Mycotoxins in Cereal and Soybean-Based Food and Feed

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:

• Plant pathogens as Fusarium graminearum and Alternaria alternata;

• Fungi that grow and produce mycotoxins on senescent or stressed plants, e.g. F. moniliforme and Aspergillus flavus;

• Fungi that initially colonize the plant and increase the feedstock’s susceptibility to contamination after harvesting, e.g. Aspegillus flavus.

• 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 verrucosum and Aspergillus ochraceus.

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 handling may contribute to increased contamination extent.

Fungal contamination can cause damage in cereal grains and oilseeds, including low germination, 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 stored 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, amylases, 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 carbohydrate, 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 microorganisms have been characterized in several review articles [2, 5].

The largest producers of soybean in the World are the United States of America, Brazil, Argentina, 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 cultivation, 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 mycotoxin 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 recorded 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.

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. European 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 significantly greater on the conventional than the transgenic cultivars in 2000, but in 1999 the difference 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 epiphytes, parasites, or pathogens. Fusarium-induced diseases of soybean have been attributed to different species: Fusarium oxysporum (fusarium blight, wilt and root rot), Fusarium semitectum (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 within 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 conventional 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].

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 Alternaria (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 finished 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 Aspergillus 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 replaced by storage fungi that are capable of growing at lower a_w. 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 psychrotolerant, mesophilic, thermophilic, xerophilic and hydrophilic species. The extremely xerophilic 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.verrucosum, mesophilic species can be represented by P.corylophilum, and thermophilic species by Talaromyces thermophilus. Among the hydrophiles, the most common are Fusarium and Acremonium species [25]. The minimum a_w for conidial formation is influenced by temperature, for instance, P.aurianogriseum produces conidia to a minimum of 0.86 a_w at 30^oC, but to 0.83 a_w at 23^oC. 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 fungal development.

2.1. Physical factors

The most important factor governing colonisation of grains and mycotoxin production is the availability of water which on the field comes mainly with rainfall. The second important factor is temperature. The moisture and temperature effects on mycotoxin production often differ from those on germination and growth. Table 2 presents the moisture and temperature requirements of most common toxigenic fungi for their growth and mycotoxin production.

It was found that optimal temperature for F.graminearum growth on soybean contained in the range 15-20^oC (in isothermal temperature) and 15/25^oC (in cycling temperature). The optimal temperature for mycotoxin production on soybean was 20^oC for deoxynivalenol (DON) and 15^oC for zearalenone (ZEA). After 15 days of incubation, the maximum levels 39 ppm and 1040 ppm for ZEA and DON, respectively, were detected. Fumonisins were produced by Fusarium graminearum only the on culture medium at 30^oC; on soybean no fumonisins were detected [31].

Most fungi need at least 1-2% of O₂ for their growth. The influence of high carbon dioxide and low oxygen concentrations on the growth and mycotoxin production by the foodborne fungal species was investigated by [32]. Three groups of species were distinguished: first, which did not grow in 20% CO₂ <0.5% O₂ (Penicillium commune, Eurotium chevalieri and Xeromyces bisporus); second, which grew in 20% CO₂ <0.5% O₂, but not 40% CO₂ <0.5% O₂ (Penicillium roqueforti and Aspergillus flavus); and third, which grew in 20%, 40% and 60% CO₂ <0.5% O₂ (Mucor plumbeus, Fusarium oxysporum, F.moniliforme, Byssochlamys fulva and B.nivea). The production of aflatoxin, patulin, and roquefortine C was greatly reduced under all of the atmospheres tested. For example, aflatoxin was not produced by A. flavus during growth under 20% CO₂ for 30 days. Patulin was produced by B.nivea in the atmospheres of 20% and 40% CO₂, but only at low levels [32].

2.2. Chemical factors

Nutritional factors such as carbonohydrate and nitrogen sources and microelements (copper, zinc, cobalt) affect mycotoxin production, but the mechanisms of this impact are still unclear. A relationship between mycotoxin production and sporulation has been documented in several toxigenic fungi. For example, chemical substances that inhibit sporulation of Aspergillus parasiticus have also been shown to inhibit the production of aflatoxin [33]. Chemical preservatives such as organic acids (sorbic, propionic, acetic, benzoic) or fungicides have been used to restrict the growth of mycotoxigenic fungi. It was found that propionic acid at the concentration of up to 0.05% inhibited the growth and ochratoxin production by Penicillium auriantogriseum. A more effective result in higher temperature was observed [34]. Inhibiting fungal growth and toxigenic properties by organic acids is connected with lowering the pH value. It was found that ammonium and sodium bicarbonate at the concentration of 2% fully inhibited the development of the cultures of Aspergillus ochraceus, Fusarium graminearum and Penicillium griseofulvum inoculated into corn. The production of ochratoxin A by Aspergillus ochraceus was reduced from 26 ppm in untreated corn to 0.26 ppm in bicarbonate-treated corn samples [35].

2.3. Biological factors

The simultaneous presence of different microorganisms, such as bacteria or other fungi, could disturb fungal growth and the production of mycotoxins. For instance, Alternaria and Fusarium are antagonistic, and Alternaria was less abundant in grain with a high incidence rate of F.culmorum. Epicoccum is a strong antagonist too [25].

At 30^oC, the ochratoxin production by Aspergillus ochraceus was inhibited by A.candidus, A.flavus, and A.niger in 0.995 a_w. At 18^oC and 0.995 a_w, the interaction between Aspergillus ochraceus and Alternaria alternata resulted in a significant stimulation of ochratoxin A production [36]. Therefore, several microorganisms were reported as effective biocontrol agents against several fungal plant pathogens [37]. It was determined that Trichoderma harzianum produces a lytic enzyme, chitinase, which manifests antifungal activity against a wide range of fungal strains. It was found that non-toxigenic T.harzianum isolates significantly reduce the production of six types of A trichothecenes in cereals [38].

According to [39], soybean is not a favourable medium for ZEA production since it possesses some features that limit the production of this toxin by Fusarium isolates. Similarly, the production of aflatoxin B₁ by Aspergillus flavus was suppressed by soybean phytoalexin – glyceollin [40].

3.1. Aflatoxins (AFs)

Aflatoxins are difuranocumarin derivatives. The main naturally produced aflatoxins based on their natural fluorescence (blue or green) are called B₁, B₂, G₁, and G₂. Aflatoxin M₁ is a monohydroxylated derivative of AFB₁ which is formed and excreted in the milk of lactating animals. AF_s are very slightly soluble in water (10–30 μg/mL); insoluble in non-polar solvents; freely soluble in moderately polar organic solvents (e.g. chloroform and methanol) and extremely soluble in dimethyl sulfoxide. They are unstable under the influence of ultraviolet light in the presence of oxygen, to extremes of pH (< 3, > 10) and to oxidizing agents [44].

Aflatoxins are produced only by a closely related group of aspergilli: Aspergillus flavus, A.parasiticus, and A.nomius strains [45]. These species are very widespread in the tropical and subtropical regions of the world. Other species such as A.bombycis, A.ochraceoroseus, and A.pseudotamari are also aflatoxin-producing species, but they are found less frequently [46, 47]. Aflatoxins constitute a problem concerning many commodities (nuts, spices), however, in terms of grain they are primarily problematic in case of maize. This is because only maize can be colonised by A.flavus and related species in the field. Out of the other grains, rice is an important dietary source of aflatoxins in tropical and subtropical areas. In regions with moderate climate, the problem is connected with imported commodities or the local crops that are wet or stored in improper conditions [45]. The carcinogenicity, mutagenicity and acute toxicology of AFB₁ have been well documented. The IARC determined it to be a human carcinogen (group 1A).

3.2. Ochratoxin A (OTA)

Ochratoxin A is a chlorinated isocumarin derivative, which contains a chlorinated isocoumarin moiety linked through a carboxyl group to L-phenylalanine via an amide bond. It is colourless, crystalline, and soluble in polar organic solvents compounds. This toxin is more stable in the environment than AFs. The studies of [45] reported that thermal destruction of OTA occurs after exceeding 250^oC. OTA is produced by Penicillium species such as P.verrucosum, P.auriantiogriseum, P.nordicum, P.palitans, P. commune, P.variabile and by Aspergillus species e.g. A.ochraceus, A.melleus, A.ostanius, as well as the aspergilli species of section Nigri. In moderate climates, the main producers of OTA are Penicillium species, while Aspergillus species dominate in tropical and subtropical climates. Ochratoxin A is often found with citrinin produced by Penicillium aurantiogriseum, P.citrinum, and P.expansum [48]. Significant human exposure comes from the consumption of grape juice, wine, coffee, spices, dried fruits and cereal-based products, e.g. whole-grain breads, and in addition to this from products of animal origin, e.g. pork and pig blood-based products. The Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) has derived an OTA tolerable weekly intake (TWI) on the level of 120ng/kg b.w. The IARC [49] determined it to be a possible human carcinogen (group 2B). Ochratoxins are the cause of urinary tract cancers and kidney damage. In ruminants, ochratoxin A is divided to non-toxic ochratoxin alfa and phenylalanine [44].

3.3. Citrinin

Citrinin is a polyketide nephrotoxin produced by several species of the genera Aspergillus, Penicillium and Monascus. Some of the citrinin-producing fungi are also able to produce ochratoxin A or patulin. Citrinin is insoluble in cold water, but soluble in aqueous sodium hydroxide, sodium carbonate, or sodium acetate; in methanol, acetonitrile, ethanol, and most other polar organic solvents. Thermal decomposition of citrinin occurs at >175 °C under dry conditions, and at > 100 °C in the presence of water. The known decomposition products include citrinin H₂ which did not show significant cytotoxicity, whereas the decomposition product citrinin H₁ showed an increase in cytotoxicity as compared to the parent compound [50].The most commonly contaminated commodities are barley, oats, and corn, but contamination can also occur in case of other products of plant origin e.g. beans, fruits, fruit and vegetable juices, herbs and spices, and also in spoiled dairy products [50].

3.4. Fumonisins (F_s)

Fumonisins are a group of diester compounds with different tricarboxylic acids and polyhydric alcohols and primary amine moiety. There are several fumonisins, but only fumonisins B₁ (FB₁) and B₂ (FB₂) have been found in significant amounts. Some technological processes hydrolyze the tricarboxylic acid chain in fumonisin B₁. The product of this reaction is more toxic than fumonisin [51].

FB₁ is produced by fungi from Fusarium genera, especially by F.moniliforme and F.proliferatum. The study of [11] suggests that the risk of contamination with Fusarium toxins is higher for maize and wheat than for soybean and pea. High concentrations of fumonisins are associated with hot and dry weather, followed by the periods of high humidity. Studies on fumonisin residues in milk, meat and eggs are incomplete [52, 53]. Human exposure assessments on fumonisin B₁ have rarely been reported. The mean daily intake in Switzerland is estimated to be 0.03 μg/kg bw/day. In the Netherlands the exposure estimates ranged from 0.006 to 7.1 μg/kg bw/day. In South Africa, the estimates ranged from 14 to 440 μg/kg bw/day, showing that the exposure to FB₁ is considerably higher than in the other countries in which exposure assessments were performed [54]. It was concluded that for F_s there was inadequate evidence in humans for carcinogenicity. Therefore, the IARC classified Fusarium monilliforme toxins, including fumonisins, as potential carcinogens to humans (group 2B).

3.5. Zearalenone

Zearalenone is a macrocyclic lactone with high binding affinity to oestrogen receptors. ZEA is produced mainly by Fusarium graminearum and F.sporotrichoides in the field and during storage of commodities such as maize, barley, sorghum, and soybean. The IARC has evaluated the carcinogenicity of zearalenone and found it to be a possible human carcinogen (group 2B). Residues of zearalenone in meat, milk and eggs do not appear to be a practical problem [53, 54].

3.6. Trichotecenes

Trichothecenes constitute a group of 50 mycotoxins produced by Fusarium, Cephalosporium and Stachybotrys genera in different commodities. There are including T-2 toxins, deoxynivalenol, nivalenol, and diacetoxyscirpenol. Beside trochothecenes, deoxynivalenol (DON, womitoxin) is probably the most widely distributed in cereal and soybean foods and feeds. In contaminated cereals, DON derivatives such as 3-acetyl DON and 15-acetyl DON can occur in significant amounts (10 – 20%) with DON. DON is produced by closely related Fusarium graminearum, F.culmorum and F.crokwellense species [55].

T-2 toxin produced mainly by F.sporotrichoides and F.poae is primarily associated with mould millet, wheat, rye, oats, and buckwheat. This toxin can be transmitted from dairy cattle feed to milk [56].

3.7. Alternaria toxins

Alternaria species, besides Fusarium, is the most isolated fungi from soybean and other cereals. Several species are known producers of toxic metabolites called Alternaria mycotoxins. The most important Alternaria mycotoxins include alternariol (AOH), alternariol monomethyl ether (AME), altertoxins I, II, and III (ATX-I, -II, III), tenuazonic acid (TeA), and altenuene (ALT). They belong to three structural classes: dibenzopyrone derivatives, perylene derivatives, and tetramic acid derivatives. Alternariol and related metabolites (AME and ALT) are produced by Alternaria alternate, A.brassicae, A.citri, A.cucumerina, A.dauci, A.kikuchiana, A.solani, A.tenuissima, and A.tomato. These strains are known as plant, especially fruit and vegetable pathogens. In cereals, soybean and oilseeds, AOH, AME and ALT are produced mainly by Alternaria alternata, A.tennuisima, and A.infectoria. AOH has been reported to possess cytotoxic, genotoxic, mutagenic, carcinogenic, and oestrogenic properties [27]. Tenuazonic acid (TeA) is a mycotoxin and phytotoxin produced primarily by Alternaria alternata and other phytopatogenic Alternaria species. The overview of the chemical characterisation, producers, toxicity, analysis and occurrence in foodstuffs was summarised by [27].

3.8. Sterigmatocystin

Sterigmatocystin (STC) is a precursor of the aflatoxins produced mainly by many Aspergillus species such as A.versicolor, A.chevalieri, A.ruber, A.aureolatus, A.quadrilineatus, A.sydowi, Eurotium amstelodami, and less often by Penicillium, Bipolaris, Chaetomium, and Emericella genera [30]. Sterigmatocystin was reported as a fungal metabolite in mouldy wheat, rice, barley, rapeseed, peanut, corn, and cheeses or salami. The STC producers, occurrence and toxic properties were reviewed by [30, 57].

5.1. Negative effects of mycotoxins on humans

Mycotoxicoses can be divided into acute and chronic. Acute toxicity usually has a rapid onset and obvious toxic response, chronic exposure is characterized by chronic doses over a long period of time and may lead to cancer and other effects that are generally irreversible. The symptoms of mycotoxicosis depend on the type, amount and duration of exposure, age, health and sex of the exposed individual, and many poorly understood synergistic effects involving genetics, dietary status, and interaction with other toxic contaminants. Thus, the severity of mycotoxin poisoning can be compounded by factors such as vitamin deficiency, caloric deprivation, alcohol abuse, and infectious disease status. Mycotoxicosis is difficult to diagnose because doctors do not have experience with this disease and its symptoms are so wide that it mimics many other conditions [74, 75].

Aflatoxicosis is toxic hepatitis leading to jaundice and, in severe cases, death. AFB₁ has been extensively linked to human primary liver cancer and was classified by the International Agency for Research on Cancer (IARC) as a human carcinogen (Group 1A - carcinogens) [49]. Although acute aflatoxicosis in humans is rare, several outbreaks have been reported. In 2004, one of the largest aflatoxicosis outbreaks in Kenya, resulting in 317 cases and 125 deaths was observed. Contaminated corn was responsible for the outbreak, and officials found the level of aflatoxin B₁ as high as 4400 ppb [76]. Research in Gambian children and adults reported a strong association between aflatoxin exposure and impaired immunocompetence suggesting that the consumption of aflatoxin reduces resistance to infections in human populations [77, 78]. In 1974, an epidemic of hepatitis in India affected 400 people resulting in 100 deaths. The death was due to consumption of corn that was contaminated with A. flavus containing up to 15000 ppb of aflatoxins [79].

Ochratoxin A was the cause of epithelial tumours of the upper urinary tract in the Balkans [80, 81]. The condition is known as Balkan endemic nephropathy. Despite the seriousness of the problem, the study did not explain the mechanism of action and the size of OTA carcinogenicity in humans [82]. Ochratoxin has been detected in blood in 6-18% of the human population in some areas where Balkan endemic nephropathy is prevalent. Ochratoxin A has also been found in human blood samples from outside the Balkan Peninsula. In some survey, over 50% of the tested samples were contaminated. A highly significant correlation was observed between Balkan nephropathy and urinary tract cancers, particularly tumours of the renal pelvis and ureter. However, no data have been published that establishes a direct causal role of ochratoxin A in the etiology of these tumours [81].

Fumonisin B₁ was classified by the IARC as a group 2B carcinogen (possibly carcinogenic for humans) [44]. Fumonisins, which inhibit the absorption of folic acid through the foliate receptor, have also been implicated in the high incidence of neural tube defects in the rural population known to consume contaminated corn, such as the former Transkei region of South Africa and some areas of Northern China [75, 83].

Trichothecenes have been proposed as potential biological warfare agents. In the years 1975-1981, T-2 toxin was implicated as a chemical agent "yellow rain" used against the Lao Peoples Democratic Republic. A study conducted from 1978 to 1981 in Cambodia revealed the presence of T-2 toxin, DON, ZEA, and nivalenol in water and leaf samples taken from the affected areas [75, 84]. Clinical symptoms proceeding to death included vomiting, diarrhoea, bleeding, and difficulty with breathing, pain, blisters, headache, fatigue and dizziness. There also occurred necrosis of the mucosa of the stomach as well as the small intestine, lungs and liver [85]. One disease outbreak was recorded in China and was associated with the consumption of scabby wheat containing 1000-40000 ppb of DON. The disease is characterized by gastrointestinal symptoms. Also, in India there took place a reported infection associated with the consumption of bread made from contaminated wheat (DON 350-8300 ppb, acetyldeoxynivalenol 640-2490 ppb, NIV 30-100 ppb and T-2 toxin 500-800 ppb). The disease is characterized by gastrointestinal symptoms and throat irritation, which developed within 15 minutes to one hour after ingestion of the contaminated bread [81].

5.2. Negative effects of mycotoxins on animal

Animals may show varied symptoms upon contact with mycotoxins, depending on the genetic factors (species, breed, and strain), physiological factors (age, nutrition) and environmental factors (climatic conditions, rearing and management). The natural contamination with mycotoxins in animal feed usually does not occur at the levels that may cause acute or overt mycotoxicosis, such as hepatitis, bleeding, nephritis and necrosis of the oral and enteric epithelium, and even death. It is often difficult to observe and diagnose the symptoms of the disease, but it certainly is the most common form of mycotoxicosis in farm animals, affecting such parameters as productivity, growth and reproductive performance, feed efficiency, milk and egg production.

The negative effects of mycotoxins on the performance of poultry have been shown in numerous studies. For example, feeding the broilers with feed containing an AF_s mixture (79% AFB₁, 16% AFG₁, AFB₂ 4% and 1% AFG₂) in the concentration of 3.5 ppm decreased their body weight and increased their liver and kidney weight [75, 86]. Feeding OTA (0.3-1 ppm) to broilers reduced glycogenolysis and dose-dependent accumulation of glycogen in the liver. These negative metabolic reactions were attributed to inhibition of cyclic adenosine 3',5'-monophosphate-dependent protein kinase, and were reflected in reduced efficiency of feed utilization and teratogenic malformations [75].

Fusarium mycotoxins proved to be harmful to poultry. In addition to reduced feed intake and weight gain, sore mouth, cheeks and plaque formation was observed after 7-day-old chicks were exposed to T-2 toxin (4 or 16 ppm) [75, 87]. Pigs are among the most sensitive species to mycotoxins. In the study by [88], pigs in response to AF_s (2 ppm), OTA (2 ppm), or both were evaluated. Compared to the control group, the body weight gains were reduced by 26, 24 and 52% for animals consuming diets containing AF_s, OTA, or both, respectively. Additional symptoms in pig ochratoxicosis were anorexia, fainting, uncoordinated movements, and increased water consumption and urination. Pigs also are susceptible to other mycotoxins, such as fumonsins and ergot alkaloids. Fumonisin B₁, for example, has been shown to cause pulmonary oedema and heart and respiratory dysfunction. The symptoms of swine pulmonary oedema included dyspnoea, cyanosis, and death [89, 90]. Mycotoxic porcine nephropathy is a serious disease, often associated with pigs consuming feed contaminated with OTA, especially in Scandinavian region. In addition to the enlarged and pale kidneys (with vascular lesions and white spots), morphological changes include a proximal tubular injury, epithelial atrophy, fibrosis and hyalinization of renal glomerular [80, 81]. Negative effects of ZEA on pigs’ reproductive function have also been demonstrated [91]. Oestrogenic effects of ZEA on gilts and sows include oedematous uterus and ovarian cysts, increased maturation of follicles, more numerous litters or decreased fertility [92].

Aflatoxins affect the quality of the milk produced by dairy cows and result in a carry-over of AFM₁ with AFB₁-contaminated feed. Ten ruminally-canulated Holstein cows received AFB₁ (13 mg per cow daily) through a hole in the rumen for 7 days. The AFM₁ levels in the milk of the treated cows ranged from 1.05 to 10.58 ng/L. The carry-over rate was higher in early lactation (2-4 weeks) compared to late lactation (34 -36 weeks) [75, 93]. The T-2 toxin causes necrosis of the lymphoid tissues. Bovine infertility and natural abortion in the last trimester of pregnancy also result from consumption of feed contaminated with T-2 toxin. Calves consuming T-2 toxin in the amount of 10-50 mg/kg of feed showed abomasal ulcers and sloughing of papillae in the rumen [75, 94, 95].

7.1. Resistance

There are inherent differences in the susceptibility of cereal species to Fusarium infections. The differences between crop species appear to vary between countries. This is probably due to the differences in the genetic pool within each country’s breeding program and the diverse environmental and agronomic conditions in which crops are cultivated [114, 115]. It was observed that oats had higher levels of DON than barley and wheat in Norway from 1996 to 1999, whereas the DON levels in wheat, barley and oats were similar when grown under the same field conditions in Western Canada in 2001 [116].

7.2. Field management

Crop rotation

Numerous studies have shown that fumonisins or DON contamination in wheat is affected by the previous crop. It was shown that a higher incidence of F_s occurred in wheat after maize and, in particular, in wheat after a succession of two maize crops and in wheat following grain maize compared to silage maize. In Ontario, Canada, in 1983, the fields where maize was the previous crop had a significantly higher incidence of fumonisins than the fields where the previous crop was a small grain cereal or soybean [117]. In a repeated study, the following year, the fields where maize was the previous crop had a 10-fold DON content than the fields following a crop other than maize [118]. The research of [119] found higher levels of fumonisins in wheat following wheat rather than wheat following fallow.

An observational study performed using commercial fields in Canada [120] identified significantly lower DON content in wheat following soybean or wheat, compared to wheat following maize. In New Zealand, an observational study determined that higher levels of DON occurred in wheat grown after maize (mean = 600 ppb) and after grass (mean = 250 ppb), compared to small grain cereals (mean = 90 ppb) and other crops (mean = 70 ppb). The highest levels were recorded in wheat-maize rotations [121].

Codex recommends that crops such as potatoes, other vegetables, clover and alfalfa that are not hosts to Fusarium species should be used in rotation to reduce the inoculum in the field [122].

7.3. Soil cultivation

Soil cultivation can be divided into ploughing, where the top 10-30 cm of soil are inverted; minimum tillage, where the crop debris is mixed with the top 10-20 cm of soil; and no till, where seed is directly drilled into the previous crop stubble with minimum disturbance to the soil structure [111]. In the 1990s, a large observational study of F_s and DON was conducted in Germany (n=1600). The DON concentration of wheat crops after maize was ten-times higher in the field that was min-tilled compared to the ploughed one [123]. In wheat the DON concentration after min-till was 1300 ppb, after no-till it was 700 ppb and after ploughing it was 500 ppb [120]. Studies in France have determined that crop debris management can have a large impact on the DON concentration at harvest, particularly after maize. The highest DON concentration was found after no-till, followed by min-till, whereas the lowest DON levels were recorded after ploughing. The reduction in DON has been linked to the reduction in crop residue on the soil surface [124]. Large replicated field trials in Germany identified that there was a significant interaction between the previous crop and the cultivation technique [125]. Following sugar beet, there was no significant difference in the DON concentration between wheat plots receiving different methods of cultivation; however, following a wheat crop without straw removal, direct drilled wheat had a significantly higher DON level compared to wheat from plots which were either ploughed or min-tilled [125].

In accordance with the guidelines contained in the Codex Alimentarius, soil should be tested to determine if there is need to apply a fertilizer and/or soil conditioners to assure adequate soil pH and plant nutrition to avoid plant stress, especially during seed development [122].

Research of [126] showed that supplementary nitrogen and a plant growth regulator increased, by up to 125%, the incidence of infection by Fusarium species in the seed of wheat, barley and triticale. Similarly, in the studies of [127], a significant increase in fumonisins and deoxynivalenol contamination in the grain of wheat and kernels was observed with increasing N fertilizer from 0 to 80 kg/ha. That research concluded that in practical crop husbandry, F_s cannot be sufficiently controlled by only manipulating the N input [111]. The study of [128] showed that the use of six different combinations of agricultural practices (sowing time, plant density, N fertilization and European corn borer (ECB) control with insecticide) can effectively lead to good control of fumonisins and deoxynivalenol in maize kernels.

7.4. Use of chemical and biological agents

In accordance with the guidelines contained in the Codex Alimentarius [122], farmers should minimize insect damage and fungal infections of the crop by proper use of registered insecticides, fungicides and other appropriate practices within an integrated pest management program.

Some studies have been conducted to examine the effectiveness of the fungicides which are applied during flowering can reduce Fusarium infections and subsequent DON in the harvested grains. The results of [129] provided that azoles, tebuconazole, metconazole and prothioconazole significantly reduced the Fusarium disease symptoms and Fusarium mycotoxin concentrations. The greatest reduction in the DON concentration occurred with prothioconazole (10-fold). Azoxystrobin had little impact on the mycotoxin concentration in the harvested grain infected by Fusarium species, but could increasing the mycotoxin concentration in grains when F. nivale was the predominant species present [130, 131]. Fungicide mixtures of azoxystrobin and azole resulted in a lower reduction of DON, compared to azole alone [120, 132]. A number of trials in Germany have indicated that some strobilurin fungicides applied before anthesis can also result in increased DON compared to unsprayed plots [133]. Reductions in DON observed in field experiments using fungicides against natural infections of Fusarium are lower and inconsistent [134]. This is probably due to the fact that during a natural infection, the infection occurs over a longer period of time.

Alternatively, a limited number of biocompetitive microorganisms have been shown useful for the management of Fusarium infections [111]. Research has demonstrated the successful use of bacteria in biocontrol of mycotoxigenic fungi. One bacterium, Enterobacter cloacae was discovered as an endophytic symbiont of corn [135]. Corn plants with roots endophytically colonized by these bacteria were observed to be fungus-free and in vitro control of F.verticillioides and other fungi with this bacterium was demonstrated. An endophytic bacterium, Bacillus subtilis showed promising for reducing the mycotoxin contamination with F.verticillioides during the endophytic growth phase [136]. Yeast antagonists such as Cryptococcus nodaensis were isolated from wheat anthers. The antagonists reduced Fusarium head blight severity by up to 93% in greenhouse and by 56% in field trials when sprayed onto flowering wheat heads [137]. The most successful antagonists reduced the DON content of grain more than 10-fold in greenhouse studies [138].

Actions to be taken during harvest in order to reduce the risk of mould contamination and mycotoxin production include [112]:

• harvest as quickly as possible

• avoid field drying

• transport the crop to the homestead as soon as possible

• if lack of labour force or time prevents removal from the field, then dry the crops on platforms raised above ground (if climate is hot and the drying crop can be left to stay on the field on a platform or cut and tied into stooks) to dry

• bundles of stover should also be placed on platforms to dry and not left lying on the soil

The post-harvest strategies include improving the drying and storage conditions together with the use of chemical, physical or biological methods.