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 to 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 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 d then examined under U.V light For T L C cultures were grown on Czapek Yeast Extract agar (Pitt 1973) for d Isolates which did not produce detectable toxins on Czapek Yeast Extract agar were grown on CCA for 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 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 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 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 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 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 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 REFERENCES Adye, J and Mateles, R.I (1964) Incorporation of labelled compounds into aflatoxins Biochimica et Biophysica Acta 86, 418420 Ambrecht, B.H., Hodges, F.A., Smith, H.R and Nelson, A.A (1963) Mycotoxins I Studies on aflatoxin derived from contaminated peanut meal and certain strains of Aspergillus flavus Journal of the Association of Official Agricultural Chemists 46, 805-8 17 Arseculeratne, S,N., De Silva, L.M., Wijesundera, S and Bandunatha, C.H.S.R (1969) Coconut as a medium for the experimental 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 estimation 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 rapid screening test for aflatoxin-synthesizing aspergilli of the flavus-oryzae group Journal of Applied Bacteriology 28, 213220 Diener, N.D and Davis, U.L (1966) Aflatoxin production by isolates of Aspergillusflavus Phytopathology 56, 1390-1393 El-Naghy, M.A., Mazen, M.B and Fadl-Allah, E.M (1991) Production of aflatoxin B, by Aspergillus flavus isolated from stored cotton seeds with different substrates World Journal of 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 cultures Applied and Environmental Microbiology 45, 581-585 Hara, S., Fennell, D.I and Hesseltine, C.W (1974) Atlatoxinproducing strains of Aspergillus flavus detected by fluorescence of agar medium under ultraviolet light Applied Microbiology 27, 1118-1 123 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 Kornerup, A and Wanscher, J.H (1978) Methuen Handbook of Colour London : Eyre Methuen Kurtzman, C.P., Horn, B.W and Hesseltine, C.W (1987) Aspergillus nomius, a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii Antonie van Leeuwenhoek 53, 147-158 Lin, M.T and Dianse, J.C (1976) A coconut-agar medium for rapid detection of aflatoxin production by Aspergillus spp Phytopathology 66, 1466-1469 Naik, M., Modi, V.V and Patel, N.C (1970) Studies on aflatoxin synthesis in Aspergillus flavus Indian Journal of Experimental Biology 8, 345-346 Pitt, J.I (1973) An appraisal of identification methods for Penicillium 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 parasiticus Journal of General Microbiology 101, 35-40 Reddy, T.V., Viswanathan, L and Venkitasubramanian, T.A (1971) High aflatoxin production on a chemically defined medium Applied Microbiology 22, 393-396 Shih, C.N and Marth, E.H (1974) Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus Applied Microbiology 27, 452456 Venkitasubramanian, T.A (1977) Biosynthesis of aflatoxin and its control In Mycotoxins in Human and Animal Health ed Rodricks, 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 produced by Aspergillusflavus in association with other fungi in autoclaved corn kernels Phytopathology 69, 589-591 Wicklow, D.T., Shotwell, O.L and Adams, G.L (1981) Use of aflatoxin-producing ability medium to distinguish aflatoxinproducing strains of Aspergillus flavus Applied and Environmental Microbiology 41, 697-699 Yabe, K., Ando, Y., Ito, M and Terakado, N (1987) Simple method for screening aflatoxin-producing molds by UV photography Applied and Environmental Microbiology 53, 23G234  ... 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... 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... detectable toxins on Czapek Yeast Extract agar were grown on CCA for d and retested by TLC RESULTS Comparison of media Comparison of coconut cream media containing 40-60% coconut cream (Table 2)