Mould infection and aflatoxin contamination ofthe peanut kernels harvested from spring andfall crops as affected by artificial inoculation ofthe seeded kernels with Aspergillus ¯avus andAspergillus nigerRobin Y-Y Chiou,1* Y-Y Wen,2 S Ferng1 and SP Learn1
1Department of Food Science, National Chiayi Institute of Technology, Chiayi, Taiwan2Department of Agronomy, National Chiayi Institute of Technology, Chiayi, Taiwan
Abstract: Spring and fall crops of peanut are grown each year in Taiwan. Mould infection and a¯atoxin
contamination of crops as affected by arti®cial inoculation of the seeded kernels with conidia of
Aspergillus ¯avus, A niger and a combination of A ¯avus and A niger (inocula>105 CFU kernelÿ1)
were determined. Three cultivars, ie Tainan 9, Tainan 11 and Tainan 12, were consecutively grown for
fall 1996, spring 1997, fall 1997 and spring 1998 crops with green vegetable pea for rotation. In crops
from uninoculated kernels (control), percentages of germination and harvested plants were higher in
spring crops than in fall crops. Inoculation with A niger alone or with A ¯avus resulted in various levels
of seed and seedling mortality and lower yields of peanut pods than yields of the other inoculation
treatments. When harvested and sized (US No 1), kernels were subjected to examination for mould
colonisation and analysis of a¯atoxin content. Fairly low percentages were colonised and a¯atoxin
contents were low or non-detectable. Colonisation and a¯atoxin content were independent of arti®cial
inoculation. Average a¯atoxin contents in the kernels harvested from all cultivars and crops ranged
from 0 to 6.1mgkgÿ1. However, the highest levels of a¯atoxin content among samples of the four crops
were 4.0, 18.2, 9.6 and 36.7mgkgÿ1, respectively.
# 1999 Society of Chemical Industry
Keywords: peanut; a¯atoxin; mould colonisation; arti®cial inoculation; A ¯avus; A niger
INTRODUCTIONSpring and fall crops of peanut are grown each year in
Taiwan. As a general practice, spring crops are planted
in spring and harvested in summer and fall crops are
planted in fall and harvested in winter. The weather
pattern during the planting period of spring and fall
crops varies in a reverse manner.1,2 As a traditional
view, mainly based on roasted peanut ¯avour pre-
ference, consumers believe that the quality of the
kernels of fall crops is superior to that of spring crops.3
From the viewpoint of food safety, a¯atoxin contam-
ination of peanuts poses a serious health hazard to
humans.4 In particular, prevention of infection of
peanuts resulting in preharvest a¯atoxin contamina-
tion (PAC) is a challenging problem for production of
peanut products of high quality. In Taiwan, peanut
seeds harvested from one crop are used as seeds for the
next crop. Thus, the quality of harvested peanut
kernels, regardless of their destination for seeding or
for food use, needs further research attention.
The presence of both Aspergillus ¯avus and A niger is
not uncommon in peanut kernels.5 Antagonistic
interaction between A ¯avus and A niger in peanuts
was reported by Joffe.6 A niger is the causal organism of
Aspergillus crown rot of peanut.7 Seedlings and young
plants are susceptible to A niger infection, which
usually results in high mortality rates. The interacting
effects of A ¯avus and A niger on peanut productivity
and PAC of the harvested kernels have received
meagre attention. Information regarding the effect of
the growing season on the interaction between A ¯avusand A niger is limited. In this study, sound peanut
kernels of three cultivars were arti®cially inoculated
with A ¯avus, A niger and a combination of A ¯avusand A niger, and seeded in an experimental ®eld with
vegetable green pea for rotation. Four crops, consist-
Journal of the Science of Food and Agriculture J Sci Food Agric 79:1417±1422 (1999)
* Correspondence to: Robin Y-Y Chiou, Department of Food Science, National Chiayi Institute of Technology, Chiayi, TaiwanE-mail: [email protected]/grant sponsor: National Science Council, Republic of China; contract/grant number: NSC86-2321-B021-013; contract/grant number:NSC 87-2321-B021-020(Received 12 October 1998; revised version received 8 March 1999; accepted 6 April 1999)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1417
ing of fall 1996, spring 1997, fall 1997 and spring
1998, were grown. Percentages of seed germination
and harvested plant in proportion to the seeded
kernels, yields expressed as production of dried peanut
pods per unit area and production of the sized (US No
1) kernels in proportion to the weight of dried peanut
pods were determined. Mould colonisation and
a¯atoxin contamination of the harvested kernels were
subsequently examined.
MATERIALS AND METHODSInoculum preparation and artificial inoculation of theseeded kernelsA ¯avus ATCC 3616 and a strain of A niger originally
isolated from peanut kernels were used as inocula.8
Inoculum suspension was prepared by transferring a
loopful of conidia from a slant culture into a ¯ask
(500ml) containing 200ml of Aspergillus ¯avus/parasiticus broth (AFPB, containing 10g litreÿ1 of
bacteriological peptone, 20g litreÿ1 of yeast extract,
0.5g litreÿ1 of ferric ammonium citrate, 0.2g litreÿ1 of
chloramphenicol and 1ml litreÿ1 of a 2g litreÿ1
dichloran stock solution in ethanol),9 followed by
incubation without agitation at 28°C for 10 days.
Then the contents of ¯asks were vigorously shaken to
prepare inoculum suspensions. For preparing the dual
inoculum of A ¯avus and A niger, two separately
incubated cultures were mixed and shaken vigorously
before use.
Along with the routine seeding practice, each kernel
was dipped in a speci®ed inoculum suspension, seeded
in a plot and fully covered with soil for cultivation.
Number of colony forming units (CFU) of the inocula
were estimated by placing the dipped kernels in a series
of 1g litreÿ1 peptone solutions and further diluting
appropriately prior to surface spreading on Aspergillus¯avus/parasiticus agar (AFPA, containing AFPB
described above and 15g litreÿ1 of agar).9 The
numbers of CFU were enumerated after incubation
at 28°C for four days. The inoculum levels among the
three cultivars and four crops of fall 1996, spring 1997,
fall 1997 and spring 1998 were 1.0±8.3�105 CFU
kernelÿ1 for A ¯avus, 2.6±7.7�105 CFU kernelÿ1 for
A niger and 1.1±6.1�105 CFU kernelÿ1 for the
combination of A ¯avus and A niger, respectively.
Peanut cultivation and assessment of agronomiccharacteristicsThree cultivars, ie Tainan 9, Tainan 11 and Tainan 12
(Spanish cv), were planted in an experimental ®eld
(0.1ha) located in Sueshan, Chiayi, for all four crops.
Vegetable green pea as a rotation crop was planted
concurrently in half of the area of the ®eld. The
cultivation periods were from 3 September to 10
December 1996; from 25 February to 15 June 1997;
from 23 September to 23 December 1997 and from 9
March to 20 July 1998. Sound and sized (1.92�0.6cm opening for US No 1 Spanish standard) kernels
harvested from the former crops without arti®cial
inoculation (control) were arti®cially inoculated with
A ¯avus, A niger or A ¯avus/A niger and planted for
®eld cultivation. During seeding, each kernel was
dipped in a speci®ed inoculum suspension and seeded
in a four-row plot (2�4m) with one seed per 10cm. In
total, 160 kernels were cultivated in a plot. Triplicate
plots for a cultivar in each treatment block with a
completely randomised design among cultivars were
conducted. The block nearest to the water source for
irrigation was seeded without arti®cial inoculation as a
control for comparison. Blocks followed in order were
seeded with kernels inoculated with A niger, A ¯avus/Aniger and A ¯avus to minimise cross-contamination of
A ¯avus during ¯ood irrigation.
Peanuts were cultivated using normal agronomic
practices and harvested 70 days after ¯owering.
Percentages of seed germination and harvested plant
in proportion to the numbers of seeded kernels were
determined by counting the emerged seedling num-
bers after 14 days of cultivation and the dug plants,
respectively. After digging, the edible peanut pods,
including yellow I, yellow II, orange, brown, and black
of Hull-scrape classes,10 were detached manually,
collected in nylonnet bags and dried in a greenhouse
for nine days for the spring crops and 15 days for the
fall crops. Dried pods were weighed and shelled
manually. Kernels were sieved and graded into US
No 1 and non-graded sublots. Yield of dried peanut
pods per unit area and weight percentage of the sized
(US No 1) kernels in proportion to the dried peanut
pods were then determined. Moisture contents of the
sized kernels collected in this study ranged from 55 to
70gkgÿ1 (dry basis) and were dependent upon crop
and cultivar.
Mould colonisation of the harvested kernelsMould colonisation of the harvested US No 1 was
determined following a standard technique for myco-
logical colonisation examination described by Pitt etal.11 Each kernel was surface disinfected by soaking in
a diluted household bleach solution (200ml litreÿ1
Clorox household bleach solution) containing 10.4g
litreÿ1 of NaOCl for 1min, transferring to 750ml
litreÿ1 ethanol for 30s and then depositing onto AFPA
(six kernels per Petri dish). Kernels were incubated at
28°C for ®ve days before examining for visible growth
of mould on the surface of kernels or in the
surrounding AFPA.
Aflatoxin analysis of the harvested kernelsFor each sublot of sized (US No 1) kernels harvested
from each replicate experiment, two 30-kernel samples
were randomly taken and ground with a cyclone mill
into meal. Moisture content of each meal was
determined by subjecting 3g of meal to heating at
70(�2)°C until constant weight was reached. For
a¯atoxin analysis, 1.0g of the meal was deposited into
a centrifuge tube and combined with 4ml of methanol
and 1ml of 0.1 M HCl for a¯atoxin extraction; quan-
titation was done following the procedure of Chiou
1418 J Sci Food Agric 79:1417±1422 (1999)
RY-Y Chiou
and Tsao.12 A¯atoxin content was expressed as
mgkgÿ1 of dry peanut meal.
StatisticsTriplicate experiments for each treatment of a cultivar
were conducted. ANOVA was applied to analyse the
variance as affected by arti®cial inoculation. The effect
of growing season was analysed by each-pair compari-
sons with Student's t-test using JMP software (SAS
Institute Inc, Cary, NC). Based on a preliminary
statistical analysis among peanut cultivars (Tainan 9,
Tainan 11 and Tainan 12), the effect of cultivar was
not signi®cant and could be ignored. Some of the data
obtained from the three cultivars were pooled to
address the effect of growing season.
RESULTS AND DISCUSSIONSPercentages of germination and harvested plantGermination percentages of the seeded kernels arti®-
cially inoculated with A ¯avus and A niger are listed in
Table 1. Germination percentage of the uninoculated
kernels (control) varied slightly depending upon year
and crop. The germination percentages of 1997 and
1998 spring crops were signi®cantly higher than of the
respective fall crops. Since the seeded kernels for each
crop were collected from the former crop, kernels of
fall crops had a higher percent germination than
kernels of spring crops. This might be related to the
traditional view that peanut quality of fall crops is
somewhat higher than that of spring crops. Taiwan is
located across tropical and subtropical areas and
between Euro-Asia and the Philippines. Due to its
unique geographical nature, weather varies from year
to year.
Except for the 1998 spring crop, inoculation of the
seeded kernels resulted in decreases of germination
percentage. In particular, lower germination percen-
tages were observed in kernels inoculated with A nigeror A ¯avus/A niger than kernels inoculated with A¯avus. A niger is the causal organism of Aspergillus
crown rot of peanut7 and usually causes high mortality
rates of young plants. It is apparent that the presence
of A niger caused mortality of the seeded kernels.
However, the mortality rates of the inoculated kernels
varied with year and crop.
During harvest of peanuts, numbers of plants in
each experimental plot were counted for estimation of
the harvested plant percentage in proportion to the
seeded kernels (Table 2). In comparison with the
initial germination percentages shown in Table 1, a
varied percentage of the emerged seedlings died during
cultivation. For uninoculated seeds (control), the
harvested plant percentages of spring crops were
signi®cantly higher than those of fall crops. In
inoculated seeds, death caused by A niger during
cultivation was observed. Even for the spring crop of
1998, in which germination percentages were fairly
high, a large portion of the seedlings died after
cultivation. An enhanced or antagonistic effect on
mortality of the seeded kernels or the emerged
seedlings in association with the interaction between
A niger and A ¯avus was not observed. This is not in
agreement with the antagonistic effect observed
between A niger and A ¯avus in peanuts reported by
Joffe.6
Agronomic characteristics of peanut productionYield of the harvested peanut pods per unit area is
summarised in Table 3. Peanut production was
affected by year. Based on the pooled data, including
yields of Tainan 9, Tainan 11 and Tainan 12, the
highest yields of dried peanut pods for inoculation
treatments were observed in 1997 spring crops. In
general, lower yields were observed in crops from
kernels inoculated with A niger than crops from
uninoculated seeds or seeds inoculated with A ¯avus.Apparently, yields were closely dependent on the
germination and, particularly, on the harvested plant
percentages (Tables 1 and 2). Since A niger is a causal
organism of peanut disease, inoculation of A nigeralone or together with A ¯avus resulted in early
Table 1. Germination percentages of the peanut kernels of three Spanishcultivars including Tainan 9, Tainan 11 and Tainan 12 during field cultivationfor peanut production as affected by growing season and artificial inoculationof the seeded kernels with Aspergillus flavus and Aspergillus niger (n=9)
Germination ratio (%)1
Arti®cial inoculation
Crops Control A ¯avus A niger
A ¯avus/
A niger
ANOVA
level 2
1996 Fall 66.0c 52.7c 43.3c 39.3c **1997 Spring 85.2a 76.0b 62.1b 62.4b **1997 Fall 77.0b 76.4b 66.6b 61.5b **1998 Spring 92.3a 94.3a 87.8a 93.8a *1 Mean values in the same column that are not followed by the same
superscript letter are signi®cantly different (p<0.05) analysed by Student's
t-test.2 ANOVA: * indicates signi®cant (p<0.05); ** indicates very signi®cant
(p<0.01).
Table 2. Harvested plant percentages of the peanut kernels of three Spanishcultivars including Tainan 9, Tainan 11 and Tainan 12 during field cultivationfor peanut production as affected by growing season and artificial inoculationof the seeded kernels with Aspergillus flavus and Aspergillus niger (n=9)
Harvested plant (%)1
Arti®cial inoculation
Crop Control A ¯avus A niger
A ¯avus/
A niger
ANOVA
level 2
1996 Fall 54.8c 44.4b 25.2c 26.2c **1997 Spring 78.2a 70.2a 54.7ab 53.3a **1997 Fall 66.4b 68.4a 45.3b 39.9b **1998 Spring 82.8a 75.3a 58.0a 60.2a **1 Mean values in the same column that are not followed by the same
superscript letter are signi®cantly different (p<0.05) analysed by Student's
t-test.2 ANOVA: * indicates signi®cant (p<0.05); ** indicates very signi®cant
(p<0.01).
J Sci Food Agric 79:1417±1422 (1999) 1419
Peanut a¯atoxin as affected by growing season
mortality for the seeded kernels and infection of the
seedlings which caused death.
Weight percentages of the sized US No 1 kernels in
proportion to the harvested dried peanut pods are
shown in Fig 1. For each crop, the difference among
inoculation treatments was minimal. The percentages
of fall 1996, spring 1997 and fall 1997 crops were
higher than that of the spring 1998 crop. According to
weather records, a long monsoon season was encoun-
tered during the cultivation of spring 1998 crop. The
abnormal weather condition may be related to the El
Nino year of 1998, resulting in lower-sized kernel
production.
Mould colonisation and aflatoxin contaminationMould colonisation of harvested kernels as affected by
crop, cultivar and arti®cial inoculation of the seeded
kernels is presented in Table 4. On average, less than
5.5% of the kernels were colonised with moulds.
Among inoculation treatments, differences in mould
infection of the harvested kernels were not observed.
For an overall comparison of the records, the highest
mould colonisation ratio was 33.3% in two of the test
Petri dishes, each containing six kernels. This indicates
that most of the harvested and sized kernels were free
of mould infection.
A fairly low or undetectable level of a¯atoxin
contamination was observed in the harvested kernels
(Table 5). In general, average a¯atoxin contents for all
crops, cultivars and inoculation treatments were less
than 6.1mgkgÿ1. The highest observed levels of
Table 3. Yields of the harvested dried peanut pods of three Spanish cultivarsincluding Tainan 9, Tainan 11 and Tainan 12 during field cultivation for peanutproduction as affected by growing season and artificial inoculation of theseeded kernels with Aspergillus flavus and Aspergillus niger (n=9)
Yield (kg dried pods haÿ1)1
Arti®cial inoculation
Crop Control A ¯avus A niger
A ¯avus/
A niger
ANOVA
level 2
1996 Fall 1950b 2075c 1436c 1361b **1997 Spring 3909a 3894a 3544a 3522a *1997 Fall 2312b 2646b 2078b 1551b **1998 Spring 2243b 2020c 1865b 1663b **1 Mean values in the same column that are not followed by the same
superscript letter are signi®cantly different (p<0.05) analysed by Student's
t-test.2 ANOVA: * indicates signi®cant (p<0.05); ** indicates very signi®cant
(p<0.01).
Figure 1. Weight percentages of the sized (US No 1) kernels in proportionto the harvested dried peanut pods as affected by artificial inoculation of theseeded kernels with Aspergillus flavus and A niger; 19962: fall 1996 crop;19971: spring 1997 crop; 19972: fall 1997 crop; 19981: spring 1998 crop.
Table 4. Mould colonisation of the harvested andsized (US No 1) kernels of Tainan 9, Tainan 11 andTainan 12 during field cultivation for peanutproduction as affected by growing season andartificial inoculation of the seeded kernels withAspergillus flavus and Aspergillus niger (n=6)
Mould colonisation of the sized kernels (%)1
Arti®cial inoculation
Crop Control A ¯avus A niger A ¯avus/A niger Highest level
1996 Fall
Tainan 9 2.8 (�3.9) 2.8 (�6.2) 4.2 (�6.4) 2.8 (�6.2) 16.7
Tainan 11 1.4 (�3.1) 1.4 (�3.1) 0 2.8 (�6.2) 16.7
Tainan 12 4.2 (�6.4) 2.8 (�6.2) 1.4 (�3.1) 0 16.7
1997 Spring
Tainan 9 0 5.5 (�12.4) 2.8 (�6.2) 5.5 (�12.4) 33.3
Tainan 11 2.8 (�6.2) 2.8 (�6.2) 0 0 16.7
Tainan 12 0 2.8 (�6.2) 11.1 (�6.4) 0 33.3
1997 Fall
Tainan 9 2.8 (�6.2) 0 2.8 (�6.2) 0 16.7
Tainan 11 0 0 0 0 0
Tainan 12 0 0 0 2.8 (�6.2) 16.7
1998 Spring
Tainan 9 4.2 (�7.2) 0 0 0 16.7
Tainan 11 0 0 0 0 0
Tainan 12 2.8 (�6.2) 0 0 0 16.7
1 Mean of determinations with standard deviation (n =6).
1420 J Sci Food Agric 79:1417±1422 (1999)
RY-Y Chiou
a¯atoxin contamination were 4.0, 18.2, 9.6 and
36.7mgkgÿ1 for fall 1996, spring 1997, fall 1997 and
spring 1998 crops, respectively. This was in agreement
with Ashworth et al13 and McDonald and Harkness14
who reported that a¯atoxin is rarely found in the
freshly harvested peanuts. However, this was not in
agreement with peanuts grown under stress conditions
resulting in extensive a¯atoxigenic mould infection
and subsequent a¯atoxin contamination of the har-
vested peanuts.15±19 Peanuts infected by a¯atoxigenic
moulds must be accompanied by suf®cient growth for
a¯atoxin contamination. A period of at least 20 days of
heat and drought stress immediately before harvest
may result in a¯atoxin production. In this study, a
severe drought stress did not occur during the
cultivation period. Therefore, extensive a¯atoxin
contamination of the harvested kernels was unlikely.
In addressing the effect of arti®cial inoculation on
a¯atoxin contamination, inoculation of a¯atoxigenic A¯avus did not increase a¯atoxin contamination. This is
in agreement with the observations of Will et al,20
Kisyombe et al21 and Azaizeh et al,19 who reported that
there is no signi®cant correlation between a¯atoxin
concentration and soil population densities of A ¯avus.When the combined effect of dual inoculation with
A niger and A ¯avus was investigated, an enhanced or
antagonistic effect on a¯atoxin contamination was not
observed. Antagonistic interaction between A ¯avusand A niger has been reported by Joffe6 for peanuts and
by Wicklow et al22 for corn. In this study, the presence
of A niger mainly affected peanut mortality (Table 1).
From the viewpoint of food safety, a¯atoxin
contamination higher than 10mgkgÿ1 in some of the
harvested kernel samples occurred (Table 5). The risk
of a¯atoxin contamination in peanut commodities still
exists. Wilson and Flowers23 reported that a¯atoxin
contamination of peanuts is unavoidable. In this study,
the extent of contamination varied and was indepen-
dent of arti®cial inoculation. This is in agreement with
the observation that subsamples of a given lot of
peanut may vary greatly in a¯atoxin content due to
extreme variability in degree of contamination of
individual kernels.24 A wide range of a¯atoxin
contamination in visibly sound unblanched peanut
kernels has been reported previously.12,25 Thus, a
continued effort is needed to identify an appropriate
and ef®cient means to eliminate a¯atoxin-contami-
nated kernels to ensure food safety.
In summary, arti®cial inoculation of the seeded
peanut kernels prior to cultivation has been conducted
for assessment of the interaction between the inocu-
lated moulds and peanut production of spring and fall
crops. The two-year experiments indicate that the
growing season (growing crop) played one of the prime
roles affecting ecological interactions. The presence of
A niger was mainly relevant to peanut mortality during
cultivation. Under normal cultivation practice, a heavy
inoculation of the seeded kernels with A ¯avus, A nigeror A ¯avus/A niger did not cause extensive mould
colonisation and a¯atoxin contamination. However,
the fact that some kernels contained over 10mgkgÿ1 of
a¯atoxin is a concern from the viewpoint of food
safety.
ACKNOWLEDGEMENTFinancial support by National Science Council,
Republic of China (NSC 86-2321-B021-013; NSC
87-2321-B021-020), valuable advice in the manu-
script preparation by Dr L R Beuchat, University of
Georgia and the helpful assistance in the laboratory by
Misses H-H Tsao, Y-S Lai and L-G Chang are
gratefully acknowledged.
Table 5P. Aflatoxin content in the harvested andsized (US No 1) kernels of Tainan 9, Tainan 11 andTainan 12 as affected by growing season andartificial inoculation of the seeded kernels withAspergillus flavus and Aspergillus niger (n=6)
A¯atoxin content (mgkgÿ1)1
Arti®cial inoculation
Crop Control A ¯avus A niger A ¯avus/A niger Highest level
1996 Fall
Tainan 9 0.5 (�1.1) nd nd nd 3.0
Tainan 11 nd 0.1 (�0.1) 0.1 (�0.1) 1.1 (�1.4) 4.0
Tainan 12 nd 0.1 (�0.1) nd nd 0.3
1997 Spring
Tainan 9 nd 0.1 (�0.2) nd nd 0.5
Tainan 11 3.0 (�3.4) nd 1.1 (�2.4) nd 11.1
Tainan 12 nd nd 3.2 (�6.7) 0.1 (�0.2) 18.2
1997 Fall
Tainan 9 nd nd nd nd nd
Tainan 11 nd nd nd nd nd
Tainan 12 1.6 (�3.6) nd nd nd 9.6
1998 Spring
Tainan 9 nd nd 6.1 (�13.7) 0.4 (�0.9) 36.7
Tainan 11 nd nd nd nd nd
Tainan 12 nd nd nd nd nd
1 Mean of the determinations with standard deviation (n =6); nd: non-detected.
J Sci Food Agric 79:1417±1422 (1999) 1421
Peanut a¯atoxin as affected by growing season
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