Mycotoxin Production by Fungi Isolated from Rice and Stored Grains in Riyadh, KSA

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Wheat and Barley are the most production and consumption grains in the world.The necrotrophic Fusarium spp is pathogen caused many diseases on plants, the major two disease caused by Fusarium on wheat is Fusarium Crown rot (FCR) and head blight (FHB), also known as scab (Matny, 2015).
Rice is the second level of cereal staples consumed food worldwide after wheat, and it consists the major part of the diets for the half of the world.It is also reported that the rice is composed of 27% of the global diet and 20% of dietary protein intake in the developing countries (Alamer, 2014).Rice is one of the most cultivated food crops globally.About 593 million tons (Mt) of rice are produced annually (FAO, 2002).It is used in variety of food products.Cooked rice, breakfast cereals, desserts, and rice flour are some important food products of rice.It is also used to prepare local beer, while rice hull is used as fuel, fertilizer, packing, and insulation (Gupta, 2012).Rice bran is also used as a suitable substrate in mushroom cultivation.Straw from the leaves and stems is used as bedding for animals.Rice floor is used to prepare many traditional foods on some special occasions (Giddel and Jivani, 2007).Toxigenic fungi can attack rice in the field and during storage resulting in increased mycotoxin levels in this commodity.Aspergillus, Fusarium, and Penicillium are the predominant fungal genera associated with food grains during storage (Makun, 2011).
The main fungi, Aspergillus flavus, produce these mycotoxins thrive under favorable conditions on a wide range of foods and feed such as maize and peanuts, and are a world-wide problem (Sanusi, 2015).Toxigenic fungi in crops have been historically divided into two more or less distinct groups, the first includes those which invade and produce their toxins before harvest which are often rather loosely called "field fungi", the second group which becomes a problem after harvest is known as storage fungi.Invasion by fungi before harvest is governed primarily by plant hostfungus and other biological interactions while growth by fungi postharvest is governed by crop physical and biotic factors (Miller, 2000).
Mycotoxins can produce at field preharvest, harvest, transportation, and storage stages while the conditions are favorable for growing the fungus resulting in the mycotoxin production.The risk of contamination by mycotoxins is an important food safety concern for grains and other field crops.Mycotoxins affecting cereals is considered to be of greater significance world over for human beings (Parvathi, 2014).

MATERIALS AND METHODS
Isolation: 30 samples of maize seed, rice, wheat, barley, and millet were collected from locations stored in the agricultural fields, and the seeds were put in plastic bags and brought to the laboratory.These samples submerged in Clorox solution concentration of 10% for 1-2 minutes to purify the outer surface of the seeds of pollution, and then the seeds were taken out and put the nomination to get rid of the excess solution papers.The seeds were washed with sterile distilled water and put on the surface of the medium.We used Sabourauds Dextrose Agar (SDA) medium by the way (Emmon et al., 1974).Samples were incubated at a temperature of 25 and the dishes were checked after 7 days of incubation in order to isolate and diagnose fungus and for pure isolated farm species.The percentage was determined to appear or occur following law: The proportion The emergence of a number of one type of appearing

= _______________________________ x 100
The total number of samples

Study The Feasibility of Species on The Secretion of Aflatoxin:
We studied the viability of the species isolated in this study on the production of aflatoxin using the two mediums Potato Dextrose Agar (PDA) by the way (Emmon et al., 1974) and Yeast Extract Sucrose (YES) by the way (Davis et al.,1966) in order to determine the impact of the type of medium to produce aflatoxin.As it has been part of the colony fungal pure transfer to the center of the dishes containing the two mediums (PDA, YES) and dishes incubated at a temperature of 25 for a period of 7 days for the purpose of obtaining full colony.For the detection of aflatoxin, ammonia solution with concentration of 25% was used by adding 0.2 ml of this solution in the middle of the glass dish and cover overturned dishes (Saito and Machida, 1999) and incubated at a temperature of 25.Check dishes control after the second day of cuddling to note the color change of the rules of the colonies.If colonial rule changed to red color or pink or yellow orange, it indicates that the fungus is capable of producing aflatoxin and otherwise the fungus is unable to produce aflatoxin (Saito andMachida 1999, Lin andDianese 1976).

Study The Effect of Temperature on The Secretion of Aflatoxin:
To study the effect of different temperatures on the viability of the species isolated during the study on the secretion of Aflatoxin, the following temperatures were selected: (20, 25, 30, 35, and 40).Fungus grown on the two mediums (PDA, YES) was due to varying temperatures mentioned above.After a week of portability, it showed cuddling fungi on the secretion of aflatoxin in each temperature test using ammonia as mentioned above.

Mycotoxin production
The medium used for this study was potato dextrose broth (PDB.).The medium was prepared routinely and sterilized.Twenty-five millilitres (25 ml) of the broth were dispensed into sterile conical flasks.The flasks were inoculated separately with 5 ml of any of the test fungi.The inoculated flasks were incubated on a shaker at a room temperature of 25 ± 1oC for 12 days.The filtrates of the test isolates were obtained and assayed for the presence of mycotoxins.

RESULTS
The results of this study indicated that the injury or contamination of crop seeds in the Riyadh region was at high rates, it He found that there are 22 sample was infected with one type or more types of Aspergillus of 30 samples collected from corn, rice, wheat, barley, millet an increase of 73.2% fungal infection.It appeared during the study that the corn crop is more fungal infection of crops compared to other crops where infection in 7 samples showed an increase of 70%, followed by wheat crop because of injury in any 6 samples appeared by 60% while the rice crop injury is the least compared to other crops, It was noted that the two samples were infected and by (20%).Was isolated and diagnosis of 5 types of Aspergillus during this study, was the type (1 A.) visible most of the other species where he appeared in (7) samples by (23.3%), followed by type (2 A.) where he appeared in (5) samples by (16.6%), two of which showed the emergence of similar proportions 10%), (Table 1) (Fig. 1).Upon detection of the ability of this species on the secretion of aflatoxin, it appeared during the study that all types of fungi isolated its ability to produce aflatoxin growth at the medium (PDA).Through color change, the rules of fungal colonies were in red-pink or yellow-orange using the ammonia solution concentration 25%.But when using the medium (YES), it was observed that all species showed the ability to secrete aflatoxin except three types of Aspergillus did not demonstrate any ability to secrete aflatoxin at this medium (Tables 2&3).(Figs.2&3).
Table 2: Detection of toxic activity (aflatoxin) of isolated fungi using the two mediums PDA, YES 25C°  Note that in (Table 4) (Fig. 4) the temperature has a clear impact on the production of aflatoxin in both the two mediums.It showed fungi isolated and developed on medium (PDA) and the ability to produce aflatoxin compared with the growth on medium (YES).This was noted during the study variation in susceptibility fungus aflatoxin production in different temperatures.So it is clear that the ability to produce aflatoxin fungus when temperatures (20, 25, and 30) were down, while this increased susceptibility at a temperature of 35 and the fungus showed the highest ability to produce aflatoxin in temperature 40.It was observed that Aspergillus sp.gave a high affinity to produce aflatoxin in temperatures (35 and 40).The Aspergiulls sp.(2) shows no ability to produce aflatoxin in temperatures (30 and 25) when growth on the medium (YES).Also types (3 and 4) of Aspergillus sp.show no ability to produce aflatoxin in temperature 25 when growth on the medium (YES).
Table 4: Effect of temperature on the production of aflatoxin in isolated fungi Fig. 4: Effect of temperature on the production of aflatoxin in isolated fungi

DISCUSSION
The study reviewed contamination of crop seeds that have been studied in this research, and that all fungi gave an account of the positive use of the medium (PDA) and this may be due to the nature of the central components of the fungus, which gave an account of the negative using the medium (YES) that does not mean they are unable to produce aflatoxin but perhaps due to the nature center components or cuddling degree heat.It is believed that the cause of seed infection with fungi is the presence of factors that help increase such incidence of insects and spiders and the quality of the crop and mixing with other materials contaminated during harvesting and storage.Toxic fungus Aspergillus flavus is responsible for the loss of 25% of the grain in the world through pollution (Alhaji Sanusi, 2015).Crops and nuts injury poisons aflatoxin is one of the problems that affect food and feed on a global level.Mold product of this poison can grow and produce the toxin when the conditions of its heat and moisture are available, and with ill-handling and storage operations for those products, it may be that those toxins formed in the products inside warehouses and stores (Ibrahim, 2014).
One of the most important ubiquitous fungal species in tropical environments is Aspergillus flavus which can be found in soil and other substrates.A. flavus is associated with many diseases of humans, most severe  2015).Aflatoxins are secondary metabolites produced by Aspergillus section flavi group and fungi, these Aflatoxins are potent carcinogenic, teratogenic, mutagenic, hepatotoxic, and immunosuppressive agents that cause significant damage to human and animal health (Srilakshmi, 2015).Aflatoxins are natural contaminants of cereals and other commodities throughout the world.Chronic dietary exposure even to low doses of aflatoxins is a known risk factor for liver cancer and effect protein metabolism and immunity (Hina, 2014).Aspergillus flavus produces the secondary metabolites aflatoxins B1 and B2 and other mycotoxins such as cyclopiazonic acid.A. flavus is the predominant species [1,2] responsible for aflatoxin contamination of crops prior to harvest or during storage (Whitelaw, 2004).The global impact of aflatoxins on trade and health was featured in a presentation which demonstrated a synergistic effect between aflatoxin and hepatitis B virus to cause liver cancer in humans (Bowman, 2015).Under unfavorable field conditions stressful to the crop, some fungi produce mycotoxins, secondary metabolites that may contaminate various agricultural commodities in the field and in storage (Hruska, 2015).Mycotoxins are low molecular weight secondary metabolites produced by filamentous fungi that are commonly resistant to a wide spectrum of environmental factors and, therefore, undergo slow degradation.They are stabile at high temperatures and at low pH values typical of the gastric juice of animals (Pfliegler and Pusztahelyi and Pócsi, 2015).

Table 1 :
Organisms isolated from stored grains.

Table 3 :
The effect of ammonia solution on isolated fungi.