Re: 아스퍼길루스 플라부스 Aspergillus flavus: the major producer of aflatoxin

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Aspergillus flavus: the major producer of aflatoxin

MAREN A. KLICH

First published:25 September 2007

 

https://doi.org/10.1111/j.1364-3703.2007.00436.x

Citations: 220

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SUMMARY

Aspergillus flavus is an opportunistic pathogen of crops. It is important because it produces aflatoxin as a secondary metabolite in the seeds of a number of crops both before and after harvest. Aflatoxin is a potent carcinogen that is highly regulated in most countries. In the field, aflatoxin is associated with drought‐stressed oilseed crops including maize, peanut, cottonseed and tree nuts. Under the right conditions, the fungus will grow and produce aflatoxin in almost any stored crop seed. In storage, aflatoxin can be controlled by maintaining available moisture at levels below that which will support growth of A. flavus. A number of field control measures are being utilized or explored, including: modification of cultural practices; development of resistant crops through molecular and proteomic techniques; competitive exclusion using strains that do not produce aflatoxin; and development of field treatments that would block aflatoxin production.

Taxonomy: Aspergillus flavus Link (teleomorph unknown) kingdom Fungi, phyllum Ascomycota, order Eurotiales, class Eurotiomycetes, family Trichocomaceae, genus Aspergillus, species flavus.

Host range: Aspergillus flavus has a broad host range as an opportunistic pathogen/saprobe. It is an extremely common soil fungus. The major concern with this fungus in agriculture is that it produces highly carcinogenic toxins called aflatoxins which are a health hazard to animals. In the field, A. flavus is predominantly a problem in the oilseed crops maize, peanuts, cottonseed and tree nuts. Under improper storage conditions, A. flavus is capable of growing and forming aflatoxin in almost any crop seed. It also is a pathogen of animals and insects. In humans it is predominantly an opportunistic pathogen of immunosuppressed patients.

Useful websites: http://www.aspergillusflavus.org, http://www.aflatoxin.info/health.asp, plantpathology.tamu.edu/aflatoxin, http://www.aspergillus.org.uk

INTRODUCTION

Few fungi have had as broad an economic impact as Aspergillus flavus. It is a pathogen of plants, animals and insects, causes storage rots in numerous crops, and it produces the highly regulated mycotoxin, aflatoxin B1. As human pathogens, Aspergillus species have become increasingly important because immunosuppressed people are very susceptible to infection by these fungi. Of the aspergilli causing mycoses in humans, only A. fumigatus is more important than A. flavus (Stevens et al., 2000). A. flavus is also an allergen causing allergic bronchopulmonary aspergillosis. As an insect pathogen, it affects a number of species, including honeybees in which it causes a disease called stonebrood (Gilliam and Vandenberg, 1990; Scully and Bidochka, 2005; Wicklow, 1990). Individual strains of A. flavus are not specialized to any particular host plant or insect (St. Leger et al., 2000). Aflatoxin B1 is the most potent naturally formed carcinogen. It is one of the few mycotoxins that has been developed for use as a biological weapon (Bennett and Klich, 2003).

Thousands of papers have been written on Aspergillus flavus and aflatoxins, so a complete review of the literature is not possible. The objective of this paper is to provide an overview of the major topics in the areas of A. flavus and aflatoxin research on plants, and to provide an entrée into the literature on those topics.

Pathogenicity in plants

Aspergillus flavus is a minor pathogen of corn, peanuts and cotton. In corn, A. flavus causes an ear rot (Taubenhaus, 1920). In peanuts, it causes a seedling disease known as yellow mould of seedlings or aflaroot. The symptoms include necrotic lesions, chlorosis on above‐ground parts and lack of development of secondary roots, ‘aflaroot’ (Pettit, 1984). The root effect may be a result of aflatoxin toxicity as it has been shown to inhibit root hair development in tobacco (McLean et al., 1994). A. flavus may also cause a rot of mature peanuts in the soil (Pettit, 1984). In cotton, A. flavus affects cotton quality by causing boll rot. Infection of the fibre is known as yellow spot disease (Marsh et al., 1955). The ‘yellow’ refers to the bright greenish yellow (BGY) fluorescence seen on the cotton fibres under long‐wave ultraviolet light. A. flavus infection of cottonseed lowers seed viability by about 60% (Klich and Lee, 1982). In the same study, aflatoxin was found in 38% of the viable seed and 71% of the non‐viable seeds, indicating that the presence of aflatoxin may lower seed viability more than infection of the fungus alone. This theory is supported by the fact that aflatoxin B1 per se has been shown to inhibit seed germination of some other crop seeds, including wheat, corn, mustard, mung and gram (McLean, 1994).

CLASSIFICATION

Aspergillus is an anamorphic genus consisting of about 250 recognized species. It is characterized by a distinctive spore‐bearing structure, the aspergillum (Fig. 1).

Figure 1

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Characteristic conidiophores of Aspergillus.

Some members of the genus produce teleomorphs (sexual states) which are always cleistothecial ascomycetes with inordinantly arranged ascospores in dehiscent asci. The genus has been divided into a number of sections. Aspergillus flavus belongs to section Flavi. This section contains the major economically important aflatoxin‐producing fungi A. flavus and A. parasiticus. Less common aflatoxin‐producing species in this section are A. nomius, A. pseudotamarii, A. bombysis and A. parvisclerotigenus. Four species not in section Flavi are known to produce aflatoxin: A. ochraceoroseus, A. rambellii, Emericella venezuelensis and E. astellata (Frisvad et al., 2005). The latter two species have Aspergillus anamorphs (asexual states). Section Flavi includes a number of other economically important species, including the food fermentation/industrial species A. oryzae and A. sojae. At the molecular level these two fungi are closely related to A. flavus and A. parasiticus, respectively, but they are morphologically distinct and do not produce aflatoxin.

In culture, Aspergillus flavus is characterized by fast‐growing yellow–green colonies, usually 65–70 mm in diameter after 7 days growth in the dark at 25 °C on Czapek yeast extract (CYA), CYA with 200 g sucrose or malt extract agars. It grows well at 37 °C. The stipe of the conidiophore is usually 400–800 µm long and rough‐walled. The vesicles are globose to elongate and usually 20–45 µm in diameter. Seriation is variable, but usually at least 20% of the aspergilla produce both metulae and phialides on CYA. Conidia are globose to ellipsoidal, mostly 3–6 µm in diameter with smooth to finely roughened walls (Fig. 2). Black globose to elongate firm‐walled structures called sclerotia are produced by some strains. These are usually 400–700 µm in diameter (Klich, 2002a; Raper and Fennell, 1965).

Figure 2

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Clockwise from upper left: colony of A. flavus after incubation for 7 days at 25 C on CYA agar; conidiophore of A. flavus; scanning electron micrograph of A. flavus conidia; conidia as seen under the light microscope at 100×.

Several species are morphologically similar to A. flavus: Aspergillus oryzae differs from A. flavus in producing colonies that are more floccose and turn brown with age on CYA as well as conidia that are larger (4–8.5 µm); Aspergillus parasiticus colonies are generally darker green, and the conidial walls are very rough; Aspergillus bombycis grows more slowly (0–37 mm) on CYA at 37 °C and has smooth‐walled stipes; A. nomius is most easily distinguished from A. flavus by its mycotoxin profile—A. flavus produces only B aflatoxins (see below) whereas A. nomius produces both B and G aflatoxins (Klich, 2002a). A. parvisclerotigenus has tiny sclerotia (< 400 µm), and often produces both B and G aflatoxins (Frisvad et al., 2005). There are also molecular means to differentiate these fungi (Peterson et al., 2001).

AFLATOXINS

Aflatoxins were first discovered and characterized in the early 1960s after more than 100 000 turkey poults in England died of apparent poisoning from mould‐contaminated peanut meal (Blout, 1961; Goldblatt, 1969). There are two general forms of the disease caused by exposure to aflatoxin, aflatoxicosis. Acute aflatoxicosis results in death. Chronic aflatoxicosis causes cancer, with the liver as the primary target organ, immune suppression, teratogenicity and other symptoms (Bennett and Klich, 2003). There is also some evidence that respiratory exposure to aflatoxin increases the occurrence of respiratory and other cancers (see Dvorackova, 1990).

Chemically, aflatoxins are difuranocoumarin derivatives produced by a polyketide pathway. There are four major aflatoxins, B1, B2, G1 and G2, with the letters referring to the colour of their fluorescence under ultraviolet light (blue or green) and the numbers indicating their relative migration distance on a thin‐layer chromatographic plate. There are many other, less common aflatoxins. Aflatoxin B1 is the most potent naturally formed carcinogen (Squire, 1981), and generally, if the term aflatoxin is used in the singular, the author is referring to aflatoxin B1 (Fig. 3).

Figure 3

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The structure of aflatoxin B1.

The genes for aflatoxin biosynthesis, like those of many secondary metabolites, are clustered (Cary, 2003). The complete cluster has been sequenced and annotated (Yu et al., 2004). The gene cluster is 82 kb in length, and contains 25 genes. Regulation of the pathway has been the subject of intensive study (see reviews by Bhatnagar et al., 2003; Price and Payne, 2005). It is hoped that understanding the biosynthesis of aflatoxin will facilitate development of control strategies and provide an understanding of how and why aflatoxin evolved. Recently, the genome of A. flavus has been sequenced (Payne et al., 2006). The information therein will provide useful tools for understanding both the fungus and aflatoxin.

Regulatory levels for aflatoxins are in place in at least 99 countries. (http://www.fao.org/docrep/007/y5499e/y5499e07.htm; van Egmond and Jonker, 2005) The regulatory levels vary from country to country. The US FDA action levels are set at 20 parts per billion (p.p.b., µg/kg) for human food, 0.5 p.p.b. for milk and up to 300 p.p.b. for corn and cottonseed in animal feed. In the EU, the levels are 4 p.p.b., whereas in India the regulatory levels are set at 30 p.p.b. for all foods.

In the United States alone, the mean economic loss from mycotoxins is estimated to be $932 million (CAST, 2003). Aflatoxin is a major contributor to this problem.

Economic costs of aflatoxin are difficult to estimate because such total estimates would need to include losses such as slow weight gain and immune suppression in farm animals, and even the loss of companion animals to aflatoxicosis. Estimating the cost of aflatoxicosis in humans worldwide is even more difficult. In terms of crop loss alone, estimates of annual costs in the US growing areas susceptible to aflatoxin were: $4 367 000 for Arizona cottonseed; $7 000 000 for Texas cottonseed; $25 000 000 for Georgia peanuts; $15 000 000 for Texas corn; $2 000 000 for Mississippi corn; $38 700 000 for California walnuts; and $23 000 000 to $47 000 000 for California almonds (Robens and Cardwell, 2005).

Aflatoxin detection methods

Even before the discovery of aflatoxin, there was a well‐known association between A. flavus contamination and a BGY fluorescence of the seed under ultraviolet light (Marsh et al., 1955). It is used as an indicator of potential aflatoxin contamination, but this method is not always reliable (Diener et al., 1987; Shotwell and Hesseltine, 1981). Contamination does occur in non‐fluorescent seed and may be absent from BGY seed. It was not until 1999 that this compound was identified as a dehydrogenator dimer of kojic acid linked through the C‐6 positions (Zeringue et al., 1999). The kojic acid component of the BGY is produced by the fungus and it interacts with host plant peroxidases to form the compound.

Once the aflatoxin hazard became apparent, there was a need for accurate assessment of aflatoxin contamination levels. Chemical methods for extraction, chromatographic methods for purification and methods for measurement of aflatoxin levels were developed (Pons and Goldblatt, 1969). Thin layer chromatography became the standard for analysis as well as screening of aflatoxins, and is still used because it is simple and inexpensive. Other methods including high‐performance liquid chromatography, gas chromatography and immunological methods such as enzyme‐linked immunosorbent assay (ELISA) are also used (CAST, 2003; Gilbert and Vargas, 2005). Official validation methods for analysis have been published by the Association of Official Analytical Chemists (2006). New methods are being developed based on hyper spectral and infra‐red imaging and PCR (Bhatnagar et al., 2004).

Aflatoxins are not evenly distributed in crop commodities; therefore, it is not surprising that the sampling step is the greatest source of variation in testing for aflatoxin (CAST, 2003; Coker, 1998). There are a number of approaches that may be taken to minimize variation (Whitaker, 2001), but as yet there is no worldwide agreement on sampling protocols for any crop.

Biogeography

A. flavus has been isolated from soils in all of the major biomes. Although A. flavus may be isolated in all climatic zones, it is isolated relatively more frequently in warm temperate zones (latitudes 26–35°) than in tropical or cooler temperate zones, and is quite uncommon in latitudes above 45° (Klich, 2002b; Manabe and Tsuruta, 1978). It is therefore not surprising that chronic aflatoxin problems are associated with crops in latitudes below 35° and are generally not a major problem in crops raised in Europe (Logrieco and Visconti, 2004). In the United States, for instance, aflatoxin is only a chronic problem in corn in the south‐eastern growing area (Payne, 1983). Field contamination by aflatoxin is associated with high temperatures and drought stress, and does occur in crops in more temperate climates in warm drought years (CAST, 2003). Studies of A. flavus spore densities in soils have shown that they are more dense in crop soils than in forest or prairie soils (Angle et al., 1982; Horn, 2005). Aflatoxins are not restricted to crop seeds. They have been found in the native legumes in the Sonoran Desert in Arizona (Boyd and Cotty, 2001).

Incidence of the aflatoxigenic species varies with crop and geographical location. For many years, researchers did not separate A. flavus from A. parasiticus in field studies, which led to some confusion regarding the distribution of the latter. Although A. parasiticus has been isolated from a wide variety of soils (Klich, 2002b), including corn fields in the Midwestern US (Angle et al., 1982; McAlpin et al., 1998; Shearer et al., 1992; Wicklow et al., 1998) and orchards in California (Doster and Michailides, 1994; Doster and Michailides, 1995), it is only an economic problem in peanuts and is isolated only rarely in other crops (Diener et al., 1987). In an elegant study involving soil samples from agricultural fields along a transect through the southern USA, Horn and Dorner (1998) demonstrated that fields from peanut‐growing areas had higher incidences of A. parasiticus than others. In that study, it was demonstrated that a variant of A. flavus with small sclerotia was more preval‎ent in cotton‐growing areas than others. This association was also reported by Cotty (1997) in a study of cotton‐producing areas in the USA.

Vegetative compatibility groups (VCGs) have been used extensively to study within‐population diversity of A. flavus (Horn, 2005). Using VCGs together with DNA fingerprinting and RAPD analysis, it has been established that there is high genetic diversity of A. flavus populations in the soil. Populations seem to extend beyond field boundaries (Orum et al., 1999). This indicates that factors other than crop per se or agronomic practices influence distribution of A. flavus populations. Populations also vary greatly from year to year (Bayman and Cotty, 1991).

FACTORS INFLUENCING AFLATOXIN FORMATION IN THE FIELD

Aflatoxin is most frequently reported in the field in oilseed crops including maize, cotton, peanuts and tree nuts. Why does it affect these crops and not others? Part of the reason for this may be biogeographical—these crops are grown in the latitudes where A. flavus is most frequently reported. Another possible reason may be the carbon utilization pattern of A. flavus. In cottonseed and corn, A. flavus first utilizes free saccharides and then oil before using starch (Mellon et al., 2000, 2005). Removal of lipids from cottonseed reduced aflatoxin production 800‐fold (Mellon et al., 2000).

Drought and temperature stress are common factors for field contamination of row crops. In peanuts, experiments with drought stress and controlled soil temperatures 85–100 days after planting demonstrated that drought stress and temperatures of 29 °C yielded the greatest number of colonized edible grade peanuts and high aflatoxin levels. Other environmental combinations led to lower colonization levels and no aflatoxin (Cole et al., 1984). A series of studies demonstrated that irrigation will control aflatoxin in peanuts (Cole et al., 1989). In cotton, moderately high drought stress (–1.6 to –1.9 MPa) at the time of anthesis led to higher A. flavus infection levels in resulting seeds than lower or higher water stress levels at flowering (Klich, 1987). In corn, as in other affected crops, high temperature and drought stress are almost always precursors to aflatoxin outbreaks (Payne, 1998).

The exact roles of high temperature and drought in field contamination have not been elucidated. High temperatures and drought stress affect the physiology of plants, and therefore stressed plants may be more susceptible to infection or aflatoxin production. For instance, drought stress induces a great increase in proline production in plants (Barnett and Naylor, 1966), and proline has been reported to enhance aflatoxin production (Payne and Hagler, 1983). Formation of some phytoalexins (antimicrobial compounds produced by some plants) is inhibited by drought stress. In immature peanuts, aflatoxin did not form until phytoalexin production ceased in drought‐stressed plants (Dorner et al., 1989). Another possibility is that the fungi that normally compete with A. flavus in the soil do not grow as readily under these conditions, giving A. flavus a competitive advantage. Even among other Aspergillus species, the temperature optimum for growth of A. flavus (25–42 °C) is higher than for many other species. A. flavus is fairly xerotolerant. Although the optimal water potential for growth is –2 MPa, it can grow at water potentials down to –35 MPa, which is lower than the minimum for many other fungal species (Klich et al., 1992).

Mode of entry

Once conditions of temperature, drought stress and inoculum levels are met, A. flavus is capable of entering the plant through a number of portals. This variability has made development of control measures more difficult. Insect damage is associated with increased aflatoxin in all affected crops. The insects damage plant tissue, thereby creating entry portals for the fungus. BGY fluorescence from Aspergillus flavus is frequently associated with insect damage, indicating that insects provide entry and/or act as vectors (Lee et al., 1977; Marsh et al., 1969). Aflatoxin does occur, however, in the absence of apparent injury.

In corn, A. flavus may be brought to the surface of developing seeds by insects or by colonizing the silks and growing down to the seed area. The ear is colonized from the tip down. Although the mould is present, the fungus does not infect the kernel until the kernel is nearly mature (Payne, 1998).

In cotton, the mould appears to enter the plant at or before flowering. The fungus does not appear to enter through the stigma, but rather enters from nectaries or other natural openings below the ovary and then moves upward into the developing boll (Klich and Chmielewski, 1985; Klich et al., 1984, 1986). Ultrastructural evidence indicates that the fungus may be entering the seeds via the vascular tissue (Huizar et al., 1990). Early season bolls and those under water stress (water potentials between –1.6 and –1.9 MPa) at anthesis are more susceptible to infection than later season bolls or those under more or less stress at anthesis (Klich, 1987, 1990). Interestingly, the degree of susceptibility seems to be determined at or before anthesis, and a susceptible boll may be inoculated at the nectaries for up to 25 days post anthesis without change in the degree of susceptibility (Klich, 1990). As with corn, the fungus is present in immature bolls, but does not seem to infect the seed until it nears maturity (Klich, 1986).

For peanuts, floral infection seems to be less important than for corn and cotton and infection is usually a result of direct penetration of the pod (Cole et al., 1986). Aspergillus flavus and A. parasiticus do invade young peanut plants systemically as seedlings from seed and soil and disseminate throughout the plant, but stems and roots are more commonly infected than leaves and petioles (Pitt et al., 1991). Recovery of these fungi was shown to be higher in root and pod tissue than other plant parts in more mature peanuts (Kisyombe et al., 1985).

FIELD CONTROL

Control of aflatoxin contamination can be achieved by either controlling the fungus or controlling aflatoxin production. Resistance to other fungi apparently does not confer resistance to A. flavus. Peanut genotypes with resistance to other fungal pathogens of peanut were not resistant to A. flavus and did not reduce aflatoxin (Holbrook et al., 1997). A large number of factors influence growth and distribution of the fungus and aflatoxin formation (for reviews see Diener et al., 1987; Klich, 2007; Payne, 1998; Scheidegger and Payne, 2003). Exploitation of these factors is leading to new field control strategies.

Cultural practices

Aflatoxin is a problem in plants under stress and can be reduced by lowering plant stress. Many of the practices growers use to obtain maximum quality and yield also reduce susceptibility to A. flavus infection and aflatoxin production, e.g. planting regionally adapted cultivars, planting at appropriate seed density, adequate fertilization (especially nitrogen), limiting insect damage, controlling weeds, irrigating when needed and harvesting as soon as the crop is mature. Unfortunately, these practices are not always possible and not always sufficient to prevent aflatoxin formation.

Competitive exclusion

The premise of this approach is that fungal strains that do not produce toxin can be used to fill the niche and displace the toxigenic strains in crop fields. The goal is to reduce aflatoxin levels, not mould contamination. Because A. flavus is a human and animal pathogen, the methods developed for competitive exclusion must keep airborne spore counts as low as possible. Caution must also be used because A. flavus produces a number of toxins other than aflatoxin. In cotton, application of non‐aflatoxigenic strains of A. flavus early in the growing season led to a large reduction of aflatoxin among BGY fluorescent bolls (Cotty, 1994). A non‐aflatoxigenic strain (AF36) is currently being used to control aflatoxin in commercial cotton fields. In peanut, a commercial competitive exclusion product called afla‐guard® has been used successfully to lower aflatoxin levels (Dorner and Lamb, 2006). A similar approach is being developed for control of aflatoxin in the peanut‐growing areas in Australia (Pitt and Hocking, 2006). Application of non‐aflatoxigenic strains have also reduced aflatoxin in corn, but these have not yet been developed for commercial use (Brown et al., 1991; Dorner et al., 1999).

Reduction of injury

In cotton, chemical control of pink boll worm (Pectinophora gossypiella) reduced aflatoxin (Henneberry et al., 1978). Transgenic cotton containing the insecticidal proteins from Bacillus thuringiensis (Bt) reduces or eliminates the part of the aflatoxin problem caused by some insects, but not other modes of entry (Cotty et al., 1997). Control of aflatoxin in Bt cotton lines has been inconsistent (Bock and Cotty, 1999). In peanut, plots treated with insecticide had lower levels of A. flavus infection (Bowen and Mack, 1993). Transgenic peanuts containing the Bt gene had significantly lower levels of aflatoxin than non‐Bt peanuts in preliminary analysis of log‐transformed data (Ozias‐Akins et al., 2002; P. Ozias‐Akins, personal communication). In corn, aflatoxin is often associated with insect damage; however, Bt corn has not consistently been resistant to aflatoxin (Zipf and Rajasekaran, 2003).

Host resistance

Early attempts at finding high yielding crop cultivars resistant to A. flavus contamination and aflatoxin formation were largely unsuccessful in peanuts (Azaizeh et al., 1989; Blankenship et al., 1985) and corn (Davis et al., 1986). There are still no commercial cultivars that are totally resistant to aflatoxin contamination. Recently, the use of proteomics has led to discovery of resistant germplasm in corn (Menkir et al., 2006). For pragmatic control, a number genes for resistance to A. flavus and/or aflatoxin formation (Chen et al., 2006) or plant stress (Chen et al., 2004) will be used to develop resistant lines with acceptable yield (reviewed by Brown et al., 2003). In cotton, which has no known naturally resistant varieties, plants are being engineered to add genes which are either antifungal or which may inhibit aflatoxin biosynthesis (Rajasekaran et al., 2006).

Molecular control of toxin production

Oxidative stress has been shown to enhance aflatoxin production (Jayashree and Subramanyam, 2000). Hydrosoluble tannins and gallic acid, known antioxidants, have been shown to inhibit aflatoxin biosynthesis (Mahoney and Molyneux, 2004). The molecular aspects of this are being investigated, and research is underway to determine if use of chemicals that weaken the oxidative stress response will reduce aflatoxin production (Kim et al., 2006; Reverberi et al., 2006).

Post‐harvest options

If pre‐harvest assessment of aflatoxin demonstrates that a field is highly contaminated, the best option may be to destroy the crop. Other options include feeding the crop to less sensitive animal species, or blending contaminated crop seed with uncontaminated seed to reduce aflatoxin to acceptable levels. It should be noted that blending is illegal in some countries and legal only under very restricted conditions in others. Aflatoxins may be chemically extracted with solvents (Dollear, 1969; Hron et al., 1992, 1994). Although this method is effective, it is not economically practical. Heat treatment may reduce (but not eliminate) aflatoxins, but the temperatures must be well above those used in normal cooking, and efficacy depends on other factors such as moisture content of the commodity (for a review, see Rustom, 1997). Interestingly, the one cooking process that does drastically reduce aflatoxin is the nixtamalization process (soaking and cooking of maize in an alkaline solution) used in making tortillas (Mendez‐Albores et al., 2004). Radiation has been used to reduce aflatoxin, with varying degrees of success (Klich, 2007; Rustom, 1997). Three methods are currently used commercially: adsorbants, ammoniation and sorting.

Adsorbants such as clays can remove aflatoxins from feeds (Masimango et al., 1979; Phillips et al., 1988). A number of such products are commercially available. The concern with this method is that treating the clays may bind nutrients as well as aflatoxin; however, reported reductions are small (CAST, 2003). A number of other sequestering agents have been investigated with varying degrees of success (reviewed by Diaz and Smith, 2005).

Ammoniation involves treating the seed to gaseous ammonia or ammonium hydroxide. This converts aflatoxin B1 to less toxic products, and can remove over 90% of the aflatoxin (Coker, 1989). The fact that some of these products are slightly toxic has prevented US FDA approval of this method; however, ammoniation is accepted in other countries and used in the US in commodities that are not shipped between states (CAST, 2003; Park et al., 1988).

Removal of aflatoxin‐contaminated seed is another method of lowering aflatoxin levels post‐harvest. A. flavus and other fungal contamination of peanuts often cause discoloration of the seed. This attribute has been exploited in peanut shelling facilities in which shelled peanuts are colour‐sorted to remove contaminated seed. The process used in Australia for removing aflatoxin‐contaminated seed is described in detail by Pitt and Hocking (2006). The process has not been commercially successful for pistachios and almonds (Campbell et al., 2005).

ACKNOWLEDGEMENTS

I would like to thank Bruce Horn and Kanniah Rajasekaran for critical review of drafts of this manuscript and Gary Payne and Jay Mellon for helpful discussions.

REFERENCES