Aflatoxin synthesis in corn fields in Guanajuato, Mexico

Rev Iberoam Micol 2001; 18: 83-87
83
Aflatoxin synthesis in corn fields in
Guanajuato, Mexico
Carlos M. Bucio-Villalobos1, Doralinda Guzmán-de-Peña2 and
Juan Jose Peña-Cabriales2
1Instituto de Ciencias Agrícolas, Universidad de Guanajuato, Irapuato, Gto., México and 2Unidad Irapuato -
CINVESTAV, Irapuato, Gto., México
Summary
Aflatoxin contamination of corn is an important problem internationally, particu-
larly in tropical and subtropical conditions that favor infection and synthesis by
Aspergillus. Environmental conditions (drought) and agronomic practices i.e.
N fertilization have been reported as favorable to aflatoxin synthesis in the field.
This study was undertaken to investigate whether the contamination of corn
commonly observed in stored conditions in this important corn producing region
of Mexico known as “El Bajío” is related to infection by Aspergillus under field
conditions. Results using three corn hybrids of recognized susceptibility to infec-
tion showed that corn ears artificially inoculated in the field with a toxigenic strain
of Aspergillus parasiticus presented a low content of aflatoxin ranging from 13.6
to 24.7 µg Kg-1. No significant differences were observed between the hybrids
tested. Similarly, N fertilization practices, 260 Kg N ha-1, applied at sowing did not
have an effect on the extent of the contamination observed of 6.2 and 19.3 mg of
aflatoxin kg-1 in natural infected and inoculated samples with A. parasiticus NRRL
2999, respectively. Our data suggest that the cases of aflatoxin contamination of
corn in this part of Mexico are not related to infection occurring during the crops
growing period but most probably to poor storage conditions of corn.
Key words
Aspergillus parasiticus, Aflatoxin in corn, Mycotoxins, Field contamination,
Preharvest contamination
Síntesis de aflatoxinas en campos de maíz en
Guanajuato, México
Resumen
La contaminación por aflatoxinas en maíz es un problema internacionalmente
importante, especialmente bajo condiciones tropicales y subtropicales donde la
infección y síntesis de Aspergillus se ven favorecidas. Las condiciones del
medio ambiente (sequía) y prácticas agronómicas, por ejemplo la fertilización
nitrogenada, han sido reportadas como favorables a la síntesis de aflatoxinas en
campo. Este estudio fue realizado para investigar si la contaminación del maíz
que comúnmente es observada en condiciones de almacenamiento en esta
importante región productora de maíz en México, conocida como “El Bajío”, está
relacionada con la infección de Aspergillus bajo condiciones de campo. Los
resultados, usando tres híbridos susceptibles, mostraron que las mazorcas de
maíz inoculadas artificialmente en el campo con una cepa toxigénica de
Aspergillus parasiticus presentaron bajo contenido de aflatoxinas, el cual estuvo
dentro de un rango de 13,6 a 24,7 µg Kg-1. No se observaron diferencias signifi-
cativas entre los híbridos evaluados. Similarmente, la práctica de fertilización
nitrogenada, 260 Kg N ha-1, aplicada al momento de la siembra, no tuvo efecto
sobre la contaminación observada de 6,2 y 19,3 µg de aflatoxina Kg-1 en mues-
tras naturales y contaminadas con A. parasiticus NRRl 2999, respectivamente.
Nuestros datos sugieren que los casos de contaminación por aflatoxinas en
maíz en esta parte de México no estan relacionados con la infección ocurrida
durante el desarrollo del cultivo, pero mas probablemente son debidos a las defi-
cientes condiciones de almacenaje del maíz cosechado.
Palabras clave
Aspergillus parasiticus, Aflatoxinas en maíz, Micotoxinas,
Contaminación en campo, Contaminación en precosecha
Dirección para correspondencia:
Dra. D. Guzmán-de-Peña
Unidad Irapuato-CINVESTAV
Km 9.6 carretera Irapuato-León,
Irapuato, Gto., México
Tel.: +52 462 39600 / 39648
Fax: +52 462 45846
E-mail: dguzman@ira.cinvestav.mx
Aceptado para publicación el 3 de Mayo de 2001
©2001 Revista Iberoamericana de Micología
Apdo. 699, E-48080 Bilbao (Spain)
1130-1406/01/10.00 Euros

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84
Rev Iberoam Micol 2001; 18: 83-87
Aflatoxin contamination of preharvest corn
red with soil. The row spacing was 0.77 m.
(Zea mays L.) has been reported in several countries [1].
Eight days after emergence, Diazinon 25 E (25%
In the United States [2], it is a chronic problem in the
Diazinon, Ciba, México) was applied to control thrips
southern states, and it appears sporadically elsewhere [3].
(Frankliniella occidentalis) and at 35 days after sowing
It may be widespread in developing countries of the tro-
Cymbush 200 (20% Cipermetrin, Zeneca, México) was
pics and subtropics in which temperature conditions are
applied to control fall army worm (Spodoptera fugiperda).
likely to favor infection of corn by Aspergillus spp. [1].
Weeds were controlled by hand during 60 days after plan-
It is well known that environmental conditions
ting. No measurements of insect damage were made.
strongly influence dispersion of fungal spores [4], pene-
The corn hybrids were H-220; H-433 and A-791
tration and establishment of hyphae in plants and on the
(early, mid and late season maturity).
production of aflatoxins; also, cultural and agronomic
Inoculation. Aspergillus parasiticus strain NRRL
conditions influence the synthesis of aflatoxin in corn [5].
2999 (from the Northeast Regional Researh Laboratory),
High temperatures and drought conditions are con-
this species naturally does not infect corn fields in
ducive to heavy aflatoxin contamination [6,7], as is insect
Guanajuato, but was chosen for its stability in aflatoxin
damage [8]. The interrelationship between soil type and
production as reported elsewere [20]. The strain was
level of aflatoxin contamination in corn requires further
maintained in potato dextrose agar slant tubes. Spores
research; certainly the soil is important as an inoculum
were harvested from 5-day-old cultures: sterile water with
source [9], and altering edaphic factors by fertilization,
0.01 % triton was added to plates to obtained suspensions
irrigation or cultivation may affect spore numbers in soil
of 1.6x106 spores ml-1. Inoculation was achieved by appl-
[1]. However, Payne et al. [10] demonstrated that deep-
ying 5 ml of suspension with a syringe to each ear through
ploughing in North Carolina reduced aflatoxin contamination.
the pollination channel at 15 days after 50% silking,
There have been several reports that high tempera-
according to each hybrids (72, 78, and 81 days for H-220,
tures and humidity favor fungal growth and aflatoxin
H-433, and A-791, respectively). Each treatment was
synthesis in stored corn [4,5,11-16]. However, since the
composed of two hundred plants with one ear per plant
1970s it has been accepted that corn kernels become
inoculated. Control plants were similarly inoculated with
infected with fungi and contaminated with aflatoxin while
5 ml of sterile water.
in the field [4]. In Tamaulipas State, northeast Mexico,
Harvest. When the ears were mature according to
early sowing and proper irrigation decreased aflatoxin
each hybrid: 104, 110, and 113 days for H-220, H-433,
contamination from 246 to 6 mg kg-1 [17]. On the other
and A-791, respectively, plants were harvest.
hand, corn stored in Tamaulipas, 1985 to 1988, revealed
Fungal population: infestation and identification.
only a 2% incidence of Aspergillus flavus with unknown
Three hundred grams kernel samples from 48 blocks were
toxigenic activity and low levels of aflatoxin B1 [18].
analyzed for fungal population. Four replicates of one
Mexico has one of the highest rates of human con-
hundred kernel subsamples were placed in 1.5 % sodium
sumption of corn in the world (120 kg/year/per capita)
hypochlorite for 3 min, rinsed with sterile water three
[19]. It also represents a mosaic of environmental condi-
times and placed on a malt salt agar medium and incuba-
tions in which corn is produced and/or stored for various
ted at 30?C for seven days. The number of forming fungal
periods of time. Yet information on aflatoxin contamina-
colonies were counted macroscopic in each grain to deter-
tion of corn in the main producing regions is scarce.
mined percentage of incidence. The fungal colonies were
In central Mexico, environmental conditions, parti-
isolated on potato dextrose agar and microscopically iden-
cularly drought seem to be favorable to aflatoxin synthesis
tification was performed according to Domsch et al. [21].
in the field. Furthermore mycotoxicosis in pigs associated
Aflatoxin determination. Samples from each of 48
to ingestion of contaminated feed are frequently reported
blocks were harvested and because 12 blocks were loosed
for this region. Therefore the present study was underta-
in the process, only 36 blocks were analyzed for aflatoxin.
ken to investigate if the contamination of corn commonly
Each treatment was harvested separately and kernels
observed in stored conditions in this part of Mexico is
weighed to obtain the following samples: 20 samples of
related to infection by Aspergillus under field conditions.
5 Kg; four of 4 Kg; 11 of 2.5 Kg; and one of 2 Kg. Each
entire sample was mixed and ground to pass through an
MATERIALS AND METHODS
0.8 mm sieve and five subsamples of 60 grams were taken
by quartering. From each of these subsamples, 10 grams
Field experiment. The experiments were conducted
were taken to obtain a 50 grams composite sample. Thirty
in the field of CINVESTAV-Irapuato (20° 44’ N,
six composite samples were analyzed for aflatoxin con-
101° 19’ W) in Guanajuato, Mexico. The soil is classified
tent; they were extracted according to the modification of
as a Pelic Vertisol with pH of 7.2 (1:2 water), organic
the method 1 AOAC as published elsewhere [22].
matter 1.83 %, and a clay texture.
Quantitative determinations of aflatoxins in the extracts,
The experiment was a 3 x 2 x 2 factorial with a
were made by high pressure liquid chromatography using
randomized block design with four replications. The first
a Zorbax LC18 column (Dupont, USA). The mobile phase
factor was corn hybrids, the second was Aspergillus para-
was a mixture of water:acetonitrile:methanol (45 : 15 : 40
siticus inoculum treatment and the third was nitrogen (N)
by volume). Elution of aflatoxins was recorded at 364 nm.
fertilization, generating twelve treatments. This experi-
Standard solutions of aflatoxins B1, B2 , G1 and G2 were
ment, sown on May 30 1996, was irrigated the next day.
run under the same conditions as described by Guzmán-
Ploughing, tilling, and leveling were done with machinery
de-Peña and Ruíz-Herrera [23].
methods.
Other variables measured. Numbers, fresh and dry
Fertilization. 260 Kg ha-1 as urea was applied in the
weights of ears were determined for all treatments. Plant
soil in two furrow application: 50 % at sowing and the rest
dry weight, grain, and moisture content were also determi-
42 days later. Phosphate also was applied at sowing as
ned.
calcium superphosphate 80 Kg ha-1 in a single furrow
Environmental data. During the experiment the
application.
maximum temperature recorded was 25.2 °C and mini-
Sowing. Two corn seeds were placed over the ferti-
mum 12 °C, total rainfall was 837.7 mm and relative
lizer applied in the soil, every 20 cm and then were cove-
humidity of 81.2 % maximum and 25.5 % minimum.

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Aflatoxins in corn fields
85
Bucio-Villalobos CM,et al.
Statistical analysis. Analysis of variance and
Table 4. Variables related to yields of three corn hybrids.
Tukey`s test were applied to data using SAS (version
____________________________________________________________
6.12; SAS Institute, Cary, NC, USA).
Hybrid
Grain yield
Ears
Ear wt.
Stover DM
(t ha-1)
(ha-1)
(t ha-1)
(t ha-1)
____________________________________________________________
RESULTS
H-220
6.5b
54.935b
7.8b
2.1b
A-791
9.1a
61.558a
11.6a
2.9a
H-433 5.9b
49.481b
7.4b
2.2b
Fungal populations. Various Fusarium species
Mean Value
7.1
55.325
8.9
2.4
____________________________________________________________
predominated in the kernels, with A. parasiticus and
Mean of four replicates of each treatment; values with the same letter in the same column are not signifi-
Penicillium spp. present to a lesser extent (Table 1). In
cantly different (Tukey at P=0.05).
fact, the values for A. parasiticus were low and there was
no statistically significant effect of corn genotype (Table
1). It is important to mention that A. flavus and A. parasi-
Table 5. Yield variables of corn naturally infected with fungus and inocula-
ticus were not found in the soil (data not showed).
ted with A. parasiticus.
____________________________________________________________
However A. parasiticus was isolated from nonioculated
Condition
Grain yield
Ears
Ears wt.
Stover DM
corn.
(t ha-1)
(ha-1)
(t ha-1)
(t ha-1)
As a consequence of inoculation, the populations
____________________________________________________________
of A. parasiticus increased significantly whereas the
Naturally infected
7.1a
55.325a
8.9a
2.4a
Inoculated
7.2a
56.364a
9.0a
2.3a
others were not affected (Table 2). It is important to note
____________________________________________________________
that the identified populations corresponded to internal
Means of four replicates of each treatment; values with the same letter in the same column are not signifi-
cantly different (Tukey at P=0.05).
infections, because the kernels were washed with sodium
hypochlorite before being placed onto the culture
medium.
Table 6. Yield of corn with and without N fertilizer.
The application of fertilizer N had no effect on the
____________________________________________________________
fungal populations as it is illustrated in Table 3.
Treatment
Grain yield
Ears
Wt. of ears
Stover DM
(t ha-1)
(ha-1)
(t ha-1)
(t ha-1)
____________________________________________________________
Table 1. Fungal populations of kernels of diferent corn genotypes.
____________________________________________________________
260 kg N ha-1
8.3a
56.623a
10.4a
2.9a
Without N
6.0b
54.026a
7.4b
1.9b
Kernels infected with
_________________________________________________
____________________________________________________________
Means of four replicates of each treatment; values with the same letter in the same column are not signifi-
A. parasiticus
Fusarium spp. Penicillium spp.
Misc.
Hybrid
cantly different (Tukey at P=0.05).
(%)
____________________________________________________________
H-220
3.3a
34a
1.1a
0.8a
A-791
1.8a
32a
0.7a
0.9a
Aflatoxin contamination. The procedures used in
H-433
3.5a
35a
1.0a
1.2a
____________________________________________________________
this study allow us to detect aflatoxins B1, B2, G1 and G2
Mean of four replicates of each treatment of 100 kernels; values with the same letter in the same column
however only B1 was observed, and the levels of aflatoxin
are not significantly different (Tukey at P=0.05).
detected in the three corn genotypes were low (Table 7).
With N-fertilizer applied, under natural infection, aflato-
xin levels were below 8.8 µg Kg-1, whereas inoculation of
Table 2. Fungal populations of kernels naturally infected with fungus and
ears with spores of A. parasiticus resulted in 13.6 to
inoculated with A. parasiticus.
____________________________________________________________
24.7 µg Kg-1 (Table 7). A similar trend prevailed with the
unfertilized corn, but with generally lower levels of aflato-
Kernels infected with
____________________________________________
xin (Table 8).
A. parasiticus Fusarium spp. Penicillium spp.
Misc.
Condition
(%)
____________________________________________________________
Table 7. Aflatoxin levels in the kernels of three corn hybrids with N applied,
Naturally infected
0.4a
37a
1.0a
1.1a
naturally infected with field isolates fungus or inoculated with A. parasiticus.
Inoculated
9.0b
32a
0.9a
0.9a
____________________________________________________________
____________________________________________________________
Aflatoxins content (µg kg-1)
Means of four replicates of each treatment of 100 corn grains; values with the same letter in the same
_______________________________________
column are not significantly different (Tukey at P=0.05).
Hybrid
Naturally infected
Inoculated
____________________________________________________________
H-220
5.7a
19.8a
Table 3. Fungal populations of kernels of corn grown with and without N
A-791
8.8a
24.7a
fertilizer.
H-433
4.1a
13.6a
____________________________________________________________
Mean Value
6.2
19.3
____________________________________________________________
Kernels infected with
____________________________________________
Means of three replicates of each treatment; values with the same letter in the same column are not signi-
ficantly different (Tukey at P=0.05).
A. parasiticus Fusarium spp. Penicillium spp.
Misc.
N treatment
(%)
____________________________________________________________
260 kg N ha-1
2.3a
35a
1.2a
0.9a
Without N
3.4a
32a
0.7a
1.1a
Table 8. Aflatoxin levels in the kernels of three corn hybrids, without fertili-
____________________________________________________________
zer N, naturally infected with field isolates fungus or inoculated with A. para-
siticus.
Means of four replications of each treatment of 100 kernels; values with the same letter in the same
____________________________________________________________
column are not significantly different (Tukey at P=0.05).
Aflatoxins content (µg kg-1)
_______________________________________
Hybrid
Naturally infected
Inoculated
____________________________________________________________
Variables related to yield. The highest yield was
H-220
3.1a
13.2a
obtained with hybrid A-791 and the other hybrids showed
A-791 10.2a
14.6a
lower similar grain production (Table 4). Inoculation with
H-433 0.6a
11.5ª
Mean Value
4.6
13.1
A. parasiticus did not affect yield (Table 5). As usually
____________________________________________________________
occurs, the application of fertilizer N had a positive effect
Means of three replicates of each treatment; values with the same letter in the same column are not signi-
on yield (Table 6).
ficantly different (Tukey at P=0.05).

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86
Rev Iberoam Micol 2001; 18: 83-87
DISCUSSION
trends were observed in the hybrids without N levels, in
natural infected and inoculated kernels, in which case the
Fungal populations. The natural fungal popula-
levels of aflatoxins were 10.2 mg Kg-1 and 14.6 mg Kg-1,
tions in the kernels were mainly species of Fusarium and
respectively. Again, no significant differences were found
Penicillium, with very low incidence of Aspergillus para-
among hybrids. It is noteworthy that hybrid H-433, which
siticus; Fusarium spp. predominated. There were no signi-
is considered as susceptible material for aflatoxin synthe-
ficant differences among the hybrids. Comparison of the
sis in the northeast of the country, by contrast, showed the
fungal populations of naturally infected kernels with ker-
lowest level of aflatoxins in this experiment at
nels from inoculated ears showed a significant difference
Guanajuato.
in the A. parasiticus population, which indicated low
No significant effect of N fertilization was obser-
natural incidence of this species and confirmed that inocu-
ved on aflatoxin synthesis in the field. Furthermore, the
lation was effective. It is noteworthy that our values were
levels were below 8.8 µg Kg-1 and below 24.7 mg Kg-1 in
low in comparison with those reported before, e.g. a 5.2 %
natural infected and inoculated samples, respectively.
natural infection rate for A. flavus [24] and 83% recorded
These results contrast with those of Wilson et al. [28] in
by Widstrom et al. [25]. Our data show only 3.5 % of
Georgia, who found that high amounts of applied N incre-
grain infected with A. parasiticus; it can be suggested that
ased aflatoxin levels in corn, and with those of Payne
this fungus is poorly infective in the field under the condi-
et al. [27] in North Carolina, who found that low amounts
tions of maximum temperature of 25.2 °C, minimum of
of fertilizer similarly increased aflatoxin.
12 °C, rainfall of 837.7 mm and relative humidity of
The low levels of aflatoxin found in this experi-
81.2 % maximum and 25.5 % minimum prevalent during
ment indicate that environmental conditions were more
the test period in this region.
important in aflatoxin synthesis than toxigenic fungal
The fungal populations of kernels from fertilized
strain or corn genotype. Payne [5] has pointed out that the
corn were not significantly different from those of plants
most favourable temperature for infection of ears through
not treated with N. These data differ from those of other
the pollenation channel is in the range of 28 to 38 °C. The
reports in which low levels of N fertilizer increased the
temperatures during this experiment were in average
incidence of A. flavus [13, 26-28]. Aspergillus flavus was
18.6 °C. By contrast, in the northeast (Tamaulipas), tem-
not isolated from any of the uninoculated samples indica-
peratures of 28 to 30 °C or higher are common during the
ting absence from the corn fields of Guanajuato.
growing period [18] and aflatoxin levels of 250 µg Kg-1
Variables related to yield. Grain yield, number of
have been recorded. Another potentially important factor
ears, weight of ears and stover dry matter of hybrid A-791
is relative humidity, which in “El Bajío” was low, around
were higher than those of H-220 and H-433. These results
56 %, when the ears were inoculated. In Guanajuato State
were expected, in view of the fact that A-791 was develo-
the relative humidity is low all year, even during the rainy
ped to perform well in optimal conditions, which were
season. Sauer [15] reported that a relative humidity of at
met in these experiments. The yield-related variables were
least 85 % is necessary for effective infection of corn.
not affected by inoculation, which is consistent with the
The results indicate that infection of corn in the
fact that there was no visible damage to the ears or plants
field by A. flavus and A. parasiticus is not common in this
as a whole. Differences in yield were observed when
region of Mexico due to the unfavorable climatic condi-
nitrogen was applied.
tions i.e. low temperature and relative humidity, even if
Aflatoxin contamination. The amounts of aflatoxin
the number of propagules of the toxigenic strain is increa-
B1 contamination were low. With natural infection and N
sed by artificial inoculation. Thus, the outbreaks of aflato-
applied, levels below 8.8 µg kg-1 were recorded. Although,
xin contaminated corn occasionally observed in this part
inoculation with A. parasiticus increased the values, they
of Mexico [29] seem to be related to poor storage condi-
never reached values higher than 24.7 µg Kg-1. No signifi-
tions and not to field contamination.
cant differences were found among the hybrids. Similar
We thank Yolanda Rodríguez for technical support with the
HPLC equipment during the aflatoxin determination and
to Dr. Allan Eaglesham for helpful suggestions, and
comments on the manuscript.

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Aflatoxins in corn fields
87
Bucio-Villalobos CM,et al.
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