**2. Mycotoxin fungi**

#### **2.1** *Aspergillus flavus*

*A. flavus* is not a single species, but a "species complex", made up of eleven species that are known to occur in many kinds of plant materials, including stored grains (Christensen, 1975; Meggs, 2009). One of the species in the complex, *A. oryzae* has long been used in the Orient to prepare various kinds of food products, such as sake, tofu and soy sauce, which in turn are used in the United States (Pitt & Miscamble, 1995; Shuaib at al., 2010).

What was determined in early research of aflatoxins is that the condition which allows for growth of *A. flavus* and aflatoxins is very narrow (Klich, 2009). *A. flavus* hardly invades stored grains alone, that is as a pure culture (Purzcki and Shain 2010). Various other species of fungi will normally grow on a substrate prior to invasion by *A. flavus*, such as. *A. glaucus*  and *Candida pseudotropicalis (Khlangwiset and Wu, 2010)*. In a preinvaded substrate, regardless of how dense the *A. flavus* invasion may be, aflatoxin will not form. Thus, in order for aflatoxin formation to occur in say a storage bin full of peanuts, *A. flavus* must be growing alone and the peanuts cannot have been previously or simultaneously invaded by other fungi, an occurrence that is rare (Lugauskas and Stakeniene; Magan et al., 2010). In the case of the Turkey-X disease, the peanuts that were responsible for the aflatoxin poisoning were from South America, where the process used to harvest and dry the peanuts was responsible for providing an environment that allowed for growth of *A. flavus* and aflatoxin (Edlayne et al., 2009). *Aspergillus flavus* does not normally contaminate grains and other crops while they are still in the field. It is only after the grains are harvested and stored does *A. flavus*, as well as other so-called "storage fungi" that have a low moisture requirement,

Ding et al., 2006

Conning, 1993

Fokunang et al., 2006

1993; Hussein et al., 2001

Purzycki & Shain 2010.

Friesen et al., 2008

Kiso et al., 2004

Wu et al., 2008.

O'Brian et al 2007

**Fungal species** Mycotoxin products Mycotoxicosis Reference

*fusiformis* clavinet alkaloids Ergotism Cocker, 1999,

*C. purpurea* Ergotamine alkaloids Ergotism Anderson, &

*C. paspali* paspalinine Paspalum staggers Van Egmond. 1989 ;

*maydis* diplodiatoxin Diplodiosis Desjardins et al 1997 ;

*leptostromiformis* phomopsin Lupinosis Takayuki, and Bjeldanes,

*atra* satratoxins Stachybotryotoxicosis Bresinky and Besl, 1990;

*legumicola* slaframine Slobber syndrome Pitt 1996;

*chartarum* sporidesmin Facial eczema Lacey, 1991;

are used in the United States (Pitt & Miscamble, 1995; Shuaib at al., 2010).

*loliae* lolitrem Ryegrass staggers Cocker et al. 1999

Table 1. Fungi species and mycotoxins of regional importance (Fokunang et al., 2006

*A. flavus* is not a single species, but a "species complex", made up of eleven species that are known to occur in many kinds of plant materials, including stored grains (Christensen, 1975; Meggs, 2009). One of the species in the complex, *A. oryzae* has long been used in the Orient to prepare various kinds of food products, such as sake, tofu and soy sauce, which in turn

What was determined in early research of aflatoxins is that the condition which allows for growth of *A. flavus* and aflatoxins is very narrow (Klich, 2009). *A. flavus* hardly invades stored grains alone, that is as a pure culture (Purzcki and Shain 2010). Various other species of fungi will normally grow on a substrate prior to invasion by *A. flavus*, such as. *A. glaucus*  and *Candida pseudotropicalis (Khlangwiset and Wu, 2010)*. In a preinvaded substrate, regardless of how dense the *A. flavus* invasion may be, aflatoxin will not form. Thus, in order for aflatoxin formation to occur in say a storage bin full of peanuts, *A. flavus* must be growing alone and the peanuts cannot have been previously or simultaneously invaded by other fungi, an occurrence that is rare (Lugauskas and Stakeniene; Magan et al., 2010). In the case of the Turkey-X disease, the peanuts that were responsible for the aflatoxin poisoning were from South America, where the process used to harvest and dry the peanuts was responsible for providing an environment that allowed for growth of *A. flavus* and aflatoxin (Edlayne et al., 2009). *Aspergillus flavus* does not normally contaminate grains and other crops while they are still in the field. It is only after the grains are harvested and stored does *A. flavus*, as well as other so-called "storage fungi" that have a low moisture requirement,

alkaloids Fescue foot Bowman, & Rand. 1990;

*Claviceps* 

*Diplodia* 

*Phomopsis* 

*Stachybotrys* 

*Rhizoctonia* 

*Acremonium* 

*Pithomyces* 

**2. Mycotoxin fungi 2.1** *Aspergillus flavus*

*Balasia spp* 

can the grain be invaded (He and Zhou, 2008). Although conditions favourable for growth of the *A. flavus* and production of aflatoxin is narrow, the fungus is common and widespread in nature. Under warm humid condition *A flavus* can invade stored gains such as corn as shown in figure 4. It can be found growing on various decaying vegetation where it may heat up the substrate to as high as 113-122°F as it consumes the material (Hedyati et al., 2007; Lee, 2009).

Fig. 4. *Aspergillus flavus* infestation on corn *Zea mays* (Hedyati et al., 2007)

The term aflatoxins was derived in the early 1960s when the death of thousands of turkeys (Turkey X' disease) ducklings and other domestic animals was attributed to the presence of *A. flavus* toxins in groundnut meals imported from South America (Nageswara et al., 2002). The amount of aflatoxin formed differs as to the substrate on which it is growing. Although the mycelial mass may be the same in each substrate, the aflatoxin produced would be far greater in peanuts than in say soybeans, where relatively very little would be produced. The growth of A. flavus producing aflatoxin in peanuts is shown in figure 5.

Fig. 5. Stored peanuts infected by *A flavus* producing aflatoxin (Kios et al., 2004)

Mycotoxins: Quality Management, Prevention, Metabolism, Toxicity and Biomonitoring 123

deterioration. However, this fungus is seldom isolated from more than a small percentage of seeds or grains that are undergoing microbiological deterioration in storage because it is evidently not a good competitor, as is also the case with *A*. *flavus*. This is a general rule, but *A. ochraceus* has been isolated from 40% or more of surface-disinfected kernels of corns from bins in which deterioration was in progress. It has also been the major organism in some lots

Production of ochratoxin, by *A. ochraceus*, was first described in South Africa (Christensen 1975), where it was isolated along with a number of other fungi. In experiments done with this isolate, the LD50 (the single dose that will kill 50 percent of the individual animals tested) of ochratoxin for rats is 22mg/kg (= 22 milligrams of the toxin per kilogram of body weight of the rat), but a lesser amount will result in severe liver damage. A single dose of 12.5 mg/kg (=12.5 milligrams of the toxin per kilogram of body weight of the rat) was administered to pregnant rats on the tenth day of gestation, and of the 88 foetuses involved, 72, or 81.8% died or were resorbed (Coker 1998; He & Zhou 2010). Ducklings seem to be

Another fungus, *Penicillium viridicatum*, can also produce ochratoxin, and is relatively common in stored corn and is a more common producer of ochratoxin than *A. ochraceus* 

This species is another storage fungus. However, it is never found as the only fungus or as the predominating fungus in deteriorating cereals. Normally, by the time a grain sample has become very mouldy, *A. versicolor*, along with other *Aspergillus* species and usually other filamentous fungi and yeasts as well. Some of the black pepper mentioned earlier, as being decayed by fungi, was very heavily invaded by *A. versicolor*, but not by this fungus exclusively. This species, under the right conditions, produces sterigmatocystin, a toxic compound given the name because the fungus once was called *Sterigmatocystis*. The toxin is known to cause lung, liver and kidney tumours in laboratory animals and has been implicated as the cause of disease in calves that have consumed feed heavily invaded by *A. versicolor (Ben-Ami et al., 2011).*. Experiments carried out in which the fungus were grown, on feed that was fed to calves, produced symptoms of the disease in the calves. However, tests were not done to detect the toxin in the calves. The toxin has also been detected in mouldy coffee beans in Africa, but no evidence indicates that even if these beans were used to brew

This particular species is known to be an animal pathogen. Infection occurs through inhalation of spores and affects the lungs. Infection may also occur in eggs and the foetuses of cows. However, it also produces a metabolic product that may be considered a toxin or an antibiotic. This species differs from the others that we have discussed in that it is said to be thermophilic, that is, it is found in substrate where there are extremely high temperatures, up to 122ºF (=50ºC). This species is usually found on material that is in the advanced stages of decomposition in which the substrate temperature has been significantly raised by microbial decomposition (Edward, 2009). Under the proper conditions, *A. fumigatus* produces fumagillin. This compound is used as an amoebicide that is, as a means to rid the body of amoebae that are human pathogens and has been used effectively in honey bees as well. However, the correct dosage of this compound is critical. A little bit more than you need to get rid of the amoebae and you will be getting rid of the patient as well (Fokunang et al., 2006).

of whole black pepper (Desjardins and Hohm, 1997; Chang et al., 2011).

equally sensitive to ochratoxin as they are to aflatoxin (Ates et al., 2011).

**2.1.2.2** *Aspergillus versicolor* **and sterigmatocystin** 

coffee that the toxin would be in the drink. **2.1.2.3** *Aspergillus fumigatus* **and fumagillin** 

*(Blumenthal, 2004)*.

Other seeds of cereal crops, wheat, corn, barley, oats and sorghum are also generally of lowaflatoxin-risk (Nageswararao et al 2002). Weather and climate were also contributing factors. The amount of toxin produced vary with the isolate of *A. flavus*. That is different sources of *A. flavus* will produce different amounts of aflatoxins. Some isolates of *A. flavus* may not even form aflatoxin (Fuchs et al., 1991; Awad et al., 2010). Although the aflatoxins are the major toxins associated with mycotoxicosis, another mycotoxin called cyclopiazonic acid (figure 6), has been associated in the aetiology of Turkey X disease (Bradburn et al., 1994; Kios et al., 2004).

Fig. 6. Cyclopiazonic acid associated in the aetiology of Turkey X disease (Bradburn et al., 1994)
