Mycoflora of Maize Harvested from Iran and Imported Maize
Ali Reza Khosravi,
Ali Reza Bahonar
The natural occurrence of fungal contamination was
evaluated in stored maize in three different agro-ecological zones (Iran,
Brazil and China). A total of 45 samples were analyzed and 685 fungal
isolates were identified. The most frequent isolated fungi from maize
originated from Iran, Brazil and China were Fusarium sp. (17.3,
17.9 and 37.1%), Aspergillus sp. (9.3, 17.4 and 19.7%), Penicillium
sp. (5.8, 15.2 and 17.6%), Rhizopus sp. (2.4, 3.2 and 3.5%),
Mucor sp. (1.1, 1.6 and 1.3%), Cladosporium sp. (1.6, 1.9
and 1.9%), Alternaria sp. (1.1, 1.6 and 1.3%), Geotrichum sp.
(0, 0 and 0.3%), Acromonium sp. (0.5, 0.8 and 0%) and Absidia
sp. (0, 0.8 and 0.5%), respectively. Significant difference was observed
between the frequency of fungal isolates of Iranian maize and foreign
products (p<0.0005). Maize mycoflora profiles showed that Fusarium
verticillioides and Aspergillus flavus prevailed in 30.7 and
13.3% of the samples from China, in 12 and 5.3% of the samples from Iran
and 11.7 and 11.5% of the samples from Brazil, respectively. There were
significant differences in the frequency of Fusarium verticillioides
in Chinese maize with other countries products (p<0.0005) and that
of Aspergillus flavus in Iranian maize with other countries (p<0.002).
The results emphasize that farmers and consumers should be alerted to
the danger of fungal contamination in maize.
The increasing worldwide concern about food safety has enhanced interest in
fungal contamination and subsequent production of mycotoxins in food products.
In this regard, attention is continuously focused on maize (Zea mays L.)
because it is one of the most important dietary staple foods and feedstuffs
in different regions of the world (FAO, 2002). Maize plays an important role
in the diet of millions of people due to its high yields per hectare, its ease
of cultivation and adaptability to different agro-ecological zones, versatile
food uses and storage characteristics (Asiedu, 1989). Its economical importance
is also relevant for its use as feedstuff, mainly in the economically developed
countries (Munkvold and Desjardins, 1997). Reports indicate that maize is prone
to fungal infection during the pre and post harvest period (Hussein and Brasel,
2001; Abarca et al., 2001). Vasanthkumar (1986) demonstrated the infection
of maize by field and storage fungi during pre- and post-harvest practices in
relation to seed-borne fungal diseases of maize. The contamination of foods
and feeds usually reflects the incidence of fungal infection on the original
crops such as maize, which is affected by factors including environmental conditions
(climate, temperature and humidity), insect infestation and pre- and post-harvest
handling (Kacaniova, 2003). In the field as well as in the store, many pests
and parasites attack maize. Insects are most often considered as the principal
cause of grain losses (Gwinner et al., 1996). However, fungi are also
second important cause of deterioration and loss of maize (Scudamore and MacDonald,
2000). Kossou and Aho (1993) reported that fungi could cause about 50-80% of
damage on farmers maize during the storage period if conditions are favorable
for their development. The major genera commonly encountered on maize in tropical
regions are Fusarium, Aspergillus and Penicillium. This
is a cause of concern because these genera have species capable of producing
a wide spectrum of compounds shown to be toxic to man and animals (Orsi et
al., 2000). Also, its nutritional characteristics expose it to the constant
attack of fungi and insect predators (Samson, 1991). Fusarium verticillioides
(F. verticillioides), F. proliferatum, F. oxysporum,
F. graminearum, Aspergillus flavus (A. flavus), A.
parasiticus, Cladosporium sp. and Penicillium sp. are the
most common seed-borne maize mycoflora, with F. verticillioides being
the most frequent fungus present in maize products (Gonzáles et al.,
1995; Kedera et al., 1999). A major problem related to fungal attack
in maize is the production of toxic secondary metabolites, particularly fumonisin,
zearalenone and aflatoxin, produced by F. verticillioides, F. graminearum
and A. flavus, respectively (Scott and Zummo, 1994). Zearalenone
is an estrogenic compound that produces infertility and other reproductive problems
in animals (Ngoko et al., 2001). Aflatoxin has powerful teratogenic,
mutagenic and hepato-carcinogenic effects (Wang et al., 2001), while
fumonisins are reported to have cancer-promoting activity, in addition to causing
several diseases in animals (Bullerman and Draughon, 1994; Rheeder et al.,
2002). The co-occurrence of fumonisin with aflatoxin B1 (AFB1) is presumed to
play an important role in the promotion of carcinogenesis (Ueno, 2000). Both
fungal and mycotoxin contamination are currently regarded as the primary concern
in the effort to reduce problems in the global trade of agricultural commodities.
Hence, there is a great need for more extensive investigations, where maize
production and consumption are predominant. Since, the naturally fungal contamination
of maize based-foods and feedstuffs from Iran had not been studied to date,
the aim of the present study, was to determine mycoflora distribution of Iranian
maize and to compare it with imported maize into Iran (Brazilian and Chinese
MATERIALS AND METHODS
Sampling: A total of 45 samples originated from 45 soils, which were suspected to visual contamination, (Iranian, No. = 15; Brazilian, No. = 15; Chinese maize, No. = 15) were collected from different locations of Iran in 2006. The samples were collected in plastic bags, transferred to Mycology Research Center and stored at 4°C until analysis.
Sample culture: One hundred grams from each sample were partially sterilized in a 0.4% sodium hypochlorite solution for 2 min. Subsequently, the supernatant solution was discarded and the sample was rinsed once in distilled water and followed to dry. Twenty-five grains of each sample were homogenized and inoculated into Dichloran Rose-Bengal Chloramphenicol (DRBC, Sigma, St. Louis, USA) agar, 5 grains in a Petri dish. The cultures were then incubated for 5 to 14 days at 25°C in a dark chamber. The colonies were exactly isolated and sub-cultured on slant (PDA) and (SGA) media. Fusarium species were isolated, transferred onto Spezieller Nahrstoffarmer agar and incubated at 25°C for 7 days. Final identification of Fusarium and Aspergillus species was conducted according to Nelson et al. (1983) and Raper and Fennell (1965) methods, respectively. The other genera were identified using PDA and SGA media. All chemicals used, unless otherwise stated, were obtained from Merck Company (Darmstadt, Germany).
Statistical analyses: Unpaired Students t-test was performed using SPSS software (Version 13.0) and differences were considered significant at p<0.05.
Of the 45 samples examined, which had been collected from Iran, 15 samples
and imported foreign samples, 15 from Brazil and 15 from China, 147 (21.5%),
225 (32.8%) and 313 (45.7%) fungal isolates were isolated, respectively. Significant
difference was observed between the frequency of fungal isolates of Iranian
maize and imported samples (p<0.0005). The most frequent isolated fungi from
maize originated from Iran, Brazil and China were F. verticillioides
(12, 11.7 and 30.7%), F. proliferatum (3.7, 4.3 and 4.5 %), other Fusarium
sp. (1.6, 1.9 and 1.9%), A. flavus (5.3, 11.5 and 13.3%), A.
ochraceous (1.6, 2.4 and 2.7%), A. niger (1.3, 1.9 and 2.1%), other
Aspergillus sp. (1.1, 1.6 and 1.6%), Penicillium sp. (5.8, 15.2
and 17.6%), Rhizopus sp. (2.4, 3.2 and 3.5%), Mucor sp. (1.1,
1.6 and 1.3%), Cladosporium sp. (1.6, 1.9 and 1.9%), Alternaria
sp. (1.1, 1.6 and 1.3%), Geotrichum sp. (0, 0 and 0.3%), Acromonium
sp. (0.5, 0.8 and 0%) and Absidia sp. (0, 0.8 and 0.5%) (Table
1). Among the Fusarium sp., F. verticillioides was the most
prevalent isolated fungus in Chinese (30.7%), followed by Iranian (12%) and
Brazilian (11.73%) maize. There was significant difference in the frequency
of F. verticillioides in Chinese maize with other countries samples (p<0.0005),
whereas no significant difference was observed between Iranian and Brazilian
maize. Also, significant differences were not observed between other Fusarium
species with different origins. Overall, Fusarium sp. were the most
prevalent isolated fungi of maize collected from Iran (17.3%), Brazil (17.9%)
and China (37.1%). Mycological analyses showed that Aspergillus sp. was
the second predominant fungal genus in maize, understudy. Aspergillus sp.
prevailed in 9.3, 17.3 and 19.7% from Iranian, Brazilian and Chinese samples,
respectively. Aspergillus flavus was the most frequent species in Chinese
(13.3%), followed by Brazilian (11.5%) and Iranian (5.3%) maize. There was only
significant difference between Iranian maize with other countries samples (p<0.002),
whereas significant differences were not observed between other Aspergillus
sp. with different origins. The incidence of Penicillium sp. was
about 5.9, 15.2 and 17.6% from Iranian, Brazilian and Chinese samples, respectively.
||The relative frequency of fungal isolates from Iranian, Brazilian
and Chinese maize
|NS: Not Significant
There was significant difference in the frequency of Penicillium sp.
in Iranian maize with other countries samples (p<0.0005), whereas no significant
difference was observed between Chinese and Brazilian maize. The results showed
that there were not significant differences between the frequency of Rhizopus
sp., Mucor sp., Cladosporium sp. and Alternaria sp.
with different origins.
Fungi are worldwide microorganisms and tropical climates stimulate the growth
of toxigenic fungi on agricultural products, with consequent risk of mycotoxin
contamination (Hell et al., 2000). Maize as a cereal crop is grown throughout
the world and plays an important role in both human and animal nutrition (Asiedu,
1989). China and Brazil has been ranked as the second and third largest world
producer, following the united state. Since, Iran is one of the importers of
maize from other countries, especially China and Brazil and regarding to the
relative high incidence of mycotoxicosis in animals and to lesser extent in
human, fungal contamination was investigated in maize collected from infected
sites with different sources. In the present study, the most important isolated
fungi were Fusarium sp., Aspergillus sp. and Penicillium sp.
in different samples. Literature reviews showed that the major genera commonly
encountered on maize in tropical regions are Fusarium, Aspergillus
and Penicillium (Orsi et al., 2000; Ghiasian et al.,
2004). The predominance of Fusarium sp., Aspergillus sp. and Penicillium
sp. in freshly harvested maize grains was also shown by Lillehoj and Zuber
(1988) in a work carried out with samples from different countries. In a study
conducted by Ghiasian et al. (2004) in Iran, Fusarium (38.5%),
followed by Aspergillus (8.7%), Rhizopus (4.8%), Penicillium
(4.5%), Mucor sp. (1.1%) and four other fungal genera were noted.
Fusarium verticillioides was the most prevalent species (83% of Fusarium
isolates and 52% of the total isolations). Among the Aspergillus sp.,
A. flavus was the most widely recovered species and 38% of samples were
contaminated with this potentially aflatoxigenic fungus. Penicillium
sp. were seen in all the samples. Almeida et al. (2000) reported that
fungal contamination of 66 samples of freshly harvested maize grains collected
in different regions of the state of São Paulo (Brazil) were Fusarium
sp. (80.0%), Penicillium sp. (40.0%), Aspergillus sp. (23.3%)
and Geotrichum sp. (23.3%). These species are natural contaminants of
cereals worldwide and are mostly found in maize and its derived products (Shephard
et al., 2000). Gonzales et al. (1995) indicated the isolation
of around 20 different species, with some of them being potentially toxigenic
fungi such as A. fumigatus, A. parasiticus, F. verticillioides
and Monascus rube from maize. Previous studies revealed that
F. verticillioides, F. proliferatum, F. oxysporum, F.
graminearum, A. flavus, A. parasiticus, Cladosporium
sp. and Penicillium sp. were the most common seed-borne maize mycoflora,
with F. verticillioides being the most frequent fungus present in maize
(Scott and Zummo, 1994; Kedera et al., 1999). As described in this study,
species of Fusarium were the most prevalent component of maize mycoflora
present in all samples. Among them, F. verticillioides (12%) and then
F. proliferatum (3.7%) were the predominant Fusarium isolated
from Iranian maize and was found at higher quantity than that observed on commercial
maize harvested in Brazil (11.7 and 4.3%) and lower quantity than Chinese maize
(30.7 and 4.5%). Fusarium verticillioides and F. proliferatum
co-occur worldwide on maize (Leslie et al., 1995), probably because they
have similar optimum growth conditions and they do not show apparent antagonism
when growing together (Logrieco and Moretti, 1995). Doko et al. (1995)
reported F. verticillioides is the most frequently isolated fungus from
maize and maize based commodities in France, Spain and Italy. Likewise, Orsi
et al. (2000) found in Brazil that F. verticillioides was the
predominant Fusarium species on maize. Also, a high occurrence of F.
verticillioides associated with natural fumonisin contamination has been
found in maize in the State of Paraná, which produces 25% of the maize
crop in Brazil (Ono et al., 2001). However, reports of surveys conducted
in some African countries showed it as the most prevalent fungus on maize (Scott
and Zummo, 1994; Allah Fadl, 1998). It has been reported that late planting
of maize with harvesting in wet conditions favors disease caused by F. verticillioides
(Abarca et al., 2001) and the prevalence of this fungus is considerably
increased in seasons with wet weather (Al-Heeti, 1987). The type of maize cultivar
and grain characteristics such as color, endosperm type, chemical composition
and stage of development may also influence fungal infection and subsequent
fumonisin production. Among the Aspergillus isolates, the species identified
was A. flavus. The presence of A. flavus in freshly harvested
maize was previously observed (Lillehoj et al., 1980; Leoni and Soares,
1994; Machinski et al., 2000). In general, it is thought that maize cultivars
with upright cobs, tight husks (Emerson and Hunter, 1980), thin grain peri carp
(Riley et al., 1993) and an increased propensity for grain splitting
(Odvody et al., 1990) are likely to be more susceptible to fungal infection.
Considering a high incidence of fungal contamination of imported maize, it seems
that the traditional methods of handling grains during harvesting in the field,
drying process in relevant country and transferring it to other countries lead
to mechanical damages of grains. In this condition, broken and ground grains
are more vulnerable to fungal attack than whole grains. On the other hand, this
contamination could be due to long-term storage of imported maize in the poor
environmental conditions including high moisture and temperature in borderlines
and barns in Iran. Maize stored for long-time periods are more vulnerable than
freshly harvested maize. Insects and rodents may also be contributed to deteriorating
the grains rapidly and increasing maize mycoflora during long-term storage (Hussein
and Brasel, 2001). It is necessary to mention that the higher fungal contamination
of Chinese maize than Brazilian samples is probably related to higher moisture,
probable contamination of maize in origin and applied traditional methods in
harvesting, drying and also improper handling grains. Rapid deterioration of
grains is a major problem related to fungal contamination in maize, leading
to the accumulation of mycotoxins, particularly fumonisin, zearalenone and aflatoxin,
produced by F. verticillioides, F. graminearum and A. flavus,
respectively (Scott and Zummo, 1994). They may cause acute toxicity or decrease
productivity in animals and occasionally caused acute intoxication in humans
(Dawson, 1991). Several surveys carried out in many parts of the world have
revealed that F. verticillioides, F. proliferatum are the fumonisin-producing
Fusarium species most frequently isolated from maize in tropical and
subtropical zones (Shephard et al., 2000). Also, various reports have
shown the presence of AFB1 in 12.3% of the maize samples taken from several
Brazilian states (Sabino et al., 1989). Although the detection of toxigenic
fungi in a substrate does not necessarily indicate that mycotoxins are naturally
occurring in the field, it alerts to the potential risk of contamination. Therefore,
it is important to keep human and animals exposure to fungal and mycotoxin contaminations
as low as possible. In this regard, the lack of proper storage facilities induces
fungal contamination and accumulation of mycotoxins during the post-harvest
period. Therefore, it is suggested that the proper handling of maize during
the post-harvest phase is crucial to preserve grains for longer periods.
This study was supported by the Research Council of University of Tehran.
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