151

Journal of Food Protection, Vol. 70, No. 1, 2007, Pages 151–156

Copyright 䊚, International Association for Food Protection

Natural Occurrence of Fusarium Species, Fumonisin Production

by Toxigenic Strains, and Concentrations of Fumonisins B1 and
B2 in Conventional and Organic Maize Grown in Spain
AGUSTÍN ARIÑO,1* TERESA JUAN,2 GLORIA ESTOPAÑAN,2 AND JOSÉ F. GONZÁLEZ-CABO1

1Veterinary Faculty, University of Zaragoza, c/Miguel Servet 177, 50013 Zaragoza, Spain; and 2Agri-Food Research and Technology Center,
Avda. Montañana 930, 50059 Zaragoza, Spain

MS 06-222: Received 18 April 2006/Accepted 29 July 2006

ABSTRACT
Sixty samples of corn from both conventional and organic farms were tested for internal fungal contamination. Molds
were identified to genus, and those belonging to the genus Fusarium were identified to species. Twenty isolates of Fusarium
verticillioides were tested with a high-performance liquid chromatography–naphthalene dicarboxaldehyde–fluorescence method
for their ability to produce fumonisins B1 and B2. The internal fungal infection in organic maize (63.20%) was significantly
higher than that in conventional maize (40.27%) (P ⬍ 0.05). However, the distribution of fungal genera indicated a significantly
higher prevalence of Fusarium in conventional (34.93%) than in organic (18.15%) maize, making Fusarium the predominant
fungus in conventional maize. This difference in mold distribution between organic and conventional maize was attributed to
the difference in cultivation system. The dominant Fusarium species in both conventional and organic samples was F. verti-
cillioides. There were no significant differences in the ability of 20 selected isolates of F. verticillioides to produce fumonisins
on conventional or organic corn. Up to 13.3% of the conventional corn samples contained fumonisins B1 and B2 at mean
concentrations of 43 and 22 ng/g, respectively. Organic corn samples had somewhat lower levels of contamination: 35 ng/g
fumonisin B1 and 19 ng/g fumonisin B2 (P ⬎ 0.05). The organic farming system, with well-balanced crop rotation, tillage,
and compost fertilization, produced corn that was less likely to be contaminated with Fusarium species, although no significant
difference in fumonisin concentrations was found between the two types of contaminated corn.

Fungal contamination of foods and feeds causes con- day for the total of FB1, FB2, and FB3, either alone or in
siderable economic losses due to direct damage to crops, combination. The European Union recently regulated fu-
discoloration, off-odors, taints, off-flavors, reduced yields, monisins (as the sum of FB1 and FB2) in maize-based prod-
and loss of nutritive value. Fungal contamination of foods ucts and unprocessed maize so that if no specific concen-
is associated with production of mycotoxins, which have tration is fixed before 1 October 2007, maximum concen-
toxic effects on both humans and animals. In the temperate trations from 200 to 2,000 ␮g/kg will apply thereafter (10).
climatic conditions prevailing in Europe, Fusarium fungi Organic products of plant origin are grown without the
are important in the cereal food chain and can reduce crop aid of chemical synthetic pesticides and largely without the
yields and contaminate grain with mycotoxins (29). Fusar- use of readily soluble mineral fertilizers within a diverse
ium includes many species that are pathogenic to plants, system of crop rotation and extensive soil tillage. One hy-
responsible for a broad range of diseases, and mycotoxi- pothesis is that organic foodstuffs are more prone than con-
genic. In Aragón (Spain), maize diseases caused by Fusar- ventional foodstuffs to contamination by mycotoxins be-
ium have been reported since the early 1980s (21). cause organic products are not treated to the same extent
Members of the Gibberella fujikuroi complex (e.g., Fu- with antifungal agents. However, this hypothesis was not
sarium verticillioides, a synonym of Fusarium moniliforme) supported by a literature review conducted in 2000 by the
are generally regarded as the most important colonizers of Food and Agriculture Organization of the United Nations
cereal grains and can produce fumonisins (27, 30). Fumo- (11). In a recent study, organic farming systems had lower
nisins have been associated with leukoencephalomalacia in
rates of Fusarium ear blight infection and lower mycotoxin
equine species, pulmonary edema in pigs, and esophageal
contamination in winter wheat (Triticum aestivum) than did
cancer in humans (33). Fumonisin B1 (FB1) is classified as
conventional farming systems (4). However, fungal attack
possibly carcinogenic to humans (group 2B) by the Inter-
and mycotoxin contamination in organically and conven-
national Agency for Research on Cancer (12). The Euro-
tionally grown produce is still an extremely controversial
pean Commission Scientific Committee for Food (27) has
evaluated fumonisins and established a provisional maxi- issue (15).
mum tolerable daily intake of 2 ␮g/kg of body weight per With the steady and rapid growth of the European and
U.S. markets for organic foods, there is a need for quality
* Author for correspondence. Tel: ⫹34-976-761543; Fax: ⫹34-976- control, including safety evaluation of the products (8). In
761612; E-mail: aarino@unizar.es. organic cereals, the main concern is the growth of myco-
152 ARIÑO ET AL. J. Food Prot., Vol. 70, No. 1

toxin-producing fungi that can cause toxic syndromes in claved for 1 h, and cooled, and the corn was stirred to separate
humans and animals. For maize, not all factors involved in the kernels. The beakers were then covered with a cotton plug,
the formation of Fusarium toxins, in particular FB1 and sealed, and autoclaved again for 1 h. After the cooling period, 1
FB2, are known. Therefore, there is need for investigations ml of phosphate-buffered saline inoculum (a suspension of conidia
from single spore cultures on CLA) was introduced with a syringe.
of the formation of these mycotoxins and the identification
The cultures were incubated in the dark for 4 weeks at ambient
of the management measures needed to prevent their pres- temperature. The culture material was then autoclaved for 5 min,
ence in foodstuffs. The present study was carried out to dried at 60⬚C, ground with a laboratory mill (IKA, Wilmington,
gain information on fungal infection and the presence of N.C.), and stored at 4⬚C until analyzed.
the mycotoxins FB1 and FB2 in conventional and organic
corn grown in Spain. Apparatus and reagents for mycotoxin analysis. High-per-
formance liquid chromatography (HPLC) grade acetonitrile and
MATERIALS AND METHODS methanol were purchased from Lab-Scan (Dublin, Ireland), and
HPLC grade acetic acid was obtained from Merck (Darmstadt,
Corn samples. Thirty samples of corn were selected from
Germany). Ultrapure water was obtained with a Milli-Q Plus ap-
farms using conventional cultivation methods, and another 30
paratus (Millipore, Milford, Mass.). The solid-phase extraction
samples were collected from organic farms; no samples were
columns were Multisep 211 Fum columns (Romer Labs, Union,
moldy in appearance. These corn samples from various harvesting
Mo.). Fumonisins standards for FB1 and FB2 were provided by
years (2001 to 2003) were collected 1 to 4 weeks after harvest,
Sigma (St. Louis, Mo.), and stock solutions (1.0 mg/ml) were
October to November, at farms in Aragón. The objective was to
prepared in acetonitrile-water (1:1, vol/vol) and stored at 4⬚C.
obtain pairs of samples (conventional and organic) from farms at
NDA reagent (2,3-naphthalene dicarboxaldehyde; Sigma) was pre-
neighboring sites. These sites differed in agricultural practices and
pared in methanol at 0.5 mg/ml and stored at 4⬚C. Reagents for
fertilizer and pesticide usage, but environmental factors such as
sodium borate buffer (0.05 M), phosphate buffer (0.05 M), and
climate and soil conditions were as comparable as possible. At
sodium cyanide solution (0.13 mg/ml) were provided by Panreac
the conventional farms, the maize seeds had been treated with
(Barcelona, Spain).
fungicides (approved treatments included 70% himexazol at 2 kg/
The liquid chromatography (LC) system consisted of a Kon-
Tm and 40% maneb at 3 liters/Tm), and insecticides and herbi-
tron model 322 pump, a model 360 autosampler, and an SFM 25
cides were sprayed as needed during the vegetation period. Min-
fluorescence detector at 420 nm (excitation) and 500 nm (emis-
eral fertilization of soil also was used on the conventional farms.
sion). The LC column was a Kromasil RP C18 (250 by 4.6 mm,
On the organic farms, by contrast, no pesticides and fungicides
5-␮m particle size; Varian Inc., Lake Forest, Calif.). The LC mo-
had been used, and agronomic techniques included tilling and crop
bile phase was a gradient of acetonitrile–acetic acid (solution A;
rotation with legumes and alfalfa plus fertilization with compost.
99:1, vol/vol) and water–acetic acid (solution B; 99:1, vol/vol)
To prepare representative samples, up to 10 250-g subsam-
with the following protocol: 60% A ⫹ 40% B for 8 min, 80% A
ples were collected from each lot of cereal. The subsamples were
⫹ 20% B for 16 min, hold for 4 min, and reequilibrate to initial
pooled, and a single 0.50-kg composite sample was taken to the
conditions. The flow rate was 1 ml/min.
laboratory and stored at ⫺21⬚C until analyzed for fungi and my-
cotoxins. Analysis of fumonisins in corn and laboratory cultures.
Isolation and identification of fungi. Fifty kernels from The technique for extraction and determination of fumonisins was
each sample were surface sanitized in a 5% aqueous solution of based on the work of Bennett and Richard (3). A representative
sodium hypochlorite (NaOCl) for 1 min, rinsed twice with sterile corn sample was ground in the laboratory mill and mixed. Twen-
distilled water, and then dried with sterile paper towels. Fifty ker- ty-five grams (corn) or 5 g (cultures) were extracted with 100 ml
nels were then plated (five kernels per plate) on dichloran rose of acetonitrile-water (50:50, vol/vol) by shaking for 60 min on a
bengal chloramphenicol agar. The plates were incubated in the wrist-action shaker. The extract was filtered through Whatman no.
dark at 25⬚C for 5 to 7 days, and the fungal colonies that devel- 1 filter paper, and the pH was adjusted to 6 to 7 with 0.5 M sodium
oped from the kernels were identified and counted. The colonies hydroxide. Cleanup was carried out with Multisep 211 Fum col-
that developed were streaked for isolation and maintained on po- umns according to the instructions of the manufacturer (Romer).
tato-dextrose agar (PDA) slants. Fusarium isolates were identified The fumonisin-containing eluate was evaporated to dryness in a
according to the method of Nelson et al. (17) using carnation-leaf heating block under a gentle stream of nitrogen, and the residue
agar (CLA) and PDA. Slide mounts were prepared and observed was redissolved in 1 ml of methanol. The content of this vial was
under a phase contrast microscope. Alternaria, Aspergillus, Pen- derivatized by adding 1 ml of 0.05 M sodium borate buffer, 0.5
icillium, and other less often isolated genera were identified by ml of sodium cyanide reagent, and 0.5 ml of NDA reagent. The
microscopic morphology and culture characteristics in malt-ex- mix was heated at 60⬚C for 15 min in a heating block, cooled,
tract agar, PDA, and Czapek agar according to the method of and diluted with 7 ml of 0.05 M phosphate buffer–acetonitrile
Samson et al. (26). The frequency distribution of the fungal genera (40:60, vol/vol). Aliquots of 20 to 100 ␮l were injected into the
is defined as the ratio of the number of kernels infected with each LC fluorescence detection system.
genus to the total number of infected kernels. The average recoveries and relative standard deviations
(RSDs) obtained by the described method for FB1 and FB2 were
Production of fumonisins by F. verticillioides isolates. For 95.0% (RSD ⫽ 4%) and 85.0% (RSD ⫽ 4%), respectively. The
farm site comparisons of F. verticillioides isolates, we selected 10 performance characteristics for the analytes FB1 and FB2 were
isolates from different conventional corn samples and 10 isolates within the acceptable margins indicated in Commission Directive
from matched organic corn samples. These 20 selected isolates 2005/38/EC (9). The study of sensitivity indicated that the limit
were grown on autoclaved corn following the technique described of quantification (LOQ) for FB1 and FB2 was 25 ng/g. The linear
by Ross et al. (23). Fifty grams of yellow corn and 50 g of water range was from LOQ to 25 times the LOQ. The samples were
were added to a 250-ml beaker and left at room temperature for diluted with phosphate buffer–acetonitrile as needed to fit within
1 h. The beakers were then covered with aluminum foil, auto- the linear range. The incidences of FB1 and FB2 were expressed
J. Food Prot., Vol. 70, No. 1 FUSARIUM AND FUMONISINS IN CONVENTIONAL AND ORGANIC CORN 153

TABLE 1. Total fungal infection and Fusarium infection in conventionally and organically grown corn by year of harvesta
% positive samples (mean ⫾ SE)a
Growing
Infection conditions 2001 2002 2003

All fungi Conventional 38.80 ⫾ 7.55 A 40.20 ⫾ 7.79 A 41.80 ⫾ 10.78 A

Organic 70.00 ⫾ 7.48 A 56.60 ⫾ 7.31 A 63.00 ⫾ 7.64 A
Fusarium Conventional 32.70 ⫾ 2.22 A 38.44 ⫾ 1.46 A 33.64 ⫾ 3.13 A
Organic 15.65 ⫾ 2.90 A 18.95 ⫾ 3.31 A 19.85 ⫾ 4.00 A
a n ⫽ 10 for each type of farm for each harvest year. Within each row, means with different letters are significantly different (P ⬍ 0.05,
ANOVA).

as the percentage of samples containing concentrations above 25 infection) followed by Alternaria (25.19%). In contrast, the
ng/g. The mean was calculated using one half of the LOQ for predominant genus isolated from organic maize was Alter-
results lower than the LOQ. naria (40.29% infection) followed by Fusarium (18.15%).
Statistical analysis. The data were analyzed with an analysis This shift in dominance between Fusarium and Alternaria
of variance for significance among groups according to the meth- could have been caused by the differences in the two farm-
od of Sachs (24). Calculations were performed on StatView ing systems. Fusarium and Alternaria are generally consid-
SE⫹Graphics (1988, Abacus Concepts, Inc., Berkeley, Calif.) for ered field fungi, infecting growing plants in the field, al-
Macintosh personal computers. though both fungi also can cause problems in stored grains.
Changes in agricultural practices can affect the incidence
RESULTS AND DISCUSSION of infection by field pathogens and the genera present in
The incidences of total fungal infection and Fusarium cereal crops. Tilling the soil between crops is indispensable
infection in conventional and organic corn samples during in organic systems as a weed control technique because use
the 3 harvest years (2001 through 2003) are shown in Table of herbicides and fungicides is prohibited (15). Compared
1. Total infection was 38.80 to 41.80% on conventional with no tillage, plough tillage reduces the incidence of Fu-
farms and 56.60 to 70% on organically managed farms. sarium attack and the concentration of deoxynivalenol in
Similarly, Fusarium infection remained essentially un- cereal crops (14). Because the use of fungicides differen-
changed through the harvest seasons, 32.70 to 38.44% on tially affects the Fusarium species that infect the ears, re-
conventional farms and 15.65 to 19.85% on organic farms. sistance to some fungicides may selectively remove domi-
There were no significant differences between harvesting nant but susceptible nontoxigenic species, allowing more
years in the mycoflora of the crops (P ⬎ 0.05). This finding active colonization by toxigenic species (28).
allowed for a multiyear comparison between conventional Fusarium infection was nearly 50% higher in conven-
and organic samples from farms at neighboring sites. tionally grown maize than in organically grown maize. In
The farm site comparison revealed distinct differences contrast, the occurrence and distribution of the postharvest
between conventionally and organically managed farms in fungi Aspergillus and Penicillium was similar in both con-
terms of total fungal infection and distribution of fungal ventional and organic corn samples. Fusarium species in-
genera. The fungal infection of conventional and organic fect the grain preharvest, and several risk factors have been
maize samples is presented in Table 2. Generally, the per- identified in connection with Fusarium infection and my-
centage of infected corn samples originating from organic cotoxin formation, e.g., climatic conditions and the com-
farms (63.20%) was significantly higher (P ⬍ 0.05) than petitiveness of different fungal species. For example, Marin
that from conventional farms (40.27%). The distribution of et al. (16) reported that the activity of several Aspergillus
fungal genera found in the samples is shown in Table 3. and Penicillium species in corn reduced the presence of F.
Identical fungal genera were isolated from the maize grown verticillioides. In our study, the concurrent presence of As-
in both farming systems. However, the predominant genus pergillus and Penicillium did not play a significant role as
identified in conventional samples was Fusarium (34.93% a control factor for Fusarium development on maize kernels.
Recently summarized data (31) provide evidence for
TABLE 2. Incidence of internal fungal infection detected in con- greater bacterial, actinomycete, and fungal abundance and
ventional and organic corn grown in Spain activity under organic management systems, and most re-
search suggests that organic farming practices have a pos-
% positive samples
Growing No. of itive, stimulating influence on the soil microbial commu-
conditions samples Mean ⫾ SEa Range nity. Knudsen et al. (13) reported that the microbial biomass
and activity of the soils on organic farms were higher than
Conventional 30 40.27 ⫾ 4.92 A 4–92 those in soils on conventional farms, although high biomass
Organic 30 63.20 ⫾ 4.29 B 16–94 and activity were not always correlated with suppression of
Total 60 51.73 ⫾ 3.56 4–94 brown foot rot disease, caused by Fusarium culmorum. In
a Means with different letters are significantly different (P ⬍ 0.05, comparison, several organic soil amendments significantly
ANOVA). reduced the incidence of Fusarium stalk rot disease of
154 ARIÑO ET AL. J. Food Prot., Vol. 70, No. 1

TABLE 3. Frequency distribution of different fungal genera identified in conventional and organic corn samples
% of total fungal infectiona

Corn type Aspergillus Penicillum Fusarium Alternaria Others

Conventional 14.19 A 9.40 A 34.93 A 25.19 A 16.29 A

Organic 14.37 A 12.34 A 18.15 B 40.29 B 14.85 A
Total 14.28 10.87 26.54 32.74 15.57
a Within a column, percentages with different letters are significantly different (P ⬍ 0.05, ANOVA).

maize, caused by F. verticillioides (18). Postma et al. (22) Fumonisins were detected above the LOQ (25 ng/g) in
reported that compost-amended soil was suppressive 13.3% of the samples of conventional corn and in 10.0%
against several fungal diseases of plants. One of the factors of the samples of organic corn. The concentrations of FB1
behind these antagonistic effects has been studied by Pal et and FB2 were 43 and 22 ng/g, respectively, in conventional
al. (20), who isolated several rhizobacteria from maize that corn samples but somewhat lower in organic corn samples,
were strongly antagonistic in vitro to F. verticillioides and 35 and 19 ng/g, respectively (Table 5). Again, these small
might be involved in the biological suppression of maize differences in fumonisin concentrations between conven-
root diseases. tional and organic corn samples were not significant (P ⬎
A representative number of isolated strains of Fusari- 0.05). Similarly, the agricultural system did not have any
um were identified to species according to the method of significant effect on the fumonisin concentrations found in
Nelson et al. (17). For current names of Fusarium species, conventional and organic cornflakes from the Belgian mar-
we have followed the Index Fungorum (1). The most fre- ket (19). However, in a study of the Italian market, Cirillo
quently identified Fusarium species was F. verticillioides. et al. (7) reported that the highest median concentration of
The prevalence of F. verticillioides as the predominant spe- FB1 occurred in conventional maize-based foods (P ⬎
cies isolated from corn kernels has been previously reported 0.05) whereas the highest median concentration of FB2 was
in Spain (5, 6, 25) and in other parts of the world (2, 32). in organic maize-based foods (P ⬍ 0.05).
Fumonisin production by cultures of 20 selected F. In our study, the fumonisin concentrations in the corn
verticillioides strains is shown in Table 4. These results samples were very low, much lower than the maximum of
were obtained with the HPLC–naphthalene dicarboxalde- 2,000 ng/g (for the sum of FB1 and FB2) proposed for un-
hyde–fluorescence method. Of the 10 strains isolated from processed maize in a recent EU regulation (10). However,
conventional maize, 80% produced FB1 and FB2. For the during this survey, a maize sample suspected to have
10 strains isolated organic maize, 80% produced FB1 and caused an outbreak of equine leukoencephalomalacia re-
70% produced FB2. The occurrence of fumonisin producing sulting in two dead ponies came to the laboratory. This
strains was much higher than that reported by Sala et al. sample was heavily contaminated with F. verticillioides and
(25) but very similar to more recent data presented by Cas- had an extremely high concentration of fumonisins at 12.5
tella et al. (6), both in Spanish corn samples. There was a ␮g/g (the sum of FB1 and FB2). Thus, high concentrations
high degree of variability in the concentration of fumonisins of Fusarium mycotoxins can occur in cereal grown in Ar-
produced by the F. verticillioides strains. In conventional agón, which is a concern because some of these cereals are
isolates, FB1 concentrations were 108 to 1,057 ␮g/g, with intended for human consumption.
a mean value of 447.9 ␮g/g, and FB2 concentrations were Maize from an organic farm had a 50% lower Fusar-
59 to 410 ␮g/g, with a mean value of 140.3 ␮g/g. The ium infection rate than did maize from a conventional farm,
fumonisin concentrations observed in organic isolates were probably mainly because of lower intensity of cultivation,
somewhat lower, with mean values of 283.3 ␮g/g for FB1 different crop rotation, plough tillage, and the distinct bio-
(range, 58 to 1,062 ␮g/g) and 98.5 ␮g/g for FB2 (63 to 307 mass activity of organic soils. However, although microbial
␮g/g). However, these small differences in fumonisin pro- interactions can lead to decreased colonization by Fusarium
duction between conventional and organic isolates were not in organically grown maize, a significant decrease in fu-
significant (P ⬎ 0.05). monisin concentration may not occur. The present study is

TABLE 4. Fumonisin production by cultures of selected F. verticillioides isolates from conventional and organic maize
Fumonisin B1 Fumonisin B2
No. of
Corn type isolates % positive cultures Concn (␮g/g)a % positive cultures Concn (␮g/g)

Conventional 10 80 447.90 ⫾ 109.06 A 80 140.30 ⫾ 37.97 A

Organic 10 80 283.30 ⫾ 100.99 A 70 98.5 ⫾ 30.48 A
Total 20 80 365.60 ⫾ 74.76 75 119.4 ⫾ 24.17
a Values are mean ⫾ standard error. Within a column, means with different letters are significantly different (P ⬍ 0.05, ANOVA).
J. Food Prot., Vol. 70, No. 1 FUSARIUM AND FUMONISINS IN CONVENTIONAL AND ORGANIC CORN 155

TABLE 5. Total fumonisin in conventional and organic corn samples

Fumonisin B1 Fumonisin B2
No. of
Corn type samples % positive samples Concn (ng/g)a % positive samples Concn (ng/g)

Conventional 30 13.33 43.19 ⫾ 15.52 A 10.00 21.92 ⫾ 5.28 A

Organic 30 10.00 35.36 ⫾ 13.74 A 6.67 18.78 ⫾ 4.90 A
Total 60 11.67 39.27 ⫾ 10.29 8.33 20.35 ⫾ 3.58
a Values are mean ⫾ standard error. For values below the LOQ (25 ng/g), the mean was recorded as one half of the LOQ. Within a
column, means with different letters are significantly different (P ⬍ 0.05, ANOVA).

only a partial risk assessment because other mycotoxins managed soils towards brown foot rot of barley. Appl. Soil Ecol. 12:
such as deoxynivalenol, zearalenone, aflatoxins, ochratoxin, 61–72.
14. Krebs, H., B. Streit, and H. R. Forrer. 2000. Effect of tillage and
and alternaria mycotoxins were not analyzed.
preceding crops on Fusarium infection and deoxynivalenol content
of wheat, p. 13. In T. Alfoldi, W. Lockeretz, and U. Niggli (ed.), The
ACKNOWLEDGMENTS world grows organic. Proceedings of the 13th International IFOAM
This research was supported by the Government of Aragón (Spain) Scientific Conference. IOS Press, Basel.
through projects DGA/Grupos de Investigación Consolidados and PIP065/ 15. Magkos, F., F. Arvaniti, and A. Zampelas. 2006. Organic food: buy-
2005. We thank L. Pitarch, M. Benito, and J. Guilmin for their assistance ing more safety or just peace of mind? A critical review of the
with the laboratory work and F. Villa and the Aragonese Committee for literature. Crit. Rev. Food Sci. Nutr. 46:23–56.
Organic Agriculture for the collection of samples. 16. Marin, S., V. Sanchis, F. Rull, A. J. Ramos, and N. Magan. 1998.
Colonization of maize grain by Fusarium moniliforme and F. pro-
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