**1. Introduction**

[11] Tiwari SP, Bhatnagar PS. Pod shattering of soybean in India. Journal of Oilseed

[12] Tiwari SP, Bhatnagar PS. Consistent resistance for pod shattering in soybean (Glycine max (L.) Merrill) varieties. Indian Journal of Agricultural Sciences 1992; 63(3) 173-174.

[13] Tukamuhabwa P, Rubaihayo P, Dashiell KE. Genetic components of pod shattering in

[14] Twizeyimana M, Ojiambo PS, Hartman GL, Bandyopadhyay R. Dynamics of Soybean Rust Epidemics in Sequential Plantings of Soybean Cultivars in Nigeria. Plant Disease

[15] Twizeyimana M, Ojiambo PS, Ikotun T, Ladipo JL, Hartman GL, Bandyopadhyay R. Evaluation of Soybean Germplasm for Resistance to Soybean Rust (Phakopsora

[16] Upadhaya and Paradkar. Pod shattering in soybean (Glycine max (L.) Merrill). Journal

[17] Yadav RK, Shukla RK, Chattopadhyay D. Soybean cultivar resistant to Mungbean Yellow Mosaic India Virus infection induces viral RNA degradation earlier than the

pachyrhizi) in Nigeria. Plant Disease 2008; 92(6) 947-952.

susceptible cultivar. Virus Research 2009;144(1-2) 89-95.

Research 1988; 5 : 92-93.

184 Soybean - Pest Resistance

2011; 95(1): 43-50.

soybean. Euphytica 2002; 125(1) 29-34.

of Oilseed Research 1991; 8 121-122.

#### **1.1. Toxigenic fungi and mycotoxins in cereal and soybean products**

Cereals and soybean are plants used extensively in food and feed manufacturing as a source of proteins, carbohydrates and oils. These materials, due to their chemical composition, are particularly susceptible to microbial contamination, especially by filamentous fungi. Cereals, soybean, and other raw materials can be contaminated with fungi, either during vegetation in the field or during storage, as well as during the processing.

Fungi contaminating grains have been conventionally divided into two groups – field fungi and storage fungi. Field fungi are those that infect the crops throughout the vegetation phase of plants and they include plant pathogens such as *Alternaria*, *Fusarium*, *Cladosporium,* and *Botrytis* species. Their numbers gradually decrease during storage. They are replaced by storage fungi of *Aspergillus*, *Penicillium*, *Rhizopus* and *Mucor* genera that infect grains after harvesting, during storage [1]. Both groups of fungi include toxigenic species. Currently, this division is not so strict.

Therefore, according to [2], four types of toxigenic fungi can be distinguished:


© 2013 Piotrowska et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Piotrowska et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**•** Fungi that are found on the soil or decaying plant material that occur on the developing kernels in the field and later proliferate in storage if conditions permit, e.g. *Penicillium ver‐ rucosum and Aspergillus ochraceus*.

**Commodities Country Fungal species Ref**

Romania *Aspergillus flavus, A.parasiticus, A.candidus, A.niger, Penicillium*

India *Aspergillus flavus, A.candidus, A.versicolor, Eurotium repens,*

USA *Diaporthe phaseolorum var. sojae, Fusarium sp., Alternaria alternata,*

Croatia *Fusarium sporotrichides, F.verticillioides, F.equiseti, F.semitecium,*

Argentina *Aspergillus flavus, A.niger, A.candidus, A.fumigatus, Fusarium*

Rice Ecuador *Aspergillus flavus, A.ochraceus, Fusarium verticillioides, F.oxysporum,*

Wheat Argentina *Aspergillus flavus, A.niger, A.oryzae, Fusarium verticillioides, Penicillium*

Maize Ecuador *Aspergillus flavus, A.parasiticus, Fusarium graminearum,*

*verticillioides, F.semitectum, Penicillium janthinellum, P.simplicissimum, Nigrospora oryzae, Cladosporium cladosporioides, Arthrinium phaeospermum*

Mycotoxins in Cereal and Soybean-Based Food and Feed

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*griseofulvum, P.variabile, Fusarium culmorum, F.graminearum, F.oxysporum*

*A.sulphureus, Fusarium sp., Alternaria sp., Curvularia sp.*

*Alternaria sp., Fusarium sp., Curvularia sp., Cladosporium sp., Fusarium equiseti, F.oxysporum, F.solani*

*F.pseudograminearum, F.chlamydosporum, F.sambucinum*

*verticillioides, F.equiseti, F.semitecium, F.graminearum, Penicillium funiculosum, P.griseofulvum, P.canenscens, Erotium sp. Cladosporium sp., Alternaria alternata, A.infectoria, A.oregonensis*

*F.proliferatum, F.semitectum, F.solani, Penicillium janthinellum, Epicoccum nigrum, Curvularia lunata, Nigrospora oryzae, Rhizopus stolonifer, Bipolaris oryzae*

*funiculosum, P.oxalicum*

*auriantogriseum, P.verrucosum, P.viridicatum, Alternaria sp.*

*moniliforme, F.verticillioides, Penicillium verrucosum, P.viridicatum P.crustosum*

*F.verticillioides, Mucor racemosus Rhizopus stolonifer, Acremonium strictum, Alternaria alternata, Cladosporium sp.*

*F.verticillioides*

*F.nygamai, Alternaria alternata, Penicillium funiculosum, P.citrinum, Aspergillus flavus*

Croatia *Fusarium graminearum, F.poae, F.avenaceum, F.verticillioides* [11]

Germany *Aspergillus candidus, A.flavus, A.versicolor, Eurotium sp., Penicillium*

Poland *Alternaria tenuis, Aspergillus aculeatus, A.parasiticus, Fusarium*

Poland *Aspergillus aculeatus, Aspergillus parasiticus, Fusarium moniliforme,*

Argentina *Fusarium verticillioides, F.proliferatum, F.subglutinans, F.dlamini,*

[6]

187

[7]

[4]

[8-10]

[11]

[12, 13]

[6]

[12]

[14]

[15]

[6]

[15]

[16, 17]

Soybean Ecuador *Aspergillus flavus, A.niger, A.ochraceus, A.parasiticus, Fusarium*

Fungal growth is influenced by complex interaction of different environmental factors such as temperature, pH, humidity, water activity, aeration, availability of nutrients, mechanical damage, microbial interaction or the presence of antimicrobial compounds. Poor hygiene, inappropriate temperature and moisture during harvesting, storage, processing and han‐ dling may contribute to increased contamination extent.

Fungal contamination can cause damage in cereal grains and oilseeds, including low germi‐ nation, low baking quality, discoloration, off-flavours, softening and rotting, and formation of pathogenic or allergenic propagules.

It may also decrease the kernel size and thus affect the flour yield. Moulds growing on stor‐ ed cereals produce a range of volatile odour compounds, including 3-octanone, 1-octen-3-ol, geosmin, 2-methoxy-3-isopropylpyrazine, and 2-methyl-1-propanol which are responsible for an earthy-musty off-odour and affect the quality of raw materials even when present in very small amounts [3]. Moulds produce a vast number of enzymes: lipases, proteases, amy‐ lases, which are able to break down food into components leading to its spoilage. Fungi growing on stored grains can reduce the germination rate and decrease the content of carbo‐ hydrate, protein and oils. During storage of soybean seed lasting 12 months, the moisture content was at the level of 10-11%. It was observed that the germination rate decreased from initial 75% to 4% prior to the lapse of a 9-month period. In prolonged storage under natural conditions, the total carbohydrate content decreased from 21% to 16.8%, and protein and the total oil contents became slightly reduced [4]. Moulds as food and feed spoilage microorgan‐ isms have been characterized in several review articles [2, 5].

The largest producers of soybean in the World are the United States of America, Brazil, Ar‐ gentina, China, and India. The climatic conditions in soybean-growing regions (moderate mean temperature and relative humidity between 50 and 80%) provide optimal conditions for fungal growth. Soybean (*Glyccine max* L.Merr.) is often attacked by fungi during cultiva‐ tion, which significantly decreases its productivity and quality in most production areas. Fungi associated with cereal grains and oilseeds are important in assessing the potential risk of mycotoxin contamination. Mycotoxins are fungal secondary metabolites which are toxic to vertebrate animals even in small amounts when introduced orally or by inhalation.

Table 1 summarises the occurrence of contamination of different raw materials in various countries. Some of them are of mycotoxicological interest.

Soybean matrix has been rarely studied compared to cereals in relation to fungal and myco‐ toxin contamination. The fungi associated with soybean seeds, pods and flowers in North America were reviewed by [20]. The most common species belong to *Aspergillus*, *Fusarium*, *Chaetomium*, *Penicillium*, *Alternaria* and *Colletotrichum* genera. Most of these fungi were re‐ corded in mature seeds prior to storage. About 10% of them are commonly referred to as storage moulds. Most of the isolated fungi are facultative parasites or saprophytes.


**•** Fungi that are found on the soil or decaying plant material that occur on the developing kernels in the field and later proliferate in storage if conditions permit, e.g. *Penicillium ver‐*

Fungal growth is influenced by complex interaction of different environmental factors such as temperature, pH, humidity, water activity, aeration, availability of nutrients, mechanical damage, microbial interaction or the presence of antimicrobial compounds. Poor hygiene, inappropriate temperature and moisture during harvesting, storage, processing and han‐

Fungal contamination can cause damage in cereal grains and oilseeds, including low germi‐ nation, low baking quality, discoloration, off-flavours, softening and rotting, and formation

It may also decrease the kernel size and thus affect the flour yield. Moulds growing on stor‐ ed cereals produce a range of volatile odour compounds, including 3-octanone, 1-octen-3-ol, geosmin, 2-methoxy-3-isopropylpyrazine, and 2-methyl-1-propanol which are responsible for an earthy-musty off-odour and affect the quality of raw materials even when present in very small amounts [3]. Moulds produce a vast number of enzymes: lipases, proteases, amy‐ lases, which are able to break down food into components leading to its spoilage. Fungi growing on stored grains can reduce the germination rate and decrease the content of carbo‐ hydrate, protein and oils. During storage of soybean seed lasting 12 months, the moisture content was at the level of 10-11%. It was observed that the germination rate decreased from initial 75% to 4% prior to the lapse of a 9-month period. In prolonged storage under natural conditions, the total carbohydrate content decreased from 21% to 16.8%, and protein and the total oil contents became slightly reduced [4]. Moulds as food and feed spoilage microorgan‐

The largest producers of soybean in the World are the United States of America, Brazil, Ar‐ gentina, China, and India. The climatic conditions in soybean-growing regions (moderate mean temperature and relative humidity between 50 and 80%) provide optimal conditions for fungal growth. Soybean (*Glyccine max* L.Merr.) is often attacked by fungi during cultiva‐ tion, which significantly decreases its productivity and quality in most production areas. Fungi associated with cereal grains and oilseeds are important in assessing the potential risk of mycotoxin contamination. Mycotoxins are fungal secondary metabolites which are toxic to vertebrate animals even in small amounts when introduced orally or by inhalation.

Table 1 summarises the occurrence of contamination of different raw materials in various

Soybean matrix has been rarely studied compared to cereals in relation to fungal and myco‐ toxin contamination. The fungi associated with soybean seeds, pods and flowers in North America were reviewed by [20]. The most common species belong to *Aspergillus*, *Fusarium*, *Chaetomium*, *Penicillium*, *Alternaria* and *Colletotrichum* genera. Most of these fungi were re‐ corded in mature seeds prior to storage. About 10% of them are commonly referred to as

storage moulds. Most of the isolated fungi are facultative parasites or saprophytes.

*rucosum and Aspergillus ochraceus*.

186 Soybean - Pest Resistance

of pathogenic or allergenic propagules.

dling may contribute to increased contamination extent.

isms have been characterized in several review articles [2, 5].

countries. Some of them are of mycotoxicological interest.


with fungi by 70.5% more and barley by 24.8% less as compared to the crops from conven‐ tional farms [24]. Similarly, the total number of fungi in Polish ecological oat products was about a hundred times higher than in conventional ones. In samples of ecological origin, the mean value of fungi was 1.1×104 CFU/g, whereas for conventional grains it was 5.0×102

Mycotoxins in Cereal and Soybean-Based Food and Feed

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189

The results obtained by [14] showed that the most common moulds isolated from whole wheat and wheat flour belong to the *Aspergillus* and *Penicillium* genera. From the whole wheat flour, 83.7% of *Aspergillus* followed by *Penicillium* (7.6%), *Eurotium* (2.9%) and *Al‐ ternaria* (2.5%) species were isolated. The white flour contained 77.3% of *Aspergillus*, 15% of *Penicillium* and 4.1% of *Cladosporium* genera. *Aspergillus candidus* was the dominant species. Among all the isolated fungal species, 93.2% belonged to the group of toxigenic fungi. Several toxin-producing *Aspergillus* species were reported to dominate on cereals, especially *A.flavus*, *A.candidus*, *A.niger*, *A.versicolor*, *A.penicillioides*, and *Eurotium* sp. at lower water activity [25]. Among *Aspergillus* species isolated from Ecuadorian soybean seeds, *Aspergillus flavus* and *A.ochraceus* were the most prevalent ones. The most frequent *Fusarium* species were *F.verticillioides* and *F.semitecium*. All the examined samples were contaminated with these species [6]. The presence of mycobiota in raw materials and fin‐ ished fattening pig feed was determined in eastern Argentina. All samples of soybean seeds were contaminated with fungi in the range from 10 to 9.0×102 CFU/g, depending on the sampling period. The most prevalent species in soybean and wheat bran were *As‐*

The fungal microflora changes during post-harvest drying and storage. The field fungi are adapted to growth at high water activity and they die during drying and storage, to be re‐ placed by storage fungi that are capable of growing at lower aw. For most grains, moisture content in the range from 10% to 14% is recommended, depending on the grain type and

A wide range of microorganisms have been isolated from storage grains, including psychro‐ tolerant, mesophilic, thermophilic, xerophilic and hydrophilic species. The extremely xero‐ philic species are *Eurotium* spp. and *Aspergillus restrictus*, the moderate xerophilic ones include *A.candidus* and *A.flavus*, and the slighty xerophilic one is *A.fumigatus*. An example of psychrotolerant species belonging to *Penicillium* genera is *P.aurantiogriseum* and *P.verruco‐ sum*, mesophilic species can be represented by *P.corylophilum*, and thermophilic species by *Talaromyces thermophilus*. Among the hydrophiles, the most common are *Fusarium* and *Acre‐ monium* species [25]. The minimum aw for conidial formation is influenced by temperature,

C. Many species belonging to *Aspergillus* and *Penicillium* genera are highly adapted

to the rapid colonisation of substrates of reduced water activity. Modifying several factors in grain storage may facilitate safe storage. Stores should be monitored for relative humidity, temperature and airflow efficiency. Moisture migration may occur during storage and create damp pockets. In addition to this, insect infestations may cause heating and the generation of moisture. Aeration with cool air may help to protect the stored commodities against fun‐

C, but to 0.83

for instance, *P.aurianogriseum* produces conidia to a minimum of 0.86 aw at 30o

CFU/g [18].

*pergillus flavus* and *Fusarium verticillioides* [12].

desired storage life [1].

aw at 23o

gal development.

**Table 1.** Fungal species dominated in cereals and cereal products

*Fusarium graminearum* is associated with cereals and soybean growing in warmer areas such as South and North America or China, and *F.culmorum* in cooler areas such as Finland, France, Poland or Germany. Mechanical damage of kernels by birds or insects, e.g. Europe‐ an corn borer and sap beetles, predisposes corn to infections caused by *Fusarium* and other "field fungi". *Fusarium moniliforme* and *F.proliferatum* are the most common fungi associated with maize. It was found that the levels of contamination with *Fusarium* sp. were significant‐ ly greater on the conventional than the transgenic cultivars in 2000, but in 1999 the differ‐ ence between the cultivars was not statistically significant. In case of *Alternaria*, a greater frequency of contamination in transgenic varieties was observed. The authors concluded that the isolation frequency can vary by years and is more dependent on the environmental and cultural practices than on varieties [9]. The isolation frequencies of fungi from seeds and pods of soybean cultivars varied annually, in part due to some differences in environmental conditions (rainfall) [8].

*Fusarium* species occur worldwide in a variety of climates and on many plant species as epi‐ phytes, parasites, or pathogens. *Fusarium*-induced diseases of soybean have been attributed to different species: *Fusarium oxysporum* (fusarium blight, wilt and root rot), *Fusarium semite‐ ctum* (pod and collar rot), *F.solani* (sudden death syndrome) [21, 22]. *Fusarium* infections are spread by air-borne conidia on the heads or by a systemic infection. The species belonging to *Fusarium* genera are of particular interest due to the formation of a wide range of secondary metabolites, many of which are toxic to humans or animals. Infections by *Fusarium* spp. were determined by [11] in different crops. The contamination expressed as the percentage of seeds with *Fusarium* colonies ranged from 5% to 69% for wheat, from 25% to 100% for maize, from 4% to 17% for soybean. The dominant species were *F.graminearum* on wheat (27% of isolates), *F.verticillioides* on maize (83 % of isolates), and *F.sporotrichioides* on soybean (34 % of isolates) [11]. This study suggested that the risk of contamination with *Fusarium* toxins is higher for maize and wheat than for soybean.

The mycological state of grain can be considered as good when the number of CFU is with‐ in the range 103 -105 per gram [23]. In our research, the contamination of feed components such as barley, maize and wheat was in the range from 102 to 104 CFU/g, depending on the crop, region and mills [15]. It was found that wheat from organic farms was contaminated with fungi by 70.5% more and barley by 24.8% less as compared to the crops from conven‐ tional farms [24]. Similarly, the total number of fungi in Polish ecological oat products was about a hundred times higher than in conventional ones. In samples of ecological origin, the mean value of fungi was 1.1×104 CFU/g, whereas for conventional grains it was 5.0×102 CFU/g [18].

**Commodities Country Fungal species Ref**

Breakfast cereals Poland *Aspergillus versicolor, A.flavus, A.sydowi, A.niger, A.ochraceus,*

Wheat flour Germany *Aspergillus candidus, A.flavus, A.niger, Eurotium sp. Penicillium*

**Table 1.** Fungal species dominated in cereals and cereal products

toxins is higher for maize and wheat than for soybean.

such as barley, maize and wheat was in the range from 102

conditions (rainfall) [8].

188 Soybean - Pest Resistance

in the range 103

Oats Poland *Cladosporium sp., Aspergillus sp., Penicillium sp.* [18]

*Fusarium graminearum* is associated with cereals and soybean growing in warmer areas such as South and North America or China, and *F.culmorum* in cooler areas such as Finland, France, Poland or Germany. Mechanical damage of kernels by birds or insects, e.g. Europe‐ an corn borer and sap beetles, predisposes corn to infections caused by *Fusarium* and other "field fungi". *Fusarium moniliforme* and *F.proliferatum* are the most common fungi associated with maize. It was found that the levels of contamination with *Fusarium* sp. were significant‐ ly greater on the conventional than the transgenic cultivars in 2000, but in 1999 the differ‐ ence between the cultivars was not statistically significant. In case of *Alternaria*, a greater frequency of contamination in transgenic varieties was observed. The authors concluded that the isolation frequency can vary by years and is more dependent on the environmental and cultural practices than on varieties [9]. The isolation frequencies of fungi from seeds and pods of soybean cultivars varied annually, in part due to some differences in environmental

*Fusarium* species occur worldwide in a variety of climates and on many plant species as epi‐ phytes, parasites, or pathogens. *Fusarium*-induced diseases of soybean have been attributed to different species: *Fusarium oxysporum* (fusarium blight, wilt and root rot), *Fusarium semite‐ ctum* (pod and collar rot), *F.solani* (sudden death syndrome) [21, 22]. *Fusarium* infections are spread by air-borne conidia on the heads or by a systemic infection. The species belonging to *Fusarium* genera are of particular interest due to the formation of a wide range of secondary metabolites, many of which are toxic to humans or animals. Infections by *Fusarium* spp. were determined by [11] in different crops. The contamination expressed as the percentage of seeds with *Fusarium* colonies ranged from 5% to 69% for wheat, from 25% to 100% for maize, from 4% to 17% for soybean. The dominant species were *F.graminearum* on wheat (27% of isolates), *F.verticillioides* on maize (83 % of isolates), and *F.sporotrichioides* on soybean (34 % of isolates) [11]. This study suggested that the risk of contamination with *Fusarium*

The mycological state of grain can be considered as good when the number of CFU is with‐

crop, region and mills [15]. It was found that wheat from organic farms was contaminated


to 104

CFU/g, depending on the

Croatia *Fusarium verticillioides, F.graminearum* [11]

*Fusarium graminearum, Penicillium chrysogenum, Eurotium repens*

*auriantogriseum, P.brevicompactum, P.citrinum, P.griseofulvum, P.verrucosum, Cladosporium cladosporioides*

[19]

[14]

The results obtained by [14] showed that the most common moulds isolated from whole wheat and wheat flour belong to the *Aspergillus* and *Penicillium* genera. From the whole wheat flour, 83.7% of *Aspergillus* followed by *Penicillium* (7.6%), *Eurotium* (2.9%) and *Al‐ ternaria* (2.5%) species were isolated. The white flour contained 77.3% of *Aspergillus*, 15% of *Penicillium* and 4.1% of *Cladosporium* genera. *Aspergillus candidus* was the dominant species. Among all the isolated fungal species, 93.2% belonged to the group of toxigenic fungi. Several toxin-producing *Aspergillus* species were reported to dominate on cereals, especially *A.flavus*, *A.candidus*, *A.niger*, *A.versicolor*, *A.penicillioides*, and *Eurotium* sp. at lower water activity [25]. Among *Aspergillus* species isolated from Ecuadorian soybean seeds, *Aspergillus flavus* and *A.ochraceus* were the most prevalent ones. The most frequent *Fusarium* species were *F.verticillioides* and *F.semitecium*. All the examined samples were contaminated with these species [6]. The presence of mycobiota in raw materials and fin‐ ished fattening pig feed was determined in eastern Argentina. All samples of soybean seeds were contaminated with fungi in the range from 10 to 9.0×102 CFU/g, depending on the sampling period. The most prevalent species in soybean and wheat bran were *As‐ pergillus flavus* and *Fusarium verticillioides* [12].

The fungal microflora changes during post-harvest drying and storage. The field fungi are adapted to growth at high water activity and they die during drying and storage, to be re‐ placed by storage fungi that are capable of growing at lower aw. For most grains, moisture content in the range from 10% to 14% is recommended, depending on the grain type and desired storage life [1].

A wide range of microorganisms have been isolated from storage grains, including psychro‐ tolerant, mesophilic, thermophilic, xerophilic and hydrophilic species. The extremely xero‐ philic species are *Eurotium* spp. and *Aspergillus restrictus*, the moderate xerophilic ones include *A.candidus* and *A.flavus*, and the slighty xerophilic one is *A.fumigatus*. An example of psychrotolerant species belonging to *Penicillium* genera is *P.aurantiogriseum* and *P.verruco‐ sum*, mesophilic species can be represented by *P.corylophilum*, and thermophilic species by *Talaromyces thermophilus*. Among the hydrophiles, the most common are *Fusarium* and *Acre‐ monium* species [25]. The minimum aw for conidial formation is influenced by temperature, for instance, *P.aurianogriseum* produces conidia to a minimum of 0.86 aw at 30o C, but to 0.83 aw at 23o C. Many species belonging to *Aspergillus* and *Penicillium* genera are highly adapted to the rapid colonisation of substrates of reduced water activity. Modifying several factors in grain storage may facilitate safe storage. Stores should be monitored for relative humidity, temperature and airflow efficiency. Moisture migration may occur during storage and create damp pockets. In addition to this, insect infestations may cause heating and the generation of moisture. Aeration with cool air may help to protect the stored commodities against fun‐ gal development.
