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.. flavus isolates on glu
Trang 1of Amlied Bacteriology 1994, 76, 75-78
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
s K D Y E R A N 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
I N T R O D U C T I O N
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 iso-
lates produce aflatoxins (Klich and Pitt 1988), so a medium
capable of detecting aflatoxin production while also differ-
entiating between A flavus and A parasiticus would have
considerable value
Media which permit detection of aflatoxins by fluores-
cence 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 con-
tained 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 con- centrations 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 Syn- thetic 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) Asper-
gillus nomius is morphologically similar to A flavus but pro- duces 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 M E T H O D S 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,
Trang 276 S O N Y A 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 deter-
mined 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 differen-
tiated 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 coconut-
based 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 approx-
imately 20 ml per Petri dish Fluorescence under long wave
U.V light (two XX15c 15 watt globes: Ultra Violet Pro-
ducts, 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
Table 1 Media tested for fluorescence in the presence of aflatoxin
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 com- pared 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, incu- bated 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 detect- able toxins on Czapek Yeast Extract agar were grown on CCA for 7 d and retested by TLC
R E S U L T S 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 fluo- rescence In these experiments Trident coconut cream was found to give the strongest fluorescence
Shredded coconut
Desiccated coconut
Adye & Mateles
Glucose salts
Synthetic
Synthetic low salts
Czapek
Semisynthetic
Malt extract
Filtrate from blended, shredded coconut Filtrate from blended, desiccated coconut Glucose, KH,PO,, (NH,),SO,, MgSO,
Glucose, (NH,),SO,, K,HPO,, KH,PO,, and six trace elements
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 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
Trang 3DETECTION OF TOXIGENIC A F L A V U S 77
~~
Table 2 Maximum fluorescence observed by isolates of Aspergillus jlavus and A parasiticus
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
+ + + + + + + + +
+ +
+ + + + + + + + +
+ + +
+ + + +
-
+ + +
+ + + + + + + + + + + + + + + + + +
+ +
+ +
+ +
+ + +
+
-
+ + + + + + + +
+ +
+ + + + + + + + + + +
+ + + + + +
+ + + + + + + + + + + + + + +
+ + + +
+ + + + + +
+ + + + +
+ + + + + + + + + + + + + + + + + + + +
+ +
+ + + + + + +
+ + +
+ + + + + +
+
+ +
+ + + +
+ +
+ + + + + + + + +
+ + +
+ +
+ + +
+ + +, 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 aflatox-
in production by all of the A parasiticus isolates but only
two of the three toxigenic A faavus isolates (Table 2) Fluo-
rescence 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 Venkitasubrama-
nian’s (1977) synthetic agar in the presence of A parasiti-
cus 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 identi- fied 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 iso- lates 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 (%) Correctly identified by spore colour (%)
Time of peak
fluorescence
Aspergillus J a w s Aspergillus parasiticus (d) Aspergillus jlavus Aspergillus parasiticus
Trang 478 SONYA K D Y E R A N D S H A R E E McCAMMON
cence 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
T h e remaining nine cultures were positive on C C A but
DISCUSSION
Coconut cream is described by manufacturers as a concen-
trated cream extract from the fresh grated kernel of
matured coconut T h e chemical basis of fluorescence on
J a w s and A parasiticus, it appears that the fluorescence is
similar when examined both by eye and microscopically
REFERENCES
Adye, J and Mateles, R.I (1964) Incorporation of labelled com-
pounds into aflatoxins Biochimica et Biophysica Acta 86, 418-
420
Ambrecht, B.H., Hodges, F.A., Smith, H.R and Nelson, A.A
(1963) Mycotoxins I Studies on aflatoxin derived from con-
taminated peanut meal and certain strains of Aspergillus flavus
Journal of the Association of Official Agricultural Chemists 46,
natha, C.H.S.R (1969) Coconut as a medium for the experi-
mental production of aflatoxin Applied Microbiology 18, 88-94
Austwick, P.K.C and Ayerst, G (1963) Groundnut microflora
and toxicity Chemistry and Zndustry 2, 55-61
Cotty, P.J (1988) Simple fluorescence method for rapid estima-
tion of aflatoxin levels in a solid culture medium Applied and
Environmental Microbiology 54, 274-276
Davis, N.D., Diener, U.L and Eldridge, D.W (1966) Production
of aflatoxins B, and G, in a semisynthetic medium Applied
Microbiology 14, 378-380
Davis, N.D., Iyer, S.K and Diener, U.L (1987) Improved
method of screening for aflatoxin with a coconut agar medium
Applied and Environmental Microbiology 53, 1593-1595
De Iongh, H., Vles, R.O and De Vogel, P (1964) The occurrence
and detection of aflatoxin in food In Mycotoxins in Foodstufs
ed Wogan, G.N pp 235-245 Cambridge: M I T Press
De Vogel, P., Van Rhee, R and Koelensmid, W.A.A.B (1965) A
805-8 17
rapid screening test for aflatoxin-synthesizing aspergilli of the
flavus-oryzae group Journal of Applied Bacteriology 28, 213-
220
Diener, N.D and Davis, U.L (1966) Aflatoxin production by iso-
El-Naghy, M.A., Mazen, M.B and Fadl-Allah, E.M (1991) Pro-
duction of aflatoxin B, by Aspergillus flavus isolated from
Microbiology and Biotechnology 7, 67-71
Filtenborg, O., Frisvad, J.C and Svendsen, J.A (1983) Simple screening method for molds producing mycotoxins in pure cul-
tures Applied and Environmental Microbiology 45, 581-585
producing strains of Aspergillus flavus detected by fluorescence
of agar medium under ultraviolet light Applied Microbiology 27,
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, 9%108
Colour London : Eyre Methuen
Kurtzman, C.P., Horn, B.W and Hesseltine, C.W (1987) Asper-
gillus nomius, a new aflatoxin-producing species related to
Aspergillus flavus and Aspergillus tamarii Antonie van Leeuwen-
hoek 53, 147-158
rapid detection of aflatoxin production by Aspergillus spp Phy-
topathology 66, 1466-1469
Pitt, J.I (1973) An appraisal of identification methods for Penicil-
lium species : novel taxonomic criteria based on temperature and
water relations Mycologia 65, 1135-1 157
Pitt, J.I., Hocking, A.D and Glenn, D.R (1983) An improved
medium for the detection of Aspergillus flavus and A parasiti-
cus Journal of General Microbiology 101, 35-40
Reddy, T.V., Viswanathan, L and Venkitasubramanian, T.A (1971) High aflatoxin production on a chemically defined
Shih, C.N and Marth, E.H (1974) Some cultural conditions that
siticus Applied Microbiology 27, 452456
Venkitasubramanian, T.A (1977) Biosynthesis of aflatoxin and its
control In Mycotoxins in Human and Animal Health ed Rod-
ricks, J.V., Hesseltine, C.W and Mehlman, M.A pp 83-98 Park Forest South, IL: Pathotox Publishers
Wicklow, D.T and Hesseltine, C.W (1979) Fluorescence produc-
ed by Aspergillusflavus in association with other fungi in auto- claved corn kernels Phytopathology 69, 589-591
Wicklow, D.T., Shotwell, O.L and Adams, G.L (1981) Use of
producing strains of Aspergillus flavus Applied and Environ-
mental Microbiology 41, 697-699
method for screening aflatoxin-producing molds by UV pho-
Biology 8, 345-346