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Growth and aflatoxin production of Aspergillus flavus in wheat and barley
[ 259 ] Trans. Br. mycol. Soc. 84 (2), 259-266 (1985)
Printed in Great Britain
GROWTH AND AFLATOXIN PRODUCTION OF ASPERGILLUS FLAVUS IN WHEAT AND BARLEY By E. V. NILES, JOANNA A. NORMAN* AND D. PIMBLEYt MAFF, Olantigh Road, Wye, Ashford, Kent, *Slough Laboratory, London Road, Slough, SL3 7HJ, Berkshire and tBlock 2 Government Buildings, Lawnswood, Leeds LS16 spy Growth rates and aflatoxin production of eight strains of Aspergillus fiavus grown in wheat and barley were studied over temperature and water activity (a w ) ranges of 10-42'5 °C and 0'80-0'975 respectively. Growth and aflatoxin production were restricted by low temperature, low a w and high temperature but not by high aw ' At 15° the lowest temperature at which growth occurred, an aw of 0.95 was needed for growth. At 42.5° growth was severely restricted and aflatoxin production altogether inhibited. The optimum temperature for growth was 35°; at lower temperatures (15°, 20°) the optimum aw was> 0.975, but at temperatures above these, it was 0'95. No single optimum temperature for aflatoxin production was determined but at aw s over 0'90 it lay between 25° and 35°. Strains showed marked differences in ability to produce aflatoxins but no equivalent differences in their growth rates. Only at the most favourable condition was a significant difference in growth demonstrated between two isolates at the extreme ends of the scale. There were not significant differences in growth or aflatoxin production between wheat and barley as substrates. For the fungi which occur in stored products those factors most significantly affecting their growth and other physiological behaviour are water activity (a w ) ' temperature and nature of the substrate (Ayerst, 1969; Horner & Anagnostopolous, 1973; Northolt et al., 1976; Diener & Davis, 1969; Dickens & Patee, 1966). Since the role of Aspergillus fiavus in the pathogenesis of Turkey X disease was identified some 22 years ago (Allcroft et al., 1961) a considerable amount of information has been gathered on the conditions necessary for growth and production of aflatoxins by the causal organisms A.fiavus and A. parasiticus. The various controlling factors interact in such a way as to make simple statements such as 'the temperature range for aflatoxin production is between 20 and 35°, (Beuchat & Lechowich, 1970) or 'maximum aflatoxin occurs by about 12 days' (Christensen, 1982) inadequate and incomplete. Lower limiting temperatures for aflatoxin production are given as ~ = 7'5° on wort agar medium (Schindler, 1971) or as 10° in malt extract glucose agar (Northolt et al., 1976). Temperatures of 13° in maize and wheat (Jemmali et al., 1969; Behere et al., 1978) and of at least 13'5° or higher in barley (Chang & Markakis, 1982) are indicated for cereals. The maximum temperature permitting aflatoxin development is generally given as 40° but Rabie & Smalley (1965) found that toxin was elaborated at 42° and Shih &
Marth (1974, 1975) claimed to have demonstrated small amounts of toxin at 45°. However, Ayerst's work showed a limiting temperature for growth of A. fiavus of 43°. Reported lower limits of a w for growth and aflatoxin development are at least as variable. Reiss (1978) found that spores of A. fiavus were unable to germinate at an a w of 0·82 and Orth (1976) determined an aw limit of 0·84 for spores of A. fiavus and A. parasiticus; yet Latsch & Trapper (1978) obtained growth in several and toxin production in three out of ten isolates of A. fiavus and A. parasiticus tested on malt yeast glucose nutrient media at an aw of 0·82. Northolt et al. (1976) were unable to detect aflatoxin at a water activity of 0'83 and temperature of 10°. Similarly, jemmali et al. (1969) found there was not growth of A. fiavus even at 0'85 a w when the temperature was 13° while Nusrath (1978) records for pigeon peas maximum yields of aflatoxin at 0·80 a w and 25°, conditions in which most reports suggest A. fiavus should not grow let alone produce aflatoxin. In general, increasing the a w promotes faster growth and greater toxin production (Boller & Schroeder, 1974; Chang & Markakis, 1982) but a number of reports suggest that the optima for growth and toxin production can be obtained at a w s less then the achievable maximum (Diener & Davis, 1967; Lotzsch & Trapper, 1978). Not only
260
Growth and aflatoxin production of A. flavus
are these optimal values dependent on the interaction of temperature and water activity but also on the strain and cultivar differences of organism and substrate (N ort holt et al., 1977; Chang & Markakis, 1982). In a previous paper Niles (1978) showed how the growth rates and aflatoxin production of a strain of A. flavus were also influenced by the method of sterilization of the wheat used as substrate. In this study growth and aflatoxin production were measured over combinations of a wide range of temperature and a w on the same isolate and other isolates grown axenically in pre-sterilized wheat and barley so as to be able to predict growth and the likelihood of toxin production in any combination of the two principal factors in cereals. Close attention was paid to achieving and maintaining target a w levels. MA TERIALS AND METHODS
Test cultures and inoculum The work was done principally with a strain of A. flavus Link: Fries (P I L 110) used in the previous study and derived originally in 1962 from Brazilian groundnuts. Partial studies were conducted on 7 other isolates: PIL 295, PIL 442 from U.K. barley which had been undertreated with propionic acid; PIL 345, PIL 378, PIL 377, PIL 365 and S 922 all from a number of groundnut products used in animal feeds. All cultures were retained as freeze dried isolates and grown as required on thin plates of 2 % malt agar for 2-4 days at 25°. For inoculum a single 5'0 mm disc of the young culture was cut with a sterile cork borer for transfer to the grain. Preparation of substrate Wheat was the principal substrate. Several kilograms of the variety Maris Widgeon, collected at the beginning of the 1980 harvest were dried to 11'0 % moisture and held in a deep freeze at -12° to - 15°. Stocks of barley of unknown variety were also collected, dried to about 12 % and similarly stored in the deep freeze. When required, quantities of grain were withdrawn and the moisture content determined on 10 g lots by drying at 113° for 4 h in a mechanically ventilated oven (Oxley, Pixton & Howe, 1960) . Grain was accordingly conditioned (Niles, 1978) to the required moisture levels by addition of appropriate volumes of water. After the equilibrium period the equilibrium relative humidity (e.r .h .) was determined using a dew point method (P ixton & Warburton, 1971), at the intended storage temperature except when the latter was 40° or 42 '5°, e.r.h. was then determined also at 35°. Between 40 and 50 g of conditioned
grain was then packed into 250 x 25'0 mm borosilicate glass tubes, plugged with cotton wool at both ends and steam sterilized in an autoclave at 121° for 1 h. After removal from the autoclave, tubes of grain were held for 48 h over solutions of KOH in equilibrium with the target relative humidity. The moisture content of grains was again determined prior to inoculation, and about halfway through and at the end of the incubation period to ensure that no significant losses in the moisture levels of the grains had occurred at any stage.
Inoculation and incubation The column of grain in the tube was inoculated centrally at one end with the 5'0 mm disc of the test culture grown for 2-4 days. A minimum of 6 replicates were inoculated each time for whatever condition was being tested. Tubes were then placed on a perforated metal platform over an equilibrium solution of KOH (Solomon, 1951) contained in a sealed glass tank measuring 440 x 300 x 280 mm. The glass tanks and contents were stored in constant temperature and relative humidity room or in incubators giving maximum temperature fluctuations of ±0'5° (mean fluctuation 0'2°). Incubation was continued usually for 30 days and during this time tanks were ventilated by removing the lids for several minutes every few days.
Observation and measurement of growth Observations were made at more or less daily intervals until mycelium arising from the growing inoculum appeared at the grain surface. The time for this to happen depended on the test condition, being 2-3 days in the most favourable situations and several weeks or not at all in less favourable ones. Once growth had appeared, it was measured at frequent intervals depending on the speed of growth using a stereoscopic microscope with a specially modified stage (Niles, 1978). The mycelium was measured along the lines previously marked on opposite sides of the glass tube and the mean length then plotted against the period of growth in days . A regression line was drawn for the period between the first and the last set of measurements and from this the slope or growth rate determined.
Analysis for aflatoxin After the final growth measurement was made analysis for the presence of the aflatoxins HI' B 2, G 1 and G 2 was carried out on replicate tube cultures. A total of between 6 and 21 analyses were carried
E. V. Niles, Joanna A. Norman and D. Pimbley out for each temperaturc/c., combination. Infected grain was weighed into a plastic bag, mixed well and a 10 g sample put into a 250 ml conical flask. Chloroform (100 ml) was added to this followed by 10 ml of water. The flask was then shaken for 30 min on a rotary shaker, The liquid was filtered off through NaS0 4 on Whatman No. 41 filter paper, The grain residue was then washed three times in 250 ml lots of chloroform. The whole extract was transferred to a rotary evaporator and reduced to dryness, This residue was taken up in chloroform and transferred to a large screw cap phial in which it was again reduced to dryness and finally made up to 5 ml with chloroform. Aliquots of the extract along with standards were spotted for z-dimensional chromatographic separation on to 10 X 10 mm cuts of Merck 5535 aluminium backed silica gel TLC plates (Merck & Co. Ltd), The spots consisting of 5 p;! of the test extract and 1, 2,4, and 6 pi of the aflatoxin standard containing 0' 5 pgjpi were run first in diethyl ether: methanol: water
Table
1.
261
(94:4'5: 1'5) then in a second solvent system
consisting of toluene, ethyl acetate and formic acid (60: 30: 10), The first tank was lined and equilibrated
the second was not. Chromatograms were developed and examined under long wave (360 nm) and short wave (250 nm) u.v. light, Quantification was obtained by matching fluorescence intensities of test extracts and standards,
RESUL TS
Effect of temperature and relative humidity on growth rates Table 1 shows for all combinations of temperature and aw the minimum, maximum and mean growth rates obtained for Aspergillus fiavus PIL 110 in wheat. These values are based on from 6 to 24 replicates. Tests for variation either within or between experiments consistently showed that there were no significant differences in growth rates
Minimum, maximum and mean growth rates (mmjd) of Aspergillus flavus PIL combinations of temperature and water activity
110
at various
Water activity Temp C°C)
0,80
0'8 25
0'85
0'875
0'90
10
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0'9 25 0 0 0
0'95 0 0 0
0'975 0 0 0
15
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0'18 0'05
0 0'21 0,86
1'95 2'21 1'93
20
0 0 0
0 0 0
0 0 0
0'10 0'40 0'25
0'97 2'3 1 1'5 0
1'7 0 2'32 2'16
2'89 3'59 3'3 6
25
0 0 0
G
1'10 2'3 0 1,66
1'97 3'40 2'15
3'08 3'56 3'41
5'82 6'7 2 6'12
30
0'00 0'44 0'17
0,66 1'3 1 0'9 8
2'7 1 3'21 2,82
3'01 3'35 3'22
9'23 10'05 9'23
8'45 9'43 8'84
35
0'24 0,80 0'36
0'44 2'18 1'41
4'5 8 5'5 0 5'08
10'25 10'93 10'60
9'30 10'20 9'75
37'5
G
G
3'59 4'12 4'00
5'15 6'20 5'79 3'86 5'63 4'79
5'50 6'39 5'99 7'20 8'20 7'73 6'23 7'16 6'74
4'66 5'66 5'09 8'7 1 9'12 8'78 9'5 1 10'40 9'85
4'00 4'66 4'27 5'18 6'10 5'52
6'32 8'04 7'25
40'0
0'00
G
0'46 2'90 1'4 6
4'44 6'12 5'20
4 2'5
0'00
0'0
G
0'0
0'0
0'7 2 1'64 1'14
0'00
3'11 3'81 3'44 2,61 3'3 6 3'01
8'54 9'00 8'70
5'13 6'08 5'57
6'22 7'00 6'73
8'57 10'70 9'5 8 6'00 7'70 6'89
2'35 3'75 3'31
1'43 3'83 2'74
2'4 1 3'12 2,80
Growth and aflato xin production of A. fiavus Table
2.
M ean levels (p .p.b.) of total afl atoxins produ ced by A. flavus PIL temperature and wa ter activ ity
110
various combinations of
Water activity Temp (0C) 10 15 20 25 30 35 37 '5 40 '0 42 '5
0·80 0 0 0 0 0 0 0 0 0
0 .825
0 '85
0 '875
0 '90
0 0 0 0 14 8 3 1 0
0 0 0 ot 261 14 12 47 8 0
0 0 0 3 33 2 1852 107 15 0
0 0 4 71 69 14 27 83 86 4 9 1
0'9 25 0 0* 33 2 1892 5884 1708 124 3 1
0 '95 0 3 3 12 3440 8042 2216 2600 2 0
0'9 75 0 33 64 18230 2010 4 1842 216 3 0
* Toxin identified after 61 days storage. t 0 = Trace (less than 1 p.p.b. ) identified after 70' days .
for anyone condition. For a number of temperature/a w combinations indicated in the table by the letter ' G', germination ofinoculum spores occurred but no further extension of mycelium took place. In other instances germination and growth took place only after 30 days, the normal duration of the storage period. In this table these instances are recorded as negative for growth even though appreciable growth may have taken place in the following weeks as occurred on rare occasions . At a given aw the growth rate increased with temperature reaching a maximum at 35° and then declined (Fig. 1). The minimum temperature that permitted growth was 15° but only when the a w was 0'92 or higher. At the highest temperature tested the aw needed to be 0'875 or higher. At the lower temperatures, 15° and 20° growth rates increased with increasing a u> up to 0'975; no maximum value was in fact demonstrated. At temperatures above 25° however, the growth rate again increased but reached a clear maximum at 0'95 a w before declining at the higher aw • A small amount ofgrowth occurred even at 0·80 a w but only at the mid range temperatures, 30° and 35°. Two typical growth rate curves are plotted in Fig. 1 (a), for an aw of 0'95 and for a temperature of 35°. From a series of such graphs the combination of temperature and r .h . giving growth rates in whole number steps of from 1'0 to 10 rum/day were calculated and used to construct the isopleth in Fig. 2 from which the conditions needed to sustain particular growth rates can be read or in terpolated .
Effect of temperature and relative humidity on aflatoxin production
For Aspergillus flauu s PIL 110 as for all other strains aflatoxins B I and B 2 were the only toxins isolated. Toxins formed at temperatures between 15° and 42 '5° depending on the a w and at 0·82 a w and above depending on the temperature. The overall mean levels of total aflatoxins are given in Table 2. At 15° aflatoxins formed within the 30 days storage period only when the a w was 0'95 or higher. At an aw of 0'925 no toxins were formed at this temperature within 30 days but they were detected after 61 days (not shown). At 20° the a w needed to be 0'90 or higher for toxin formation . At temperatures between 25° and 40° toxin formed at a w s above 0'825 but at 42 '5° only traces formed between 0 '90 and 0'95 a w ' At 10° and at 0·80 a w ' no toxin formed at all. Typical effects of temperature and aware illustrated in Fig. 1 (b). Maximum toxin yields were obtained at differing temperatures between 25 and 35° and in general the optimal a w s are those nearest 1 ' 0 . Barley as substrate. Growth rates and aflatoxin levels were similar and followed similar trends in wheat and barley in all those conditions in which barley was also used as substrate. Table 3 shows the results obtained for the two substrates at 0'95 aw ' Unlike wheat, the maximum mean growth in barley at this a w was obtained at 37'5° although the rate is almost ident ical with that at 35°. Temperatures at which max imum aflatoxin accumulated also differed, 25° in wheat and 30° in barley .
E. V. Niles, Joarina A. Norman and D. Pimbley , Growth rate at 35° , Growth rate at 0·95 a w
(a)
,Aflatoxin (log + I p.p.b.) at 35° , Aflatoxin (log + 1 p.p.b.) at 0·95 aw
(b)
11 10
4·0
v\
\
9
/
i
I
.
\ \
I
!
\ \
I I I
\
;
i
\
I I I
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3
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1·0
2
i i
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-_........i 5 10 15 20 25 30 35 40 45°C 0·800·820·840·860·880'900·92 0·940·960·98 aw
Fig.
I
\I
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i : \ I I
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:
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5 lO 15 20 25 30 35 40 45°C 0·80 0·82 0·84 0·86 0·880·900·92 0·940·96 0·98 aw
Growth rates (a) and aflatoxin levels (b) of A.fiavus PIL 110 in wheat at 35°C and various aw s and at 0'95 aw and various temperatures.
1.
1·00
0·95
o :Eo
...'"
~
~
0·90
0·85
10
IS
20
25 30 35 40 45 Temperature (OC) Fig. 2. Interpolated growth rates (solid line) of A. fiavus PIL 110 as influenced by temperature and aw ' Numbers refer to the daily rates in mm. Only the two points joined by the solid line formed the isopleth for 10 mmjday.
Growth and aflatoxin production of A. flavus Table 3. Growth rate (mmjd) and aflatoxin yields (p.p.b.) of A. flavus PIL 110 in wheat and barley at 0'95 a w Storage temperature
Growth rate in:
and 8'43 mmjday respectively, proved to be significantly different. DISCUSSION
Total aflatoxin in:
(0C)
Wheat
Barley
Wheat
Barley
10 15 20 25 30 35 37 4°'0 4 2'5
0 0,86 3"36 6'12 9'23 10,60 9'5 8 6'89 2,80
0 0,66 3'23 6"47 8'24 9'37 9"43 6'7 2 2,64
° 3 312 9 186 8042 2216 2600 2 T
0 T* 737 493 8 1133° 2158 1468 T T
* T = trace, less than 1 p.p.b,
Effect of temperature and relative humidity on different strains of A. flavus Growth rates and aflatoxin yields of the eight strains were compared in wheat and barley at a number of different temperature-relative humidity conditions. The means, obtained from 6 replicates are shown in Table 4 for three conditions in which all strains were tested simultaneously. Since there were no significant differences between wheat and barley as substrates, results for all eight fungal strains on either substrate are combined in Table 4,
Although PIL 110 consistently produced most toxin and PIL 442 consistently grew slowest, there were no other trends amongst the isolates. Analysis of variance for data in Table 4 showed no significant differences between the growth rates of any of the strains, However, in a separate test to compare growth ofPIL 110 and PIL 442 at 0'95 a w 35°, rates of 11'08,11'05 and 11'23 and of8'72, 8'77
The minimum temperature that permitted growth of Ai fiaous was 15° which was in fact extrapolated backwards to 13° but this required the aw to be over 0'90. This agrees with the findings of Northolt et al. (1976, 1977) and with Ayerst (1969) who grew this same isolate on wort agar strips. At 0'95 a w a limiting upper temperature was above the highest test temperature (42'5°) and could be extrapolated to 46°. For all isolates and for both substrates, whatever the relative humidity, the optimum growth rate was at 35°; only in barley at 0'95 a w was the growth at 37'5° marginally higher but almost identical with the rate at 35°. At the lower temperatures (15° and 20°) maximum growth occurred at the highest humidities but at temperatures between 25° and 42'5° maximum growth occurred at 0'95 a w ' Lotzsch & Trapper (1978) also noted that at temperatures between 25° and 35° A. flauus grew better at a lower aw (0'92) than at a higher one (0'99)· Since these inversions occur at favourable temperatures, including optimal temperature, as well as at limiting high temperatures, it is not at once apparent what feature if any of the water balance may be responsible. The obvious possibilities such as pH, texture or nutritional characteristics of the medium do not adequately explain the finding. Nor does it appear to be due to the method of growth since the phenomenon has been noticed whether growth has been determined as two dimensional radial growth, as dry weight from liquid media or as in our case extension of mycelium within a tube. A possible explanation lies in postulating that with increasingly favourable relative humidity-temperature conditions, additional hypal tips arise in the
Table 4. Comparison of growth rates and aflatoxin yields in eight strains of A. flavus PIL 110
PIL 365
PIL 378
PIL 295
PIL 377
P1L442
GRa AF
3'20
3'57
4'26
PIL 345 2,88
2'94
2'7 1
GRb AF
5'9° 2750
4'94 1
5'18 1116
5'88 2650
4'44 31
5'97 1
3"42 128
3'5 1 1250
GRc AF
7'99 2567
6'70 1
7"77 24
6,88 2000
6'94 29
6'74 1
5'11 146
7'14 2330
GRd AF
5'7° 2659
5'07 1
5'74 570
5'21 2325
4'77 30
5'14 1
3'59 137
4'52 1791
2'9 1
GR, Growth rate in mm per day; AF, total aflatoxin (B, + B 2) in p.p.b.; a and b, grown in wheat stored at 0,875 aw and 30°C and 0'85/35°, respectively; c, grown in barley stored at 90%/30°; d, average.
E. V. Niles, Joanna A. Norman and D. Pimbley (a)
(b)
_ _ _ , Growth rates in wheat _ - - - - , Growth rates in barley
II
Aflatoxin levels in wheat Aflatoxin levels in barley
4·0
10 9
>: '" "0
E -5 2: ...ee
8
,
! !
7
6
I
~ 5
\
I
..c
8
o
\
!
4
I I I
3
!
1·0
\I \ 10
15 20
25
30
35
40
45
Temperature (oC)
10
15
20
25
30
35
40
45
Temperature (oC)
Fig. 3. Growth rates (a) and aflatoxinlevels (b) of A.fiavus PIL 110 in wheat and barley ato'95 a w and various temperatures.
growing culture so as to maintain a steady growth rate. In time the 'density' of the culture limits oxygen diffusion which results in a slowing down of the growth. The effect of temperature and relative humidity operated differently on aflatoxin yields. The low temperatures (10°, 15°) and the high (40°, 42'50) restricted toxin production at all a w levels and optimum toxin production occurred between 25 and 35° although no single optimum temperature could be demonstrated as it could for growth. Lotzsch & Trapper (1978) also found optimum temperatures of 20° and 25° at 0'92 a w and Diener & Davis (1967) found in peanuts an optimum aw of 0'95 although Northolt et al. (1976, 1977) found in liquid media an optimum value of 0'99. It would seem, therefore, from our work and from these other reports that at a w s above about 0'90, any of the temperatures between 25 0 and 35° may stimulate maximum aflatoxin production. There was no equivalent reduction in toxin levels when the relative humidity increased from 95 to 97'5 % or higher. However, the combination ofhigh temperatures and any aw drastically reduced the toxin level (Fig.1b). Altogether, eight strains of A. fiavus were examined at some combination of temperature and relative humidity. There was no consistency in
ranking by growth rates and by aflatoxin yields. Two of the eight strains, PIL 365 and PIL 377 are weak producers of aflatoxin but this is not reflected in their growth rates. In wheat and barley, PIL 110 and PIL 378 have almost identical growth rates but their average aflatoxin yields differ markedly. In other isolates the toxin yields were, on the whole, consistent and similar in wheat and barley which could suggest that the depression of PIL 378 level at 0'90/30° was a substrate effect. There is not sufficient evidence to make this a strong assertion however, for although substrate is frequently mentioned as a factor influencing growth rate and aflatoxin production, there are few studies that contrast the effects of wheat and barley. Chang & Markakis (1982) found maximum elaboration of aflatoxin at 25° and 30° but not at 35° for barley with moisture contents of 20 and 25 %. The slight differences observed here in temperature optima for growth and aflatoxins in barley compared with wheat at 0.95 aw (Fig. 3) are probably not significant in view of the closeness of the two temperature optima and of the earlier assertion that above 0'90 a w ' maximum aflatoxin may be produced at any of the temperatures between 25 and 35°. Nevertheless, this observation could repay further study.
266
Growth and aflatoxin production of A. flavus
We would like to thank Mrs S. Henderson of Slough Laboratory for making the measurements of moisture contents of grains.
REFERENCES
ALLCROFT, R, CARNAGAN, R B. A., SARGEANT, K., & O'KELLY, J. (1961). A toxic factor in Brazilian groundnut meal. Veterinary Record 73, 428---9. AYERST, G. (1969). The effects of moisture and temperature on growth and spore germination in some fungi. Journal of Stored Products Research 5, 127-141. BEHERE, A. G., SHARMA, A., PADWALDESAI, S. R. & NADKARNI, G. B. (1978). Production of aflatoxins during storage of gamma-irradiated wheat. Journal of Food Science 43, 1102-1103. BEUCHAT, L. R. & LECHOWICH, R. V. (1970). Aflatoxin production on beans as affected by temperature and moisture content. Journal of Milk and Feed Technology 33, 373-376. BOLLER, R. A. & SCHROEDER, H. W. (1974). Influence of relative humidity on production of aflatoxin in rice by Aspergillus parasiticus. Phytopathology 64, 17-21. CHANG, H-G. & MARKAKIS, P. (1982). Effect of temperature on aflatoxin production. Korean Journal of Food Science Technology 14, 162-163. CHRISTENSEN, C. M. (Ed.) (1982). Storage of Cereals and their Products. American Association of Cereal Chemists Inc., St. Paul, Minnesota. DICKENS,J. W. & PATEE, H. E. (1966). The effects of time, temperature and moisture on aflatoxin production in peanuts inoculated with a toxic strain of A. ftavus. Tropical Science 8, 11-22. DIENER, U. L. & DAVIS, N. D. (1967). Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids by Aspergillus ftavus in sterile peanuts. Journal of the American Oil and Chemical Society 44, 259-263. DIENER, U. L. & DAVIS, N. D. (1969). Production of aflatoxin on peanuts under controlled experiments. Journal of Stored Products Research 5, 251-258. HORNER, K. J. & ANAGNOSTOPOULOS. (1973). Combined effects of water activity, pH and temperature on the growth and spoilage potential of fungi. Journal of Applied Bacteriology 36, 427-436. JEMMALI, M., POISSON, J. & GUILBOT, A. (1969). Aflatoxin production in cereal products. Effects of various conditions. Annales de la Nutrition et de I'alimentation 23, 151-166. LOTZSCH, R & TRAPPER, D. (1978). Production of aflatoxin and patulin as a function of water activity. Die Fleishsoirtschaft 12, 2001-2007.
NILES, E. V. (1978). Growth rate and aflatoxin production of Aspergillus ftavus in wheat sterilized by gammairradiation,ethyleneoxideandautoclaving. Transactions of the British Mycological Society 70, 239-247. NORTHOLT, M. D., VERHULSDONK, C. A. H., SOENTORO, P. S. S. & PAULSCH, W. E. (1976). Effect of water activity and temperature on aflatoxin production by Aspergillus parasiticus. Journal of Milk and Food Technology 39, 170-174. NORTHOLT,M. D., VANEGMOND,H. P.&PAULSCH, W. E. (1977). Differences between Aspergillusftavus strains in growth and aflatoxin B, production in relation to water activity and temperature. Journal of Food Protection 40, 778-781. NUSRATH, M. (1978). Effect of moisture and incubation period on aflatoxin B, formulation in pigeon pea. National Academy of Science Letters 1,431-32. ORTH, R (1976). The influence of water activity on spore germination of aflatoxin, sterigmatocystin and patulin producing moulds. Lebensmittel- Wissenschaft und Technologie 9, 156-159. OXLEY, T. A., PIXTON, S. W. & HOWE, R. W. (1960). Determination of moisture contents in cereals. I. Interaction of type of cereal and oven method. Journal of the Science of Food and Agriculture 11, 18-25. PIXTON, S. W. & WARBURTON, S. (1971). Moisture content/relative humidity equilibrium of some cereal grains at different temperatures. Journal of Stored Products Research 6, 283-293. RABIE, C. J. & SMALLEY, E. B. (1965). Influence of temperature on the production of aflatoxin by A. ftavus. In Symposium on Mycotoxins in Foodstuffs. South African Department of Agriculture and Technical Services, Pretoria. REISS, J. (1978). Mycotoxins in foodstuffs. XII. The influence of water activity aw of cakes on the growth of moulds and the formation of mycotoxins. Zeitschrift fur Lebensmittel und Forschung 167,419-422. SCHINDLER, A. F. (1977). Temperature limits for production of aflatoxin by twenty five isolates of Aspergillus ftavus and Aspergillus parasticus. Journal of Food Protection 40, 39-40. SHIH, C. N. & MARTH, E. H. (1975). Production of aflatoxin and its partition between the medium and the mycelium of A. parasiticus during incubation under various conditions. Zeitschrift fur Lenbensmittelsuchung und Forschung 158, 215-224. SHIH, C. N. & MARTH, E. H. (1974). Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus. Applied Microbiology 27,452-456. SOLOMON, M. E. (1951). Control of humidity with potassium hydroxide, sulphuric acid, or other solutions. Bulletin of Entomological Research 42, 543-554.
(Received for publication 15 September 1983; revised 3 July 1984)
Printed in Great Britain
GROWTH AND AFLATOXIN PRODUCTION OF ASPERGILLUS FLAVUS IN WHEAT AND BARLEY By E. V. NILES, JOANNA A. NORMAN* AND D. PIMBLEYt MAFF, Olantigh Road, Wye, Ashford, Kent, *Slough Laboratory, London Road, Slough, SL3 7HJ, Berkshire and tBlock 2 Government Buildings, Lawnswood, Leeds LS16 spy Growth rates and aflatoxin production of eight strains of Aspergillus fiavus grown in wheat and barley were studied over temperature and water activity (a w ) ranges of 10-42'5 °C and 0'80-0'975 respectively. Growth and aflatoxin production were restricted by low temperature, low a w and high temperature but not by high aw ' At 15° the lowest temperature at which growth occurred, an aw of 0.95 was needed for growth. At 42.5° growth was severely restricted and aflatoxin production altogether inhibited. The optimum temperature for growth was 35°; at lower temperatures (15°, 20°) the optimum aw was> 0.975, but at temperatures above these, it was 0'95. No single optimum temperature for aflatoxin production was determined but at aw s over 0'90 it lay between 25° and 35°. Strains showed marked differences in ability to produce aflatoxins but no equivalent differences in their growth rates. Only at the most favourable condition was a significant difference in growth demonstrated between two isolates at the extreme ends of the scale. There were not significant differences in growth or aflatoxin production between wheat and barley as substrates. For the fungi which occur in stored products those factors most significantly affecting their growth and other physiological behaviour are water activity (a w ) ' temperature and nature of the substrate (Ayerst, 1969; Horner & Anagnostopolous, 1973; Northolt et al., 1976; Diener & Davis, 1969; Dickens & Patee, 1966). Since the role of Aspergillus fiavus in the pathogenesis of Turkey X disease was identified some 22 years ago (Allcroft et al., 1961) a considerable amount of information has been gathered on the conditions necessary for growth and production of aflatoxins by the causal organisms A.fiavus and A. parasiticus. The various controlling factors interact in such a way as to make simple statements such as 'the temperature range for aflatoxin production is between 20 and 35°, (Beuchat & Lechowich, 1970) or 'maximum aflatoxin occurs by about 12 days' (Christensen, 1982) inadequate and incomplete. Lower limiting temperatures for aflatoxin production are given as ~ = 7'5° on wort agar medium (Schindler, 1971) or as 10° in malt extract glucose agar (Northolt et al., 1976). Temperatures of 13° in maize and wheat (Jemmali et al., 1969; Behere et al., 1978) and of at least 13'5° or higher in barley (Chang & Markakis, 1982) are indicated for cereals. The maximum temperature permitting aflatoxin development is generally given as 40° but Rabie & Smalley (1965) found that toxin was elaborated at 42° and Shih &
Marth (1974, 1975) claimed to have demonstrated small amounts of toxin at 45°. However, Ayerst's work showed a limiting temperature for growth of A. fiavus of 43°. Reported lower limits of a w for growth and aflatoxin development are at least as variable. Reiss (1978) found that spores of A. fiavus were unable to germinate at an a w of 0·82 and Orth (1976) determined an aw limit of 0·84 for spores of A. fiavus and A. parasiticus; yet Latsch & Trapper (1978) obtained growth in several and toxin production in three out of ten isolates of A. fiavus and A. parasiticus tested on malt yeast glucose nutrient media at an aw of 0·82. Northolt et al. (1976) were unable to detect aflatoxin at a water activity of 0'83 and temperature of 10°. Similarly, jemmali et al. (1969) found there was not growth of A. fiavus even at 0'85 a w when the temperature was 13° while Nusrath (1978) records for pigeon peas maximum yields of aflatoxin at 0·80 a w and 25°, conditions in which most reports suggest A. fiavus should not grow let alone produce aflatoxin. In general, increasing the a w promotes faster growth and greater toxin production (Boller & Schroeder, 1974; Chang & Markakis, 1982) but a number of reports suggest that the optima for growth and toxin production can be obtained at a w s less then the achievable maximum (Diener & Davis, 1967; Lotzsch & Trapper, 1978). Not only
260
Growth and aflatoxin production of A. flavus
are these optimal values dependent on the interaction of temperature and water activity but also on the strain and cultivar differences of organism and substrate (N ort holt et al., 1977; Chang & Markakis, 1982). In a previous paper Niles (1978) showed how the growth rates and aflatoxin production of a strain of A. flavus were also influenced by the method of sterilization of the wheat used as substrate. In this study growth and aflatoxin production were measured over combinations of a wide range of temperature and a w on the same isolate and other isolates grown axenically in pre-sterilized wheat and barley so as to be able to predict growth and the likelihood of toxin production in any combination of the two principal factors in cereals. Close attention was paid to achieving and maintaining target a w levels. MA TERIALS AND METHODS
Test cultures and inoculum The work was done principally with a strain of A. flavus Link: Fries (P I L 110) used in the previous study and derived originally in 1962 from Brazilian groundnuts. Partial studies were conducted on 7 other isolates: PIL 295, PIL 442 from U.K. barley which had been undertreated with propionic acid; PIL 345, PIL 378, PIL 377, PIL 365 and S 922 all from a number of groundnut products used in animal feeds. All cultures were retained as freeze dried isolates and grown as required on thin plates of 2 % malt agar for 2-4 days at 25°. For inoculum a single 5'0 mm disc of the young culture was cut with a sterile cork borer for transfer to the grain. Preparation of substrate Wheat was the principal substrate. Several kilograms of the variety Maris Widgeon, collected at the beginning of the 1980 harvest were dried to 11'0 % moisture and held in a deep freeze at -12° to - 15°. Stocks of barley of unknown variety were also collected, dried to about 12 % and similarly stored in the deep freeze. When required, quantities of grain were withdrawn and the moisture content determined on 10 g lots by drying at 113° for 4 h in a mechanically ventilated oven (Oxley, Pixton & Howe, 1960) . Grain was accordingly conditioned (Niles, 1978) to the required moisture levels by addition of appropriate volumes of water. After the equilibrium period the equilibrium relative humidity (e.r .h .) was determined using a dew point method (P ixton & Warburton, 1971), at the intended storage temperature except when the latter was 40° or 42 '5°, e.r.h. was then determined also at 35°. Between 40 and 50 g of conditioned
grain was then packed into 250 x 25'0 mm borosilicate glass tubes, plugged with cotton wool at both ends and steam sterilized in an autoclave at 121° for 1 h. After removal from the autoclave, tubes of grain were held for 48 h over solutions of KOH in equilibrium with the target relative humidity. The moisture content of grains was again determined prior to inoculation, and about halfway through and at the end of the incubation period to ensure that no significant losses in the moisture levels of the grains had occurred at any stage.
Inoculation and incubation The column of grain in the tube was inoculated centrally at one end with the 5'0 mm disc of the test culture grown for 2-4 days. A minimum of 6 replicates were inoculated each time for whatever condition was being tested. Tubes were then placed on a perforated metal platform over an equilibrium solution of KOH (Solomon, 1951) contained in a sealed glass tank measuring 440 x 300 x 280 mm. The glass tanks and contents were stored in constant temperature and relative humidity room or in incubators giving maximum temperature fluctuations of ±0'5° (mean fluctuation 0'2°). Incubation was continued usually for 30 days and during this time tanks were ventilated by removing the lids for several minutes every few days.
Observation and measurement of growth Observations were made at more or less daily intervals until mycelium arising from the growing inoculum appeared at the grain surface. The time for this to happen depended on the test condition, being 2-3 days in the most favourable situations and several weeks or not at all in less favourable ones. Once growth had appeared, it was measured at frequent intervals depending on the speed of growth using a stereoscopic microscope with a specially modified stage (Niles, 1978). The mycelium was measured along the lines previously marked on opposite sides of the glass tube and the mean length then plotted against the period of growth in days . A regression line was drawn for the period between the first and the last set of measurements and from this the slope or growth rate determined.
Analysis for aflatoxin After the final growth measurement was made analysis for the presence of the aflatoxins HI' B 2, G 1 and G 2 was carried out on replicate tube cultures. A total of between 6 and 21 analyses were carried
E. V. Niles, Joanna A. Norman and D. Pimbley out for each temperaturc/c., combination. Infected grain was weighed into a plastic bag, mixed well and a 10 g sample put into a 250 ml conical flask. Chloroform (100 ml) was added to this followed by 10 ml of water. The flask was then shaken for 30 min on a rotary shaker, The liquid was filtered off through NaS0 4 on Whatman No. 41 filter paper, The grain residue was then washed three times in 250 ml lots of chloroform. The whole extract was transferred to a rotary evaporator and reduced to dryness, This residue was taken up in chloroform and transferred to a large screw cap phial in which it was again reduced to dryness and finally made up to 5 ml with chloroform. Aliquots of the extract along with standards were spotted for z-dimensional chromatographic separation on to 10 X 10 mm cuts of Merck 5535 aluminium backed silica gel TLC plates (Merck & Co. Ltd), The spots consisting of 5 p;! of the test extract and 1, 2,4, and 6 pi of the aflatoxin standard containing 0' 5 pgjpi were run first in diethyl ether: methanol: water
Table
1.
261
(94:4'5: 1'5) then in a second solvent system
consisting of toluene, ethyl acetate and formic acid (60: 30: 10), The first tank was lined and equilibrated
the second was not. Chromatograms were developed and examined under long wave (360 nm) and short wave (250 nm) u.v. light, Quantification was obtained by matching fluorescence intensities of test extracts and standards,
RESUL TS
Effect of temperature and relative humidity on growth rates Table 1 shows for all combinations of temperature and aw the minimum, maximum and mean growth rates obtained for Aspergillus fiavus PIL 110 in wheat. These values are based on from 6 to 24 replicates. Tests for variation either within or between experiments consistently showed that there were no significant differences in growth rates
Minimum, maximum and mean growth rates (mmjd) of Aspergillus flavus PIL combinations of temperature and water activity
110
at various
Water activity Temp C°C)
0,80
0'8 25
0'85
0'875
0'90
10
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0'9 25 0 0 0
0'95 0 0 0
0'975 0 0 0
15
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0'18 0'05
0 0'21 0,86
1'95 2'21 1'93
20
0 0 0
0 0 0
0 0 0
0'10 0'40 0'25
0'97 2'3 1 1'5 0
1'7 0 2'32 2'16
2'89 3'59 3'3 6
25
0 0 0
G
1'10 2'3 0 1,66
1'97 3'40 2'15
3'08 3'56 3'41
5'82 6'7 2 6'12
30
0'00 0'44 0'17
0,66 1'3 1 0'9 8
2'7 1 3'21 2,82
3'01 3'35 3'22
9'23 10'05 9'23
8'45 9'43 8'84
35
0'24 0,80 0'36
0'44 2'18 1'41
4'5 8 5'5 0 5'08
10'25 10'93 10'60
9'30 10'20 9'75
37'5
G
G
3'59 4'12 4'00
5'15 6'20 5'79 3'86 5'63 4'79
5'50 6'39 5'99 7'20 8'20 7'73 6'23 7'16 6'74
4'66 5'66 5'09 8'7 1 9'12 8'78 9'5 1 10'40 9'85
4'00 4'66 4'27 5'18 6'10 5'52
6'32 8'04 7'25
40'0
0'00
G
0'46 2'90 1'4 6
4'44 6'12 5'20
4 2'5
0'00
0'0
G
0'0
0'0
0'7 2 1'64 1'14
0'00
3'11 3'81 3'44 2,61 3'3 6 3'01
8'54 9'00 8'70
5'13 6'08 5'57
6'22 7'00 6'73
8'57 10'70 9'5 8 6'00 7'70 6'89
2'35 3'75 3'31
1'43 3'83 2'74
2'4 1 3'12 2,80
Growth and aflato xin production of A. fiavus Table
2.
M ean levels (p .p.b.) of total afl atoxins produ ced by A. flavus PIL temperature and wa ter activ ity
110
various combinations of
Water activity Temp (0C) 10 15 20 25 30 35 37 '5 40 '0 42 '5
0·80 0 0 0 0 0 0 0 0 0
0 .825
0 '85
0 '875
0 '90
0 0 0 0 14 8 3 1 0
0 0 0 ot 261 14 12 47 8 0
0 0 0 3 33 2 1852 107 15 0
0 0 4 71 69 14 27 83 86 4 9 1
0'9 25 0 0* 33 2 1892 5884 1708 124 3 1
0 '95 0 3 3 12 3440 8042 2216 2600 2 0
0'9 75 0 33 64 18230 2010 4 1842 216 3 0
* Toxin identified after 61 days storage. t 0 = Trace (less than 1 p.p.b. ) identified after 70' days .
for anyone condition. For a number of temperature/a w combinations indicated in the table by the letter ' G', germination ofinoculum spores occurred but no further extension of mycelium took place. In other instances germination and growth took place only after 30 days, the normal duration of the storage period. In this table these instances are recorded as negative for growth even though appreciable growth may have taken place in the following weeks as occurred on rare occasions . At a given aw the growth rate increased with temperature reaching a maximum at 35° and then declined (Fig. 1). The minimum temperature that permitted growth was 15° but only when the a w was 0'92 or higher. At the highest temperature tested the aw needed to be 0'875 or higher. At the lower temperatures, 15° and 20° growth rates increased with increasing a u> up to 0'975; no maximum value was in fact demonstrated. At temperatures above 25° however, the growth rate again increased but reached a clear maximum at 0'95 a w before declining at the higher aw • A small amount ofgrowth occurred even at 0·80 a w but only at the mid range temperatures, 30° and 35°. Two typical growth rate curves are plotted in Fig. 1 (a), for an aw of 0'95 and for a temperature of 35°. From a series of such graphs the combination of temperature and r .h . giving growth rates in whole number steps of from 1'0 to 10 rum/day were calculated and used to construct the isopleth in Fig. 2 from which the conditions needed to sustain particular growth rates can be read or in terpolated .
Effect of temperature and relative humidity on aflatoxin production
For Aspergillus flauu s PIL 110 as for all other strains aflatoxins B I and B 2 were the only toxins isolated. Toxins formed at temperatures between 15° and 42 '5° depending on the a w and at 0·82 a w and above depending on the temperature. The overall mean levels of total aflatoxins are given in Table 2. At 15° aflatoxins formed within the 30 days storage period only when the a w was 0'95 or higher. At an aw of 0'925 no toxins were formed at this temperature within 30 days but they were detected after 61 days (not shown). At 20° the a w needed to be 0'90 or higher for toxin formation . At temperatures between 25° and 40° toxin formed at a w s above 0'825 but at 42 '5° only traces formed between 0 '90 and 0'95 a w ' At 10° and at 0·80 a w ' no toxin formed at all. Typical effects of temperature and aware illustrated in Fig. 1 (b). Maximum toxin yields were obtained at differing temperatures between 25 and 35° and in general the optimal a w s are those nearest 1 ' 0 . Barley as substrate. Growth rates and aflatoxin levels were similar and followed similar trends in wheat and barley in all those conditions in which barley was also used as substrate. Table 3 shows the results obtained for the two substrates at 0'95 aw ' Unlike wheat, the maximum mean growth in barley at this a w was obtained at 37'5° although the rate is almost ident ical with that at 35°. Temperatures at which max imum aflatoxin accumulated also differed, 25° in wheat and 30° in barley .
E. V. Niles, Joarina A. Norman and D. Pimbley , Growth rate at 35° , Growth rate at 0·95 a w
(a)
,Aflatoxin (log + I p.p.b.) at 35° , Aflatoxin (log + 1 p.p.b.) at 0·95 aw
(b)
11 10
4·0
v\
\
9
/
i
I
.
\ \
I
!
\ \
I I I
\
;
i
\
I I I
\ I
3
I ;
!
1·0
2
i i
I
-_........i 5 10 15 20 25 30 35 40 45°C 0·800·820·840·860·880'900·92 0·940·960·98 aw
Fig.
I
\I
\
!
\
i : \ I I
i
:
\
\
5 lO 15 20 25 30 35 40 45°C 0·80 0·82 0·84 0·86 0·880·900·92 0·940·96 0·98 aw
Growth rates (a) and aflatoxin levels (b) of A.fiavus PIL 110 in wheat at 35°C and various aw s and at 0'95 aw and various temperatures.
1.
1·00
0·95
o :Eo
...'"
~
~
0·90
0·85
10
IS
20
25 30 35 40 45 Temperature (OC) Fig. 2. Interpolated growth rates (solid line) of A. fiavus PIL 110 as influenced by temperature and aw ' Numbers refer to the daily rates in mm. Only the two points joined by the solid line formed the isopleth for 10 mmjday.
Growth and aflatoxin production of A. flavus Table 3. Growth rate (mmjd) and aflatoxin yields (p.p.b.) of A. flavus PIL 110 in wheat and barley at 0'95 a w Storage temperature
Growth rate in:
and 8'43 mmjday respectively, proved to be significantly different. DISCUSSION
Total aflatoxin in:
(0C)
Wheat
Barley
Wheat
Barley
10 15 20 25 30 35 37 4°'0 4 2'5
0 0,86 3"36 6'12 9'23 10,60 9'5 8 6'89 2,80
0 0,66 3'23 6"47 8'24 9'37 9"43 6'7 2 2,64
° 3 312 9 186 8042 2216 2600 2 T
0 T* 737 493 8 1133° 2158 1468 T T
* T = trace, less than 1 p.p.b,
Effect of temperature and relative humidity on different strains of A. flavus Growth rates and aflatoxin yields of the eight strains were compared in wheat and barley at a number of different temperature-relative humidity conditions. The means, obtained from 6 replicates are shown in Table 4 for three conditions in which all strains were tested simultaneously. Since there were no significant differences between wheat and barley as substrates, results for all eight fungal strains on either substrate are combined in Table 4,
Although PIL 110 consistently produced most toxin and PIL 442 consistently grew slowest, there were no other trends amongst the isolates. Analysis of variance for data in Table 4 showed no significant differences between the growth rates of any of the strains, However, in a separate test to compare growth ofPIL 110 and PIL 442 at 0'95 a w 35°, rates of 11'08,11'05 and 11'23 and of8'72, 8'77
The minimum temperature that permitted growth of Ai fiaous was 15° which was in fact extrapolated backwards to 13° but this required the aw to be over 0'90. This agrees with the findings of Northolt et al. (1976, 1977) and with Ayerst (1969) who grew this same isolate on wort agar strips. At 0'95 a w a limiting upper temperature was above the highest test temperature (42'5°) and could be extrapolated to 46°. For all isolates and for both substrates, whatever the relative humidity, the optimum growth rate was at 35°; only in barley at 0'95 a w was the growth at 37'5° marginally higher but almost identical with the rate at 35°. At the lower temperatures (15° and 20°) maximum growth occurred at the highest humidities but at temperatures between 25° and 42'5° maximum growth occurred at 0'95 a w ' Lotzsch & Trapper (1978) also noted that at temperatures between 25° and 35° A. flauus grew better at a lower aw (0'92) than at a higher one (0'99)· Since these inversions occur at favourable temperatures, including optimal temperature, as well as at limiting high temperatures, it is not at once apparent what feature if any of the water balance may be responsible. The obvious possibilities such as pH, texture or nutritional characteristics of the medium do not adequately explain the finding. Nor does it appear to be due to the method of growth since the phenomenon has been noticed whether growth has been determined as two dimensional radial growth, as dry weight from liquid media or as in our case extension of mycelium within a tube. A possible explanation lies in postulating that with increasingly favourable relative humidity-temperature conditions, additional hypal tips arise in the
Table 4. Comparison of growth rates and aflatoxin yields in eight strains of A. flavus PIL 110
PIL 365
PIL 378
PIL 295
PIL 377
P1L442
GRa AF
3'20
3'57
4'26
PIL 345 2,88
2'94
2'7 1
GRb AF
5'9° 2750
4'94 1
5'18 1116
5'88 2650
4'44 31
5'97 1
3"42 128
3'5 1 1250
GRc AF
7'99 2567
6'70 1
7"77 24
6,88 2000
6'94 29
6'74 1
5'11 146
7'14 2330
GRd AF
5'7° 2659
5'07 1
5'74 570
5'21 2325
4'77 30
5'14 1
3'59 137
4'52 1791
2'9 1
GR, Growth rate in mm per day; AF, total aflatoxin (B, + B 2) in p.p.b.; a and b, grown in wheat stored at 0,875 aw and 30°C and 0'85/35°, respectively; c, grown in barley stored at 90%/30°; d, average.
E. V. Niles, Joanna A. Norman and D. Pimbley (a)
(b)
_ _ _ , Growth rates in wheat _ - - - - , Growth rates in barley
II
Aflatoxin levels in wheat Aflatoxin levels in barley
4·0
10 9
>: '" "0
E -5 2: ...ee
8
,
! !
7
6
I
~ 5
\
I
..c
8
o
\
!
4
I I I
3
!
1·0
\I \ 10
15 20
25
30
35
40
45
Temperature (oC)
10
15
20
25
30
35
40
45
Temperature (oC)
Fig. 3. Growth rates (a) and aflatoxinlevels (b) of A.fiavus PIL 110 in wheat and barley ato'95 a w and various temperatures.
growing culture so as to maintain a steady growth rate. In time the 'density' of the culture limits oxygen diffusion which results in a slowing down of the growth. The effect of temperature and relative humidity operated differently on aflatoxin yields. The low temperatures (10°, 15°) and the high (40°, 42'50) restricted toxin production at all a w levels and optimum toxin production occurred between 25 and 35° although no single optimum temperature could be demonstrated as it could for growth. Lotzsch & Trapper (1978) also found optimum temperatures of 20° and 25° at 0'92 a w and Diener & Davis (1967) found in peanuts an optimum aw of 0'95 although Northolt et al. (1976, 1977) found in liquid media an optimum value of 0'99. It would seem, therefore, from our work and from these other reports that at a w s above about 0'90, any of the temperatures between 25 0 and 35° may stimulate maximum aflatoxin production. There was no equivalent reduction in toxin levels when the relative humidity increased from 95 to 97'5 % or higher. However, the combination ofhigh temperatures and any aw drastically reduced the toxin level (Fig.1b). Altogether, eight strains of A. fiavus were examined at some combination of temperature and relative humidity. There was no consistency in
ranking by growth rates and by aflatoxin yields. Two of the eight strains, PIL 365 and PIL 377 are weak producers of aflatoxin but this is not reflected in their growth rates. In wheat and barley, PIL 110 and PIL 378 have almost identical growth rates but their average aflatoxin yields differ markedly. In other isolates the toxin yields were, on the whole, consistent and similar in wheat and barley which could suggest that the depression of PIL 378 level at 0'90/30° was a substrate effect. There is not sufficient evidence to make this a strong assertion however, for although substrate is frequently mentioned as a factor influencing growth rate and aflatoxin production, there are few studies that contrast the effects of wheat and barley. Chang & Markakis (1982) found maximum elaboration of aflatoxin at 25° and 30° but not at 35° for barley with moisture contents of 20 and 25 %. The slight differences observed here in temperature optima for growth and aflatoxins in barley compared with wheat at 0.95 aw (Fig. 3) are probably not significant in view of the closeness of the two temperature optima and of the earlier assertion that above 0'90 a w ' maximum aflatoxin may be produced at any of the temperatures between 25 and 35°. Nevertheless, this observation could repay further study.
266
Growth and aflatoxin production of A. flavus
We would like to thank Mrs S. Henderson of Slough Laboratory for making the measurements of moisture contents of grains.
REFERENCES
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(Received for publication 15 September 1983; revised 3 July 1984)