Aspergillus Fr.: Fr.
Syn.: Sterigmatocystis, Stilbothamnium
Colonies usually growing rapidly, white, pink, yellow,
yellow-brown, brown to black or shades of green, mostly
consisting of a dense felt of erect conidiophores. Conidi-
ophores of an (usually aseptate) unbranched stipe, with
a swollen apex (vesicle). Phialides borne directly on the
vesicle (uniseriate) or on metulae (biseriate). Vesicle,
phialides, metulae (if present) and conidia form the
conidial head. Conidia in dry chains forming compact
columns (columnar) or diverging (radiate), one-celled,
smooth or ornamented, hyaline or pigmented. Species
may produce Hülle cells (single or in chains, thick- and
smooth-walled cells) or sclerotia (firm, usually globose,
masses of hyphae). Many species of Aspergillus have a
teleomorph, and recently the teleomorph of the
important species A. fumigatus, A. flavus and A. para-
siticus have been found.
Teleomorphs 1 holomorphs: Eurotium, Chaetosartorya,
Petromyces, Neopetromyces, Fennellia, Emericella, Sclero-
cleista, Neocarpenteles, and Neosartorya.
The placement of Warcupiella (= Raperia), Dichlaena and
Hemicarpenteles is still under discussion. Dichotomomyces
Fig. 78. Morphological structures of the Aspergillus conidio-
phore.
106
with its Polypaecilum anamorphic state appear to be phyl-
ogenetically close to Aspergillus (Varga et al., 2007)
Aspergillus species are common contaminants of various
substrates. In subtropical and tropical regions their
occurrence is more common than the Penicillia. Several
species have attracted attention as human and animal
pathogens or because of their ability to produce toxic
metabolites (mycotoxins). Others are important for
their role in fermentation of oriental food products or
industrial application in the production of organic acids,
secondary metabolites or enzymes or in biotransforma-
tions of chemicals.
The classification is mainly based on morphological
characters. Raper and Fennell (1965) divided the genus
into 18 groups and accepted 132 species with 18 vari-
eties. Samson (1979, 1992, 1994 a and b) provided a
compilation of the species and varieties described since
1965 with a critical review of the validity of the published
taxa. The genus now contains more than 180 species,
with about 70 named teleomorphs.
Typification of Aspergillus names has now been achieved
because the majority of the Aspergillus names used by
Raper and Fennell (1965) were typified by Samson and
Gams (1986) and Kozakiewicz (1989). In order to protect
Aspergillus names in current use from being threatened
or displaced by names that are no longer in use, and in
order to eliminate uncertainties regarding their appli-
cation spelling, gender, typification date and place. or
valid publication, Pitt and Samson (1993, 2000) proposed
a list of species names in current use in the family
Trichocomaceae (Fungi, Eurotiales). All published names
and synonyms are listed in Samson and Pitt (2000).
Raper and Fennell (1965) subdivided Aspergillus into
"Groups". This infrageneric classification has no nomen-
clatural status under the ICEN and Gams et al. (1986)
replaced the group names by names of subgenera and
sections (Table 11).
Only the most common Aspergillus species are briefly
described and keyed out in this guide, particularly those
on food products and in indoor environments. However,
for a more exact identification of important isolates the
taxonomic literature (Raper and Fennell, 1965; Samson,
1992, 1994 a and b; Frisvad and Samson (2000); Sam-
son and Varga (2007)) should be consulted. As already
stated above Aspergilli are very common contaminants.
Therefore we have not listed every substrate from which
each species has been isolated. Data of habitats of many
species are given by Domsch et al. (2007). However, it
should be noted that these references might be based on
incorrect identifications.
Practicallaboratory guides for identi:fication of common
Aspergilli which are primarily based on morphology and
CBS LABORATORY MANUAL SERIES
 Table 11. Subdivision of the genus Aspergillus in subgenera and sections.

Subgenus Section Teleomorph Species in this book

Aspergillus Aspergillus Eurotium Eur. amstelodami, Eur. chevalieri, Eur. herbariorum, Eur. rubrum
Restricti Eurotium Asp. restrictus, Asp. penicillioides
Asp. fumigatus, Neo. fischeri, Neo. glabra, Neo. hiratsukae, Neo.
Fumigati Fumigati Neosartorya
spinosa,
Clavati Neocarpenteles, Dichotomomyces Asp. clavatus

Cervini (Asp.cervinus)*
Circumdati Circumdati Neopetromyces Asp. westerdijkiae
Nigri Asp. acidus, Asp.carbonarius, Asp. niger
Asp. arachidicola, Asp. flavus, Asp. minisclerotigenes, Asp. oryzae,
Flavi Petromyces
Asp. parasiticus, Asp. tamarii
Cremei Chaetosartorya Asp. wentii
Candidi Candidi Asp. candidus
Terrei Terrei Asp. terreus
Flavipedes Fennellia (Fen. flavipes)*
Nidulantes Nidulantes Emericella Asp. sydowii, Asp. versicolor, Em. nidulans
Usti Emericella Asp. calidoustus, Asp. ustus
Sparsi (Asp. sparsus)*
Warcupi Warcupi Warcupiella (War. spinulosa)
Zonati Penicilliopsis (Asp. zonatus)*
Ornati Ornati Sclerocleista (Scl. ornata)*
* examples, not relevant for this book.

conidia

/
>-:--:~
~

¡...

1"
stipe ~
\ )-1>-' Jl.r]

radiate column
uniseriate biseriate
1

Fig. 79. Morphological structures in Aspergillus.

ASPERGILLUS 107
 Table 12. Producers of aflatoxin (Varga et al., 2009)
Species Types of aflatoxin Common on foods and feeds
Aspergillus flavus B1, B2 verycommon
Aspergillus parasiticus Bl, B2, Gl, G2 quite common
Aspergillus minisclerotigenes Bl, B2, Gl, G2 verycommon
Aspergillus nomius Bl, B2, Gl, G2 quite common (Brazil nuts)
Aspergillus arachidicola Bl, B2, Gl, G2 quite common
Aspergillus pseudotamarii Bl,B2 rather rare
Aspergillus parvisclerotigenus Bl, B2, Gl, G2 apparently rather rare
Aspergillus bombycis Bl, B2, Gl, G2 notcommon
Aspergillus pseudocaelatus Bl,B2,Gl,G2 notcommon
Aspergillus ochraceoroseus Bl,B2 notcommon
Aspergillus rambellii Bl,B2 notcommon
Emericella olivicola Bl,B2 notcommon
Emericella astellata Bl,B2 notcommon
Emericella venezuelensis B2 notcommon

colony characters have been published by Tzean et al.
(1990), Klich (2002) and Pitt and Hocking (2009). Many
books have been published on Aspergillus 9f which that
of Machida and Gomi (2010) is the most recent.
IDENTIFICATION OF AFLATOXIGENIC
ASPERGILLI
Sorne specialized media are used for the rapid recogni-
tion of potentially aflatoxigenic Aspergilli. For the detec-
tion of A flavus andA. parasiticus within 3 days Aspergil-
lus Differential Medium (Bothast and Fennell, 1974) or
Aspergillus Flavus and Parasiticus Agar (AFPA) (Pitt et
al., 1983) can be used. Another medium containing the
antibiotic bleomycin helps to distinguish A. parasiticus
from A. sojae (Klich and Mullaney 1989). On this me-
dium growth of both species is somewhat reduced, but
A. sojae produces very restricted colonies while A. para-
siticus colonies are at least 3 mm in diameter within six
days. These selective media are especially appropriate for
the rapid detection of mycotoxinogenic isolates in food
commodities.
Profiles of secondary metabolites, including mycotoxins,
are of great use in Aspergillus systematics. Frisvad and
Samson (2000) and Pildain et al. (2008) examined the re-
lationship between current species concepts and second-
ary metabolite production in sect. Flavi and found a good
correlation between the production of aflatoxins B1 + B2 ,
G1 +G 2 and cyclopiazonic acid and the morphological char-
acteristics of the species (see also under A flavus and A.
oryzae). Table 12 shows the list of Aspergillus and Emeri-
cella spp which can produce a:flatoxins, while Table 13lists
the distinguising characters for the important taxa.

Table 13. Distinguishing characters of Aspergillus flavus and sorne related species (see Pildain et al., 2008 for
further differences, especially in extrolite profiles)
Species Colony (coni- Conidial orna- AFPA* Sclerotial Sclerotia Cyclopiazonic acid
dium) colour mentation (reverse) size

A arachidicola (fl.occose) Grass green Rough Cream 1 orange None

A bombycis (fl.occose) Grass green Finely rough Orange None

A caelatus Brown Cream None
A flavus Grass green Finely rough Orange Large Frequent +1-
(Smooth), finely
A minisclerotigenes Grass green Orange Small Always present +
mllgh to rm1gb
Frequent (bullet
A nomius Grass green Finely rough Orange

A (fl.occose) Brown Smooth Cream +1-

A parvisclerotigenus Grass green Finely rough Orange Small Always present +
A parasiticus Dark grass green Rough Orange Large Rare
A pseudotamarii Darkbrown Very rough (double Darkbrown None
+1-
wall)
A sojae Pale brown Rough Orange Non e
A tamarii Darkbrown Very rough (double Darkbrown Large Rare
+1-
wall)

*AFPA = Aspergillus flavus and parasiticus agar (Pitt et al., 1983).

108 CBS LABORATORY MANUAL SERIES

Fig. 80. Various morphological structures inAspergillus. A-D. Vesicles A-B. Globose. C. Subclavate. D. Clavate. E. Columnar conidial
chains. F. Hülle cells. G-I. Conidial heads. G-H. Radiate. I. Columnar.

ASPERGILLUS 109
CULTIVATION FOR IDENTIFICATION
Isolates are inoculated at three points on CYA and MEA
and incubated at 25 °C. Inoculation at 37 oc and culti-
vation on CREA may be helpful. For xerophilic species
such as A. penicillioides and Aspergillus species with Euro-
tium teleomorphs, CYA and MEA with 20-40 % sucrose
should be used. Colour and structure of the conidial head
(columnar or radiating) are best observed with a dissect-
ing microscope. Microscopic mounts are made in lactic
acid with or without aniline blue and a drop of alcohol
is added (70-90 %) to remove air bubbles and excess of
conidia. As a few Aspergilli are pathogenic toman (e.g. A.
fumigatus), inhalation of conidia must be avoided.

KEY TO THE SPECIES TREATED

la. Colonies white, black or in yellow, brown or grey colours 2

lb. Colonies in sorne shade of green 9

2a. Conidial heads white, often wet .......................... . ............... A. candidus

2b. Conidial heads yellow, sorne shade of brown or black .................................. 3

3a. Conidial heads dark brown to black :................. . ······································· 4

3b. Conidial heads not dark brown to black, but olive, yellow-brown or other
shades of brown .................. . ................. 5

4a. Conidia measuring 3-6 IJ.m, fast growth rate on CYA at 37 A. niger 1 A. acidus
4b. Conidia measuring 6-9 IJ.m, colonies 0-35 mm on CYA at 37 oc ................ . ................... A. carbonarius

5a. Conidial heads columnar, often cinnamon-brown to pinkish-brown .................... . A. terreus

5b. Conidial heads not columnar, colour yellow or brown ..................... 6

6a. Conidial heads olive to light brown; stipe brown. Hülle cells often produced ..................... A. calidoustus*
6b. Conidial heads not olive; stipe hyaline or yellowish. Hülle cells absent 7

7a. Conidial heads pure yellow, conidia smooth to finely roughened ............................................ A. westerdijkiae**
7b. Conidial heads yellow-brown, conidia ornamented .................................................................. 8

8a. Colonies dark green-brown, conidia conspicuously ornamented with warts and
tubercles, outer and inner wall can be distinguished, good growth on CYA at 37 oc .............. A. tamarii
8b. Colonies grayish-yellow to olive brown, conidia mostly roughened, outer
and inner wall cannot be distinguished, no growth on CYA at 37 °C ....................................... A. wentii

9a. Conidiophores typically brown, Hülle cells and Emericella teleomorph

mostly present ............................................................................................................................ Emericella (p. 66)
9b. Conidiophores not typically brown, Emericella teleomorph absent ........................................ 10

lOa. Colonies on CYA mostly restricted (colony diameter usually less than 25 mm
within one week) ............... . 11
lüb. Colonies on CYA growing fas ter with a diameter usually larger than 25 mm .............. . 15

lla. Conidial heads biseriate .............................................................................................................. 12

llb. Conidial heads uniseriate ........................................................................................................... 13

12a. Colonies grey-blue to blue-grey ("Delft blue green"), conidia rough walled ................. A. sydowii
12b. Colonies variably coloured, sometimes Hülle cells sometimes present,
conidia smooth to fin ely roughened ............................. . A. versicolor

13a. Yellow Eurotium teleomorph produced in old cultures or on low water activity
media, colonies spreading on low water activity media (eg DG18) .......................................... Eurotium (p. 68)
13b. Yellow Eurotium teleomorph absent .......................................................................................... 14

110 CBS LABORATORY MANUAL SERIES

14a. Conidial heads columnar, phialides restricted to the upper third of the vesicle,
conidia cylindrical A. restrictus
14b. Conidial heads radiate, phialides covering more than half of the vesicle, conidia
subglobose or ellipsoid ............................................................................................................... A. penicillioides

15a. Colonies yellow-green to dark yellow green 16

15b. Colonies blue to dark blue green 19

16a. Conidial heads predominantly uniseriate, conidia dark yellow green,

conspicuously echinulate ............................................................................................................ A. parasiticus
16b. Conidial heads uní- and biseriate ............................................................................................... 17

17a. Conidia roughened with dark-coloured tubercles, colonies brownish-green .......................... A. tamarii
17b. Conidia echinulate, irregularly roughened or smooth, colonies yellow green or
greenish olive .................. . 18

18a. Conidia minutely echinulate, yellow green ................ A. flavus***

18b. Conidia irregularly roughened or smooth, greenish olive ...................... . A. oryzae

19a. Conidial heads columnar, vesicles broadly clavate, conidia rough to echinulate ..................... A. fumigatus****
19b. Conidial heads radiate, splitting into several columns with age, vesicles
narrowly clavate, smooth-walled ............................................................................................... A. clava tus

Important notes:
*Aspergillus insuetus can also be common on food and indoor environments
** Several yellow Aspergilli are described, butA. westedijkiae is the most common species
***A. archidicola, A. minisclerotigenes, A.jlavus andA. oryzae are closely related species. Morphological identification
is difficult and additional characters, such as extrolites patterns and/ or sequence data, are recommended. See also Table
12 and 13.
**** For species which produce an ascomycete state, consult Neosartorya (page 78-81).

ASPERGILLUS 111