Genetic Diversity of Fungi Producing Mycotoxins in Stored Crops

Mycotoxins are a variety of critical secondary metabolites for the defense, that produced by multiple types of fungi. These metabolites are toxins where metabolic pathways that produce these toxins are found in adjacent gene groups in the fungal genome when they have adequate environmental and dietary conditions. Mainly, they found in commodities stored by the wrong ways. Mycotoxins are the most potent known toxins that cause serious diseases with minimal concentrations. Genetic diversity was detected using polymorphic randomized amplification technique for DNA fragments between fungal isolates from different crops. This review article aims to review the current status of genetically diverse of mycotoxigenic fungi in various contaminated food. Several studies that have focused on the determination of prevalence and frequency of varies types of toxic fungi were reviewed. Also, the articles that study the toxicity of stored crops such as cereals and oilseeds were considered. The high contrast between findings of these works was presented in terms of the genetic diversity of fungal isolates produced toxins. Aspergillus, Fusarium, and Penicillium were observed among the most common fungus producing toxins. This study which derived from previous researches observed that Aflatoxin was the most toxin produced by most fungi. Aspergillus was the most genetically modified fungus, carrying the most genes responsible for producing the fungal toxins.


INTRODUCTION
Mycotoxins are secondary metabolic products (SM) produced by some fungi which are genetically capable of producing toxins when they have adequate environmental and nutritional conditions.The fungal toxins are the most potent known toxins that cause severe diseases with small concentrations of less than 10 ppm.This potency is due to the fungal toxins are heat resistant to the extent that they cannot be destroyed by conventional heat treatments used in manufacturing and cooking.The second reason is that they spread quickly from fungus colonies to food.Therefore, removing the fungal parts of food, as many people do, does not lead to the complete elimination of the fungal toxins produced in these foods and therefore the growth of fungus on these foods should be avoided 1,2 .
The most important toxins are produced by Aspergillus, Fusarium, Penicillium, and Alternaria.One fungus may produce more than one poison, and currently, there are more than 200 known types of fungal toxins that cause health risks to humans and animals.The most common toxins are Aflatoxins, Ochratoxins, Fumonisins, Trichothecene, Patulin, Rubratoxin, Citrinin and Zearalenone.The effect of these toxins does not appear quickly, but has a cumulative effect that appears after 10-20 years of eating contaminated food.The other problem is that it does not stimulate the immune system in the body to be detected and have no drug treatments to reduce the impact and thus constitute a health disaster in the world 3 .Aflatoxins, a group of about 20 metabolic compounds, are the most important fungal toxins.Aflatoxins B1, B2, G1 and G2 are usually found in foods and are present in a wide range of food commodities including cereals, nuts, spices, figs, and dried fruits 4 .
The diagnosis of toxin-producing fungus was based on phenotypic characteristics and microscopic structure such as colony color, shape, pigmentation, as well as reproductive traits such as spores, type and shape, and size of produced stone bodies 5,6 .However, these characteristics are unstable and can change under environmental conditions, as well as require considerable effort and time.The recently tended emergence of heterozygous strains within the same type drive the scientists to rely on the molecular diagnosis, which is based on Polymerase Chain Reaction (PCR) to give results and delicate accuracy in diagnosis 7 .The most important feature of the PCR-RAPD technique; it is a fast, lowcost and straightforward technique.The main disadvantages are the amplification process which either occurs or does not occur due to technical randomization.In addition, it reveals the presence of sovereignty and results in non-replicable [8][9][10] .The results of the PCR-RAPD analysis of a sample cannot be compared with the same conditions in two laboratories 11 .Ribosomal DNA is amplified to determine the taxonomic characteristics and relationship of evolution between fungi.The DNA sequence is often used to study taxonomic and developmental studies because they exist in living cells with important functions and therefore their evolution may be reflected in the evolution of the whole genome 12 .This review article aims to review the current status of genetically diverse of mycotoxigenic fungi in various contaminated food.

Prevalence and Frequency of Fungi Producing Toxins
Cereals and products stored as oil crops accompany many microorganisms such as fungi, yeast, and bacteria.These microorganisms multiply when the conditions for their growth are suitable causing damage to stored materials.In turn, it causes a reduction in the quality and chemical changes in the product 13 .Fungi play a particularly dangerous role during storage operations compared to other microorganisms.The toxins produced by these fungi have significant economic effects in many agricultural crops, especially wheat, maize, field pistachios, nuts, cotton seeds, and tea.Twenty-five percent of the world's crop production is contaminated with fungal compounds 14 .
In general, toxins reach the food of humans and animals through the contamination of food with fungi which produce these toxins (the process of the formation of toxins and their secretion depends on the type of fungi and the nature of the food and the availability of appropriate environmental conditions).The nutrient encourages the growth of the fungus either during the different stages of production or transportation or storage period.The most important species responsible for the secretion of more than two-thirds of Mycotoxins are Aspergillus, Fusarium, and Penicillium 15 .The presence and spread of these toxic fungi were confirmed by isolating them from these agricultural products.Table (1) summarized the results of prevalence and frequency of mycotoxin fungi that isolated from different crops from previous studies.
Studies have elucidated the isolation of different types of fungi with different propagation percentages, where the most toxic fungi produced in grain and seed crops are Aspergillus, Fusarium, and Penicillium, which produce SM of high risk to human and animal health.Most of these dangerous toxins are Aflatoxins.
In terms of relative dominance of species, Aspergillus was found to be the most frequent and widespread fungus [16][17][18][19][20]23 . The eason is that this fungus can form large numbers of breeding units that are resistant to inappropriate environmental conditions, which form plankton in the air and thus reach many places.As well as their growth in wide ranges of heat and humidity conditions, as some species of Aspergillus grow at temperatures ranging from 5 to 45 °C.
According to the above-mentioned studies 16,18,19,22 , frequency and sovereignty indicators illustrate that some species in the environment have been confirmed and replicated, such as Aspergillus, Fusarium, and Penicillium, despite the different environmental conditions of each study.This evidence shows the extent of these species of fungi to tolerate the various environmental changes.Also, it shows their physiological activity, rapid growth, producing large numbers of reproductive units, and enzymatic and toxic activity compared to other species.

The Concentration of Mycotoxins in Crops
SM products of fungi are biologically active compounds.They are non-antigenic toxins and most of them are toxic to humans, animals, plants, and microorganisms 24 .Mycotoxic fungi divided into three groups; field fungus, storage fungus, advanced decomposition fungi.Mycotoxins, in turn, classified according to their secretion time.Direct pollution for the Mycotoxins secreted during the stages of production and circulation of food.Whereas, the indirect pollution that results of contamination of food by feeding humans on animal products produced from animals that have been fed on contaminated food with fungal toxins and this type is the most dangerous 25 .Table 2 shows the concentration of toxins (by ppb) according to the different crops samples.
Fungi varied in the production of fungal toxins and the variation in proportions was attributed to the genetic ability of different fungal isolates as shown in many studies 22,28,32,34 .
The findings of the chemical analysis of fungal isolates from many crop types using different techniques showed various types of fungal toxins which produced in different proportions 29,31,33,35 .The difference in toxin production is due to the ability to produce toxins according to the genetic diversity of the fungal isolates.Aflatoxin is the most frequently reported Mycotoxin 26,30,37 .Aflatoxin is produced in the poor stored agricultural products especially in the tropics and sub-tropical regions where the appropriate climatic conditions as high temperature and humidity.These conditions allow the growth of a broad spectrum of fungal species on the water agricultural thoroughbred especially species producing these toxins 38 .
The concentration of toxins may also be attributed to the techniques used to estimate the quantity of toxin such as the HPLC / HPTLC techniques that don't estimate toxin values.Also, some works have shown a conclusion that, their tests cannot be appropriate to estimate the amount of all toxins 26 .Alternatively, the number of genes may be related to the amount of toxin, and the method of estimate the toxin can affect the amount of poison 39 .Previous study also showed that the rate of production of toxins on oilseeds crops (exceeding 300 ppb) was more than the production it on grain 22 .
Although different conditions and areas of studies have been conducted, most have confirmed that Aspergillus strains can proliferate rapidly on nutrients such as peanuts and some other species with high moisture content 40 .Fungi do not grow evenly on all nutrients.Different species of the same type of food differ in their susceptibility to fungi and the production of Mycotoxins 41,42 .The increase in the production of Mycotoxins in oilseeds is expected to be due to the internal structure of seeds, and moisture content within it.In addition to the conditions of humidity and temperature in the storage field 43 .
On the other hand, some studies have shown high concentration rates for the production of fungus growing on cereals indicating that the toxicity of Mycotoxins due to the storage of such types of crops at very high humidity limits [43][44][45] .At these levels of moisture are considered dangerous.Thus, the safe storage limits for the grain will depend on the initial or primary content of the moisture.

Genetic Diversity
Since the initial reports of DNA amplification using PCR, the number of different applications of this technique has increased dramatically.One of the first applications of the PCR in 1990 was described by White and his coworker and dealt with the amplification and direct sequencing of ribosomal DNA (rDNA) to establish taxonomic and formative relationships between fungi 46 .The emergence of PCR has allowed the development of reliable molecular markers for the detection and differentiation of fungi, both at the species and strain level.Extensive applications have been found in the science of mycology including classification, plant composition, and diagnosis.PCR detection of pathogenic fungi has been reported for numerous vital genes such as Phytophtora sp., Fusarium sp., and Colletotrichum sp 47 .DNA-based PCR techniques are specific, sensitive and fast compared to many other detection methods.There is a wealth of methodologies for detecting microorganisms, including traditional quantification of fruiting structures, disease symptom record, and biochemical and microbiological methods.Recently, PCR techniques have gained remarkable popularity in diagnosis, due to sensitivity, quality, and ease of implementation 48 .
Table 3 showed the different genes responsible for the production of Mycotoxins from different fungal isolates from different crop types.This genetic diversity or genetic variation may be due to the effects of climate, various environmental factors, storage conditions, pollution, the effect of certain chemicals and the biological composition of the seed, or the PCR pattern that may be affected by several factors 20,51 Genetic variation may also be attributed to the method of breeding fungi or the way of coexistence with fungi and other organisms 20,52 .Some studies have indicated that there is a lack of toxic genes in some isolates 20 .This could be due to the inability of the isolate to produce the fungal toxins, the environmental conditions (such as the storage medium), or to inhibition of the PCR reaction by cell wall components during the process of reaction 53 .However, the use of ways based on PCR-targeted methods for DNA is an excellent choice and quick option to diagnose fungi because they are highly specialized, sensitive and better than other techniques.

CONCLUSION
Genetic diversity was observed between the mycotoxigenic fungi, and various genes are responsible especially avfA, omtA, and omtB for the production of fungal toxins.One of the most toxic fungi is Aspergillus, and Aflatoxin was most common Mycotoxin produced by this fungus.We recommend for studies that determine the role of each Mycotoxic gene/loci responsible for Mycotoxin production.

Table 1 .
Prevalence and frequency of Mycotoxigenic fungi isolated from different types of crop

Table 2 .
The concentration of Mycotoxins in different types of crops

Table 3 .
Genes responsible for producing mycotoxins by different fungal isolates from varies crop types