Fungi Producing Significant Mycotoxins

CHAPTER 1 CHAPTER

chapter 1. Fungi producing significant mycotoxins

Summary Aspergillus species, occurring in and zearalenone, is pathogenic warmer climates, are A. flavus on maize, wheat, and barley and Mycotoxins are secondary metabolites and A. parasiticus, which produce produces these toxins whenever it of microfungi that are known to aflatoxins in maize, groundnuts, infects these grains before harvest. cause sickness or death in humans tree nuts, and, less frequently, other Also included is a short section on or animals. Although many such commodities. The main ochratoxin Claviceps purpurea, which pro- toxic metabolites are known, it is A producers, A. ochraceus and A. duces sclerotia among the seeds generally agreed that only a few carbonarius, commonly occur in in grasses, including wheat, barley, are significant in causing disease: grapes, dried vine fruits, wine, and and triticale. The main thrust of the aflatoxins, fumonisins, ochratoxin A, coffee. Penicillium verrucosum also chapter contains information on deoxynivalenol, zearalenone, and produces ochratoxin A but occurs the identification of these fungi and ergot alkaloids. These toxins are only in cool temperate climates, their morphological characteristics, produced by just a few species from where it infects small grains. F. as well as factors influencing their the common genera Aspergillus, verticillioides is ubiquitous in maize, growth and the various susceptible Penicillium, Fusarium, and Claviceps. with an endophytic nature, and commodities that are contaminated. All Aspergillus and Penicillium produces fumonisins, which are Finally, decision trees are included species either are commensals, generally more prevalent when crops to assist the user in making informed growing in crops without obvious are under drought stress or suffer choices about the likely mycotoxins signs of pathogenicity, or invade excessive insect damage. It has present in the various crops. crops after harvest and produce recently been shown that Aspergillus toxins during drying and storage. niger also produces fumonisins, In contrast, the important Fusarium and several commodities may be and Claviceps species infect crops affected. F. graminearum, which is before harvest. The most important the major producer of deoxynivalenol

Chapter 1. Fungi producing significant mycotoxins 1 1. Introduction and aflatoxicosis, which killed 100 000 it), or a storage fungus, and it young turkeys in the United Kingdom produces mycotoxins under all three Mycotoxins have been defined as in 1960 and has caused disease conditions. “fungal metabolites which when in- and death in many other animals, While not of worldwide signif- gested, inhaled or absorbed including humans (Rodricks et al., icance, a fourth genus is of sufficient through the skin cause lowered 1977; Lubulwa and Davis, 1994). importance to be included here. performance, sickness or death in By general consent, the name Claviceps is pathogenic on a wide man or animals, including birds” “mycotoxin” is usually restricted to toxic variety of cereals and other crops, (Pitt, 1996). This definition is widely compounds produced by microfungi producing resting structures called accepted, although interpretation and excludes toxins formed by the ergots, which often contain toxins. of “animals” is still under discussion Basidiomycetes, the mushrooms or in some quarters. The definition as macrofungi consumed as foods in 2.2 Which mycotoxins are it stands may be taken as including many parts of the world. important? lower (invertebrate) animals; as it Many thousands of metabolites seems likely that some mycotoxins have been described from microfungi; As noted above, several hundred are indeed aimed at insects, such even the large number dealt with compounds are known to be toxic as the mites that prey on fungi, this in extensive reviews (Cole and to humans or animals. However, interpretation is reasonable. However, Schweigert, 2003a, 2003b; Cole et many of these cause little concern most mycotoxicologists consider that al., 2003) are only a fraction of those because they are produced by mycotoxins are of relevance only when known. It seems likely that many fungi that are rarely encountered they affect humans and domestic of these metabolites are produced in foods or feeds. Many species of animals, and this much narrower not randomly but in attempts to alter Penicillium, for example, are found definition is used here. the ecology surrounding the fungus, almost exclusively in soils and rarely, It has been known for a long time by inhibiting growth of competitor if ever, in foods or feeds. Therefore, that it is hazardous to eat some species microorganisms, insects, etc. Only a many highly toxic compounds of the larger fungi, i.e. mushrooms and limited number of compounds, a few produced by Penicillium species “toadstools”, but until comparatively hundred, are known to be toxic to have not been found in foods or feeds recently the occurrence of common humans or domestic animals. in appreciable quantities. These moulds on foods has generally been include verruculogen, produced considered an aesthetic problem, 2. Mycotoxigenic fungi by P. simplicissimum and related not a health hazard. The realization species; janthitrems, produced by P. that metabolites of some common 2.1 Which genera are janthinellum; rugulosins, produced foodborne fungi were responsible important? by P. rugulosum and P. variabile; and for animal disease and death came many others. only in the 1960s, despite a few If we look at the worldwide occurrence In a second category are com- excellent studies in the first half of fungi in foods, and at which might pounds that are demonstrably toxic of the 20th century. It is now well be capable of mycotoxin production, under some test conditions, e.g. established that mycotoxins have three genera stand out: Aspergillus, by injection, but are not toxic when been responsible for major epidemics Penicillium, and Fusarium (Pitt and taken by a natural route. Compounds in humans and animals during recent Hocking, 2009). Fusarium species of this type may be inactivated by historical times. The most important are destructive pathogens on cereal stomach acids or are so insoluble epidemics have been ergotism, which crops and other commodities, and pro- as to be excreted without harm. has killed hundreds of thousands duce mycotoxins before, or immediately Sterigmatocystin, produced by the quite of people in Europe during the last after, harvest. Certain species of common storage fungus Aspergillus millennium (Smith and Moss, 1985); Aspergillus and Penicillium are also versicolor, is so insoluble in water, or alimentary toxic aleukia (ATA), which plant pathogens or commensals, but acid, that its true toxicity to mammals was responsible for the deaths of at these genera are more commonly has been difficult to measure, and it least 100 000 people in the USSR associated with commodities and has not been known to cause illness. between 1942 and 1948 (Joffe, 1978); foods during drying and storage. In a third category, of potentially stachybotryotoxicosis, which killed Aspergillus flavus is an exception: greater human health significance, tens of thousands of horses in the it can be a pathogen, a commensal are compounds that are produced by USSR in the 1930s (Moreau, 1979); (growing in a plant without affecting fungi known to occur in foods but that

2 CHAPTER 1 CHAPTER under normal conditions are present Some other mycotoxins are im- 3. Taxonomic overview of in such low concentrations that they portant in limited areas of the world. the fungal genera producing present no real hazard, i.e. their Sporidesmin is a mycotoxin that important mycotoxins effects, if any, are not measurable. causes facial eczema in sheep. It is In most cases the reason is that produced in pasture by the fungus The genera Aspergillus, Fusarium, although the fungi are readily isolated Pithomyces chartarum in some areas and Penicillium all reproduce by from some types of foods, they do not of New Zealand and Australia, and asexually produced spores, known normally grow to the extent required can cause large economic losses in as conidia, which are formed from to produce hazardous levels of toxin. local areas. The fungus Phomopsis specialized cells called phialides, Many examples exist: cyclopiazonic leptostromiformis produces the my- where mitosis takes place and from acid, from Aspergillus flavus; citrinin, cotoxin phomopsin in lupin plants which conidia are generated rapidly from Penicillium citrinin and several and seeds in Western Australia, and and in great numbers. Some species other species; citreoviridin, produced phomopsin is of great importance in each genus also produce a sexual by P. citreonigrum and Eupenicil- to the cattle raising and lupin seed stage defined by the production of lium ochrosalmoneum; roquefortine, export industry in that state. However, asci, specialized cells that result produced by P. roqueforti and its global impact is minimal. from meiosis, usually in well-defined related species; penitrem A, Patulin is sometimes included in macroscopic bodies (up to 1 mm in from P. crustosum; many of the lists of important mycotoxins, and diameter) called cleistothecia (Kirk trichothecenes produced by various concentrations in foods are subject to et al., 2001). A few species produce Fusarium species; and tenuazonic regulatory control in some countries. hard resting cells called sclerotia, acid, from Alternaria species. Under Patulin is produced by the growth of essentially immature cleistothecia. favourable growth conditions, how- Penicillium expansum in apples and In Aspergillus and Penicillium, ever, the fungi in this category are pears. The production of significant phialides are borne in clusters, while capable of extensive growth and levels of patulin is accompanied by conidia are single-celled, more or significant toxin production, so these visible rotting of the fruit, so patulin is less spherical, and very small, usually and some other toxins should be primarily of concern in juices. Nearly not exceeding 5 µm in diameter. The kept in mind when fungal spoilage of all the toxin can be removed if rotting two genera are closely related and foods and feeds occurs. fruit are rejected by visual inspection are distinguished by the way in which Some toxins are produced by or rotten parts are removed by hand phialides are grouped. In Aspergillus, rare species. For example, the trimming or by washing them out with phialides are always borne in tight species that produces rubratoxins is high-pressure water jets. Hence, clusters around the swollen apices known from only a few isolates and patulin in apple juices and other (vesicles) of long stalks (stipes), does not even have a recognized products is controllable by simple with or without an intermediate row name. Rubratoxin A is known to food technological procedures, and of supporting cells called metulae have caused one disease outbreak, its occurrence does not warrant (Raper and Fennell, 1965; Pitt and in two people who consumed mouldy consideration here. Hocking, 2009; Samson et al., 2010); home-made rhubarb wine (Richer The mycotoxins treated in detail see Figs 1.1–1.5. In Penicillium, et al., 1997). However, rubratoxin in this book are based on those phialides are usually borne in finger- can be ignored when overviewing considered by Miller (1995) to be like clusters on more diminutive mycotoxin occurrence worldwide. the most important on a worldwide stipes, again with or without one or A few mycotoxins are considered basis: aflatoxins, ochratoxin A, two intermediate rows of supporting to be significant in feeds but not fumonisins, specific trichothecenes cells (metulae and rami) (Pitt, 1979; foods. These are of known toxicity (deoxynivalenol and nivalenol), and Pitt and Hocking, 2009); see Fig. to birds, in particular, and are mainly zearalenone. These toxins are pro- 1.6. Colonies of Penicillium species water-soluble toxins. The reason duced in foods and feeds by species of grown on identification media in Petri appears simple: whereas mammals Aspergillus, Penicillium, and Fusarium. dishes are usually green, the colour excrete water-soluble toxins, often A limited taxonomic treatment of these of Penicillium conidia, and often have with little ill effect, birds excrete fungi and the species producing other pigments from the mycelium only solid waste, so are unable to important mycotoxins is given in this or excreted from the colonies. In get rid of these toxins so readily. In chapter. Also included is Claviceps Aspergillus, colony colours are this category are cyclopiazonic acid, purpurea, the species that produces those of the conidia, which may be citrinin, and tenuazonic acid. ergot and ergot toxins in small grains. black, yellow, brown, white, or green.

Chapter 1. Fungi producing significant mycotoxins 3 Colonies of most Aspergillus species 4. Genus Aspergillus bullet-shaped sclerotia. A. nomius show no other colours. Fusarium is associated with insects, and not colonies generally consist of loose, Aspergillus is a large genus, with 100 usually with foods, but recently has fluffy mycelium, coloured white, pink, or more recognized species, most been shown to be a common cause or purple, and often show similar of which grow and sporulate well on of aflatoxin production in Brazil nuts colours in the colony reverse. In common synthetic or semisynthetic (Olsen et al., 2008). Fusarium, phialides are not clustered, media. The most widely used tax- Enumeration. Satisfactory enu- and conidia may be of two types: onomy is by Raper and Fennell (1965), meration of A. flavus and A. para- those characteristic of the genus are although some of their concepts siticus can be achieved on any large and crescent-shaped (although are out of date. For more modern antibacterial enumeration medium sometimes formed only under natural taxonomic concepts, see Samson and that contains appropriate inhibitors to conditions or on special media), Pitt (1990) or Klich (2002). A minority reduce colony spreading. Dichloran whereas the second type, produced of Aspergillus species make a sexual rose bengal chloramphenicol agar by only some species, are small stage (known as a teleomorph) in (DRBC) or dichloran 18% glycerol and usually cylindrical (Marasas et which spores (ascospores) are borne agar (DG18) are recommended (Pitt al., 1984; Pitt and Hocking, 2009; in asci, in turn borne in cleistothecia. and Hocking, 1997, 2009; Samson et Samson et al. 2010); see Figs 1.7–1.9. Species with teleomorphs are correctly al., 2010). Relatively rapidly growing, The taxonomy of all three genera classified in teleomorph genera, of moderately deep, yellow green is complex. Overall taxonomies for which Eurotium, Neosartorya, and colonies exhibiting “mop-like” fruiting mycotoxigenic fungi occurring in Emericella are the best known. Few of structures under the stereomicro- foods are given in Fungi and Food the important mycotoxigenic species scope can be presumptively counted Spoilage (Pitt and Hocking, 2009) produce teleomorphs. as A. flavus plus A. parasiticus. and Food and Indoor Fungi (Samson The most significant mycotoxigenic Microscopic examination of colonies et al., 2010). The most useful intro- species in Aspergillus are A. flavus and can provide supporting evidence, ductions to the major species in each A. parasiticus, which make aflatoxins, but representative colonies must genus are found in laboratory guides: and the species that make ochratoxin be grown on standard identification A Laboratory Guide to Common A: A. ochraceus and related species, media for confirmation. Penicillium Species (Pitt, 2000), the black species A. carbonarius, and A more effective medium for enu- Identification of Common Aspergillus (uncommonly) A. niger. A. flavus and merating these species is Aspergillus Species (Klich, 2002), and The A. parasiticus are very closely related flavus and parasiticus agar (AFPA; Fusarium Laboratory Manual (Leslie and are treated together. Pitt et al., 1983), a medium formulated and Summerell, 2006). specifically for this purpose. AFPA has Claviceps differs from the three 4.1 Fungi producing aflatoxins: two major advantages: enumeration genera mentioned above because A. flavus and A. parasiticus can be carried out after incubation at most species cannot be cultivated in 30 °C for 2 days, and both species are the laboratory. Species of Claviceps Taxonomy. Aflatoxins are now readily recognized, even by untrained grow on a wide variety of grasses, known to be produced by at least eyes, by intense orange yellow colours where they infect only the ovaries, 10 Aspergillus species. However, in the reverses of the colonies. Under forming hard bodies called sclerotia most are rare or are rarely found in the incubation conditions specified that replace normal seed heads. foods: the principal fungi producing for AFPA (30 °C for 42–48 hours), Conidia are released in droplets aflatoxins remain A. flavus and A. the presence of the bright orange called honeydew, attractive to insects, parasiticus. Of some importance is yellow reverse is diagnostic for these which then disseminate the fungus a new species found in groundnuts species. throughout the crop. in the southern hemisphere, called Descriptions. The descriptions of The taxonomy (classification) of A. minisclerotigenes. This species Aspergillus species given here are these genera is described in more looks like a variant of A. flavus that taken from Fungi and Food Spoilage detail below. produces unusually small sclerotia, (Pitt and Hocking, 2009). The fungi but like A. parasiticus it produces both are grown as colonies on Czapek B and G aflatoxins. The other species yeast extract agar (CYA) and malt of some importance is A. nomius, extract agar (MEA) at 25 °C and which also makes B and G aflatoxins CYA at 37 °C for 7 days. Fungi are and looks like A. flavus but produces inoculated onto these plates at three

4 CHAPTER 1 CHAPTER points, equidistant from each other Fig. 1.1. Aspergillus flavus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; and midway between the rim and (b, c) heads, bars = 20 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), Fig. 8.13, p. 305; reproduced with kind permission from Springer Science+Business centre of the Petri dish. Inoculation Media B.V. is facilitated by first dispersing a needle point of conidia in small vials containing 0.2 mL of 0.2% agar and 0.05% Tween 80 or similar detergent (Pitt and Hocking, 2009), as this reduces colonies from stray spores. Formulations for these and other media are given in the Annex (p. 29). Aspergillus flavus Link. See Fig. 1.1. Colonies on CYA 60–70 mm in diameter; conidial heads usually borne uniformly over the whole colony but sparse or absent in areas of floccose (cotton wool) growth or sclerotial production, characteristically coloured greyish green but sometimes pure yellow, becoming greenish in age; sclerotia produced by about 50% of isolates, at first white, becoming deep reddish brown, density varying from inconspicuous to dominating colony appearance and almost entirely suppressing conidial production. Colo- nies on MEA 50–70 mm in diameter, similar to those on CYA, although usually less dense. At 37 °C, colonies usually 55–65 mm in diameter, similar to those on CYA at 25 °C. Sclerotia spherical, usually 400– 800 μm in diameter. Teleomorph developing from sclerotia, but only Aspergillus parasiticus Speare. three quarters of the surface, mostly after selected isolates are mated. See Fig. 1.2. Colonies on CYA 50– bearing phialides only, but in some Structures bearing conidia 400 μm to 70 mm in diameter, conidial heads isolates up to 20% of heads bearing 1 mm or more long; vesicles (terminal in a uniform, dense layer, coloured metulae as well; conidia spherical, swellings) spherical, 20–45 μm in dark yellowish green; sclerotia mostly 4.0–6.0 μm in diameter, with diameter, fertile over three quarters occasionally produced. Colonies distinctly roughened walls. of the surface, typically bearing on MEA 50–65 mm in diameter, The teleomorph is Petromyces both phialides and metulae (cells generally similar to those on CYA. flavus (Horn et al., 2011), but in culture supporting phialides), but in some At 37 °C, colonies covering the is seen only after suitable strains are isolates a proportion of, or even most, available area, similar to those on mated (Horn et al., 2009a). heads bear phialides alone; conidia CYA at 25 °C. Distinctive features. Aspergillus spherical or nearly, usually 3.5–5.0 μm Sclerotia occasionally produced, flavus and A. parasiticus together are in diameter, with relatively thin walls, white at first, becoming black, distinguished by their rapid growth at finely roughened or, rarely, smooth. spherical, 400–800 μm in diameter. both 25 °C and 37 °C and their bright The teleomorph is Petromyces Teleomorph developing from sclerotia, yellow green (or, less commonly, flavus (Horn et al., 2011), but in culture but only after selected isolates are yellow) conidial colour. The definitive is seen only after suitable strains are mated. Structures bearing conidia difference between the two species mated (Horn et al., 2009b). 250–500 μm long; vesicles spherical, is that A. flavus produces conidia that 20–35 μm in diameter, fertile over are rather variable in shape and size,

Chapter 1. Fungi producing significant mycotoxins 5 Fig. 1.2. Aspergillus parasiticus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; predictive model for A. flavus growth (b, c) heads, bars = 10 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), in relation to aw and temperature was Fig. 8.18, p. 321; reproduced with kind permission from Springer Science+Business Media B.V. derived from those data (Gibson et al., 1994). Growth of A. flavus occurred over the pH range 2.1–11.2 (the entire range examined) at 25, 30, and 37 °C, with optimal growth over a broad pH range of 3.4–10 (Wheeler et al., 1991). The heat resistance of A. flavus has been studied under various conditions by several authors. The

most reliable figures indicate a D45 value (the time required at 45 °C to kill 90% of the population) of more

than 160 hours, a D50 of 16 hours, a

D52 of 40–45 minutes, and a D60 of 1

minute, at neutral pH and high aw, with z values (the increase in temperature required to reduce the D value by 90%) of 3.3–4.1 C° (ICMSF, 1996). The addition of phosphine, used to control insects, to grain at 0.80

or 0.86 aw reduced growth of A. flavus while having little effect on the survival of conidia (Hocking and Banks, 1991, 1993). Available data indicate that the influence of physical factors on the growth of A. parasiticus is very similar to that on A. flavus (Pitt and Hocking, 2009). However, A. parasiticus grows at somewhat lower temperatures, up

to 42 °C. The effect of aw is similar to that found for A. flavus (Pitt and have relatively thin walls, and range coconut cream agar (Dyer and Miscamble, 1995). from smooth to moderately rough, McCammon, 1994). Recently, the the majority being finely rough. In use of a cyclodextrin, incorporated 4.1.1 Commodities and foods at contrast, conidia of A. parasiticus are into any standard medium, has risk from aflatoxin contamination spherical and have relatively thick, been proposed (Jaimez Ordaz et rough walls. In addition, vesicles of al., 2003). Visualization of aflatoxin Aspergillus flavus and to a lesser A. flavus are larger, up to 50 μm in production is by examination of the extent A. parasiticus have been diameter, and usually bear metulae, reverse of colonies on Petri dishes isolated from a very wide range of whereas vesicles of A. parasiticus under ultraviolet (UV) light. food commodities (Pitt and Hocking, rarely exceed 30 μm in diameter and Factors influencing growth. 2009). Indeed A. flavus may be metulae are uncommon (Klich and Growth temperatures for A. flavus regarded as truly ubiquitous in foods Pitt, 1988). The teleomorphs are not most often reported are a minimum of produced in tropical and subtropical seen in pure culture of single isolates. 10–12 °C, a maximum of 43–48 °C, countries. Although evidence of A. Differentiating toxigenic from and an optimum of about 33 °C (Pitt flavus at low levels in foods cannot be non-toxigenic isolates of A. flavus or and Hocking, 2009). The minimum taken as an indicator of the presence

A. parasiticus can also be of value. A water activity (aw) permitting growth is of aflatoxins, high levels of infection, variety of media have been proposed 0.82 at 25 °C, 0.81 at 30 °C, and 0.80 i.e. plate counts of greater than to achieve this, one of which is at 37 °C (Pitt and Miscamble, 1995). A 105/g, or infection levels of more than

6 CHAPTER 1 CHAPTER 50% of grains when direct plated, Aspergillus flavus is capable of factors are drought stress (Sanders provide reasonable presumptive causing spoilage of some kinds of et al., 1981) and soil temperatures evidence that aflatoxin may be fresh fruit and vegetables, including around 30 °C (Blankenship et al., present. However, quantification of citrus, tomatoes, peppers, litchis, 1984; Sanders et al., 1984; Cole et al., the association between levels of pineapples, and pomegranates 1985; Cole, 1989; Dorner et al., 1989) A. flavus infection and aflatoxin (Snowdon, 1990, 1991), but aflatoxin during the last 30–50 days before production is not possible, so these production is unlikely. harvest (Sanders et al., 1985). figures can only serve as guidelines. Maize. Maize is usually infected Although it is possible to induce 4.1.2 Formation of aflatoxins in only by A. flavus, not by A. parasiticus aflatoxin production in a very wide susceptible crops (Lillehoj et al., 1980; Angle et al., 1982; range of foods or raw materials under Horn et al., 1995). It appears probable experimental conditions, research Groundnuts. Groundnuts are sus- that the most important route for entry and experience have shown that only ceptible to infection by both A. flavus of A. flavus to maize is through insect certain commodity types are likely to and A. parasiticus (Hesseltine et damage (Lillehoj et al., 1982; Bilgrami contain aflatoxin in the absence of al., 1973; Diener et al., 1987; Pitt et al., 1992). Invasion down the silks obvious signs of fungal growth or and Hocking, 2009). The primary is also possible (Marsh and Payne, other deterioration in appearance. source of these fungi is soil, where 1984; Diener et al., 1987). High Based on the results of many high numbers may build up because temperature stress increases infection surveys, commodities most at risk in some groundnuts are not harvested (Jones et al., 1980), the critical time international trade are groundnuts, but remain in the ground and act as for infection being between 16 and 24 maize, and cottonseed. Lesser, but a nutrient source (Griffin and Garren, days after inoculation at silking (Jones still substantial, risk is associated 1976a). Uncultivated soils contain very et al., 1980; Payne, 1983). with tree nuts of all types, especially low numbers of A. flavus, but soils Cottonseed. A. flavus is also a Brazil nuts, pistachio nuts, and semi- in groundnut fields usually contain commensal in the cotton plant (Klich processed coconuts (i.e. copra). 100–1000 propagules/g (Pitt, 1989). et al., 1984). Infection occurs through Walnuts, hazelnuts, and cashews Under drought stress conditions, this the nectaries, natural openings in are only occasionally affected. number may rise to 104–105/g (Horn the cotton stem that are important in Spices from tropical countries are et al., 1995). Large numbers of A. pollination (Klich and Chmielewski, also a frequent source of aflatoxin, flavus spores are also airborne over 1985), or through cotyledonary leaf but these spices are usually present susceptible crops (Holtmeyer and scars (Klich et al., 1984). Upward at only low levels in foods. Oilseeds Wallin, 1981). movement occurs in the stem towards of all kinds are affected from time to Direct entry to developing ground- the boll, but not downwards from boll time (Pohland and Wood, 1987), and nuts through the shell by A. flavus to stem (Klich et al., 1986). Insect figs may carry a substantial risk (Le in the soil appears to be the main damage is also a potential cause of Bars, 1990). method for nut infection (Diener et infection (Lee et al., 1987), but insects Aflatoxins have been reported al., 1987). Infection can also occur are often well controlled in cotton from smoked and dried or cured fish through the pegs and flowers (Wells crops. As in groundnuts and maize, in Sierra Leone (Jonsyn and Lahai, and Kreutzer, 1972; Griffin and temperature appears to be a major 1992) but are not considered to be a Garren, 1976b; Pitt, 1989). A. flavus environmental factor in pre-harvest problem in salted dried fish produced sometimes grows within groundnut infection of cottonseed (Marsh et under South-East Asian conditions plants themselves: growth in plant al., 1973; Simpson and Batra, 1984). (Pitt and Hocking, 1996). Other tissue is not pathogenic but High minimum temperatures, above meat products, including prepared commensal. The seedpod (Lindsey, 24 °C, appear to lead to high aflatoxin hams (Rojas et al., 1991), are not 1970) or the plant (Pitt, 1989; Pitt formation (Diener et al., 1987). considered to be at risk. et al., 1991) show no visible sign of Cereals, legumes, and pulses colonization by the fungus. 4.1.3 Formation of aflatoxins in may also be infected with A. flavus A variety of factors influence other crops (Pitt and Hocking, 2009), but invasion of developing groundnuts unacceptable levels of aflatoxin occur by A. flavus. Infection before harvest With other crops, Aspergillus flavus is only under poor storage conditions occurs only if substantial numbers not associated with the plant, so entry and are rarely of concern. of fungal propagules (perhaps 103/g) to nuts or seeds or other food parts exist in the soil. Other important is opportunistic and usually occurs

Chapter 1. Fungi producing significant mycotoxins 7 only after the crop matures. Entry of low, and oil content high. Sorption reports refer to OTA production by A. flavus into pistachio nuts depends isotherms of these commodities are other, often unspecified, Penicillium on the time of splitting of hulls. Nuts similar (Iglesias and Chirife, 1982). species, but these reports are known in which hull splitting occurs early A. flavus and A. parasiticus cannot to be erroneous (Frisvad 1989; are much more susceptible to A. grow below about 0.80 aw, equivalent Frisvad and Filtenborg, 1989; Pitt flavus invasion on the tree (Doster to about 10% moisture content and Hocking, 2009). and Michailides, 1994). It is known in these commodities. However, Recently, Aspergillus carbon- that some cultivars are more prone storage above 8% moisture content arius has been identified as a third to early splitting than others, and this (about 0.7 aw) can lead to fungal major source of OTA, together with is especially important where nuts spoilage. Fungal growth may result a low percentage of isolates of the are harvested from the ground, after in a moisture increase, creating closely related species A. niger contact with the soil. conditions under which A. flavus (Abarca et al., 1994; Téren et al., Brazil nuts are harvested from can grow, so 8% moisture must be 1996). It is now clear that OTA is the ground, beneath trees growing considered the safe moisture content produced by two closely related naturally in Amazonian forests. Har- for these commodities. Penicillium species, P. verrucosum vesting is intermittent, up to a month Such a low moisture content can and P. nordicum, and by a rather apart, providing time for the ever be difficult to maintain in practice. remarkable range of Aspergillus present A. flavus and other related Shipment of nuts in containers species. The following sections deal species to infect the nuts (Johnsson et across the tropics is a particular with these species in more detail. al., 2008). A. nomius appears to be an hazard as unsuitable stowage, on important source of aflatoxins in Brazil decks or near engines, can lead 4.2.2 Aspergillus ochraceus and nuts (Olsen et al., 2008). to moisture migration sufficient to related species In other tree nuts, formation cause sporadic spoilage or even total of aflatoxins occurs sporadically, loss. Cases of rampant growth of A. Taxonomy. Recent work has shown usually as the result of insect damage flavus accompanied by high aflatoxin that Aspergillus ochraceus is not or poor storage practices. production have been observed a common producer of OTA. Most Figs are sometimes infected by under these conditions. OTA in foods originally attributed to A. flavus. The unique structure of the Storage of nuts in tropical A. ochraceus is now known to be due fruit evolved to enable fertilization countries is sometimes inadequate, to A. westerdijkiae and A. steynii, by insects, and insects may carry A. again leading to spoilage or aflatoxin newly described species very similar flavus spores into the seed cavity. production. morphologically to A. ochraceus Also, figs are harvested from the (Frisvad et al., 2004). Apart from ground in some countries. Immature 4.2 Ochratoxin A these species, this group of OTA figs are not colonized by A. flavus, but producers includes two ascosporic once they are ripe infection occurs 4.2.1 Formation of ochratoxin A fungi, Neopetromyces muricatus readily and fungal growth continues (asexual state A. muricatus) and during drying (Buchanan et al., 1975; Ochratoxin A (OTA) was originally Petromyces alliaceus (asexual Le Bars, 1990). The proportion of described as a metabolite of state A. alliaceus), and two that the crop infected is low, 1% or less Aspergillus ochraceus from labora- do not produce a teleomorph, A. (Steiner et al., 1988). The problem tory experiments (van der Merwe sclerotiorum and A. sulphureus. N. has been serious for some exporting et al., 1965), and subsequently muricatus is the correct name for countries (Sharman et al., 1991) but is from several related Aspergillus isolates that produce OTA, previously now well controlled by examination of species. However, the first report of identified as A. melleus. Both N. individual figs under UV light. natural occurrence, and the potential muricatus and P. alliaceus are importance, of OTA was from a uncommon species. A. sclerotiorum 4.1.4 Formation of aflatoxins in Penicillium species (Scott et al., isolates produce OTA only rarely, and storage 1970; Krogh et al., 1973), reported as although isolates of A. sulphureus are P. viridicatum, but later corrected to usually OTA producers, this is a rare Crops susceptible to aflatoxin P. verrucosum (Pitt, 1987). Larsen et species. Apart from A. westerdijkiae formation are mostly nuts and al. (2001) reported that P. nordicum, and A. steynii, all of these species oilseeds, where soluble solids closely related to P. verrucosum, are very uncommon in foods and are (sugars) in the dried commodity are is also a producer of OTA. Many not known to cause food spoilage.

8 CHAPTER 1 CHAPTER Fig. 1.3. Aspergillus ochraceus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; sparsely sporing. At 37 °C, colonies of (b) heads, bar = 20 µm; (c) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), 25–30 mm in diameter produced. Fig. 8.17, p. 318; reproduced with kind permission from Springer Science+Business Structures bearing conidia 1.0– Media B.V. 1.5 mm long, with yellowish to pale brown walls, finely to conspicuously roughened; vesicles spherical, 25– 50 μm in diameter, bearing tightly packed metulae and phialides over the entire surface; conidia spherical or near, 2.5–3.5 μm in diameter, with smooth to finely roughened walls. Distinctive features. Aspergillus ochraceus and closely related spe- cies producing ochratoxin all grow moderately slowly on standard identification media such as CYA and MEA (Pitt and Hocking, 2009). Colonies are coloured pale brown to yellow brown from the conidia. A. ochraceus grows strongly at 37 °C, whereas the closely related species A. westerdijkiae and A. steynii do not grow at that temperature. Apart from that distinction, A. westerdijkiae produces spherical, finely roughened conidia, whereas those of A. steynii are smooth-walled and ellipsoidal, not spherical. Factors influencing growth. Asper- gillus ochraceus and the closely related species described here are mesophilic xerophiles. Growth occurs between 8 °C and 37 °C, with the optimum at 24–31 °C (Pitt and Hocking, 2009). Optimal conditions

Enumeration. Aspergillus ochra- by growth in pure culture is necessary. for growth are 0.95–0.99 aw, while ceus, A. westerdijkiae, and A. steynii Satisfactory enumeration should the lower limit for growth is 0.79 aw on grow slowly on media of high aw as usually be possible also on DRBC, media containing sugars and down they are all xerophilic. Enumeration a selective medium of higher aw to 0.81 aw on media based on NaCl. on a medium of reduced aw, such (King et al., 1979). Dilute media, such A. ochraceus grows slowly at pH 2.2 as DG18, is recommended (Pitt and as potato dextrose agar (PDA) or and well between pH 3 and 10 (Pitt Hocking, 2009). bacteriological enumeration media, and Hocking, 1977). Colonies of A. ochraceus and and incubation temperatures above Commodities and foods at risk. closely related species can be 25 °C, are unsatisfactory. Aspergillus ochraceus has been presumptively recognized by relative- Aspergillus ochraceus Wilhelm. reported from a wide range of food ly deep colonies, uniformly coloured See Fig. 1.3. Colonies on CYA 40– products, more commonly in dried pale brown to yellow brown, that under 55 mm in diameter; conidial heads and stored foods than elsewhere. the low-power stereomicroscope closely packed, coloured light to golden However, it is likely that many of exhibit long fruiting stalks bearing yellow; sclerotia sometimes produced, these reports relate to the recently radiate Aspergillus heads, with spore white when young, later pink to purple. described A. westerdijkiae or A. chains splitting into two or three dense Colonies on MEA 40–55 mm in diameter, steynii. Stored foods from which these columns in age. Confirmation of identity plane, similar to those on CYA but quite species have been isolated include

Chapter 1. Fungi producing significant mycotoxins 9 smoked or salted dried fish and meat, that Aspergillus ochraceus, and no japonicus can be achieved on any beans, chickpeas, and nuts, especially doubt its close relatives A. westerdijkiae antibacterial enumeration medium pecans and pistachios. These species and A. steynii, are major sources of that contains appropriate inhibitors have been reported (usually as A. OTA in coffee (Taniwaki et al., 1999, to reduce colony spreading. DRBC ochraceus) from cereals and cereal 2003; Pitt et al., 2001; Batista et al., or DG18 is recommended (Pitt products but, rather infrequently, also 2003). Other known OTA producers, and Hocking, 1997; Samson et al., from cheese, spices, black olives, A. niger and A. carbonarius, have 2010). Rapidly growing, very dark and cassava. However, these species also been isolated from coffee (Frank, brown to black colonies exhibiting rarely cause spoilage, and are often 2001; Pitt et al., 2001). As detailed “mop-like” fruiting structures under found in foods at only low levels, so mycological studies have not yet the stereomicroscope can be their presence is not a good indicator been conducted in some major coffee presumptively counted as A. niger of significant mycotoxin production growing areas, the relative importance plus A. carbonarius. Microscopic ex- (Pitt and Hocking, 2009). of A. ochraceus and A. carbonarius amination of colonies can provide Several studies have detected as the main source of OTA in coffee is supporting evidence, but repre- A. ochraceus in green coffee beans difficult to assess. sentative colonies must be grown (Levi et al., 1974; Cantafora et al., Available evidence indicates that on standard identification media for 1983; Tsubouchi et al., 1984; Micco the sources of these fungi are environ- confirmation. et al., 1989; Studer-Rohr et al., 1994). mental and that entry to cherries is Aspergillus carbonarius (Bainier) Coffee cherries are usually picked gained during picking and drying Thom. See Fig. 1.4. Colonies on by hand, or sometimes mechanically (Taniwaki et al., 1999). OTA is produced CYA 60 mm or more in diameter, on large farms, and are usually during drying (Taniwaki et al., 1999; usually covering the whole Petri dried in the sun. The beans may be Bucheli et al., 2000; Teixera et al., dish; conidia black or nearly black. dried directly and separated from 2001). Coffee picked and dried under Colonies on MEA 50–60 mm in the hull afterwards, or mechanically good agricultural practice appears to diameter, usually smaller than those dehulled and dried, or dehulled by contain OTA only rarely (Taniwaki et on CYA, otherwise similar. At 37 °C, fermentation before drying. Coffee al., 1999, 2003). colonies 10–20 mm in diameter. beans are stored after drying (as Structures bearing conidia 1.0– “green” coffee), then graded and 4.2.3 Aspergillus carbonarius 3.0 mm long, with heavy, hyaline, shipped to manufacturers. and related species smooth walls; vesicles spherical, Picking cherries and spreading usually 60–85 μm in diameter, bearing them on drying yards frequently Taxonomy. Aspergillus carbonarius closely packed metulae and phialides causes damage, allowing ingress was recognized as a source of OTA over the whole surface; conidia of fungi. If cherries are picked from relatively recently (Horie, 1995; Téren spherical, 7–10 μm in diameter, black, the ground, contamination is likely et al., 1996; Wicklow et al., 1996). It is with walls extremely roughened. to be high. Drying is often a slow now known that most, if not all, isolates Aspergillus niger Tiegh. See Fig. 1.5. process, in particular because of of A. carbonarius produce OTA when Colonies on CYA 60 mm or more in the environment in which coffee grown in pure culture (Heenan et al., diameter, usually covering the whole is grown. Coffee trees will not 1998; Taniwaki et al., 1999), although Petri dish; conidia black. Colonies flower above 19 °C but require high the extent of production is variable. on MEA varying from 30 mm to temperatures to mature, so coffee is The closely related species A. niger 60 mm in diameter, usually smaller than commonly grown on upland areas in has also been reported reliably as a those on CYA and often quite sparse. the tropics. In consequence, drying producer (Ueno et al., 1991; Abarca At 37 °C, colonies 60 mm or more in is often conducted under less than et al., 1994; Heenan et al., 1998; diameter, covering the available space. ideal conditions, with morning mists Taniwaki et al., 1999). However, all Structures bearing conidia 1.0– or rain common in some growing reports agree that OTA production by 3.0 mm long, with heavy, hyaline, areas (Teixera et al., 2001). Fungal A. niger is very uncommon; OTA is smooth walls; vesicles spherical, growth frequently occurs. formed under pure culture conditions usually 50–75 μm in diameter, bearing Although the possibility of signifi- by only 1–2% of isolates. closely packed metulae and phialides cant levels of OTA being present in coffee Enumeration. Satisfactory enumer- over the whole surface; conidia beans has been known for some time, ation of A. niger and A. carbonarius spherical, 4–5 μm in diameter, brown, the fungal cause remained elusive. and the closely related (but with walls conspicuously roughened Only recently has it been established always non-toxigenic) species A. or sometimes striped.

10 CHAPTER 1 CHAPTER Fig. 1.4. Aspergillus carbonarius (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; optimum of 35–37 °C, and has been (b, c) heads, bars = 40 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), reported to germinate down to 0.77 Fig. 8.10, p. 300; reproduced with kind permission from Springer Science+Business a (Pitt and Hocking, 2009). Media B.V. w Commodities and foods at risk. Some black Aspergillus species, i.e. A. niger, A. carbonarius, and A. japonicus, are common inhabitants of vineyards, as these fungi grow rapidly at relatively high temperatures (above 30 °C) and their pigmentation renders them highly resistant to the deleterious effects of sunlight and UV light. These species appear to have no pathogenicity towards grapes and to be unable to penetrate an intact grape skin. Entry to maturing grapes results from attack by other pathogenic fungi (e.g. Rhizopus stolonifer, Botrytis cinerea, or powdery mildews), from mechanical damage due to cultivating or harvesting equipment, or, in some cultivars, from the splitting of berry skins that results from rain near harvest time. Once entry to a berry is gained, these fungi thrive in the acid, high-sugar environment. Where grapes are dried, the black Aspergilli enjoy a considerable ecological advantage, and will continue to grow and produce OTA until the

grapes dry below 0.8 aw. As grapes are normally dried in the sun, this usually takes several days, allowing ample time for OTA production to occur (Hocking et al., 2003). Distinctive features. Differen- carbonarius and A. japonicus grow None of the three species of tiation of Aspergillus niger, A. much more slowly (less than 20 mm) black Aspergilli appears to enjoy a carbonarius, and A. japonicus from (Mitchell et al., 2003). particular ecological advantage, at nearly all other species is not difficult. It is important to remember that least in Australia, as all three species These species grow rapidly and very few isolates of A. niger produce are commonly recorded from maturing produce very dark brown to black OTA. A. niger is an exceptionally grapes, with proportions of each varying conidia. A. niger and A. carbonarius common species, and recovery of with seasonal factors (Leong et al., produce metulae, whereas A. this species from foods should not 2004). For OTA formation, Aspergillus japonicus does not. A. niger produces be regarded as evidence that OTA is carbonarius is the significant species: conidia that are 4–5 µm in diameter, likely to be present. only a small proportion of A. niger whereas those of A. carbonarius are Factors influencing growth. As- isolates are capable of producing larger, on average 7 µm or more in pergillus carbonarius can grow down OTA, and A. japonicus isolates do not diameter. If these species are grown to 10 °C, with an optimum near 30 °C produce this toxin. on CYA for 7 days at 37 °C, separation and a maximum near 41 °C. The In grapes intended for wine- on colony diameters can be very optimal aw for growth is 0.96–0.98, making, the time interval is usually useful: A. niger grows very quickly with a minimum near 0.85 at 25 °C. short between infection and crushing, (60 mm or more), whereas A. A. niger grows up to 45 °C, with an when fermentation stops fungal growth

Chapter 1. Fungi producing significant mycotoxins 11 Fig. 1.5. Aspergillus niger (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; The classification of Pitt (1979, (b) head, bar = 15 µm; (c) heads, bar = 10 µm; (d) conidia, bar = 5 µm. Source: Pitt 2000) includes four subgenera: and Hocking (2009), Fig. 8.15, p. 314; reproduced with kind permission from Springer Aspergilloides, in which phialides are Science+Business Media B.V. borne directly on the stalk cells without intervening supporting elements; Furcatum and Biverticillium, in which phialides are supported by metulae; and Penicillium, in which both metulae and rami are usually present. The majority of important toxigenic and food spoilage species are found in subgenus Penicillium. Enumeration. Enumeration pro- cedures suitable for all common Penicillium species are similar. Any effective antibacterial enumeration medium can be expected to give satisfactory results. However, some Penicillium species grow rather weakly on dilute media, such as PDA or dichloran chloramphenicol peptone agar (DCPA), so DRBC is recommended. Penicillia can also be effectively enumerated on DG18 (Pitt and Hocking, 1997; Samson et al., 2010). Identification. For a comprehen- sive taxonomy of Penicillium, see Pitt (1979). Keys and descriptions to common species are provided by Pitt (2000) and to foodborne species by Pitt and Hocking (2009) and Samson et al. (2010). and toxin production ceases. So 5. Genus Penicillium Identification of Penicillium isolates control of OTA in wines relies on good to species level is not easy, preferably vineyard management, i.e. control Taxonomy. Penicillium is a large being carried out under carefully of bunch rots and skin splitting, and genus, with more than 200 recognized standardized conditions of media, short time intervals between harvest species, of which 50 or more are of incubation time, and temperature. and crushing. common occurrence (Pitt, 2000). All As well as microscopic morphology, Occasional contamination of figs common species grow and sporulate gross physiological features, including with OTA has been reported (Özay and well on synthetic or semisynthetic colony diameters, colours of conidia, Alperden, 1991). In a study of Turkish media, and usually can be readily and colony pigments, are used to figs sampled during different stages recognized at genus level. Most distinguish species. of processing, only 3 of 100 samples Penicillium species grow slowly, and contained OTA, and in each case levels have green conidia. 5.1 Ochratoxin A production were between 5 μg/kg and 10 μg/kg. Classification within Penicillium by Penicillium verrucosum Aspergillus niger and A. carbon- is based primarily on microscopic arius also occur in a wide variety of morphology: the genus is divided into Soon after the discovery of OTA from other fruits (Snowdon, 1990, 1991). subgenera based on the number and Aspergillus ochraceus, the formation Generally, other fruits are handled in arrangement of phialides (elements of OTA by a Penicillium species, ways that minimize fungal infection, or producing conidia) and metulae P. viridicatum, was reported (van damaged fruit is discarded, not eaten, and rami (elements supporting Walbeek et al., 1969) and natural so OTA formation is not a hazard. phialides) on the main stalk cells. occurrence confirmed (Krogh et al.,

12 CHAPTER 1 CHAPTER Fig. 1.6. Penicillium verrucosum (a) colonies on CYA (left) and MEA (right), 7 days, mycelium white; conidial production 25 °C; (b, c, d) penicilli, bars = 10 µm; (e) conidia, bar = 5 µm. Source: Pitt and moderate, coloured as on CYA; Hocking (2009), Fig. 7.48, p. 260; reproduced with kind permission from Springer reverse dull brown or olive. No growth Science+Business Media B.V. at 37 °C. Structures bearing conidia 200–500 μm long, with walls finely to conspicuously roughened; fruiting structures variable, with two or three supporting cells beneath phialides; conidia usually spherical, 2.5–3.0 μm in diameter, with smooth walls. Distinctive features. Penicillium verrucosum is characterized by slow growth on CYA and especially on MEA, by conidia coloured relatively bright green, by the absence of other conspicuous pigmentation, and by rough walls on the stalk cells (Pitt, 2000). It is similar in general appearance to P. viridicatum and P. solitum. P. verrucosum and P. viridicatum produce a distinctive violet brown reverse on DRYS (Frisvad, 1983). It should be noted that recognition of this species requires specialist knowledge, or detailed comparison with known cultures (of this and other related species). Only one other species of Penicillium is known to produce ochratoxin A: P. nordicum. This species was segregated from P. verrucosum by small physiological differences (it produces a yellow 1973). The view that P. viridicatum was Enumeration. The media specified reverse on DRYS; Larsen et al., a major source of OTA contamination above for general enumeration of 2001), but it is ecologically distinct, in foods and feeds in some parts of Penicillium species are effective for occurring mainly on meat and the world was accepted for more P. verrucosum. On dichloran rose cheese. Its significance in terms of than a decade. The species involved bengal yeast extract sucrose agar human health is unknown. was later correctly identified as P. (DRYS), a selective medium for the Factors influencing growth. P. ver- verrucosum (Pitt, 1987), and this was enumeration of P. verrucosum and P. rucosum grows from 0 °C to 31 °C, with confirmed (Frisvad, 1989; Frisvad viridicatum, P. verrucosum produces the optimum at 20 °C. The minimum and Filtenborg, 1989). P. viridicatum a violet brown reverse colouration aw for growth is about 0.80 (Pitt and does produce mycotoxins, but these (Frisvad, 1983). Isolation and identi- Hocking, 2009). Growth occurs have only rarely been implicated in fication of P. verrucosum in pure over the pH range 2.1–10.0 at least animal health. culture is essential for confirmation. (Wheeler et al., 1991). The ability of Penicillium verrucosum, and the Penicillium verrucosum Dierckx. P. verrucosum to produce significant closely related P. nordicum, are the only See Fig. 1.6. Colonies on CYA 15– levels of OTA at 4 °C and aw as low Penicillium species that produce OTA. 25 mm in diameter; mycelium white; as 0.86 is noteworthy (Northolt et al., P. verrucosum commonly occurs in conidial formation light to moderate, 1979). The physiology of P. nordicum cereals in temperate climates, whereas grey green to dull green; reverse is likely to be very similar. P. nordicum has been isolated, yellow brown to deep brown. Colonies uncommonly, from processed meats. on MEA 12–15(–20) mm in diameter;

Chapter 1. Fungi producing significant mycotoxins 13 Commodities and foods at risk. 6. Genus Fusarium Taxonomy. The signature micro- The major food habitat for P. morphological characteristics of Fu- verrucosum is cereal crops grown Fusarium is one of the most important sarium species are uncoloured, in cool temperate climates, ranging genera of plant pathogenic fungi, with multiseptate, large (25 µm to 50 across northern and central Europe a record of devastating infections µm or more) curved conidia called and Canada. The occurrence of this in many kinds of economically macroconidia, which are produced species in European cereals has important plants. Fusarium species from phialides. Most species produce two consequences: OTA is present are responsible for wilts, blights, macroconidia in cushion-like structures in many kinds of European cereal root rots, and cankers in legumes, called sporodochia. In some species, products, especially bread and flour- coffee, wheat, maize, carnations, macroconidia are sparsely formed based foods, and in animals that eat pine trees, and grasses. The in Petri dish culture and recognition cereals as a major dietary component. importance of Fusarium species in of these species as belonging to OTA was detected in Danish pig the current context is that infection Fusarium requires experience. meats nearly 40 years ago (Krogh may sometimes occur in developing In addition to macroconidia, some et al., 1973), and its implications seeds, especially in cereals, and also Fusarium species can make one or for human and animal health were in maturing fruits and vegetables. two kinds of smaller, one- or two- recognized at the same time. As An immediate potential for toxin celled conidia called microconidia. bread and other cereal products and production in foods is apparent. Microconidia are usually produced in pig meats are major components The very important role of the aerial mycelium in culture. Most of the European diet, the further Fusarium species as mycotoxin often, microconidia are produced in consequence is that most Europeans producers appears to have remained slimy heads, but some species produce who have been tested have shown largely unsuspected until the them in chains or singly. Microconidia appreciable concentrations of OTA in 1970s. Research has now strongly are also produced from phialides, their blood (WHO, 2007). associated alimentary toxic aleukia which may have a single spore- Recent information indicates that (ATA) with Fusarium species. An bearing opening (monophialides) or P. verrucosum is not a commensal epidemic of this human mycotoxicosis multiple openings (polyphialides). on cereal crops, i.e. it does not grow in the USSR killed at least 100 000 The taxonomy of Fusarium has in grain crops before harvest, but people between 1942 and 1948 been difficult, with several competing its presence is due to post-harvest (Joffe, 1978). ATA outbreaks are taxonomic schemes, recognizing from contamination. The primary sources also known to have occurred in the 9 to 60 species in the genus. The of infection of the grain appear to Russian Federation in 1932 and taxonomy of Nelson et al. (1983), which be from harvesting, processing, 1913, and there is little doubt that accepted 30 species, has met with and storage equipment (Magan and outbreaks occurred in earlier years widespread approval, and is still widely Olsen, 2004; Olsen et al., 2004). as well (Joffe, 1978). Matossian used in conjunction with the laboratory A maximum growth temperature (1981, 1989) has argued persuasively manual of Leslie and Summerell near 30 °C restricts Penicillium that ATA outbreaks occurred in other (2006). Recent molecular studies verrucosum geographically. It ap- countries, including England, in the have suggested that Nelson et al. pears to be uncommon, indeed 16th to 18th centuries at least. (1983) greatly underestimated species almost unknown, in warm temperate Research since 1970 has shown numbers in Fusarium. For example, or tropical climates, or in other kinds that Fusarium species are capable O’Donnell et al. (1998) recognized of foods. P. verrucosum is never a of producing a bewildering array of 36 phylogenetic species (species source of OTA in foods from warmer mycotoxins. Foremost among these recognizably different by molecular climates, such as coffee, wines, or are the trichothecenes, of which at techniques) in a grouping corresponding other grape products. least 50 are known; the majority are to Fusarium section Liseola in which Penicillium nordicum has been produced by Fusarium. The most four species had been recognized by isolated quite commonly from meats, notorious trichothecene is T-2 toxin, Nelson et al. (1983). Nirenberg and especially refrigerated products. which was linked to ATA. Of no less O’Donnell (1998) described 10 new importance in modern times are the species in this section. However, for fumonisins, which are especially practical identification of the species toxic to horses, and are suspected important for mycotoxin production, the to be responsible for chronic human manual of Leslie and Summerell (2006) diseases also. is recommended.

14 CHAPTER 1 CHAPTER Table 1.1. Media of value for isolation and enumeration of Fusarium species advocated the use of PDA made from old potatoes, rather than commercial Medium Advantages Disadvantages Reference formulations, but many laboratories use commercial PDA with satisfactory Pentachloronitrobenzene Often used Carcinogenic; no spore Snyder and results. Oatmeal agar is used in some (PCNB) agar production by Fusarium Hansen species (1940) laboratories. The most commonly Dichloran chlorampheni- Sporulation allows Little pigmentation for Andrews used weak media are carnation col peptone agar recognition of differentiating Fusarium and Pitt leaf agar (CLA) and Synthetischer (DCPA) Fusarium species species (1986) Nährstoffarmer Agar (often now Czapek–Dox iprodione Pigmentation helpful Sterile colonies do Abildgren called synthetic nutrient agar [SNA]). dichloran agar in distinguishing not permit ready et al. (CZID) Fusarium species identification of (1987) SNA has the advantage of being a Fusarium species defined medium, but CLA supports Dichloran 18% glycerol Sporulation allows Low-aw medium, not Hocking superior sporodochial production agar (DG18) recognition of ideal for Fusarium and Pitt Fusarium species growth (1980) in some species. However, use of CLA requires access to a source of gamma-irradiated carnation leaves. A direct consequence of confusion carcinogen. DCPA is to be preferred Banana leaf agar (autoclave-sterilized in taxonomy has been confusion over as it contains pentachloronitroaniline banana leaves on half-strength corn- species–mycotoxin associations. Fu- (dichloran), a molecule with similar meal agar) is also a good medium for sarium isolates producing a particular properties to PCNB but that is not stimulation of sporulation, but is not yet toxin have often been given different carcinogenic. widely used. To maintain uniformity for names. However, Desjardins (2006) Recognition of Fusarium col- descriptions of all foodborne fungi, Pitt has provided a comprehensive onies on these media requires and Hocking (2009) provided Fusarium clarification of the important myco- careful observation and experience. descriptions on PDA, together with toxigenic species and the mycotoxins Presumptive identification to genus media used in Aspergillus and they each produce. The species judged level can usually be made from colony Penicillium identification. Some readily to be most important from the viewpoint appearance: low to floccose colonies, prepared media of value in Fusarium of human health are discussed here. coloured white, pink, or purple, with identification are given in Table 1.1. Enumeration and isolation. Growth pale to red or purple reverses, are Opinions differ regarding the of Fusarium species is favoured by indicative of Fusarium. Confirmation necessity to make a single conidium dilute media of high aw. Enumeration requires microscopic examination, isolate of Fusarium species before of Fusaria can be effectively carried in which the crescent-shaped mac- identification. Some laboratories out on media such as PDA provided roconidia characteristic of the genus make a new single conidium isolate chloramphenicol or other broad- should be observed. However, at every transfer of the culture. This spectrum antibiotics are added to these are not always produced on ensures a highly reproducible growth suppress bacteria. However, acidified enumeration media, especially PDA. rate. Other laboratories prefer not to PDA, a frequently used antibacterial Differentiation of some species on make single spore cultures because medium, is not recommended enumeration media is possible, but this may decrease the vigour of the because it may inhibit sensitive cells. also requires experience. culture. Because Fusarium species DCPA (Andrews and Pitt, 1986) and Identification. All contemporary often grow in mixed colonies when Czapek–Dox iprodione dichloran agar identification schemes based primarily isolated from soil or plant material, (CZID) (Thrane, 1996) are effective on morphology use two media: a weak at least one generation of single enumeration and isolation media for medium for stimulation of sporulation conidium isolates is advisable for most foodborne Fusarium species. and a richer medium for measuring cultures intended for experimental In addition, half-strength PDA is growth rates and for stimulation of use, especially genetic studies. used by many laboratories isolating diagnostic pigment production. The Traditionally, Fusarium cultures directly from plant tissue, where the most commonly used rich medium is have been cultivated for 7–10 days number of unwanted fungi is much PDA. Potato sucrose agar (PSA) was under mixed fluorescent/near UV lower than in soil or plant debris. It used in the manual of Booth (1971) light at room temperature, near 25 °C. should be pointed out that, although and is still used in some laboratories Recently, particularly for the group pentachloronitrobenzene (PCNB) agar instead of, or in addition to, PDA. of species producing fumonisin, is still widely used, PCNB is a known Nelson et al. (1983) among others the importance of also growing the

Chapter 1. Fungi producing significant mycotoxins 15 same cultures in darkness to allow Fig. 1.7. Fusarium verticillioides (a) colonies on PDA (left) and DCPA (right), 7 days, the development of some diagnostic 25 °C; (b) phialides bearing chains of microconidia, bar = 50 µm; (c) phialides, features has been emphasized. bar = 10 µm; (d) macroconidia and microconidia, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.36, p. 120; reproduced with kind permission from Springer The only definitive taxonomy Science+Business Media B.V for Fusarium is that of Nelson et al. (1983). The most useful and up-to- date guide to important species is that provided by Leslie and Summerell (2006). Pitt and Hocking (2009) and Samson et al. (2010) provide keys and descriptions for common foodborne species. The descriptions of species given here have been provided by Dr K.A. Seifert (Agriculture and Agri-Food Canada, Ottawa) and are based on the protocols and media described above.

6.1 Fumonisin production by F. verticillioides and F. proliferatum

Fusarium verticillioides (Sacc.) Niren- berg. See Fig. 1.7. Colonies on rich media (PDA or PSA) at 25 °C grow moderately rapidly, 3.5–5.5 cm in diameter in 4 days. Abundant aerial mycelium is produced, and the reverse usually has rays or large patches of violet or purple. On weak media (CLA or SNA) at 25 °C, sporodochia are The teleomorph of F. verticillioides name F. moniliforme is predated by F. sparsely produced or not present in is Gibberella moniliformis. verticillioides and also cannot reliably most isolates. When present they are Distinctive features. Fusarium be linked to modern species concepts. inconspicuous and almost colourless, verticillioides is recognized by the Fusarium thapsinum (teleomorph: on the agar surface beneath the often combination of purplish colours on Gibberella thapsina) is a closely related dense aerial mycelium. Macroconid- PDA, and the production of long, species, found primarily on sorghum. It ia are usually 3–5 septate, mostly curly chains of microconidia from has similar micromorphology, but PDA 30–45 µm long, straight or variably monophialides in the aerial mycelium. colonies lack purplish pigmentation curved, with more or less parallel walls Although macroconidia are found and tend to be yellow to yellow brown. and with the widest point near the in some cultures, many strains do Both F. verticillioides and F. thapsinum middle. Microconidia are abundantly not produce them. Therefore, an produce longer microconidial chains produced in the aerial mycelium on experienced eye can be necessary to and lack the polyphialides that highly branched conidiophores. The recognize those strains as belonging to characterize F. proliferatum. conidiogenous cells are monophialides Fusarium. The teleomorph, Gibberella Factors influencing growth. The that often collapse before the cultures moniliformis, is produced in culture maximum temperature for growth are about 10 days old. Microconidia only when strains of opposite mating of Fusarium verticillioides has been are produced in long, dry chains type are crossed under appropriate reported as 32–37 °C, the minimum visible with the stereomicroscope; conditions. as 2.5–5 °C, and the optimum near these chains, which are often coiled, Until recently, Fusarium verticil- 25 °C. The minimum aw for growth is give the colonies a distinctive texture lioides was known as F. moniliforme, about 0.87 (Pitt and Hocking, 2009). similar to curly hair. Individual conidia a name that is no longer used on Values of these parameters for F. are ellipsoidal, 0–1 septate, 4–19 × the recommendation of an expert proliferatum are essentially identical. 1.5–4.5 µm. committee (Seifert et al., 2003). The

16 CHAPTER 1 CHAPTER Fig. 1.8. Fusarium proliferatum (a) colonies on PDA (left) and DCPA (right), 7 days, proliferatum. Although macroconidia 25 °C; (b) phialides bearing microconidia in chains and false heads in situ, bar = 50 µm; are found in some cultures, many (c) macroconidia and microconidia, bar = 10 µm; (d) polyphialides, bar = 10 µm; strains do not produce them. (e) monophialides, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.31, p. 111; reproduced with kind permission from Springer Science+Business Media B.V. Therefore, an experienced eye can be necessary to recognize those strains as belonging to Fusarium. The teleomorph, Gibberella intermedia, is produced in culture only when strains of opposite mating type are crossed under appropriate conditions. Fusarium proliferatum is distin- guished from F. verticillioides by the shorter chains of microconidia and the occurrence of polyphialides in the aerial mycelium. Another frequently isolated species that occupies the same ecological niche is F. subglutinans, which does not produce fumonisins. It produces slimy heads rather than chains of microconidia from a mixture of monophialides and polyphialides; strains of this species are more likely to produce macroconidia than either F. verticillioides or F. subglutinans. F. nygamai is a similar species that also produces fumonisin in some strains; it can be distinguished from F. proliferatum by the production of chlamydospores. Other species making fumonisins. Several other Fusarium species are known to produce fumonisins, including F. anthophilum, F. beo- miforme, F. dlamini, F. globosum, Fusarium proliferatum (Matsush.) near the middle. Microconidia are F. napiforme, F. nygamai, F. Nirenberg. See Fig. 1.8. Colonies on abundantly produced in the aerial oxysporum, F. polyphialidicum, and rich media (PDA or PSA) at 25 °C mycelium on divergently branched F. subglutinans (IARC, 2002). None grow moderately rapidly, 3.5–5.5 cm in conidiophores. The cells producing is of major importance in fumonisin diameter in 4 days. There is abundant conidia are predominantly mono- production in foods. white to pink or slightly orange aerial phialides, but up to 20% may be Commodities and foods at risk. mycelium, and the reverse usually has polyphialides. Microconidia are pro- Fusarium verticillioides and F. rays or large patches of violet or purple. duced in short, dry chains visible with proliferatum, the major sources of On weak media (CLA or SNA) at 25 °C, the stereomicroscope, ovoid to ellip- fumonisins, are the most common sporodochia are usually sparsely soidal, 0–1 septate, 7–12.5 × 2–3 µm. fungi associated with maize. F. produced and often are not present. The teleomorph of F. proliferatum verticillioides has been known for When present they are inconspicuous is Gibberella intermedia. many years to occur systemically in and almost colourless, on the agar Distinctive features. The production leaves, stems, roots, and kernels of surface beneath the often dense aerial of purplish pigments on PDA, and the maize (Foley, 1962). These fungi can mycelium. Macroconidia are usually occurrence of short, dry chains of be recovered from virtually all maize 3–5 septate, 30–45 µm long, straight microconidia in the aerial mycelium kernels worldwide, including those or variably curved, with more or less and the sometimes sparse occurrence that are healthy (e.g. Hesseltine parallel walls and with the widest point of polyphialides characterize F. et al., 1981; Pitt et al., 1993; Miller,

Chapter 1. Fungi producing significant mycotoxins 17 1994; Miller et al., 1995; Ramirez European locations and results in the Canada, counties with the highest and et al., 1996; Logrieco et al., 2002). accumulation of moniliformin (Logrieco lowest average concentration after the F. verticillioides has been reported et al., 2002). From limited data, 1993 harvest were 1.4 and 0.4 times to suppress the growth of other ear moniliformin is not commonly found in the state average, respectively. The fungi (Reid et al., 1999), and kernels United States (Gutema et al., 2000) or average temperatures in the counties heat-treated to destroy the fungus Canadian maize (Farber et al., 1988). were similar, at 104% and 107% of germinate but do not thrive (Foley, After genotype susceptibility, the 30-year average, respectively. 1962). Strains of F. verticillioides temperature is the primary determining However, rainfall in the county with isolated from maize have the factor for maize diseases caused the highest fumonisin B1 level was potential to produce fumonisins, by Fusarium species (Miller, 1994; only 49% of normal, whereas in the even for maize from regions where Munkvold, 2003). F. graminearum county with the lowest level, it was fumonisin accumulations in maize are has a very narrow temperature 95% of normal (Miller et al., 1995). historically uncommon. These include window for growth in maize. The After experimental inoculation of Africa, Asia, Europe, North America optimal temperature is between 26 °C 14 maize genotypes in Poland, the (Canada, the USA, and Mexico), and and 28 °C. Its growth rate at 24 °C is average temperature in the year with

South America (WHO, 2000). one quarter that at 26–28 °C, while at the highest fumonisin B1 accumulation Fusarium kernel rot in maize 30 °C it is about one half. In contrast, was 117% of the 30-year average; that due to the growth of F. verticillioides F. verticillioides grows well above 26 °C in the year with the lowest fumonisin and related species causes the (Miller, 2001; Munkvold, 2003). B1 accumulation was 102% of normal. formation of fumonisins, whereas In culture, fumonisin B1 is pro- Rainfall in the year with the highest

Gibberella ear rot or pink ear rot, duced under conditions known to fumonisin B1 accumulation was 6% of mainly caused by F. graminearum, is favour the production of polyketides normal and in the year with the lowest, associated with deoxynivalenol and and sesquiterpenes. The toxin is 65% of normal (Pascale et al., 1997). zearalenone production (see Section optimally produced in media that A study of fumonisin occurrence

6.3). These fungi grow under different have moderate aw and are nitrogen- in hybrids grown in the USA indicated environmental conditions but with limited. Fumonisin is produced under that fumonisins are produced in higher overlap. F. proliferatum, which causes relatively high oxygen tensions concentrations in hybrids grown kernel rot, concurrently produces but apparently has an unusual outside their area of adaptation. fumonisin and moniliformin. Slightly requirement for low pH (about 2) Fumonisin concentrations were in- different environmental conditions for optimal production (Miller et al., versely proportional to June rainfall appear to favour one or the other of 1995); such conditions arise only (Shelby et al., 1994), again suggesting the principal species that produce when the plant is dead or dying. the important role of drought stress fumonisins. For these reasons, it is Fusarium kernel rot is associated (Munkvold, 2003). Data from samples sensible to consider the two diseases with warm, dry years and insect collected in Africa, Italy, and Croatia Fusarium kernel rot and pink ear damage and is caused by F. subglu- also indicate fumonisin accumulation rot together. In North America and tinans (teleomorph: Gibberella subgluti- in lines grown outside their area of Europe, fumonisins can occur in maize nans), F. verticillioides (teleomorph: adaptation, which includes tolerance crops in some seasons, but in others, Gibberella moniliformis), and F. to moisture stress (Doko et al., 1995; deoxynivalenol can occur, sometimes proliferatum (Logrieco et al., 2002; Visconti, 1996). accompanied by zearalenone. In Munkvold, 2003). In warmer parts of Hybrids with an increased other regions, such as Africa or the USA and in the lowland tropics, likelihood of kernel splitting show South-East Asia, all three toxins F. verticillioides is one of the most higher levels of Fusarium kernel can be seen together (Yamashita important ear diseases (De Leon and rot, and kernel splitting is generally et al., 1995; Doko et al., 1996; Ali Pandey, 1989). worse under drought conditions. et al., 1998). These differences in Studies of the occurrence of Drought stress also results in greater mycotoxin occurrence have important fumonisin from natural occurrence insect herbivory on maize; hence toxicological implications. and experimental infections clearly it is not possible to totally separate Fusarium subglutinans is also demonstrate the importance of drought these variables. Further, in studies of common in maize kernels in North stress and insect damage at the same experimental inoculation methods, the America (Miller, 1994; Munkvold, time as temperatures are favourable. severity of Gibberella ear rot is related

2003), but this species apparently The fumonisin B1 concentrations to wound size (Drepper and Renfro, causes a higher level of ear rot in some found in maize from the two Ontario, 1990). It was observed early on that

18 CHAPTER 1 CHAPTER a strong relationship exists between Factors that control insects, confer fumonisins produced by A. niger can be insect damage and Gibberella ear rot resistance to other ear diseases, and expected to be widespread if more than (Lew et al., 1991). Transgenic Bt maize adaptations, including drought and a few isolates are producers. Recent genotypes, which contain a gene from temperature tolerance, are important information indicates that fumonisin the soil bacterium Bacillus thuringiensis in reducing the risk of fumonisin production by A. niger is indeed that results in the accumulation of accumulations in maize. common. In one study of A. niger proteins toxic to key insect pests of Fumonisins are very uncommon in strains from a sample of Californian maize, had lowered levels of recovery commodities or foods other than maize raisins, 50 of 66 strains (77%) produced of F. verticillioides and fumonisin and maize products (IARC, 2002). fumonisins (Mogensen et al., 2010b). (Munkvold et al., 1997, 1999; Bakan In a second study, where isolates were et al., 2002; Munkvold, 2003). Under 6.2 Fumonisin production taken from 13 samples of dried vine conditions of high disease pressure, by Aspergillus niger and fruits from several countries, 20 of 30 the Bt hybrids can make the difference Alternaria arborescens (67%) produced fumonisins (Varga et between a crop being fit or unfit for al., 2010). Of an unstated number of human consumption (Hammond It has been known for 20 years that isolates from Thai coffee, 67% were et al., 2004; De La Campa et al., one particular race of Alternaria able to produce fumonisins (Noonim 2004). In an examination of fumonisin alternata, described as Alternaria et al., 2009). A. niger isolates do not concentration in relation to various alternata f. sp. lycopersici, produces usually produce fumonisin B1; the major climate variables under moderate fumonisins (Chen et al., 1992). This metabolite is fumonisin B2, sometimes insect pressure, most of the variation particular race is a host-specific with lower amounts of fumonisin B4. was explained by temperatures above pathogen that causes a stem canker So far, only a few studies have 30 °C (about 40%), followed by insect disease on tomato plants. For that examined food products that are pressure (about 20%) and hybrid reason, fumonisin production by this frequently infected by A. niger for the (about 10%; De La Campa et al., 2004). species has been largely ignored in presence of fumonisins. Low levels Maize infected by other patho- general discussions of fumonisins (1–9.7 µg/kg) were found in 7 of 12 gens that damage ears (such as F. in foods and feeds. This taxon has coffee samples (Noonim et al., 2009). graminearum) may be predisposed been re-identified as Alt. arborescens Levels of up to 7.8 mg/kg were found to F. verticillioides damage and fumo- (Frisvad et al., 2007). in inoculated dried fruits (Mogensen nisin accumulation. Ears inoculated Recently, however, the picture et al., 2010b). More alarming, seven with F. graminearum, F. verticillioides, has become more complicated. commercial dried fruit samples posi- and F. subglutinans by wounding Studies on the genome sequence tive for A. niger were all positive for produced visible symptoms on a 1–9 of Aspergillus niger showed, totally fumonisins B1 to B4: the average total scale of 7.3, 4.4, and 4.7, respectively. unexpectedly, the presence of the fumonisin level was 7.2 mg/kg, with the Despite the fact that F. graminearum genes for fumonisin (Baker, 2006), range 4.6–35.5 mg/kg (Varga et al., and F. subglutinans do not produce and this was independently verified by 2010). It is not surprising that wines can fumonisin, ears inoculated with Pel et al. (2007). It was soon confirmed also contain fumonisins. Of 51 market these fungi contained 42 µg/g and that this gene cluster (consisting of at samples of Italian wines, 9 (18%)

3 µg/g fumonisin B1, respectively least 15 genes) was active and that contained fumonisin B2, with levels (Schaafsma et al., 1993). at least some strains of A. niger can ranging from 0.4 µg/L to 2.4 µg/L Breeding for resistance to indeed produce fumonisins (Frisvad (Logrieco et al., 2010). Seventy-seven Fusarium kernel rot has not been et al., 2007). wine samples from 13 countries effective. Within areas of adaptation, The implications are vast. As were examined by Mogensen et al. there are apparent differences in described in Section 4.2.3, A. niger is (2010a), and 18 (23%) were found to symptom response (Miller, 2001; a very common fungus, of which a few contain fumonisins, with a range of Munkvold, 2003). In a large trial at strains produce ochratoxin A. Foods 1 µg/L to 25 µg/L. These levels are the International Maize and Wheat in which OTA is found, produced by A. low, but wine has a high consumption Improvement Center, in Mexico, niger and the closely related species rate in some areas. slight improvements in symptom A. carbonarius, include grapes, dried It has been found that fumonisin expression in some tropical maize vine fruits, wines, and coffee. A. niger production in culture by A. niger genotypes were observed after is also common in some fresh fruits, is enhanced at a slightly reduced many cycles of selection (De Leon particularly berries, and on onions aw, about 0.99, by the addition of and Pandey, 1989). (Pitt and Hocking, 2009). Therefore, 5% NaCl to CYA (Mogensen et

Chapter 1. Fungi producing significant mycotoxins 19 al., 2010c). This additive reduced Fig. 1.9. Fusarium graminearum (a) colonies on PDA (left) and DCPA (right), 7 days, fumonisin production by Fusarium 25 °C; (b) Gibberella zeae perithecium and ascospores, bar = 25 µm; (c) macroconidia, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.27, p. 103; reproduced with kind species. Fumonisin was produced permission from Springer Science+Business Media B.V. optimally by A. niger at 25–30 °C, whereas optimal temperatures for production were lower in Fusarium species (20–25 °C). Clearly, some evolution has occurred since the genes were transferred to A. niger (Mogensen et al., 2010c). At this time, it is difficult to assess the relative importance of fumonisin production by A. niger, but the range of commodities in which fumonisins are found has been extended considerably. However, given the very high consumption of maize in some countries, production by A. niger will have lesser importance.

6.3 Deoxynivalenol, nivalenol, and zearalenone production by Fusarium graminearum and related species

The most important Fusarium spe- cies that produce the trichothecenes deoxynivalenol (DON) and (less commonly) nivalenol (NIV) in small grains are F. graminearum, F. Fusarium graminearum Schwabe. Sexual fruiting structures (peri- culmorum, and, less frequently, F. See Fig. 1.9. Colonies on rich thecia) are often produced on CLA crookwellense. media (PDA or PSA) at 25 °C grow or SNA as the medium begins to The name Fusarium roseum rapidly, 7.5–9 cm in diameter in desiccate, almost black, about as used by Snyder and Hansen 4 days. There is abundant white, 200 µm in diameter, with a warty (1940) has caused a great deal of reddish or yellowish brown aerial wall, and exuding a light orange confusion in the literature as their mycelium, and the reverse is usually cloud of ascospores. Ascospores very broad species concept included distinctly red. About 5% of isolates are generally fusiform to allantoid several well-known and important have an orange brown reverse. On (slightly bean-shaped), light brown, Fusarium species, including F. weak media (CLA or SNA) at 25 °C, and 20–30 µm long. Ascospores graminearum. The literature before almost colourless sporodochia are are forcibly discharged and can be the mid-1980s is therefore unreliable, produced under a sparse layer of found on the agar surface away from both taxonomically and with respect white aerial mycelium. Structures the perithecia, or on Petri dish lids. to mycotoxin production. Since the bearing conidia have 1–2 levels The teleomorph of F. graminearum is publications by Nelson et al. (1983) and of branching, terminating with Gibberella zeae (Schw.) Petch. Marasas et al. (1984), F. graminearum 1–4 phialides. Macroconidia are Distinctive features. Fusarium and F. culmorum have become well usually 4–6 septate and abundantly graminearum produces straight mac- established and species–mycotoxin produced in fresh isolates, mostly roconidia with parallel walls, which in relationships have become clear. 40–60 µm long, more or less straight combination with rapid growth and F. crookwellense is a widespread with parallel walls. No microconidia usually red pigments on PDA, are species first described in 1982. are produced, although sometimes relatively distinctive. The frequent immature macroconidia are seen and occurrence of perithecia of the could be confused with microconidia. teleomorph in culture is also a

20 CHAPTER 1 CHAPTER reliable characteristic, occurring in is abundant aerial mycelium, white but F. graminearum appears to be about 90% of fresh isolates under to pink, and the reverse is usually displacing it (Waalwijk et al., 2003). appropriate lighting conditions. F. distinctly red. On weak media (CLA Concerning the other species pseudograminearum (teleomorph: or SNA) at 25 °C, sporodochia are involved in Fusarium head blight, Gibberella coronicola) is an ecologi- usually abundant and distinctly F. avenaceum is also common in cally and phylogenetically distinct reddish brown. Structures bearing wheat from all regions studied. F. species that causes crown rot of wheat. conidia, arising from the sporodochia, crookwellense is relatively common The macroconidia are similar to those have 1–4 levels of branching, in Australia and South Africa, but of F. graminearum, but are reportedly terminating with 1–6 phialides. is rare in wheat from Canada and broadest above the middle; the growth Macroconidia are usually 5 septate, the USA. F. poae, F. langsethii, F. rate on PDA is slower. Diagnostic PCR 35–60 × 4.5–6.5 µm, fairly uniform equiseti, and F. sporotrichioides are primers for F. pseudograminearum in shape, with a more or less straight also isolated from wheat kernels at were designed by Aoki and O’Donnell ventral wall and curved dorsal wall, low to moderate frequencies, more (1999) based on β-tubulin sequences. with the widest point near the middle, commonly under cooler conditions This species also produces DON and of medium size. Microconidia are (Bottalico and Peronne, 2002). zearalenone (ZEA). not produced, although sometimes The distribution of head blight Fusarium culmorum (W. G. Sm.) immature macroconidia give this species is affected by pathogenicity, Sacc. Colonies on rich media (PDA impression. with a relative pathogenicity of F. or PSA) at 25 °C grow rapidly, 7.5– Distinctive features. Distinctive graminearum > F. culmorum >> F. 9 cm in diameter in 4 days. There is macroconidia, with straight inner walls crookwellense > F. avenaceum. The abundant white to slightly orange, and curved outer walls, characterize regional and annual variation of the brown or reddish aerial mycelium, Fusarium crookwellense. pathogenic species is most affected and the reverse is usually distinctly Factors affecting growth. The op- by temperature; species ranked red. About 5% of isolates have timal temperature for growth of from coldest to warmest areas are an orange brown or tan reverse. Fusarium graminearum is 25 °C, F. culmorum > F. crookwellense > F. On weak media (CLA or SNA) at and the maximum below 37 °C. avenaceum > F. graminearum (Miller,

25 °C, reddish brown or orange The minimum aw for growth is near 1994; Bottalico and Peronne, 2002). sporodochia are produced under a 0.90 (Pitt and Hocking, 2009). F. Isolates of F. graminearum and sparse layer of white aerial mycelium. culmorum is a psychrotroph, growing F. culmorum produce a fairly large Structures bearing conidia have up down to 0 °C but up to only 31 °C (Pitt number of other compounds as well to 4 levels of branching, terminating and Hocking, 2009). as DON and ZEA. Isolates from with 1–4 phialides. Macroconidia are Trichothecenes in small grains: North and South America produce usually 3–6 septate and abundantly Fusarium graminearum and related 15-acetyl deoxynivalenol, the precur- produced in fresh isolates, mostly species. Fusarium head blight is an sor to DON. If isolates from Asia 30–45 µm long, with the widest important plant disease in temperate and Europe produce DON or NIV, point above the middle and hence regions that affects small grains, they also produce the respective somewhat wedge-shaped, often mainly wheat, but also barley and 3-acetate, i.e. deoxynivalenol mono- appearing short and fat. No micro- triticale. Five or six Fusarium species acetate or fusarenon-X. Strains of F. conidia are produced. are consistently isolated from small crookwellense produce NIV regardless Distinctive features. Broad, grains affected by this disease, and of geographical origin (Miller et al., wedge-shaped macroconidia with the most pathogenic species, F. 1991; Bottalico and Peronne, 2002, short apical cells and basal cells graminearum and F. culmorum, are under the name F. cerealis). Some are distinctive for F. culmorum. The the most common. These two species of the minor metabolites are found in related species F. sambucinum are closely related and produce DON small grains along with DON. produces narrower macroconidia or NIV and ZEA, depending on the Although increased rainfall than F. culmorum. The wedge geographical origin of the isolate promotes Fusarium head blight, shape of the conidia distinguishes F. (Miller et al., 1991; Waalwijk et al., incidence is most affected by moisture culmorum from F. crookwellense. 2003). F. graminearum is common at anthesis as long as the temperature Fusarium crookwellense Burgess in wheat from North America, remains in the favourable range et al. Colonies on rich media (PDA or South America, and China (Miller, for growth (Miller, 1994). Cultivar PSA) at 25 °C grow rapidly, 7.5– 1994). In cooler parts of Europe, susceptibility and rainfall at anthesis 9 cm in diameter in 4 days. There F. culmorum has been dominant, explain most variability in infection,

Chapter 1. Fungi producing significant mycotoxins 21 but crop rotation also has a large to the membrane-damaging effects of poisonous to humans and animals, effect. Growing wheat following maize DON than susceptible cultivars (Miller which have also found major use in increases disease under favourable and Ewen, 1997). pharmaceuticals. weather conditions (Schaafsma et al., Many Claviceps species are 2002). Reduced tillage is an equivocal 7. Genus Claviceps restricted to a few grass genera source of variation in the amount of as hosts. However, the species disease observed (Miller et al., 1998; The following descriptions of Clav- most important from the mycotoxin Schaafsma et al., 2002). iceps species are taken from viewpoint, C. purpurea, has a host Fusarium graminearum is a Alderman et al. (1999). range of more than 200 grass species. necrotrophic pathogen, i.e. it invades The genus Claviceps, an Asco- It is distributed worldwide in temperate plants by killing host cells in advance. mycete with a conidial state, includes climates and is responsible for the This was reported by the earliest several species that are parasitic disease called ergotism in humans investigators (see Schroeder and on grasses, including cultivated and domestic animals. Christensen, 1963). Trichothecenes cereals throughout the temperate Identification. Species of Claviceps were recognized to be phytotoxic world. Claviceps species infect only cannot be grown in culture, so must be compounds at the time of their the flowers of susceptible hosts. identified by natural characteristics. C. discovery (Brian et al., 1961). It Infection involves replacement of the purpurea is a pathogen on grasses, was realized much later that large ovary by a specialized structure that but not on sorghum or maize, whereas differences exist in the responses develops into a sclerotium, a hard, C. africana, also of importance in of wheat cultivars to Fusarium head compact mass of fungal tissue. The terms of mycotoxins, is a cause of a blight. Coleoptile tissue of cultivars that sclerotia are usually white, black, or serious disease resulting in male- were resistant to Fusarium head blight tan and are 1–4 times as large as sterile sorghum seed. was 10 times as resistant to necrosis the seeds they replace. The sclerotia Claviceps purpurea (Fr.: Fr.) Tul. in the presence of DON than was that and diseases caused by Claviceps The sclerotium of Claviceps purpurea of disease-susceptible cultivars (Wang species go by the general name ergot. comprises a compact mass of fungal and Miller, 1988). This difference was Ergots formed by the most important tissue encased in a dark pigmented shown to be due to the presence species, C. purpurea, are dark purple outer rind. This overwintering stage of a modified peptidyl transferase to black, and are most prevalent on apparently requires 2 months of involved in protein synthesis (Miller rye, but also occur to some extent on cold weather (0–10 °C) to induce and Ewen, 1997) and to unknown barley, oats, and wheat, as well as germination. In warmer regions, functional changes in the membranes wild and cultivated grasses. sclerotia do not survive well. of more resistant types (Snijders and During ergot development, conidia In spring, the sclerotia germinate, Krechting, 1992; Cossette and Miller, are produced by the fungus and are producing stalked ascocarps, in 1995; Miller and Ewen, 1997). Earlier immersed in plant sap to produce a which small, thread-like ascospores studies had shown that cultivars of sugary liquid known as honeydew, are produced. The ascospores are wheat in the field appeared to be able which drips from the infected plant ejected forcibly from the ascocarp to metabolize DON, and this was later as large drops. These are attractive and are carried by air currents to shown to be the case in vitro in cultivars to insects, which act as vectors for grass flowers. resistant to head blight (Miller and dispersing the conidia and spreading The period of susceptibility for Arnison, 1986). Strains that produce infection throughout the crop. most grasses is very brief, from flower high concentrations of DON in the field Sclerotia are the resting stage of opening to fertilization, as fertilized were more virulent (Snijders, 1994; Claviceps species between seasons. ovaries are resistant to infection. Mesterhazy et al., 1999). This implied Under favourable conditions scle- Environmental conditions that delay that one component of resistance rotia germinate, producing the pollination, such as cool temperatures, to Fusarium head blight is related ascomycete stage of the fungus. increase the infective period. Male- to reducing the phytotoxic impact of Ascospores are formed in closed sterile lines of grasses are especially DON. In addition, DON has been bodies on stalks arising from the susceptible to Claviceps infection found to appear in wheat kernels in sclerotia, and in many species because they are not pollinated. advance of fungal mycelia (Snijders these provide the initial inoculum for Within a week after infection, and Perkowski, 1990; Snijders and infection of the next season’s crop. conidia are produced in abundance, Krechting, 1992). The wheat cultivar The sclerotia of many Claviceps present in the sticky honeydew that Frontana is substantially more resistant species contain toxic alkaloids, drips from the flowers. The honeydew

22 CHAPTER 1 CHAPTER acts as the main infective stage, being 8. Decision trees spread by insects, rain splash, or contact with uninfected flower heads. The major commodities susceptible Within about two weeks after to mycotoxin formation are sum- infection, sclerotia begin to appear. marized in Figs 1.10 and 1.11, Maturity of the sclerotia coincides together with the major fungal with maturity of the infected grass species involved. seed heads.

Fig. 1.10. Decision tree for directing risk management decisions or actions based on environmental considerations and probability of fungal contamination in warm climates. Expected toxic effects in susceptible animals are given for each group of mycotoxins.

Warm to hot humid conditions

Sorghum Small grains Maize Tree nuts Cottonseed Groundnuts

F. graminearum F. verticillioides A. flavus Claviceps spp. A. ochraceus? A. flavus F. culmorum F. proliferatum A. parasiticus

Ergot alkaloids Deoxynivalenol Fumonisins Ochratoxin A Aflatoxins and zearalenone

Fig. 1.11. Decision tree for directing risk management decisions or actions based on environmental considerations and probability of fungal contamination in cool climates. Expected toxic effects in susceptible animals are given for each group of mycotoxins.

Cool to warm temperate conditions

Grapes, Coffee, Small grains Maize Small grains Tree nuts and figs wine cocoa

F. culmorum F. verticillioides A. carbonarius A. ochraceus Claviceps spp. A. ochraceus? A. flavus F. graminearum F. proliferatum (A. niger) A. carbonarius

Ergot alkaloids Deoxynivalenol Fumonisins Ochratoxin A Aflatoxins and zearalenone

Chapter 1. Fungi producing significant mycotoxins 23 References

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26 Pascale M, Visconti A, Pronczuk M et al. Pitt JI, Hocking AD, Bhudhasamai K et al. Schaafsma AW, Hooker DC, Baute TS, CHAPTER 1 CHAPTER (1997). Accumulation of fumonisins in maize (1993). The normal mycoflora of commodities Illincic-Tamburic L (2002). Effect of Bt-corn hybrids inoculated under field conditions from Thailand. 1. Nuts and oilseeds. Int J Food hybrids on deoxynivalenol content in grain at with Fusarium moniliforme Sheldon. J Sci Microbiol, 20:211–226. doi:10.1016/0168- harvest. Plant Dis, 86:1123 –1126. doi:10.1094/ Food Agric, 74:1–6. doi:10.1002/(SICI)1097- 1605(93)90166-E PMID:8110599 PDIS.2002.86.10.1123 0010(199705)74:1<1::AID-JSFA752>3.0.CO;2-5 Pitt JI, Taniwaki MH, Teixiera AA, Iamanka BT Schaafsma AW, Miller JD, Savard ME, Ewing Payne GA (1983). Nature of field infection of corn (2001). Distribution of Aspergillus ochraceus, R (1993). Ear rot development and mycotoxin by Aspergillus flavus. In: Diener UL, Asquith A. niger and A. carbonarius in coffee in four production in corn in relation to inoculation RL, Dickens JW, eds. Aflatoxin and Aspergillus regions in Brazil. In: Proceedings of the 19th method and corn hybrid for three species of flavus in Corn. Auburn, AL: Department of International Scientific Colloquium on Coffee, Fusarium. Can J Plant Pathol, 15:185–192. Research Information, Alabama Agricultural Trieste, Italy, 14–18 May, 2001 [compact disc]. doi:10.1080/07060669309500821 Experiment Station, Auburn University Lausanne, Switzerland: International Coffee Scott PM, Lawrence JW, van Walbeek W (Southern Cooperative Series, Bulletin 279), pp. Science Association. (1970). Detection of mycotoxins by thin-layer 16–19. Pohland AE, Wood GE (1987). Occurrence of chromatography: application to screening of Pel HJ, de Winde JH, Archer DB et al. mycotoxins in food. In: Krogh P, ed. Mycotoxins fungal extracts. Appl Microbiol, 20:839–842. (2007). Genome sequencing and analysis in Food. London: Academic Press, pp. 35–64. PMID:5485087 of the versatile cell factory Aspergillus niger Ramirez ML, Pascale M, Chulze S et al. Sharman M, Patey AL, Bloomfield DA, Gilbert CBS 513.88. Nat Biotechnol, 25:221–231. (1996). Natural occurrence of fumonisins and J (1991). Surveillance and control of aflatoxin doi:10.1038/nbt1282 PMID:17259976 their correlation to Fusarium contamination in contamination of dried figs and fig paste im- Pitt JI (1979). The Genus Penicillium and commercial corn hybrids growth in Argentina. ported into the United Kingdom. Food Addit Its Teleomorphic States Eupenicillium and Mycopathologia, 135:29–34. doi:10.1007/ Contam, 8:299–304. doi:10.1080/026520391093 Talaromyces. London: Academic Press. BF00436572 PMID:20882450 73979 PMID:1778266

Pitt JI (1987). Penicillium viridicatum, Penicillium Raper KB, Fennell DI (1965). The Genus Schroeder HW, Christensen JJ (1963). Factors verrucosum, and production of ochratoxin Aspergillus. Baltimore, MD: Williams and affecting resistance of wheat to scab caused by A. Appl Environ Microbiol, 53:266–269. Wilkins. Gibberella zeae. Phytopathology, 53:831. PMID:3566267 Reid LM, Nicol RW, Ouellet T et al. (1999). Seifert KA, Aoki T, Baayen RP et al. (2003). The Pitt JI (1989). Field studies on Aspergillus Interaction of Fusarium graminearum and F. name Fusarium moniliforme should no longer flavus and aflatoxins in Australian groundnuts. moniliforme in maize ears: disease progress, be used. Mycol Res, 107:643–644. doi:10.1017/ In: McDonald D, Mehan VK, eds. Aflatoxin fungal biomass, and mycotoxin accumulation. S095375620323820X Phytopathology, 89:1028–1037. doi:10.1094/ Contamination of Groundnut: Proceedings of Shelby RA, White DG, Burke EM (1994). PHYTO.1999.89.11.1028 PMID:18944658 the International Workshop, 6–9 October l987, Differential fumonisin production in maize ICRISAT Center, India. Patancheru, India: Richer L, Sigalet D, Kneteman N et al. (1997). hybrids. Plant Dis, 78:582–584. doi:10.1094/ International Crops Research Institute for the Fulminant hepatic failure following ingestion PD-78-0582 Semi-Arid Tropics, pp. 223–235. of moldy homemade rhubarb wine (Abstract). Simpson ME, Batra LR (1984). Ecological Gastroenterology, 112:A1366. Pitt JI (1996). What are mycotoxins? Aust relations in respect to a boll rot of cotton caused Mycotoxin Newsletter, 7:1. Rodricks JV, Hesseltine CW, Mehlman ME, by Aspergillus flavus. In: Kurata H, Ueno Y, Pitt JI (2000). A Laboratory Guide to Common eds (1977). Mycotoxins in Human and Animal eds. Toxigenic Fungi: Their Toxins and Health Penicillium Species, 3rd ed. North Ryde, NSW: Health. Park Forest South, IL: Pathotox Hazard. Amsterdam: Elsevier, pp. 24–32. Publishers. 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Mycotoxin Contamination Snijders CHA, Krechting CF (1992). Inhibition in Grains. Canberra: Australian Centre for Samson RA, Hoekstra ES, Frisvad JC, of deoxynivalenol translocation and fungal International Agricultural Research, pp. 5–10. eds (2010). Food and Indoor Fungi. colonization in Fusarium head blight resistant wheat. Can J Bot, 70:1570–1576. doi:10.1139/ Pitt JI, Hocking AD (1997). Fungi and Food Utrecht, Netherlands: Centraalbureau voor b92-198 Spoilage, 2nd ed. London: Blackie Academic Schimmelcultures. and Professional. Sanders TH, Hill RA, Cole RH, Blankenship Snijders CHA, Perkowski J (1990). Effects of head blight caused by Fusarium culmorum Pitt JI, Hocking AD (2009). Fungi and Food PD (1981). Effect of drought on occurrence of on toxin content and weight of wheat kernels. Spoilage, 3rd ed. New York: Springer. Aspergillus flavus in maturing peanuts. J Am Oil Chem Soc, 58:966A–970A. doi:10.1007/ Phytopathology, 80:566–570. doi:10.1094/ Pitt JI, Miscamble BF (1995). Water relations of BF02679302 Phyto-80-566 Aspergillus flavus and closely related species. J Snowdon AL (1990). A Colour Atlas of Post- Food Prot, 58:86–90. Sanders TH, Blankenship PD, Cole RJ, Hill RA (1984). Effect of soil temperature and drought harvest Diseases and Disorders of Fruits and Pitt JI, Hocking AD, Glenn DR (1983). An on peanut pod and stem temperatures relative Vegetables. 1. General Introduction and Fruits. improved medium for the detection of Aspergillus to Aspergillus flavus invasion and aflatoxin London: Wolfe Scientific. flavus and A. parasiticus. J Appl Bacteriol, contamination. Mycopathologia, 86:51–54. Snowdon AL (1991). A Colour Atlas of Post- 54:109–114. doi:10.1111/j.1365 -2672.1983. doi:10.1007/BF00437229 PMID:6429541 harvest Diseases and Disorders of Fruits and tb01307.x PMID:6406419 Sanders TH, Cole RH, Blankenship PD, Hill RA Vegetables. 2. Vegetables. London: Wolfe Pitt JI, Dyer SK, McCammon S (1991). Systemic (1985). Relation of environmental stress duration Scientific. invasion of developing peanut plants by to Aspergillus flavus invasion and aflatoxin Aspergillus flavus. Lett Appl Microbiol, 13:16– production in preharvest peanuts. Peanut Sci., 20. doi:10.1111/j.1472-765X.1991.tb00558.x 12:90–93. doi:10.3146/pnut.12.2.0011

Chapter 1. Fungi producing significant mycotoxins 27 Snyder WC, Hansen HN (1940). The species Tsubouchi H, Yamamoto K, Hisada K, Sakabe Wang Y-Z, Miller JD (1988). Effects of concept in Fusarium. Am J Bot, 27:64 – 67. Y (1984). A survey of occurrence of mycotoxins Fusarium graminearum metabolites on wheat doi:10.2307/2436688 and toxigenic fungi in imported green coffee tissue in relation to Fusarium head blight beans. Proc Jpn Assoc Mycotox, 19:14–21. resistance. J Phytopathol, 122:118–125. Steiner WE, Rieker RH, Battaglia R (1988). doi:10.1111/j.1439- 0434.1988.tb00998.x Aflatoxin contamination in dried figs: distribution Ueno Y, Kawamura O, Sugiura Y et al. (1991). and association with fluorescence. J Agric Food Use of monoclonal antibodies, enzyme-linked Wells TR, Kreutzer WA (1972). Aerial invasion Chem, 36:88–91. doi:10.1021/jf00079a022 immunosorbent assay and immunoaffinity of peanut flower tissue by Aspergillus flavus column chromatography to determine under gnotobiotic conditions (Abstract). Studer-Rohr I, Dietrich DR, Schlatter J, ochratoxin A in porcine sera, coffee products Phytopathology, 62:797. Schlatter C (1994). Ochratoxin A and coffee. and toxin-producing fungi. In: Categnaro Mitteil Gebiete Lebesmittel Hyg, 85:719 –727. M, Plestina R, Dirheimer G et al., eds. Wheeler KA, Hurdman BF, Pitt JI (1991). Influence of pH on the growth of some Taniwaki MH, Pitt JI, Urbano GR et al. (1999). Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. Lyon: International Agency toxigenic species of Aspergillus, Penicillium Fungi producing ochratoxin A in coffee. In: and Fusarium. Int J Food Microbiol, 12:141– Proceedings of the 18th International Scientific for Research on Cancer (IARC Scientific Publications Series, No. 115), pp. 71–75. 149. doi:10.1016/0168-1605(91)90063-U Colloquium on Coffee, Helsinki, Finland, PMID:2049282 2–6 August, 1999. Lausanne, Switzerland: PMID:1820356 International Coffee Science Association, pp. van der Merwe KJ, Steyn PS, Fourie L et Wicklow DT, Dowd PF, Alfatafta AA, Gloer JB 239 –247. al. (1965). Ochratoxin A, a toxic metabolite (1996). Ochratoxin A: an antiinsectan metabolite from the sclerotia of Aspergillus carbonarius Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT produced by Aspergillus ochraceus Wilh. Nature, 205:1112–1113. doi:10.1038/2051112a0 NRRL 369. Can J Microbiol, 42:1100 –1103. (2003). The source of ochratoxin A in Brazilian doi:10.1139/m96-141 PMID:8941986 coffee and its formation in relation to processing PMID:5833211 methods. Int J Food Microbiol, 82:173– van Walbeek W, Scott PM, Harwig J, WHO (2000). Environmental Health Criteria 179. doi:10.1016/S0168-1605(02)00310-0 Lawrence JW (1969). Penicillium viridicatum 219: Fumonisin B1. Marasas WFO, Miller JD, PMID:12568757 Westling: a new source of ochratoxin A. Can J Riley RT, Visconti A, eds. Geneva: United Nations Environment Programme, International Teixera AA, Taniwaki MH, Pitt JI, Martins CP Microbiol, 15:1281–1285. doi:10.1139/m69-232 PMID:5358203 Labour Organization, World Health (2001). The presence of ochratoxin A in coffee Organization. Available at http://libdoc.who.int/ due to local conditions and processing in four Varga J, Kocsubé S, Suri K et al. (2010). ehc/WHO_EHC_219.pdf. regions in Brazil. In: Proceedings of the 19th Fumonisin contamination and fumonisin International Scientific Colloquium on Coffee, producing black Aspergilli in dried vine fruits of WHO (2007). Ochratoxin A. In: Evaluation of Trieste, Italy, 14–18 May, 2001 [compact disc]. different origin. Int J Food Microbiol, 143:143– Certain Food Additives and Contaminants: Lausanne, Switzerland: International Coffee 149. doi:10.1016/j.ijfoodmicro.2010.08.008 Sixty-eighth Report of the Joint FAO/WHO Science Association. PMID:20826035 Expert Committee on Food Additives. Geneva: Food and Agriculture Organization of the Téren J, Varga J, Hamari Z et al. (1996). Visconti A (1996). Fumonisins in maize United Nations, World Health Organization Immunochemical detection of ochratoxin A genotypes grown in various geographic (WHO Technical Report No. 947), pp. 169–180. in black Aspergillus strains. Mycopathologia, areas. Adv Exp Med Biol, 392:193–204. 134:171–176. doi:10.1007/BF00436726 PMID:8850617 Yamashita A, Yoshizawa T, Aiura Y et al. (1995). PMID:8981783 Fusarium mycotoxins (fumonisins, nivalenol, Waalwijk C, Kastelein P, de Vries I et al. (2003). and zearalenone) and aflatoxins in corn from Thrane U (1996). Comparison of three selective Major changes in Fusarium species in wheat in Southeast Asia. Biosci Biotechnol Biochem, media for detecting Fusarium species in foods: the Netherlands. Eur J Plant Pathol, 109:743– 59:1804–1807. doi:10.1271/bbb.59.1804 a collaborative study. Int J Food Microbiol, 754. doi:10.1023/A:1026086510156 PMID:8520126 29:149–156. doi:10.1016/0168-1605(95)00040-2 PMID:8796416

28 CHAPTER 1 CHAPTER annex. media adaptation of the original published Add minor ingredients and agar to formulation (Abildgren et al., 1987), about 800 mL of distilled water. Steam The formulations given below are made from basic ingredients rather to dissolve agar, then make up to 1 L from Pitt and Hocking (2009). than using commercial Czapek–Dox with distilled water. Add glycerol; note broth. Chloramphenicol (100 mg/L) that the final concentration is 18% Aspergillus flavus and replaces the original combination w/w, not w/v. Sterilize by autoclaving parasiticus agar (AFPA) of chlortetracycline (50 mg) and at 121 °C for 15 minutes. Final aw is Peptone, bacteriological: 10 g chloramphenicol (50 mg). 0.955; final pH is 5.5–5.8. Yeast extract: 20 g Ferric ammonium citrate: 0.5 g Czapek yeast extract Dichloran rose bengal Chloramphenicol: 100 mg agar (CYA) chloramphenicol agar (DRBC)

Agar: 15 g K2HPO4: 1 g Glucose: 10 g Dichloran (0.2% in ethanol, 1.0 mL): Czapek concentrate: 10 mL Peptone, bacteriological: 5 g

2 mg Trace metal solution: 1 mL KH2PO4: 1 g

Water, distilled: 1 L Yeast extract, powdered: 5 g MgSO4.7H2O: 0.5 g Sucrose: 30 g Agar: 15 g Sterilize by autoclaving at 121 °C for Agar: 15 g Rose bengal (5% w/v in water, 15 minutes. Final pH is 6.0–6.5. Water, distilled: 1 L 0.5 mL): 25 mg Dichloran (0.2% w/v in ethanol, Czapek concentrate Refined table grade sucrose is 1.0 mL): 2 mg

NaNO3: 30 g satisfactory for use in CYA provided Chloramphenicol: 100 mg KCl: 5 g it is free of sulfur dioxide. Sterilize by Water, distilled: 1 L

MgSO4.7H2O: 5 g autoclaving at 121 °C for 15 minutes.

FeSO4.7H2O: 0.1 g Final pH is 6.7. Sterilize by autoclaving at 121 °C Water, distilled: 100 mL for 15 minutes. Final pH is 5.5–5.8. Dichloran chloramphenicol Store prepared medium away from Czapek concentrate will keep indef- peptone agar (DCPA) light; photoproducts of rose bengal initely without sterilization. The pre- Peptone: 15 g are highly inhibitory to some fungi, cipitate of Fe(OH)3 that forms in time KH2PO4: 1 g especially yeasts. In the dark, the can be resuspended by shaking MgSO4.7H2O: 0.5 g medium is stable for at least 1 month before use. Chloramphenicol: 0.1 g at 1–4 °C. The stock solutions of Dichloran (0.2% in ethanol, 1.0 mL): rose bengal and dichloran need no Czapek–Dox iprodione 2 mg sterilization, and are also stable for dichloran agar (CZID) Agar: 15 g very long periods. Sucrose: 30 g Water, distilled: 1 L Yeast extract: 5 g Dichloran rose bengal yeast Chloramphenicol: 100 mg Sterilize by autoclaving at 121 °C for extract sucrose agar (DRYS) Dichloran (0.2% in ethanol, 1.0 mL): 15 minutes. Final pH is 5.5–6.0. Yeast extract: 20 g 2 mg Sucrose: 150 g Czapek concentrate: 10 mL Dichloran 18% glycerol Dichloran (0.2% in ethanol, 1.0 mL): Trace metal solution: 1 mL agar (DG18) 2 mg Agar: 15 g Glucose: 10 g Rose bengal (5% w/v in water, Water, distilled: 1 L Peptone: 5 g 0.5 mL): 25 mg

Iprodione suspension: 1 mL KH2PO4: 1 g Chloramphenicol: 50 mg

MgSO4.7H2O: 0.5 g Agar: 20 g Sterilize by autoclaving at 121°C for Glycerol, A.R.: 220 g Water, distilled: to 1 L 15 minutes. Add iprodione suspen- Agar: 15 g Chlortetracycline (l% in water, filter- sion (0.3 g Roval 50WP [Rhône- Dichloran (0.2% w/v in ethanol, sterilized, 5.0 mL): 50 mg Poulenc Agrochimie, Lyon, France] 1.0 mL): 2 mg in 50 mL sterile water, shaken Chloramphenicol: 100 mg Sterilize all ingredients except before addition to medium) after Water, distilled: 1 L chlortetracycline by autoclaving autoclaving. This formulation is an at 121 °C for 15 minutes. Add

Chapter 1. Fungi producing significant mycotoxins 29 chlortetracycline after tempering should be made for the additional Potato dextrose agar (PDA) to 50 °C. Chloramphenicol at twice water. Sterilize by autoclaving at Potatoes: 250 g the concentration specified (i.e. 121 °C for 15 minutes. Final pH is 7.0. Glucose: 20 g 100 mg/L) adequately controls Agar: 15 g bacteria in most situations, and Malt extract agar (MEA) Water, distilled: to 1 L this avoids the need for a second Malt extract, powdered: 20 g antibiotic that must be filter- Peptone: 1 g PDA prepared from raw ingredients is sterilized. Glucose: 20 g more satisfactory than commercially Agar: 20 g prepared media. Wash the potatoes, 25% Glycerol nitrate Water, distilled: 1 L which should not be of a red skinned agar (G25N) variety, and dice or slice, unpeeled,

K2HPO4: 0.75 g Commercial malt extract used for into 500 mL of water. Steam or boil Czapek concentrate: 7.5 mL home brewing is satisfactory for for 30–45 minutes. At the same Yeast extract: 3.7 g use in MEA, as is bacteriological time, melt the agar in 500 mL of Glycerol, analytical grade: 250 g peptone. Sterilize by autoclaving water. Strain the potato through Agar: 12 g at 121 °C for 15 minutes. Do not several layers of cheesecloth into Water, distilled: 750 mL sterilize for longer as this medium the flask containing the melted will become soft on prolonged or agar. Squeeze some potato pulp Glycerol for G25N should be of high repeated heating. Final pH is 5.6. through also. Add the glucose, mix quality, with a low (1%) water content. thoroughly, and make up to 1 L If a lower grade is used, allowance with water if necessary. Sterilize by autoclaving at 121 °C for 15 minutes.

30