
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
29
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.
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
32
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.
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.
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
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
mycotoxins and other secondary metabolites. Applied and Environmental Microbiology 46: 1301-1310.
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.!.
Pitt, pp. 373-384. New York and London: Plenum Press.
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.
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
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.
MATERIAL AND METHODS
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.
41
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%
~m.
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.
45
46
P. Staugaard et al.
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
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
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
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
Relative frequency
(%)
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
288/3800
10/3800
6/3800
198/3800
7/3800
5/3800
66/3800
90/3800
73/3800
8.88
12.11
9.82
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
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.
Variant 2 (MUCL 30224)
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.
Variant 3 (MUCL 30225)
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.
Variant 5 (MUCL 30227)
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.
Variant 6 (MUCL 30228)
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.
Variant 7 (MUCL 30229)
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.
Variant 8 (MUCL 30230)
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.
Variant 9 (MUCL 30231)
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
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
59
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
00 0
60
i)
of P. expansum
Variants of
61
Penicillium expansum
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
and some allied species. Studies in Mycology, Baarn 11: 1-47.
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.
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
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
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; 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.
69
P. viridicatum: Meat pie (2); cheese (4); raw peanuts; maize (2); banana; suet.
Other Penicillia
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.
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.
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
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).
Problems and prospects for improving the stability of names in Aspergillus and Penicillium
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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BENNETT, J.W. 1985. Taxonomy of fungi and biology of the Aspergilli. In Biology of Industrial
Microorganisms eds. A.L. Demain and N.A. Solomon, pp. 359-406. Melno Park, Calif.:
Benjamin/Cummings Publishing.
BRIDGE, P.O., HAWKSWORTH, 0.1., KOZAKIEWICZ, Z., ONIONS, A.H.S., PATERSON, R.R.M.,
SACKIN, M.I. and SNEATH, P.H.A. 1990. A reappraisal of the terverticillate Penicillia using biochemical,
physiological and morphological features. In Modern Concepts in Penicillium and Aspergillus
classification, eds. R.A. Samson and J.I. Pitt, pp. 139-147. New York and London: Plenum Press.
BRUMMITT, R.K., HAWKSWORTH, D.L. and McNEILL, J. 1986. Proposals for a method of defining
effective publication by means of approved publications. Taxon 35: 823-826.
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83
IDept of Biotechnology
Technical University of Denmark
2800 Lynglly, Denmark
2CAB International Mycological Institute
Kew, Surrey, TW9 3AF, United Kingdom
3CSIRO
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
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.
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
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.
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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Seances de l'Acadbnie des Sciences 184: 136-137.
WINTER, G. 1884. Rabenhorst Kryptogamen Flora. Die Pilze Deutschlands, Oesterreichs und der Schweiz
1(2): 62.
91
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.
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.
Pitt, pp. 75-82. New York and London: Plenum Press.
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
York and London: Plenum Press.
SAMSON, R. A., STOLK, A. C. and HADLOK, R. 1976. Revision of the subsection Fasciculata of Penicillium
and some allied species. Studies in Mycology, Baarn 11: 1-47.
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
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
HENNEBERT: Is the rank of the name protected? For example, could the varietal names in
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.
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
These sentiments were endorsed by the workshop, but no formal wording was proposed.
4
TAXONOMIC SCHEMES OF PENICILLIUM
103
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
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
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
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.
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.
CONCEPT.
106
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.
107
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).
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.
109
DIAGNOSIS.
110
CONCEPT.
OfHER SYNONYMS. P.
SYNONYMS.
111
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.
112
DiAGNOSIS.
113
114
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.
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.
DESCRIPTIONS. Pitt (1979); Pitt (1988).
CONCEPT.
116
2.
3.
4.
5.
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.
8.
9.
Soluble pigment and/or reverse yellow; sometimes growth at 370 ........................................ P. chrysogenum
Soluble pigment and reverse not yellow; no growth at 370.............................................................................. 10
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
agricultural commodities. Applied Microbiology 20: 761-764.
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.
CIEGLER, A., LEE, L.S. and DUNN, J.J. 1981. Production of naphthoquinone mycotoxins and taxonomy of
Penicillium viridicatum. Applied and Environmental Microbiology 42: 446-449.
CRUICKSHANK, R.H. and PITT, J.I. 1987a. The zymogram technique: isoenzyme patterns as an aid in
Penicillium classification. Microbiological Sciences 4: 14-17.
--1987b. Identification of species in Penicillium subgenus Penicillium by enzyme electrophoresis. Mycologia
79: 614-620.
DOMSCH, K.H., GAMS, W. and ANDERSON, T.-H. 1980. Compendium of Soil Fungi. London: Academic
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EL-BANNA, A.A., LEISTNER, L. and PITT, J.!. 1987. Production of mycotoxins by Penicillium species.
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FILTENBORG, O. and FRISVAD, J.c. 1980. A simple screening method for toxigenic moulds in pure culture.
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FRlSVAD, J.c. 1983. A selective and indicative medium for groups of Penicillium viridicatum producing
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FRISV AD, J.e. 1985. Profiles of primary and secondary metabolites of value in classification of Penicillium
viridicatum and related species. In Advances in Penicillium and Aspergillus Systematics, eds. RA. Samson
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FRISVAD, J.e. and FILTENBORG, 0.1983. Classification of terverticillate Penicillia based on profiles of
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FRISV AD, J.e. and VUIF, B.T. 1986. Comparison of direct and dilution plating for detecting Penicillium
viridicatum in barley containing ochratoxin. In Methods for the Mycological Examination of Food, eds.
AD. King, J.I. Pitt, L.R Beuchat and J.E.L. Corry, pp. 45-47. New York: 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 spectabi/is).
Canadian Journal of Botany 65: 765-773.
ONIONS, A.H.S., BRIDGE, P.D. and PATERSON, RR 1984. Problems and prospects for the taxonomy of
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PATERSON, R.RM. and HAWKSWORTH, D.L. 1985. Detection of secondary metabolites in dried cultures
of Penicillium preserved in herbaria. Transactions of the British Mycological Society 85: 95-100.
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- - 1987. Penicillium viridicatum, Penicillium verrucosum, and production of ochratoxin A. Applied and
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--1988. A Laboratory Guide to Common Penicillium Species. North Ryde, N.S.W.: CSIRO Division of
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PITT, J.I. and HAWKSWORTH, D.L. 1985. The naming of chemical variants in Penicillium and Aspergillus. In
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Plenum Press.
PITT, J.I. and HOCKING, AD. 1985. Fungi and Food Spoilage. Sydney: Academic Press.
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natural occurrence, toxicity and control. I. Toxigenic Fungi, J. E. Smith, ed. Boca Raton, Florida: CRC
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PITT, J.I., CRUICKSHANK, R.H. and LEISTNER, L. 1986. Penicillium commune, P. camemberti, the origin of
white cheese moulds, and the production of cyclopiazonic acid. Food Microbiology 3: 363-371.
POLONELLI, L., MORACE, G., ROSA, R., CASTAGNOLA, M. and FRISVAD, J.e. 1987. Antigenic
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RAPER, K.B. and THOM, e. 1949. A Manual of the Penicillia. Baltimore, Maryland. Williams and Wilkins.
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Aspergillus Systematics, eds. RA. Samson and J. I. Pitt, pp. 461-463. New York: Plenum Press.
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and allied species. Studies in Mycology, Baam ll: 1-47.
SAMSON, RA, HADLOK, R and STOLK, A.e. 1977a. A taxonomic study of the Penicillium chrysogenum
series. Antonie van Leeuwenhoek 43: 169-175.
SAMSON, R.A., 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, RA. 1985. The genus Coremium and the synnernatous Penicillia. In Advances
in Penicillium and Aspergillus Systematics, eds. RA. Samson and J. I. Pitt, pp. 143-154. New York: Plenum
Press.
SODERSTROM, B. and FRISVAD, J.e. 1984. Separation of closely related asymmetric Penicillia by pyrolysis
gas chromatography and mycotoxin production. Mycologia 76: 408-419.
STOLK, A.e. 1969. Four new species of Penicillium. Antonie van Leeuwenhoek 35: 261-274.
THOM, C. 1910. Cultural studies on species of Penicillium. Bulletin of the Bureau of Animal Industries of the
United States Department of Agriculture ll8: 1-109.
--1930. The Penicillia. Baltimore, Maryland: Williams and Wilkins.
VINCENT, M.A. and PITT, J.I. 1989. Penicillium allii, a new species from Egyptian garlic. Mycologia 81:
300-303
119
WILLIAMS, A.P. and PITI, J.I. 1985. A revised key to Penicillium subgenus Penicillium. In Advances in
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Press.
change?
PITI: Occasionally a band disappears, but in general terms the isoenzyme patterns remain
unchanged. The concentrations of the enzymes may decrease.
121
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
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
123
Table 2. Accepted species of the terverticillate Penicillia and the production of mycotoxins.
P. atramentosum Thorn
P. aurantiogriseum Dierckx var. aurantiogriseum
P. brevicompactum Dierckx
P. camemberti Thorn
P. chrysogenum Thorn
P. clavigerum Dernelius
P. commune Thorn
P. crustosum Thorn
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
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
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)
125
The following list outlines the accepted taxa with a short discussion of their placement:
Series LANOSA Stolk et Samson
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.
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
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.
127
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.
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 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
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
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
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
130
P.lanosovirideThom 1930
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
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. 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
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
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
133
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.
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
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
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
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.
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SEIFERT, K.A and SAMSON, R.A 1985. The genus Coremium and the synnematous Penicillia. In Advances
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Londen: Plenum Press.
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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
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
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
0.'
0.'
0.'
P. expansum
P. aurantiogriseum
P. soli tum
P. 8ucantiogriseum
P. chrysogcnwn
P. cchinulatum
p.
ali vinoviridc
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.'
141
142
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).
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
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
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.
REFERENCES
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.
Transactions of the British Mycological Society 88: 569-571.
145
--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
and London: Plenum Press.
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.
Morphological and biochemical variation in single isolates of Penicillium. Transactions of the British
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.
Investigation of variation in phenotype and DNA content between single-conidium isolates of single
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
enzyme electrophoresis. Mycologia 79: 614-620.
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
Penicillium. Microbiological Sciences 1: 185-189.
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
series. Antonie van Leeuwenhoek 43: 169-175.
SMITH, G. 1949. The effect of adding trace elements to Czapek Dox medium. Transactions of the British
Mycological Society 32: 280-283.
SNEATH, P.H.A. and SOKAL, R.R. 1973. Numerical Taxonomy. San Francisco: W.H. Freeman and Sons.
146
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.
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
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.
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
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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.
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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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.
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.
daleae (lMI 89338) and with IFJM 3004. P. daleae is very close to P. janczewskii. When mature
154
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156
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.
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?
159
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.
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).
RESULTS AND DISCUSSION
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
161
Table 2. Accepted species, synonyms and isolates examined in Penicillium subgen. Furcatum.
1.
CBS 241.56(T), CBS 243.56, IMI 61836ii, IMI 161964, IMI 103148, CBS 240.65
2.
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.
NRRL 184l(T), NRRL 1843, NRRL 783, NRRL3463, IMI 92267, CBS 232.38, IMI 92229ii, CCM F391, 1M! 276863, NRRL 2148
7.
NRRL 802m, CBS 320.59, CBS 330.79, NRRL A-23347, CBS 254.37
9.
162
Table 2 (cont.)
11. P. dierckxii Biourge 1923
?P. cinerascens Biourge 1923 (a)
P. charlesii G. Smith 1933
IJFM 7852(T)
163
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
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.
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
P. atrovenetum
P. brasilianum
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
P. dierckxii
P. echinulonalgiovense
P. estinogenum
1. Estin (c)
2. Geodin, dihydrogeodin, erdin (c)
P. flavidostipitatum
P. griseopurpureum
P. herquei
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
P. mariaecrucis
P. matriti
P. megasprum
l.
2.
3.
4.
5.
6.
P. melinii
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
P. piscarium
1. Brefeldin A (a,b,c)
2. Janthitrem B (a,b,c)
P. pulvillorum
P. raciborski
P. raistricki
P. rollsii
P. rubefaciens
P. sclerotigenum
l. Griseofulvin (a,b)
2. Roquefortine C (a,b)
P. simplicissimum
= P. janthinel/um
P. sizovae
(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
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
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.
CRUICKSHANK, R.H. and PITT, J.1. 1987. Identification of species in Penicillium subgenus Penicillium by
enzyme electrophoresis. Mycologia 79: 614-620.
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
misidentified isolates. Archives of Environmental Contamination and Toxicology 18: 452-467.
FRISVAD, J.e. and FILTENBORG, 0.1989. Terverticillate Penicillia: chemotaxonomy and mycotoxin
production. Mycologia 81 (in press).
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.
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
anamorphs. Studies in Mycology, Baarn 23: 1-149.
- - 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.
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
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
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.
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
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. rubrisclerotium'"
P. d. funiculosum
"P. rubrisclerotium'"
"P. rubrisclerotium'"
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
"P. rubrisclerotium'"
175
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
var. rubrisclerotium"
P. purpurogenum
var. rubrisclerotium"
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
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).
RESULTS AND DISCUSSION
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
Species
Secondary metabolites
P. allahabadense
P. aurantiacum
P. diversum
Alternariol monomethylether"
Austinol* and isoaustin
Diversonols
Lichexanthone
P. funiculosum
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 (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.
177
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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.
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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00
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181
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.
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.
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
()
0
100 fJ.ffi
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
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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
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).
Representative isolates examined:
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.
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
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188
189
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.
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
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
ACKNOWLEDGEMENTS
The authors thank the NATO Scientific Affairs Division (Brussel, Belgium) for the research
grant (0216/86) for international collaboration.
REFERENCES
FIlTENBORG, 0., FRISVAD, J.e. and THRANE, U. 1983. Simple screening method for molds producing
intracellular mycotoxins in pure culture. Applied and Environmental Microbiology 45: 581-585.
FRISVAD, J.e. and THRANE, U. 1987. Standardized high-performance liquid chromatography of 182
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
et al.
193
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.
MATERIAL AND METHODS
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
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.
195
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
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
197
P. janthinellum
P. cremeogriseum
"P. trimorphum"
(BBA6S203)
P. ochrochloron
(Biourge, 1923)
P. ochrochloron
(Pitt, 1979)
P. ochrochloron
O?
P. ochrochloron
(BBA64184)
P. janthinellum
(Biourge, 1923)
P. janthinel/um
(Pitt, 1979)
P. cremeogriseum
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
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.
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
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
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).
RESULTS AND DISCUSSION
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
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.
204
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.
205
Fumigati.
Species
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
Brief descriptions follow of the morphology and secondary metabolites of the species
studied.
206
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
grant (0216/86) for international collaboration.
207
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YAMAMOTO, Y., NlTTAI, K., OOHATA, Y. and FURUKAWA, T. 1972. Studies of the metabolic products of
Aspergillus fumigatus DH 413. V. A new metabolite produced by ethionine inhibition. Chemical and
Pharmaceutical Bulletin 20: 931-935.
YAMAZAKI, M., SUZUKI, S. and MIYAKI, K. 1971. Tremorgenic mycotoxins from Aspergillus fumigatus.
Chemical and Phamaceutical Bulletin 19: 1739-1740.
YAMAZAKI, M., FUJIMOTO, H. and OKUYAMA, E. 1976. Structure determination of six tryptoquivaline
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Chemical and Pharmaceutical Bulletin 25: 2554-2560.
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209
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
210
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. glabra
N. fischeri var. spinosa
N. fischeri var. spinosa
N. quadricincta
N. quadricincta
N. spathulata
N. spathulata
N. slramenia
212
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
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. fumigatus
A. fumiga tus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumiga tus
A. fumigatus var. acolumnaris
A. fumigatus var. ellipticus
A. fumigatus var. sclerotiorum
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. fischeri var. glabra
N. fischeri var. glabra
N. fischeri var. spinosa
N. fischeri var. spinosa
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
10
15
20
25
3D
40
35
:10
',...."
"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:
'"S!.
er
0<:
"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.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
O+-~~-+~~~+-~~-+~~~+-~~-+~~~+-~~-+-r~~+-~~-+-r~
0.5
1.5
2.5
;'.5
;,
m
)(
-oJ
D>
Cl.
(I)
1(iI
.Cl.
:::>
D>
D>
~.
2'
cO
Q.
l>o
g.
:::>.
:::T
D>
"8
er
&
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.
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>
c:
:b
(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.
220
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
Aspergillus brevipes
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. fischeri var. glabra
N. fischeri var. glabra
N. fischeri var. spinosa
N. fischeri var. spinosa
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
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
REFERENCES
ASHWELL, G. 1966. New colorimetric methods of sugar analysis. Methods in Enzymology 8: 85-95.
AZUMA, I., KIMURA, H., HIRAO, F., TSUBURA, E., YAMAMURA, Y. and MISAKI, A. 1971. Biochemical
and immunological studies on Aspergillus III.Chemical and immunological properties of glycopeptide
obtained from Aspergillus fumigatus. Japanese Journal of Microbiology 15: 237-246.
BARDALAYE, P.C and NORDIN J.H. 1977. Chemical structure of the galactomannan from the cell wall of
Aspergillus niger. Journal of Biological Chemistry 252: 2584-2591.
BARRETO-BERGTER, E.M., and TRAVASSOS, L.R. 1980. Chemical structure of the galacto-D-mannan
component from hyphae of Aspergillus spp. Carbohydrate Research 86: 273-285.
BARRETO-BERGTER, E.M., GORIN, P.A.J. and TRAVASSOS, L.R. 1981. Cell constituents of mycelia and
conidia of Aspergzllus fumigatus.Carbohydrate Research 95: 205-218.
BAZIN, H. 1987. Rat hybridoma formation and rat monoclonal methods. In Methods of Hybridoma
Formation, eds. A.H. Bartal and Y. Hirsault, pp. 337-348. Oifton: Humana Press,
BENNETT, J.E., BHATTACHARJEE, A.K. and GLAUDEMANS, c.P.J. 1985. Galactofuranosyl groups are
immunodominant in Aspergillus fumigatus galactomannan. Molecular Immunology 22: 251-254.
BRADFORD, M.M. 1976. Rapid sensitive method for the quantitation of protein utilizing the principles of
protein dye-binding. Analytical Biochemistry 72: 248-254.
DUBOIS, M., GILLES, K.A., HAMILTON, J.K., REBERS, P.A. and SMITH, F. 1956. Colorimetric method for
determination of sugars and related substances. Analytical Chememistry 28: 350-356.
GANDER J.E., JENTOFT N.H., DREWS L.R. and RICK D.P. 1974. The 5-O-a-D-galactofuranosyl-containing
exocellular glycopeptide of Penicillium charlesii. Journal of Biological Chemistry 249: 2063-2072.
GORIN, P.A.J. AND SPENSER. J.F.T. 1968. Structural chemistry of fungal polysaccharides. Advances in
Carbohydrate Chemistry 23: 367-414.
HEARN, V. 1988. Serodiagnosis of Aspergillosis. In Aspergillus and Aspergillosis, eds. H. Vanden Bossche,
D.W.R. Mackenzie and G. Cauwenbergh, pp. 43-71. New York and London: Plenum Press.
HEARN, V., MOUTAOUAKIL, M. and LATGE, J.P. 1990. Analysis of components of Aspergillus and
Neosartorya mycelial preparations by gelelectophoresis and Western blotting techniques. In Modem
Concepts In Penicillium and Aspergillus classification, eds. R.A.Samson and J.I. Pitt, pp. 235-245, New
York and London: Plenum Press.
HUPPERT, M., 1983. Antigens for measuring immunological reactivity, In Fungi pathogenic for human and
animal. Part B. Pathogenicity and detection", ed. D. Howard, p. 219-301. New York & Basel: Marcel
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JOHNSTON, I.R. 1965. The composition of the cell wall of Aspergillus niger. Biochemical Journal 96: 651-658.
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NOTERMANS, S. and HEUVELMAN, c.J. 1985. Immunological detection of moulds in food by using the
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NOTERMANS, S. and SOENTORO, P.S.S. 1986. Immunological relationship of extracellular polysaccharide
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Penicillium and Aspergillus species. Molecular Immunology 25: 975-979.
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SUZUKI, S. and TAKEDA, N. 1975. Serologic cross-reactivy of the D-galacto-D-mannans isolated from
several pathogenic fungi against anti-Hormodendrum pedrosoi serum. Carbohydrate Research 40: 193-197.
223
WEBSTER, S.F. and McGINLEY, KJ. 1980. Serobiological analysis of an extractable carbohydrate antigen of
Pityrosporum ovale. Microbios 28: 41-45.
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225
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.
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. fumigatus var. sclerotiorum
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. fischeri var. fischeri
N. fischeri var. glabra
N. fischeri var. glabra
N. fischeri var. spinosa
N. fischeri var. spinosa
N. quadricincta
N. spathulatR
N. spathulatR
Collection
number
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
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
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 of Aspergillus fumigatus and related taxa by the yeast killer system
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).
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
ft.spergillus isolates
9 110
Aspergillus isolates
study nullber (see table I)
nlnlnlHI~IHlnlulBlm
Aspergillus Isolates
study nullber (see table I)
3tJ!2
I 33 I 34 I 35 I 36 137 138
Aspergillus isolates
study "mber (see table I)
'"o
:-
Q)
::l
(5
Biotyping of Aspergillus fumigatus and related taxa by the yeast killer system
231
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.
Table 6. Grouping of killer biotypes among teleomorph and anamorph of Aspergillus sect.
Fumigati isolates.
16
1
1
1
1
1
1
1
2
4
2
2
2
1
2
38
1
1
2
1
1
1
1
1
2
4
Biotyping of Aspergillus fumigatus and related taxa by the yeast killer system
233
REFERENCES
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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
Microbiology 24: 866-869.
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.
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York and London: Plenum Press.
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
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WEEMS, J.J. jr. ANDERMONT, A., DAVIS, B.J. TANCREDE, C.H., GUIGET, M., PADHYE, A.A.,
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WELLENS, F., POTULIEGE, c., DEUVART, F.E. and PRIMO, G. 1982. Aspergillus osteochondritis after
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235
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
Species examined.
The origin of the Aspergillus and Neosartorya species studied are shown in Table 1.
V.M. Hearn
236
et at.
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
A. fumigatus var. acolumnaris
N. fischeri var. spinosa
N. fischeri var. spinosa
N. fischeri var. glabra
N. quadricincta
A. duricaulis
N. aurata
A. fumigatus
N. stramenia
N. aureola
A. unilateralis
A. fumigatus
A. fumigatus
A. fumigatus var. sclerotiorum
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
samples studied
a+b
a
b
+
+
+
+
+
+
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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.
238
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
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- _
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. 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
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
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
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
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
247
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
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
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
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
TAXONOMY
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.
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.
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.
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
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
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
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.
PITT, J.I. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
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
and London: Plenum Press.
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
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
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
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.
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
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
10 11 12 13 14
15 16 17
18
19 20 21 22 23
9 10 11 12 13 14 15 16 17 18
19 20 21
263
22 23
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
Penicillium classification. Microbiological Sciences 4: 14-17.
--1987b. Identification of species in Penicillium subgenus Penicillium by enzyme electrophoresis. Mycologia
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.
New York and London: Plenum Press.
- - 1988. Differentiation of Aspergillus flavus from A. parasiticus and other closely related species.
Transactions of the British Mycological Society 91: 99-108.
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.
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.
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
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
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.
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
272
M.A. Klich
Co~uter
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
et ai., 1976).
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
276
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.
REFERENCES
ADEY, M. E., ALLKIN, R., BISBY, F. A, WHITE, R. J. and MACFARLANE, T. D. 1984. The Vicieae database:
an experimental taxonomic monograph. In Databases in Systematics, eds. R. AIlkin and F. A. Bisby.
Orlando, Florida: Academic Press, pp. 175-188.
AL-MUSALLAM, A. 1980. Revision of the black Aspergillus species. Ph.D Thesis. Utrecht, Netherlands:
University of Utrecht.
ALLKIN, R. and BISBY, F. A., eds. 1984. Databases in Systematics. Orlando, Florida: Academic Press.
ANDERSON, T. H., BODENSTEIN, J. and OOMSCH, K. H. 1976. A partially computerized documentation
system for microscopic soil fungi. Canadian Journal of Botany 54: 1709-1713.
BARNETT, J. A and PANKHURST, R. J. 1974. A New Key to the Yeasts. New York: Elsevier Press.
BRIOCE, P. D. 1990. Identification of terverticillate Penicillia from a matrix of percent positive test results. In
Modem Concepts in Penicillium and Aspergz1lus Classification, eds. R. A Samson and J. I. Pitt, pp. 283-287.
New York and London: Plenum Press.
BRIDGE, P. D., HAWKSWORTH, D. L., 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. I. Pitt, pp. 281-309. New York and London: Plenum
Press.
BRYANT, T. N. 1983. A microcomputer-based information storage and retrieval system for the maintenance
of records for a culture collection. Journal of Applied Bacteriology 54: 101-107.
CHRISTENSEN, M. 1981. A synoptic key and evaluation of species in the Aspergillus flavus group. Mycologia
73: 1056-1084.
CROVELLO, T. J., HAUSER, L. A and KELLER, C. A 1984. BRASS BAND (The Brassicaceae databank at
Notre Dame): an example of database concepts in systematics. In Databases in Systematics, eds. R. AlIkin
and F. A Bisby, pp. 219-233. Orlando, Florida: Academic Press.
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.
--1976b. Strain variation in Aspergillus niger. Microbios Letters 3: 183-185.
DOMSCH, K. H., GAMS, W. and ANDERSON, T. H. 1980. Compendium of Soil Fungi. London: Academic
Press.
FRANK, H. M. and ZWANZIGER, H. 1988. Macrochemical color reactions of macromycetes. VI. Cluster
analysis of color data. Mycotaxon 33: 91-96.
277
GAMS, W., HENNEBERT, G. 1., STALPERS, J. A., JANSSENS, D., SCHIPPER, M. M. A., SMITH, J.,
YARROW, D. and HAWKSWORTH, D. 1. 1988. Structuring strain data for storage and retrieval of
information on fungi and yeasts in MINE, the Microbial Information Network Europe. Journal of General
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GORE, M. and STUBBE, J. 1983. Elements of Systems Analysis. 3rd ed. Dubuque, Iowa: Wm. C. Brown.
GOWER, J. C. 1975. Relating classification to identification. In Biological Identification with Computers, ed.
R. J. Pankhurst, pp. 251-263. New York: Academic Press.
HALL, A. V. 1970. A computer-based system for forming identification keys. Taxon 19: 12-18.
HEYWOOD, V. H. 1984. Electronic data processing in taxonomy and systematics. In Databases in
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HOGEWEG, P. 1976. Iterative character weighing in numerical taxonomy. Computers in Biology and Medicine
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IBRAHIM, F. M. and THRELFALL, R. J. 1966. The application of numerical taxonomy to some
graminicolous species of Helminthosporium. Proceedings of the Royal Society of London, Series B, 165: 362-388.
JOHNSTON, B. C. 1980. Computer programs for constructing polyclave keys from data matrices. Taxon 29:
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KENDRICK, W. B. 1965. Complexity and dependence in computer taxonomy. Taxon 14: 141-154.
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KENDRICK, B. W. and WERESUB, 1. K. 1966. Attempting neo-Adansonian computer taxonomy at the
ordinal level in the Basidiomycetes. Systematic Zoology 15: 307-329.
KLICH M. A. and pm, J. I. 1985. The theory and practise 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.
- - 1988. A computer-assisted synoptic key to common Penicillium species and their teleomorphs. New
Orleans, Louisiana: privately published.
LEE, J. D., STRETTON, R. J. and DART, R. K. 1976. Classification of Aspergillus and Penicillium species.
Microbios Letters 2: 163-167.
LEE, J. D., DART, R. K. and STRETTON, R. J. 1977. Computerized classification of Aspergillus. Transactions of
the British Mycological Society 69: 137-141.
MARGOT, P., FARQUHAR, G. and WATLING, R. 1984. Identification of toxic mushrooms and toadstools
(Agarics) - an on-line identification program. In Databases in Systematics, eds. R. Allkin and F. A. Bisby,
pp. 249-261. Orlando, Florida: Academic Press.
McNEILL, J. 1984. Taximetrics To-day. In Current Concepts in Plant Taxonomy, eds. V. H. Heywood and D.
M. Moore, pp. 281-299. Systematics Special Vol. 25. Orlando, Florida: Academic Press.
MURAKAMI, H. 1971. Classification of the koji mold. Journal of General and Applied Microbiology 17: 281-309.
PAYNE, R. W. and PREECE, D. A. 1980. Identification keys and diagnostic tables: a review. Journal of the
Royal Statistical Society, Ser. A, 143: 253-292.
PANKHURST, R. J., Ed. 1975. Biological Identification with Computers. Systematics Association Special Vol.
7. London: Academic Press.
pm, J. I. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
--1988. A Laboratory Guide to Common Penicillium Species. North Ryde, N.S.W.: CSIRO Division of
Food Research.
- - 1990. PENNAME, a new computer key to common Penicillium species. In Modem Concepts in
Penicillium and Aspergillus Oassification, eds. R. A. Samson and J. I. Pitt, pp. 279-281. New York and
London: Plenum Press.
RAPER, K. B. and THOM, C. 1949. A Manual of the Penicillia. Baltimore, Maryland: Williams and Wilkins.
RHOADES, F. 1988. PC-TAXON: a Taxonomic Database. Wentworth, New Hampshire: COMPress.
SAMSON, R. A., STOLl<, A. C. and HADLOK, R. 1976. Revision of the subsection Fasciculata of Penicillium
and some allied species. Studies in Mycology, Baarn 11: 1-47.
SNEATH, P. H. A. and SOKAL, R. R. 1973. Numerical Taxonomy: the Principles and Practice of Numerical
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SOKAL, R. R. and SNEATH, P. H. A. 1963. Principles of Numerical Taxonomy. San Francisco: Freeman and
Sons.
278
M.A. Klich
STRETI'ON, R. J., LEE, J. D. and DART, R. K. 1976. Relationship of Aspergillus species based on their long
chain fatty acids. Microbios Letters 2: 89-93.
THIERS, B. M. 1988. Herbarium Manager User's Manual "e" Version 1.0. Privately published.
WETMORE, C. M. 1979. Herbarium computerization at the University of Minnesota. Systematic Botany 4:
339-350.
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. 289-294. New
York and London: Plenum Press.
WOLFF, B. R., GUCK, B. R. and PASTERNAK, J. J. 1986. A microcomputer program for establishing and
maintaining a database for laboratory culture collections. Letters in Applied Microbiology 3: 4548.
279
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.
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.
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.
PITT, J.I. 1979. The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.
- - 1988. A Laboratory Guide to Common Penicillium Species. 2nd ed. North Ryde, N.S.W.: CSIRO
Division of Food Processing.
283
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
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
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
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
290
A.P. WiIUams
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
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
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.
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
Penicillium
Aspergillus
Penicillium
Aspergillus
293
Current name
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)
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
Neosartorya fischeri
Eurotillm amstelodami
Eurotium chevalieri
Eurotium herbariorum
Eurotium repens
Eurotium rubrum
Emericella nidulans
Aspergillus candidus
Aspergillus clavatus
Aspergt7lus flavus
Aspergillus fumigatus
(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)
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
Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 311-325. New York and London: Plenum Press.
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
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. 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.
295
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
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.
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
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
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
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
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
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
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
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.
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics
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.
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).
314
.'
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
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.
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics
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
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,
316
A.nidulans
oxiA
XhoI
cobA
oxiB
URF1 URF4
~.-A.unguis
oxiA
XhoI
-=~~----.--'
~--~~----------~!----------~
~---
A.heterothallicus
XhoI URF's 1.4
cobA
oxiA
DxiB
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
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics
Lanes
3
317
isolates
37 26
l1kb
6kb
..................
4kb
2.2kb
1.75kb
1.7kb
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.
318
--
---- -
10
lOkb
5kb
3kb
1.75kb
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
RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus systematics
319
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321
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.
322
Table 1. Species concepts of the black Aspergilli according to AI-Musallam (1980) and Raper
and Thom (1965)
Al-Musllllam (1980)
A CQrbonarius
A carbonarius
A heteromorphus
A heteromorphus
A ellipticus
A helicothrix
A ellipticus
A japonicus
A aculfilfus
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
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
323
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
324
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,
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
326
10
11
11
11
1.
..
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
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
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.
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
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.
REFERENCES
AL-MUSALLAM, A 1980. Revision of the black Aspergillus species. Thesis, State University Utrecht.
DE GRAAFF, L., VAN DEN BROEK, H. and VISSER, J. 1988. Isolation and expression of the Aspergillus
nidu/ans pyruvate kinase gene. Current Genetics 13,315-321.
GAMS, W., CHRISTENSEN, M., ONIONS, AH.S., PIIT, J.1. and SAMSON, R.A. 1985. Infrageneric taxa of
Aspergillus. In Advances in Penicillium and Aspergillus systematics. eds., R.A.Samson and J.1. Pitt,
pp. 55-62. New York and London: Plenum Press.
GODDARD, J.M., CAPUT, D., WILLIAMS, S.R. and MARTIN, D.W. 1983. Cloning of human purinenucleoside phosphorylase cDNA sequences by complementation in E.coli. Proceedings of the National
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LAEMMLI U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 227: 680-685.
LOGRlECO, A., PETERSON, S.w. and WICKLOW, D.T. 1990. Ribosomal RNA comparisons among taxa of
the terverticillate Penicillia. In. Modem Concepts in Penicillium and Aspergillus Systematics, eds. R.A.
Samson and J.1. Pitt. pp. 343-354. New York and London; Plenum Press.
MANIATIS, T., FRITSCH, E.F. and SAMBROOK, J. 1982. Molecular cloning, a laboratory manual. New
York: Cold Spring Harbor Laboratory Press.
MOSSERAY, R. 1934 a. Sur la systematique des Aspergillus de la section 'niger' Thorn & Church. Annals de
Societe Science de Bruxelles Ser. 2, 54: 72-85
-1934 b. Les Aspergillus de la section niger Thorn et Church. Cellule 43: 203-285.
334
PONTECORVO, G., ROPER, J.A., HEMMONS, L.J., MACDONALD, K.O. and BUFfON, A.W.J. 1953. The
genetics of Aspergillus nidulans. Advances in Genetics 5: 141-238.
RAPER, K.B. and FENNELL, 0.1. 1965. The genus Aspergillus. Baltimore: Williams & Wilkins.
THOM, C. and RAPER, K.R. 1945. A manual of the Aspergilli. Baltimore: Williams & Wilkins.
VAN HOUOENHOVEN, F.E.A. 1975. Studies on pectin lyase. Thesis, Agricultural University Wageningen.
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
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
336
J. Dupont et al.
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
337
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
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
------T--- ----------
---------G
--A-
-------A-- ---------T
-------A-- ----------
-----T----
-O--GG-GCA
--A-A--
---------c
--A-
------T--- ----------
--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- ----------
--------A- ---------- ---------- ---------- ---------- ------T--- ---------- CT--C----- -----T---- --O-GA---O ---------G --A-
--------A- ---------- ---------- ---------- ---------- ------T--- --T------- CT--C----- -----T---- --O-----CG ---------G --1\-
--------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)
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:
!'-
'"~
341
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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Stilbothamnium togoense for Aspergillus f1avus group mycotoxins. Mycotaxon 34: 249-252.
WOESE, C.R, STACKBRANDT, E., MACKE, T.J. and FOX, C.E. 1985. A phylogenetic definition of the major
eubacterial taxa. Systematic Applied Microbiology 6: 143-151.
343
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
344
A. Logrieco et al.
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.
345
346
A. Logrieco et al.
(375-470)
P. pubQrulum
P.
P.
n~$tioulo.';um
P.
hil'sutum
P.
gJ:anulatmn
"lscnil
rc-'quefol'tl
2032
echinulatum
r.
P.
l151
--- . . . . . -- . "
.. N--NG--NNN
.N ..
'OS
. N
.AGe....
. . . . . . NN ..
... N ..
983
l'lt1Vl-<:.'ompac.'tullI
2011
P.
elavlg'H'um
1003
f.,nn,;illl-B6>
,;,lav.lforme
atramentc';!'um
3697
.NGC..
.N
-.u
963
l.,-,aI'lfo.ul1s
Q.L'atel'iwn
--NNN
"
N . --NNN
UNNUC
.N-NN.N . N N . N.--NNU
. --GGN
OA.N.U
--!'INN
1'..1'. .
195
971
cnlstao9um
hellCUI/J
--NNG
NNGC.
.. , .UU ..
aU.I:al1tl0g.t-.l-Seum
. . . ---N8
.N, ,
N NU
A.A..
A.A..
ale/dccla
~f.
,N.A,.
13058
P.
F.
P.
P.
E.
1'.
F,
N-G.C...
r. 111..r.z.dicatum
F.
S.
CG---~CUCG
P.
P . . . xpansum
f'.
845
877
2021
camembertl.i
2638
2106
.UNNN.--..
GGCAUU.GAU .AGA.1'.UGG.
.UCU.CUCC. UGT) . 0---1) 1'.G.GGA-AI)---00 -. A 1'..1'. UNGGUG1'..1'.. GO.1'.oo1'.G.G GUU.CUU N C.C.GU-GUA
c'el'eVlSOla"
1411-510)
p, pubel'ulwn
F. camembel'tii
!-'. re,::tlculo:::um
P. hll'::-l.Itl.lm
P. echlnulatum
glanl.llatum
P. ol.socnll
r. roquefol'tl.
:2032
1151
:2036
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9155
983
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P.
r.
P.
f'.
P.
F.
f' .
P.
f'.
F.
1'.
P.
P.
P.
.NN . . . . . N.
.N1'...
NGNG.
A..
GGO....
GGNN......
GGUN..
.N.
.. . . . . . N
.. .... U .. G ,-,
.-N ..
.-N ..
tl.
. . - -.!'tN.CG.
.N .. NG.
-----.C..
.NN.
.N.
.-A . . . . . N.
..N...
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. .. mL
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N ... UNN...
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20.31
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3332
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. .N..
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. .N,.
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N.NC.CU U ONU.
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. . . . N . . . . . . . U .. N ----A.c.CC
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(571-614)
I'. pl.Iberulum
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, ... N..
. . . N..
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s.
[/.
916
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2032
1151
hll':;:utufll
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gl"Bnl.llBtum
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roquafortl
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ital.l<:,ufII
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2036
13058
P. axpansum
P.
1'.
V1'6v.J.-canp<wtum
clavlgerum
2011
1003
P. vir!dicatum
P.
P.
P.
clav.lfonna
Btl'am .. ntoSIJIII
E.
P.
crustacaum
arenicolB
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P. halicum
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.. N
aUl'antiogr.lSelJfII
c'l'aterlum
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N ... N .
. N .N . . . . . . . U . . . . . --.
.. G.NN . . . N N . . . . . . NN. - . . . . . . --.
.. G N G -U --.
G . . . . . . . . C -C . . . . . U-.
.UG .. A .. UG G . . . U..
GCG.NN G CN N
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
P. ",chl-Ilulatum
P. grBnulatum
2021
2032
P.
s.
1./.
.... NN ..
13056
""C'qUSfOl-t1
849
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965
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explllllSUm
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<::lavl-gerum
v1T1(1icatum
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F.
E.
P.
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.A . N
.N .... N .. .
NN . . . . 0 .. .
.. N . N
ltalicum
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F.
P.
. N
11~1
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P.
AAAGGGA-AN CCGGUUUACU UUCCGGUNCC UAGAU-UGGA UUCUCCACGG CNACGUNACU GAACGCGGAG ACGOCGGCGG GNNUC(:OGGG AAGAGTJTJCOC
.. -.0
. . . . . . A .
.N .. N .
. . . . . . . 0 .
.tL ... N ...
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clavifcnne
atl:alllentosllm
976
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2011
. . . N .. N
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. . . . . . . 1'1 ..
.... N.
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"3
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. . . . NN.
"
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. . . . 0 ..
. . . . 0 ..
195
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3332
3392
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. .N
.. A.
.AGe.
--- --GC,.
cereVl-.'Sl-ae
craterium
.G.
.G.
(1681-1780)
F.
P.
puherulum
P.
resticulosum
F.
F.
P.
P.
P.
P.
eC'hlnulatum
granulatum
P.
P.
P.
UUU1JCUUCUU GACAGCCUAU C-ACCCUGAA AUCGGUOUGU CCGGAGCUAG GGUUCUA-UG GCUN---GCA G-ACGCACUU UUar:GG-NNIJ CCGrmG':G'.C
1151
l"c'quefoL-tlVGrrucC',>,um
849
965
.. N-.
ltal:lcum
983
. . . . . . N-.
e:-:pansum
b.ulv.l-compa ct urn
-NN.
. N--- ..
. -NN .
NN--r~AG.
.-NNN
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13058
c-l:;;C'nl-l-
-UN
' .. N
'"
NN.
2011
cla<Tl-gQrum
.-MM.
v.l.l"idicatum
P. renneillae
., .... -AA.
?6!
3697
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2031
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3392
2638
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.. N---.
877
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. . . . - . . . . . A.
A .
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pubgrullDll
P.
F.
P.
P.
re.S'tit;.'ulo"um
h1r:::utum
877
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2032
1151
r:.'amemb'Ht~l
ach~nulatum
granulatum
P. 01S01111
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P.
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13058
849
965
98'
976
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P.
P.
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bn~vi-compactum
claviger-um
2011
1003
v~ridiaatum
t'ennellii!l6
.NNNN"
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.NNNN
.NNN ....
3697
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3332. NN
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2638
2106
'f-12632
.NH' U.
... N.
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.u ..
. N ..
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
.n
P. IiIcbinulatulII
2032
1151
P.
2036
P.
P.
P.
P.
P.
P.
E.
P.
P.
roquat'orti
brtllvi-cClJlpactiJDI
clavigll'rtUII
v.lcidioatwn
t'ennelliae
clavit'onllo
atramento.$'um
aurant.iogriselJ1l
.."..3
......... -
3697
2031
CN -.
.VA . -.
.N ...... -.
-. NNNU -.
A AG
........ -
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AG . . . . . . . . . . . . N .. A . . . . . . . . . . . NN........
.. . . . . . . . ON . . . . . . . . . . . . N N . . . . . . . . . . . NN . . . . . . . . . . . . N A . . . . . . . . . . . NG........
.. . . . . . . . C
971
845
8"
96.
vercucoSUIJI
italicum
clavigerum
C18vit'onna
crU3'tacliIUIII
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... N. N ........ .
.. A . . . . . . . . . . . . . N .. N NN ....... .
. --.11.
. N. N .
N N. U
. NN. U ..
.N . . . . . . N. N . . . . . . . . .
.. .... A .. N
.U . . . . . . ..
...... N .. N .N ...... ..
97'
. N--.N .
--.A
.. --.A
. . . . . . . NN. N . . . . . . . . .
. --NA
. . . . . . . . G. '0' . . . . . . . . .
.. --.A
.. . . . . . . G. U oo
NNN N
. . . . . . A .. G ... CC ... ..
971
3332
. N--.N N
.0 . . . N . . . . . . . . . H .. G . . . . . . . . . .
3392
. . --.11. G C
. .. --.N . . -G . N .. CC
.... --.A . . . . . . . GG . . . . . . . . . C .. .
C.AG--UC.A 00 .
705
aurantiogrisetUII
N ..
N ...
~-.A
....... -
10033697
2031
fallnelliae
"
....... NN. N . . . . . . . . .
N. N . . . . . . . . .
....... NN. N
2011
.63
ViL-idicatWII
UN . . . . . . . . . . . . N U . . . . . . . . . . . -
..N . . . . . . . . . . . . NN .. N
..N . . . . . . . . . . . . . NNNN
..N . . . . . . . . . . . . . N .. N
..A . . . . . . . . . . . . . A .. G
..11. A G
..A . . . . . . . . . . . . . A .. G
983
ft;t;pansum
br9vl -COIIIpact um
i;;:arit'o.l1lle
heliaWII
S. a&.I'liVisia&
I). arat"rium
. . . . . . N ..
. . . . N .. U
U O.U
.. N .. O ..
.C -.
N CN . . . . . -.
. . . . . . . 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
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 . . . . . . -.
NO........
........ AN . . . . . . . . . . . . 0' A . . . . . . . . . . . NN........
.. . . . . . . . NN . . . . . . . . . . . . O N . . . . . . . . . . . -
1003
3332
arenicola
.. . . . . . 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.
P.
P.
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.
.'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
P.
P. viridicatulII
2021
2032
1151
... - .. N ...
. A-UA.O
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
24
:4
2031
79'
971
19
ZZ
3332
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677
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1151
:::036
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96'
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11
10
10
11
12
14
13
10
11
13
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963
10
11
:::031
10
10
795
11
11
'"
13
3392
19
:::63e
24
10
10
11
11
12
11
11
10
14
14
13
14
12
15
16
13
16
11
16
13
13
10
11
20
20
20
23
24
3332
19
,.
24
13
14
14
19
17
24
"'
25
26
25
31
117
118
116
25
"
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
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.
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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
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CRUICKSHANK, R. H. and PITI, J.I. 1987. Identification of species in Penicillium subgenus Penicillium by
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ELWOOD, H. J., OLSEN, G.L. and SOGIN. M.L. 1985. The small subunit ribosomal RNA gene sequences
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RAMIREZ, O. 1977. A taxonomic study of Penicillium series Expansa Thorn emend. Fassatiova. Acta
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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
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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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MALLOCH, D. 1985. The Trichocomaceae: relationships with other Ascomycetes. In Advances in Penicillium
and Aspergillus Systematics. eds. RA. Samson and J. l. Pitt. pp. 365-382. New York and London: Plenum
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MCCARROLL, R., OLSEN, G.J., STAHL, Y.D., WOESE, C.R and SOGIN, M.L. 1983. Nucleotide sequence of
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evolutionary implications. Biochemistry 22: 5858-5968.
MCNAUGHTON, S. J. 1981. Niche: Definition and generalizations. InThe Fungal Community: Its
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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
characterization of Penicillium camemberti and related common cheese contaminants. Applied and
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RUBSTOV, P. M., MUSAKHANOV, M. M., ZAKHARYEV, V.M., KRAYEV, A.s., SKRYABIN, K.G. and
BAYEV, A.A. 1980. The complete structure of yeast ribosomal RNA genes. I. The complete nucleotide
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SAMSON, R A. 1985. Taxonomic considerations of harmful and beneficial moulds in food. In Filamentous
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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
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
358
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
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).
MATERIAL AND METHODS
360
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
~1938
1741
1823, 1630
FRR 1061, 1147
~1095,
~ 833,
~1036,
~1048,
1399
1055
1563
~1658,3054
FRR2670
FRR 3583, 2015
FRR 895, 1079
361
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
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
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
0.08 "
0.57
0.04
2080 Tstr
0.04
0.000
0.200
0.400
0.600
0.800
1.000
364
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
365
366
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
Talaromyces/Penicillium
Penicillium
subgenus Biverticillium
I 1265 Tlia
J2417 Tlia
[J
0.56
0.12
1.00
I 1791
Tthr 0.04
I
0.000
0.200
0.400
0.600
O.BOO
1.000
368
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).
REFERENCES
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FoRSTER, H., K1NSCHERF, T. G., LEONG, S. A. and MAXWELL, D. P. 1988. Estimation of relatedness
between Phytaphthura species by analysis of mitochondrial DNA. Mycologia 80: 466-478.
FREE, S. J., RICE, P. W. and METZENBERG, R. L. 1979. Arrangement of the genes coding for ribosomal
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GYLLENSTEN, U. B. and ERLICH, H. A. 1988. Generation of single stranded DNA by the polymerase chain
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HOOPES, B. C. AND McCLURE, W. R. 1981. Studies on the selectivity of DNA precipitation by spermine.
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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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MALLOCH, D. 1981. The Plectomycete centrum. In "Ascomycete Systematics: The Luttrellian Concept". D.
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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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369
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
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REED, K. R. and MANN, D. A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic
Acids Research 13: 7207-7221.
RHOLF, F. J. 1988. NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 1.4. Setauket,
New York: Applied Biostatistics.
RIGBY, P. W. J., DIECKMANN, M., RHODES, C. and BERG, P. 1977. Labeling deoxyribonudec acid to high
specific activity in vitro by nick translation with DNA polymerase I. Journal of Molecular Biology 113: 237251.
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1985. Enzymatic amplification of Il-globin genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science, N. Y. 230: 1350-1354.
SAMSON, R. A. 1974. Paecilomyces and some allied Hyphomycetes. Studies in Mycology, Baarn 6: 1-119.
SOKAL, R. R. and SNEATH, P.H.A. 1%3. Principles of Numerical Taxonomy. Freeman; San Francisco.
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STOLK, A. C. and SAMSON, R. A. 1972. The Genus Talaromyces. Studies on Talaromyces and Related Genera
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SUBRAMANIAN, C. V. 1979. Phialidic Hyphomycetes and their teleomorphs - an analysis. In "The Whole
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370
TAYLOR:
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
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
(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.
MATERIAL AND METHODS
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.
375
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.
376
Penicillium.
Mycotoxin
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
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.
377
Isolate
CBS 477.75
CBS 473.75
FRR1403
NRRL 13627
IMI285526
IMI321500
IMI293196
IMI321501
ATCC64629
Griseofulvin
Roquefortine C
Meleagrin
Oxaline
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
378
Rltenborg
4121121
'iic
11.
3121121
2-
.
11.
0
>.
2121121
c ii
..
>.
...
L
")
P.
.
~
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.
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
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
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.
17a.
17b.
18a.
18b.
18c.
18d.
18e.
19.
20.
21.
22.
23a.
23b.
24a.
24b.
25.
(26.
(27.
(28.
381
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
383
REFERENCES
ABARCA, M.L., BRAGULAT, M.R., BRUGUERA M.T. and CABANES, F.J. 1988. Comparison of some
screening methods for aflatoxigenic moulds. Mycopathologia 104: 75-79.
BETINA, V. 1989. Mycotoxins. Amsterdam: Elsevier.
BLASER, P. and SCHMIDT-LORENZ, W. 1981. Aspergillus flavus contamination von Niissen, Mandeln, mais
mit bekannten Aflatoxin-gehalt. Lebensmittel Wissenschaft und Technologie 14: 252-259.
BRIDGE, P.O., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A.H.S. and PATERSON, R.R.M. 1986.
Morphological and biochemical variation in single isolates of Penicillium. Transactions of the British
BRIDGE, P.O., HUDSON, L., KOZAKIEWICZ, 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 Microbiolgy 133: 995-1004.
CIEGLER, A., FENNELL, 0.1., SANSING, G.A., DETROY, R.W. and BENNETT, G.A. 1973. Mycotoxinproducing strains of Penicillium viridicatum: classification into subgroups. Applied Microbiology 26: 271-278.
CRUICKSHANK, R.H. and PITT, J.1. 1987. Identification of species in Penicillium subgenus Penicillium by
enzyme electrophoresis. Mycologia 79: 614-620.
EL-BANNA, A.A. and LEIS1NER, L. 1987. Quantitative determination of verrucosidin produced by
FILTENBORG, O. and FRISVAD, J.e. 1980. A simple screening-method for toxigenic moulds in pure
cultures. Lebensmittel Wissenschaft und Technologie 13: 120-130.
FILTENBORG, 0., FRlSVAD, J.e. and SVENDSEN, J.A. 1983. Simple screening method for molds producing
intracellular mycotoxins in pure culture. Applied and Environmental Microbiology 45: 581-585.
FILTENBORG, 0., FRlSVAD, 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.
384
FRISVAD, J.C 1981. Physiological criteria and mycotoxin production as aids in identification of common
asymmetric Penicillia. Applied and Environmetal Microbiology 41: 568-579.
FRISVAD, J.C 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.C 1985. Classification of asymmetric Penicillia using expressions of differentiation. In Advances
in Penicillium and Aspergillus Systematic, eds. R.A. Samson and J.I. Pitt, pp. 327-333. New York and
London: Plenum Press.
FRISVAD, J.C 1988. Fungal species and their specific production of mycotoxins. In Introduction to Foodborne Fungi, eds. R.A. Samson and E.5. van Reenen-Hoekstra, pp. 239-249. Baarn, Netherlands:
Centraalbureau voor Schimrne1cultures.
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
mycotoxins and other secondary metabolites. Applied and Environmental Microbiology 46: 1301-1310.
FRISVAD, J.C and FILTENBORG, O. 1989. Chemotaxonomy of and mycotoxin production by the
terverticillate Penicillia. Mycologia 81 (in press).
FRISV AD, J.C and THRANE, U. 1987. Standardized high-performance liquid chromatography of 182
mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VlS spectra
(diode array detection). Journal of Chromatography 404: 195-214.
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
chromatography. Archives of Environmental Contamination and Toxicology 18: 331-335.
FRISVAD, J.c., FILTENBORG, 0, and WICKLOW, D.T. 1987. Terverticillate Penicillia isolated from
underground seed caches and cheek pouches of the banner-tailed kangaroo rat (Dipodomys spectabilis).
KLICH, M.A. and PITT, J.I. 1988. Differentiation of Aspergillus flavus from A. parasiticus and other closely
related species. Transactions of the British Mycological Society 91: 99-108.
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.
LAND, CJ. and HULT, K. 1987. Mycotoxin production by some wood-associated Penicillium spp. Letters in
PATERSON, R.R.M., SIMMONDS, M.S.J. and BLANEY, W.M. 1987. Mycopesticidal effects of characterized
extracts of Penicillium isolates and purified secondary metabolites (including mycotoxins) on Drosophila
melanogaster and Spodoptora lilloralis. Journal of Invertebrate Pathology 50: 124-133.
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 Agriculturae Scandinavica 38: 215-222.
THOM, C 1930. The Penicillia. Baltimore: Williams and Wilkins.
TURNER, W.E. and D.C ALDRIDGE. 1983. Fungal metabolites II. London: Academic Press.
385
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.
386
Table 1. Genera, species and ubiquinone systems in the Aspergillus omatus group and in
Aspergillus subgen. Oman.
A. omatus group:
Raper & Fennell (1965)
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.
Species
Isolate"
Source
Hemicarpente/es acanthosporus
IF0949O(T)
IF096CY7
JCM2268
JCM2269
Sclerae/eistJz ornata
NRRL 2256(1')
= JCM 2354
JCM2263
CBS 425.68
NRRL 2162(1')
=IF08172
= JCM 2355
IF09127
Sc/eraeleistJz thaxteri
JCM2264(1')
JCM2350
Warcupie/la spinu/osa
IFO 31800(1')
= JCM 2358
JCM2270
Chaetosartorya chrysella
NRRL 5084(1')
NRRL5085
Chaetosartorya CTemea
NRRL 5081(1')
Chaetosartorya stromatoides
NRRL4519(1')
= IFO 9652
Soil,Panama
Aspergillus apica/is
CBS 236.81(1')
Aspergillus brunneo-uniseriatus
IF06993(1')
=JCM2348
Soil, India
JCM2349(1')
Aspergillus ivoriensis
JCM2353(1')
IF06416(1')
=JCM2356
JCM2266
Hemicarpenteles paradoxus
Sarbhoy & Elphick
Subram.
Subram.
Soil, Singapore
>(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
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
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
G6PDH
MDH
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
54,57
48,52
58
84
Aspergillus ivoriensis
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
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[
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
Aspergillus ivoriensis
JCM
Chaetosartorya s tromatoi des NRRL
IFO
Hemicarpenteles acanthosporus IFO
IFO
JCM
JCM
Chaetosartorya cremea
Chaetosartorya chryse 11 a
Sclerocleista thaxteri
Sclerocleista ornata
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
London: Plenum Press.
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
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/.
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')
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).
RESULTS AND DISCUSSION
K. Yamatoya et al.
398
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)"
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
A. oryzae v. magnasporus
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.
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
A. oryzae v. magnasporus
~: ~~;~~ari us
lAM 2138
NRRL 13139
NRRL
429
l-b I
A. paras i ti cus
A. fl avus soj ae
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
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.
KLICH, M.A. and MULLANEY, E.J. 1987. DNA restriction enzyme fragment polymorphism as a tool for
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
related species. Transactions of the British Mycological Society 91: 99-108.
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 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
London: Plenum Press.
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.
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
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.
MATERIAL AND METHODS
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
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
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
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
Aspergillus
A. brunneo-uniseriatus Singh et Bakshi
!FO 6993, JCM 2348(T)
CBS 236.81
Q-lO
Q-9
Q-9
Q-9
Q-10
Q-9
Q-10(H2)
Q-10
JCM 2353(T)
Q-IO(H2)
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)
Q-IO(H2)
Q-(10(52%)+ 10(H2)(48%
411
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
Q-IO(H2)
IF04096
Q-IO(H2)
lF04097
Q-IO(H2)
lF04114
Q-IO(H2)
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
IFO 6357
Q-I0(H2)
Q-IO(H2)
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
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
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
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~
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.)
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
Q-9
Q-9
Q-9
Q-9
Q-9
Q-10(H2)
Q-10(H2)
Q-10(H2)
Q-10(H2)
H. Kuraishi et af.
416
5.6
52%
48%
88%
12%
7.0
46%
54%
89%
11%
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
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.
r-------------------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
\~~.L-------------
A. cremeUB group
-.....:::::~C:::;::;:::;.>"
A. ochraceus group----______________~
A. spars us group
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).
r------------------- Sec t.
~,__-----------
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
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:::::-"""--------
' - - - - - - - - - - - - Sect.
Ornati I
Circumdati I
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
Aspergillus Systematics, eds. R.A. Samson and J.I. Pitt, pp. 195-209. New York and London: Plenum Press.
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.
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 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.
421
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
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
424
B. Fuhrmann et al.
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%.
425
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.
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
427
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.
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
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
431
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
Aspergilloides
A. versicolor
B3
73 strains
A8
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pp. 153-165. Ottawa: National Museums of Canada.
MORACE, G., ORSINI, D., CASTAGNOLA, M. and POLONELU, L. 1984. Exoantigen studies of
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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
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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
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POLONELLI, L. and MORACE, G. 1986. Specific and common antigenic determinants of C.albicans isolates
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POLONELLI, L., MORACE, G., ROSA, R., CASTAGNOLA, M. and FRISVAD, J.e. 1987. Antigenic
characterization of Penicillium camemberti and related common cheese contaminants. Applied and
Environmental Microbiology 53: 872-878.
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.
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identification of Coccidioides immitis cultures. Journal of Clinical Microbiology 5: 149-153.
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
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
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.
435
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
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
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.
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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.
9
TAXONOMIC STUDIES ON THE TELEOMORPHS OF
PENICILLIUM AND ASPERGILLUS
445
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
446
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
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
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
450
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
452
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
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
The authors thank the NATO Scientific Affairs Division (Brussel, Belgium) for the research
grant (0216/86) for international collaboration.
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
intracellular mycotoxins in pure culture. Applied and Environmental Microbiology 45: 581-585.
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
misidentified isolates. Archives of Environmental Contamination and Toxicology 18: 452-467.
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.
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
anamorphs. Studies in Mycology, Baarn 23: 1-149.
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.
454
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
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
N. aurata
N. quadricincta
N. stramenia
456
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
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).
The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
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.
MATERIAL AND METHODS
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).
RESULTS AND DISCUSSION
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
The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
459
460
A.A. Samson
et at.
The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
461
462
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
The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
213'11
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.
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
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
N. fischeri var. fischeri
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
+
+
+
+
+
+
+
+
+
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The genus Neosartorya: differentiation by scanning electron microscopy and mycotoxin profiles
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
grant (0216/86) for international collaboration.
466
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467
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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
The Technical University of Denmark
Food Technology Lab., Building 221
2800LYNGBY
Dr O. Filtenborg
The Technical University of Denmark
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
MN.H.N.
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
Centraalbureau voor Schimmelcultures
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
C.A.B. Mycological Institute
Ferry Lane,
KEW, Surrey TW9 3AF
Dr. Z. Lawrence
C.A.B. Mycological Institute
Ferry Lane,
KEW, Surrey TW9 3AF
Dr. Agnes H. S. Onions
The nineteenth, North Road
Dunbar,
EASTLOTHIANEH421 AY
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
471
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
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
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
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
478
Warcupiella
spinulosa 388, 392, 417
Yersinia
enterocolitica 212
Zacintha
verrucosa 186
Zea
mays 179, 182,351