Detection of toxigenic isolates of aspergillus flavus and related species on coconut cream agar

Journal of Amlied Bacteriology 1994, 76, 75-78

Detection of toxigenic isolates of Aspergillus f l a w s and
related species on coconut cream agar
Sonya K. Dyer and Sharee McCammon
CSIRO Division of Food Science and Technology,North Ryde, NSW, Australia
4573/05/93: accepted 30 July 1993

AN D s. M cc A M M o N . 1994. A new readily-prepared medium, coconut cream agar, was
developed for the detection of aflatoxin production by isolates of Asp_ergillusJavus a n d related
species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%),
detected isolates of A. jlavus more effectively than the synthetic media tested a n d was as
effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on
coconut cream agar could be used to differentiate A. flavus from A . parasiticus a n d A . nomius.
In addition, conidial colour of A.Javus a n d A . nomius was quite distinct from that of A .
parasiticus.

s. K . D Y E R

INTRODUCTION

Detection of Aspergillus flavus Link and A. parasiticus
Speare is readily accomplished by plating on Aspergillus
flavus and parasiticus agar (AFPA) (Pitt et al. 1983). T h e
two species can then be distinguished microscopically
(Klich and Pitt 1988). However, only 40% of A . flavus isolates produce aflatoxins (Klich and Pitt 1988), so a medium
capable of detecting aflatoxin production while also differentiating between A . flavus and A . parasiticus would have
considerable value.
Media which permit detection of aflatoxins by fluorescence under long wave ultraviolet (u.v.) radiation have been
the subject of research since the 1960s. Early work with
natural media used peanuts or coconut in a Czapek-Dox

medium with Hyflo-Supercel added to produce a white
background (de Iongh et al. 1964; de Vogel et al. 1965;
Arseculeratne et al. 1969). Preparation of these media in
large volumes is time-consuming.
Aflatoxin Producing Ability medium (APA) (Hara et al.
1974), a modified Czapek agar, contained corn steep liquor
which is not universally available. Wicklow et al. (1981)
found that APA did not give false positives, but
occasionally gave false negatives. More recent media contained coconut in the form of coconut meat, or desiccated
or shredded coconut (Lin and Dianse 1976; Davis et al.

1987).
Austwick and Ayerst (1963) produced a synthetic
medium that fluoresced in the presence of aflatoxin. T h e
synthetic liquid medium of Adye and Mateles (1964)
(A&M) has been widely used to assess aflatoxin production.
Correspondence t o : Miss S . K . Dyer, CSIRO Division of Food Science and
Technology, PO Box 52, North Ryde, NSW 2113, Australia.

Addition of agar enabled use of a scanning densitometer
with a fluorometry attachment to determine aflatoxin concentrations directly (Cotty 1988). El-Naghy et al. (1991)
reported better aflatoxin production in A&M medium than
on natural substrates. Naik et al. (1970), however, showed
increased aflatoxin production in A&M liquid by adding
peanuts or coconut. Venkitasubramanian (1977) also found
that A&M medium did not support high yields of aflatoxin
and so modified it by adding asparagine and increasing the
concentration and number of trace elements to form Synthetic Low Salts medium.
Yabe et al. (1987) grew A . flavus isolates on glucose (2%)
yeast (0.5%) extract agar then photographed the reverse

sides of colonies under U.V. light. This method has the
drawback of needing a u.v.-transparent camera lens.
Aspergillus nomius, a third species capable of producing
aflatoxins, was described by Kurtzman et al. (1987). Aspergillus nomius is morphologically similar to A . flavus but produces distinctive bullet-shaped sclerotia. Like A .
parasiticus, it produces G aflatoxins. Its behaviour on media
for detecting aflatoxin production has not been assessed.
This paper reports a modification of coconut extract agar
which is easily prepared, effective for detecting aflatoxin
production, useful for distinguishing A . flavus from A .
parasiticus and may aid in the detection of A . nomius.
M A T E R I A L S AND METHODS
Fungi

Six toxigenic fungal isolates were studied: A. flavus FRR
2746, FRR 2754 and FRR 2882; A . parasiticus FRR 2744,
FRR 2752 and FRR 3385; and a nontoxigenic A. flavus,


76 SONYA K. DYER A N D S H A R E E M c C A M M O N

FRR 2879. FRR denotes the culture collection of the
CSIRO Division of Food Science and Technology, North
Ryde, NSW, Australia. Production of toxins was determined by thin layer chromatography (TLC) (Filtenborg et
al. 1983).
Six A. nomzus isolates, FRR 3543, FRR 3544, FRR 3545,
FRR 3546, FRR 3547 and FRR 3673, were examined on
coconut cream agar to determine if they could be differentiated from A. JEavus.
Spores of all isolates were inoculated at three points on
three plates of each medium, then incubated at 30°C in
darkness.

Media

Media reported to support aflatoxin production (Table 1)
were compared for their ability to detect aflatoxin under
long wave U.V. radiation. With the exception of coconutbased media and A&M medium, these formulations have
not been examined previously for in situ fluorescence.
Several other experimental media were prepared during the
course of this work and also studied. These were 100%
coconut milk (Leecan, Penang, Malaysia), 40, 50 and 60%
coconut milk powder (NestlC, Sri Lanka) and 30, 40, 50
and 60% coconut cream (Trident, Thailand). Agar (1.5%)
was added to all media, which were dispensed at approximately 20 ml per Petri dish. Fluorescence under long wave
U.V. light (two XX15c 15 watt globes: Ultra Violet Products, San Gabriel, USA) was observed from 3 to 7 d after
inoculation.
Coconut cream agar vs Adye and Mateies agar

A comparative study was made of fluorescence on the most
effective of the synthetic and natural media. These were

50% coconut cream agar (CCA) and A&M agar. Media
were made, inoculated and observed as outlined above.
Fifteen isolates each of A. fIavus and A. parasiticus from
the FRR culture collection were studied. Spore colour was
also noted after incubation for 7 d.

Comparison with chromatography

The effectiveness of CCA for aflatoxin detection was compared with T L C using 95 toxigenic isolates of A. JEavus.
These had been isolated from peanut plants and soils from
Australia and grains from Thailand or were from the FRR

culture collection. Cultures were inoculated on CCA, incubated at 30°C for 4 d then examined under U.V. light. For
T L C cultures were grown on Czapek Yeast Extract agar
(Pitt 1973) for 7 d. Isolates which did not produce detectable toxins on Czapek Yeast Extract agar were grown on
CCA for 7 d and retested by TLC.

RESULTS
Comparison of media

Comparison of coconut cream media containing 40-60%
coconut cream (Table 2) indicated that 50% was optimal:
this concentration was used to produce coconut cream agar
(CCA). CCA (50% coconut cream) was found to be as
effective for detecting A. Jlavus and A. parasiticus as media
made with dried coconut, and has the advantage of being
rapidly and readily prepared. Trials with six brands of
coconut cream showed they could all be used to detect fluorescence. In these experiments Trident coconut cream was
found to give the strongest fluorescence.

Table 1 Media tested for fluorescence in the presence of aflatoxin

Medium

Composition

Reference

Shredded coconut
Desiccated coconut
Adye & Mateles

Filtrate from blended, shredded coconut
Filtrate from blended, desiccated coconut
Glucose, KH,PO,, (NH,),SO,, MgSO,
and six trace elements
Glucose, (NH,),SO,, K,HPO,, KH,PO,,
glycine, glutamic acid, MgSO, and
three trace elements
Sucrose, asparagine, KH,PO,, (NH,),SO,,
MgSO, and six trace elements
Sucrose, asparagine, (NH,),SO,
and eight trace elements
Sucrose, yeast extract, K,HPO,
and four trace elements
Sucrose, yeast extract
Sucrose, yeast extract, KNO, , MgSO,
Glucose, malt extract, peptone

Davis et al. 1987
Davis et al. 1987
Adye and Mateles 1964

Glucose salts
Synthetic
Synthetic low salts
Czapek
Yeast extract

Semisynthetic
Malt extract

Shih & Marth 1974
Venkitasubramanian 1977
Reddy et al. 1971
Pitt 1973; Ambrecht et al. 1963
Davis et al. 1966
Diener and Davis 1966
Wicklow and Hesseltine 1979


DETECTION OF TOXIGENIC A . FLAVUS 77
~~

Table 2 Maximum fluorescence observed by isolates of Aspergillus jlavus and A. parasiticus
~~~~

~

Aspergillus fIavus

Medium
Shredded coconut
Desiccated coconut
Coconut cream 30%
Coconut cream 40%
Coconut cream 50%
Coconut cream 60%
Coconut milk powder 40%
Coconut milk powder 50%
Coconut milk powder 60%
Coconut milk

Adye & Mateles
Glucose salts
Synthetic low salts
Synthetic

Aspergdlus parasiticus

FRR
2746

FRR
2754

FRR
2882

++
+++
++
++
++
+++
+++
+++
+++
++
+
+

+++

+++
+++
+++
+++
+++
+++
++
++
++
++
+
+

++
++
++
++
++
+++
+
++
++
++
+

-

FRR
2879

FRR
2744

FRR
3385

FRR
2752

++
++
++
+++
+++
+++
+++
+++
++
++
+++
+++
++

+++

+++
+++
+
++
++

++
++
++
++
++
+++
+++
++
+++

+++

-

+++
+
++
+++

+++
+++
++

++
+
+++
+++
++
+

+ + +, Very strong fluorescence; + +, strong fluorescence; +, detectable fluorescence; -, no fluorescence.
Coconut milk did not induce very strong fluorescence.
Coconut milk powder produced stronger fluorescence than
coconut milk and at the 40% concentration compared
favourably with desiccated and shredded coconut and
coconut cream agars. When grown on coconut milk powder
agar, A . parasiticus colonies produced copious exudate.
Exudate production was also observed on shredded and
desiccated coconut agars, but to a lesser extent.
Synthetic media were not as effective as CCA. Synthetic
Low Salts, Glucose Salts and A&M media detected aflatoxin production by all of the A. parasiticus isolates but only
two of the three toxigenic A. faavus isolates (Table 2). Fluorescence by A. parasiticus was more intense on A&M and
Glucose Salts agars than on Synthetic Low Salts agar.
Conidia of A. faavus and A. parasiticus on Glucose Salts
agar were yellow in contrast with those on the other media
tested. Fluorescence was only detected in Venkitasubramanian’s (1977) synthetic agar in the presence of A . parasiticus. Colonies grown on this agar were wrinkled and broke
the agar surface. No fluorescence was observed on Semi-

synthetic, Yeast Extract Sucrose, Czapek or Malt Extract
agar.
Coconut cream agar vs Adye and Mateles agar

I n tests that compared CCA and A&M, all of the toxigenic
A. parasiticus isolates examined fluoresced on both media
(Table 3). Toxigenic isolates of A. faavus were detected
more frequently on CCA than on A&M. After incubation
for 7 d, isolates of A. flavus and A. parasiticus were identified by conidial colour with greater accuracy on CCA than
on A&M.
It was found that after incubation on CCA for 4 d, 93%
of the A. flavus isolates and 80% of the A. parasiticus isolates examined were correctly identified by the colour of

their fluorescence. Aspergillus flavus fluoresced pastel blue
(20-21A4) in a ring around each colony while A . parasiticus
fluoresced bluish white (2&23A2)(Kornerup and Wanscher
1978) over all of each colony. T h e A. nomius isolates also
fluoresced bluish white and could not be differentiated by

Table 3 A comparison between Adye & Mateles agar and coconut cream agar using 15 isolates of AspergillusfIavus and A . parasiticus

Toxigenic isolates which fluoresced (%)

Adye & Mateles agar
Coconut cream agar (50%)

Correctly identified by spore colour (%)

Aspergillus J a w s

Aspergillus parasiticus

Time of peak
fluorescence
(d)

53
100

100
100

5

4

Aspergillus jlavus

Aspergillus parasiticus

88
100

53
93


78 SONYA K . D Y E R A N D S H A R E E McCAMMON

fluorescence from A. parasiticus. Very bright white fluorescence by a few cultures has been observed. I t is not clear
whether this is due to aflatoxin or a masking compound.

Comparison with chromatography
Detection of toxigenic isolates by CCA was found to correspond to TLC results for 86 of the 95 cultures of A. flavus.
T h e remaining nine cultures were positive on CCA but
negative on TLC plates.

DISCUSSION
Coconut cream is described by manufacturers as a concentrated cream extract from the fresh grated kernel of
matured coconut. T h e chemical basis of fluorescence on
CCA has not been investigated. Because of the high correlation between results on CCA and TLC plates and the
difference in colour of the fluorescence produced by A.
J a w s and A . parasiticus, it appears that the fluorescence is
due to aflatoxins. A s p e r g i l l u s f l a v u s and A. nomius are very

similar when examined both by eye and microscopically.
The difference in the colour of their fluorescence can be
used as a tool in the identification of A. nomius.

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