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The Fungal Infection of Agricultural Produce and the Production of Mycotoxins

Dr Peter Wareing, Principal Food Safety Advisor, Leatherhead Food Research

Introduction

Fungi are destructive agents causing losses of agricultural commodities in many zones of the world, ranking alongside insects and weeds for crop loss or yield reduction. They can occur on growing in-field crops as well as harvested commodities, leading to damage ranging from rancidity, odour, flavour changes, loss of nutrients, and germ layer destruction. This can result in a reduction in the quality of grains, as well as gross spoilage and possible mycotoxin production. Fungi are often referred to as moulds in this context, to differentiate them from single celled yeasts.

Estimates on crop losses that can be directly attributed to fungi vary. For example, Oecke and Dehne (2004) estimated that, worldwide, weeds caused up to 32% losses, animal pests (18% and pathogens about 15%, For individual crops, fungal losses can be 100% if a susceptible cultivar is planted or the climate is favourable in any year, and down to 0%, if resistant varieties are planted, and fungicides used, and good husbandry employed. Losses tend to be higher in the humid tropics than in cooler temperate zones. For example, losses in rice and maize were higher than losses for wheat and barley. Losses in fleshy staples, for example potatoes, sweet potatoes, yams, were higher than for cereal crops. Losses in barley due to pathogens were 9% worldwide, with 5% in North West Europe, and 15% in South East Asia. For potatoes, losses in Europe were about 25% in North West Europe, and 50% in Africa. Mycotoxin production is always a food safety, as opposed to a food spoilage issue, although they will still contribute to losses as the crop is downgraded, reprocessed or destroyed.

Direct crop losses in the field are caused by plant pathogens by reducing crop yields. Some plant pathogens may produce mycotoxins, either as incidental products, or as a chemical associated with the infection process, for example, some species of Fusarium. Plant pathogens may also render the crop a lower grade by causing blemishes, blights or other quality problems. Alternaria and Fusarium may do this. Spoilage fungi may not be able to attack crops in the field, but cause problems once the crop is harvested, if conditions allow. Some spoilage fungi can also produce mycotoxins, for example Penicillium, although many penicillia associated with grains are pathogenic.

Aspergillus, Penicillium, Alternaria and Fusarium are amongst the most common mycotoxin-producing fungal species associated with growth in and damage to food crops in the field, and in store, if poor storage conditions prevail after harvest, or previously dried commodities become rewetted.

Mycotoxin-producing fungi are often sub-divided into field and storage moulds. This is intended to differentiate between those fungi that attack primarily pre-harvest and those that cause damage and mycotoxin production post-harvest, or once the crop is in store. It has been recognised that that this definition, although holding true in many cases, has some flaws.

For example, Aspergillus flavus can attack crops pre harvest if insect damage, drought stress, or weed competition cause plant stress. It can also attack in the field immediately after harvest, if drying delays occur due to poor weather. Conversely, Fusarium graminearum, often thought of as a field fungus, can continue to grow in store if maize cobs are stored in cribs to continue drying. This can be a particular problem in the tropics.

In many cases, infection occurs at a low level in the field, and continues in store if conditions are favourable. Byssochlamys nivea, for example, may attack apples in the orchard, but continues to grow in store, growing from apple to apple. The problem is mainly exhibited when apples are pressed for juice; individual apples contaminate a larger batch. The effective control measure is to press soon after harvest, or to carefully select apples going for pressing, and filter the juice, if fresh juice is pressed in the storage period.

Mycotoxin contamination of crops has been a worldwide problem for thousands of years. However, the significance of the mycotoxins present in foods, the effect on human health and the impact on the economy has been assessed only over the last few decades. Plant fungal pathogens or food spoilage moulds are the source of this type of toxin. The Food and Agriculture Organisation (FAO) estimates that 25% of the world's food crops are affected by mycotoxins during growth and storage. Various different types of health problems are linked to the ingestion of different types of mycotoxins by humans or animals. Many outbreaks related to food contamination by mycotoxins have occurred all over the world, which is a global concern as a health hazard.

Food mycology is a complex subject; many food spoilage moulds have sexual and asexual growth phases; both or either phase may be seen in a spoiled product. The nomenclature has changed considerably over the years; it is vital to make sure that any papers citing crops or products in which particular moulds are found use the up to date names. The same applies with respect to mycotoxin production; for example, there have been numerous false reports of aflatoxins being produced by species other than those noted in the Basic Aflatoxin Factsheet.

Common Mycotoxigenic Fungi

The number of mycotoxins produced by fungi continues to rise, as new toxic metabolites are discovered, but over 300 have been noted to date.

Eurotium species are probably the most common fungi that grow on improperly stored grain, causing rancidity and damage to the embryo, especially in malting barley, and grain to be used as seed stock for the next year. They are xerophilic (prefer to grow in drier conditions, including semi-dry grain) They are not notably toxigenic, with the exception of Eurotium chevalieri, which has been associated with feed refusal and death in livestock. Toxic metabolites include echinulin, auroglaucin and flavoglaucin.

As noted above, Aspergillus, Penicillium, Fusarium and Alternaria species are some of the most common mycotoxigenic fungi associated with damage to field crops; they can also grow in store if poor storage conditions prevail after harvest, or previously dried commodities become rewetted.

The role of Aspergillus species in food spoilage is well-established. Mycotoxins produced by Aspergillus flavus include aflatoxins and cyclopiazonic acid. Other important mycotoxins from aspergilli include ochratoxin A and patulin.

Some aspergilli have an ascomycete teleomorphic (sexual) stage; for example, Eurotium, Neosartorya, and Emericella; Many aspergilli are xerophilic and present particular problems during commodity harvest, and during subsequent drying and storage.

About 30 species of Aspergillus or their teleomorphs are associated with food spoilage, these include: Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus ochraceus, Aspergillus candidus, Aspergillus restrictus, Aspergillus penicillioides, Aspergillus niger, Aspergillus carbonarius, Aspergillus fumigatus, Aspergillus clavatus, and Aspergillus versicolor.

Aspergillus

Aspergillus species tend to be associated more with tropical and warm temperate crops, for example oilseeds and nuts, since they prefer to grow at relatively high temperatures.

Fig 1: Aspergillus flavus-contaminated peanuts


Penicillium

Penicillium is a large genus containing 150 recognised species, of which 50 or more occur commonly. Many species of Penicillium are isolated from foods causing spoilage; in addition, some may produce bioactive compounds. Penicillium taxonomy is a veritable minefield for the inexperienced, and is best left to those working in the field; whereas many aspergilli can be crudely identified on the basis of colony morphology and colour, most penicillia are some shade of bluish or grey-green to green. Penicillium species can be divided into subgenera based on the degree of branching of the conidiophores: subgenus Aspergilloides has one branch point, Furcatum and Biverticillium two, and in subgenus Penicillium, three branch points. These latter are most "Penicillium-like".

 Fig 2: Penicillium digitatum-contaminated peanuts

 

Important mycotoxins produced by Penicillium include ochratoxin A, patulin, citrinin and penitrem A. Some of the most important toxigenic species in foods are Penicillium expansum, Penicillium citrinum, Penicillium crustosum and Penicillium verrucosum.

A much larger number of Penicillium species are mainly associated with food spoilage. Those covered here include Penicillium aurantiogriseum, Penicillium chrysogenum, Penicillium digitatum, Penicillium griseofulvum, Penicillium italicum, Penicillium oxalicum and Penicillium viridicatum; some of these produce mycotoxins.

Penicillium species are associated more with cool temperate and temperate crops, mainly cereals, since most species do not grow very well above 25-30°C.

Mucor and Rhizopus typically affect fruits and vegetables, since they can only grow at relatively high water activities.

Alternaria species are plant pathogens that can produce toxins in both pre- and post-harvest commodities. They are characterised by very large brown conidia with a characteristic "beak" at the tip. The most common species is Alternaria alternata, others include Alternaria tenuissima, Alternaria infectoria, Alternaria citri, Alternaria brassicicola and Alternaria brassicae. The species Aternaria alternata and Alternaria tenuissima are pathogenic to a wide range of crops; the other species have more limited host ranges.

Fusarium species are mainly plant pathogens and normally occur in association with plants and cultivated soils. Infection may occur in developing seeds, and in maturing fruits and vegetables. Typically able to grow only at higher water activities, damage is usually confined to pre-harvest, for cereals, or immediately post-harvest until drying is well under way and the water activity is below about 0.90. Vegetables can continue to be spoiled in store, due to their higher water activity.

Fusaria can be divided broadly into temperate and tropical/subtropical types, the former mainly affecting cereals, the latter, tropical cereals and fruits and vegetables. Some temperate species are able to grow over the winter on snow-bound cereal crops.

Examples of species are Fusarium chlamydosporum, Fusarium culmorum, Fusarium solani, Fusarium equiseti, Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Fusarium poae, Fusarium semitectum, Fusarium subglutinans, Fusarium sporotrichioides and Fusarium verticillioides (alternative name (synonym) F. moniliforme).

There are a large number of other moulds that have been isolated from food and feeds, particularly cereals, oilseeds, herbs and spices. These include Cladosporium, Geotrichum, Mucor, Rhizopus, Moniliella, Paecilomyces, Wallemia, Byssochlamys, Talaromyces, Eupenicillium, Claviceps, Phoma, Phomopsis, Curvularia, Chaetomium, Xeromyces and Chrysosporium. Some of these produce mycotoxins, to some of which legislative restrictions may apply (patulin from Byssochlamys, for example), others do not.

Foods and feeds affected

The list of crops affected by fungi and mycotoxins is very large; too long to catalogue here. Broadly, cereals, oilseeds, fruits and vegetables, tree crops, and animal feeds can be affected. Some examples of major crops and important mycotoxins are noted below.

Aspergillus flavus, Aspergillus parasiticus and aflatoxins typically affect oilseeds, including groundnuts, soya, tree nuts, maize and various oilseed-based animal feedstocks - cotton seed cake, copra, sunflower, but can also affect rice, wheat, sorghum, figs, coffee and sweet potatoes, for example. Aflatoxins are also noted in milk, via contaminated animal feed.

Fusarium species are responsible for wilts, blights, root rots and cankers in legumes, coffee, pine trees, wheat, corn, carnations and grasses.

How do moulds produce mycotoxins?

Different mycotoxins may affect humans, laboratory and farm animals. Tissues affected include the liver (aflatoxins), kidneys (ochratoxin A, patulin), the blood (trichothecenes), brain, lungs, oesophagus (fumonisins, depending upon the target animal), nervous system (penitrem A), gastrointestinal system (trichothecenes) and generalised organ haemorrhage (trichothecenes).

Mycotoxins are often produced when the fungus is under stress, for example, when the temperature, water activity or amount of oxygen becomes less favourable.

Studies in groundnuts have shown that A. flavus lies dormant in the field, and attacks when the plant is under stress, for example, drought stress, either due to a lack of rainfall or irrigation, or competition from weeds or crop overcrowding. Insect damage is another important risk factor in infection and aflatoxin production.

In the tropics, the high ambient temperatures and high humidities combine to cause major problems. Studies in maize in Thailand have shown that aflatoxin levels at harvest can be relatively low, even in affected crops, but that poor post-harvest practices quickly allow an increase in the levels seen. If maize remains on the cob in the field, levels can remain low, but once shelled, levels can increase. If maize is shelled above 23% moisture content (mc), then aflatoxin levels can increase rapidly, until the crop is below 14% mc. (Cutler, 1991).

A management regime for the humid tropics for minimising aflatoxin contamination in maize was developed from the above studies in Thailand:

 1. Leave the crop in the field to dry to 22% mc.
 2. Shell field-dried maize within 48 hours of harvest
 3. Mechanically dry shelled maize within 48 hours to 16% max., preferably 14%
 4. Further dry to <14% within 1 week, if not done so at 3.

Climate and environmental conditions for toxin production

Some growth factors are much more important than others; water activity and temperature will determine which moulds grow and how fast growth will be, whereas pH has little effect, since most moulds are very tolerant of low pH, being able to grow down to pH 2-3.

Several terms are used with respect to moisture content; water activity (aw) and Equilibrium Relative Humidity (ERH). ERH tends to be used for field crops and by grain storage specialists, whereas aw is used more by the food industry, mycologists and other research scientists. The key factor is that as the lipid content rises in a seed, so the moisture content for safe storage must fall. Table 1 illustrates this point. Note that these data are cultivar-dependant.

Table 1 Relationship Between Moisture Content and % lipid for Different Foods at aw 0.7 at 25°C

Food Type

% Lipid

% Moisture Content

Dried fruit

0.3

18-25

Wheat

1-2

13-15

Maize

3-4

13-14

Soybeans

19

8.5

Groundnut

46

7

Almond

56

4.2

Brazil nuts

68

2.8

Most fungi cannot grow below an aw of 0.7, but some extreme xerophiles can grow to aw 0.6, but they are not able to produce mycotoxins. 

In general, the minimum water activity for growth is lower than the minimum for mycotoxin production. What we have termed storage fungi tend to be able to grow at lower water activities than field fungi, with each species having particular limits.

Fungi are aerobes, and do not grow well under conditions of lower oxygen tension. Fungi can be effective oxygen scavengers, however, and so may be able to grow where there is little oxygen, for example, with the limited oxygen in the headspace of bottled waters and fruit juices. Paecilomyces and Byssochlamys, for example, can tolerate an oxygen content as low as 0.5%. Some can also tolerate very high levels of CO2, although most cannot.

For example, Penicillium roqueforti is stimulated by high (15%) levels of carbon dioxide, and can tolerate oxygen levels of 1 - 2%. Some isolates can grow in conditions of 80% CO2, 4.2% O2, and 15.8% N2. It can grow in O2 levels around 0.5%.

As with bacteria, fungi can also be categorised according to their response to temperature. As noted earlier, Aspergillus species tend to grow at higher temperatures than many penicillia; the former would be regarded as mesophiles, whereas the latter contain many psychrotolerant species (tolerating lower temperatures).

For example Aspergillus. flavus grows to a minimum of 10 - 12°C and a maximum of 43 - 48 °C, with an optimum around 33 - 35 °C. Aflatoxins are produced over a temperature range of 13 - 15°C minimum, up to 37 °C, with maximum production at 33 °C.

Optimum growth of Fusarium. graminearum occurs between 24 - 26°C, it can grow as low as 5°C and as high as 37°C. Optimum deoxynivalenol toxin and zearalenone production by Fusarium. graminearum occurs in temperatures of 24°C and 25 - 30°C, respectively.

Optimal growth for Penicillium crustosum occurs at 25°C with a minimum of <-2 - 2°C and a maximum of 30°C. Optimal penitrem A production occurs between 20 - 26°C with a minimum of <17°C and a maximum of 30°C.

Many fungi are able to scavenge nutrients, and do not require particularly rich food sources for growth; they probably will not be able to produce mycotoxins, however.

One point to note with respect to fungal growth is that xerophiles may initiate spoilage at relatively low aw, if crops are not stored under optimal conditions. This then generates water from the breakdown of the seed, which then allows less xerophilic fungi to grow, which may lead to mycotoxin production; fungal succession.

Control of Fungal Growth and Mycotoxin Production

Fungal growth in the field can be limited by using resistant cultivars, correct planting density, and irrigation where practicable, or other drought-limiting measures, by weed control, and control of insect and pest damage.

Other control methods include harvesting at the correct time, without damaging the crop, drying rapidly, avoiding rewetting, and controlling insects in store to help to reduce the risk of fungal growth.

For fruit and vegetables, again, correct harvest practices, avoiding damage, and correct storage conditions reduce the risk of fungal growth.

If crops are damaged, then removal of the outer layers by milling will remove much of the contaminated material. Note that this material is much more contaminated compared with the whole grain, and probably cannot be used for animal feed. In the case of fruits, pressure washing can blast out the fungal damaged rotted parts, reducing the overall level of contamination.

Colour sorting has been used for many years in the USA, to reduce the aflatoxin levels in peanuts, and is very effective. Infected peanuts tend to be shrivelled or discoloured, and are automatically removed by visual sorters coupled with high speed compressed air jets.

Some chemical detoxification measures have been used, for example ammoniation, but such treated commodities can only be used for animal feed due to organoleptic changes.

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