mould infection and aflatoxin contamination of the peanut kernels harvested from spring and fall...

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


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



(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


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



(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


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


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 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



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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