Introduction

Rice is one of the most famous and important cereals worldwide. According to¹ the cultivated area was estimated as 156 million hectars producing about 721 million metric tons (MMT) of rice. The most important world rice producers are China (202.3 MMT), and India (154.5 MMT). Rice is one of the most important commercial crops planted in Egypt. It is a privileged source of carbohydrates and proteins. It is used for different food and nonfood products. The foods include cooked rice, rice flour, breakfast cereals, and desserts. The inedible rice hull is used as fertilizer, fuel, and others, while the bran is a source of cooking oil. Straw from the stems and leaves is used as feeding or bedding for animals and for making roofs, bricks, hats, sandals and baskets. Rice bran and straws are also used as suitable substrates in mushroom cultivation.²

Fungal contamination of cereals is an important issue for grain quality and from consumer’s health point of view. Rice is one of the famous cereals which favor mycotoxin contamination.³ In recent years, there have been many studies from various countries on the occurrence of high levels of aflatoxins by fungal rice contamination.^4,5,6 In 2001, Reddy and Sathyanarayana⁷ listed 143 fungal species from rice. Reddy et al.⁸ published a review containing the important groups of mycotoxins  including aflatoxins (AFS), fumonisins, ochratoxin A(OTA), deoxynivlenol, and zearalenone (ZEN), which contaminated rice in different countries. Sempere & Santamarina⁹ confirmed that the majority of mycotoxins were produced by Aspergillus, Penicillium, and Fusarium, Ferre¹⁰ further found that AFS, citrinin, deoxynivalenol, fumonisins, sterigmatocystin, ZEN, cyclopiazonicacid, gliotoxin, patulin and some trichothecenes are the main mycotoxins that have been identified in rice with a high variable of contaminated varieties and at different infected levels.

In Egypt, several studies on fungi and mycotoxins have been focused on food grains including rice since rice is the main food of most people in the world, the presence of mycotoxin contamination can be a serious health risk, corn, soybean and peanuts.^11,12 Therefore, the present study was carried out for identification of species of fungi contaminating rice grains as well as for evaluation of mycotoxin levels in the tested samples and in cultures of toxinogenic fungi.

Materials and Methods

Collection of Rice Samples

A total   of  51 rice  samples  were  collected  from  the market  at  different  localities of El-Minia governorate covering El-Edwa, Maghagha, Bni Mazar, Mattay, Samalott, Minia, Abu-Qurqas, Mallawi, Deir Mawas between March 2015 and March 2016 (Fig.1). Collected samples kept in plastic bags were transported immediately to laboratory and kept at 5-7°C in the refridgerator till mycological and mycotoxin analysis were carried out on them.

Figure 1: Map of El-Minia Governorate showing different places from which rice samples were collected.   Click here to view figure

Isolation of Fungi

The method of seed-plate (direct plating) was utilized to determine the seed borne fungi on the rice grains. The grains were then plated on a suitable isolation media at a plating rate of 5 rice  grains per plate and four replicates for each rice sample.¹³

A general purpose enumeration agar medium, Dichloran Rose Bengal Chloramphenicol Agar: DRBC, which contained (g/l of distilled water): glucose 10, peptone 5, potassium dihydrogen   phosphate 1, magnesium sulphate 0.5, dichloran 0.002 (0.2% in ethanol 1ml), rose bengal 0.025, chloramphenicol 0.1, agar 15, pH 5.6.

A selective isolation medium, Dichloran 18 % Glycerol agar, (DG18) was used to detect and isolate xerophilic fungi, the medium DG18 containing (g/l of distilled water): peptone 5, glycerol 220, chloramphenicol 0.1, glucose 10, potassium dihydrogen phosphate 1, magnesium sulphate 0.5, dichloran 0.002 (0.2% in ethanol 1 ml), agar 15, final pH 5.6. Both DRBC and DG18 media were prepared as described by ¹³.

All plates were inoculated with rice grains (5 grains/plate). Cultures in quadriplicates were incubated for 7-8 days at 30°C but plates containing DG18 were incubated for 14 days to allow growth of slow growing fungi.

Identification of Isolated Fungi

Fungi isolated from rice grain samples were transferred to fresh Czapek`s Dox medium in Petri dishes and slant media bottles for identification. Morphological and cultural characteristics of the  growing fungi were evaluated for preliminary identification. Then fungal colonies were subjected to microscopic identification according to.^14-20

Analysis of Mycotoxins

The isolated fungi were screened for mycotoxin production by growing on Potato Dextrose Broth (PDB). Erlenmeyer flasks (250 ml) containing 50 ml aliquots of Potato Dextrose Broth were autoclaved, inoculated with fungi, and incubated for 7 days at 30°C. After incubation period, the extraction of mycotoxins was carried out according to.^21-23 The flask contents were blended using surface sterilized hand free blender and 50 ml of chloroform was added to flasks which were shaken for 24 hours. Cultures were filtered using what man NO.1 filter paper. Fifty ml of this filtrate   was shaken with an equal volume of chloroform for 30 min. The chloroform layer was separated using a separating funnel and filtered again over a bed of anhydrous sodium sulfate. Porcelain chips were added to flasks containing filtrates and were steam evaporated.

Estimation of Mycotoxins

Thin layer chromatography (TLC) was used for detection of mycotoxins.²⁴ About 50 µl of chloroform extract of the mycotoxin was applied on silica gel plates together with specific standards   developed with mobile phase methanol: chloroform (4:96) and observed under long wave length UV light(365 nm) in a UV chamber CN-15. LC Vilber Lourmat, France. Qualitative detection of     mycotoxins was done on the basis of their fluorescence and retention factor (RF) values.²⁵

Mycotoxin Detection in Rice Samples

Twenty four samples of rice grains were chosen for this part of study. Samples were selected on the basis of their content of potentially toxinogenic fungi.

One hundred grams of rice grains and 150 ml chloroform were mixed in 250 ml Erlenmeyer flasks. Flasks were shaken for 24 hours then filtered through What man NO.1 filter paper. Detection of mycotoxins in the rice extract was done as mentioned above using TLC plates.

Results

Using two isolation media (DRBC and DG18) it was possible to isolate and identify 54 fungal species attributed to 21 genera from the tested rice samples.

It was observed that on the on DRBC medium as shown in Table 2 the total gross fungal population reached 653 colonies per 20 grains in all samples. The mycological analysis of 51 samples revealed the isolation of 52 species related to 20 genera of fungi. Aspergillus was the most dominant genus, being isolated from 49 samples matching 96.07% of rice samples and 63.08% of total fungal population. Nineghteen species of Aspergillus were isolated of which A. flavus was the most dominant species (62.74% of samples). Two species of Aspergillus occurred in moderate incidence and these were A. candidus and A. niger (35.29% and 45.09% of samples matching 8.57% and13.93% of total fungal population respectively). A. terreus was isolated in low frequency being recovered from 17.64% of rice samples accounting for 2.14% of total fungal count.

Table 1: Total count (colonies/20 grains) and number of species (NS) of fungal species isolated from rice grains.

Grade 1= less than 5%, grade 2 = from 5% to 10% and grade 3  = more than 10%.

The genus Penicillium appeared in high incidence (54.90% of samples) giving rise to 21.89% of total fungi. Eleven species of Penicillium were recovered, among which P. chrysogenum and P. islandicum were of low incidence (17.64% and 19.60% of rice samples respectively).

Each of Alternaria and Cladosporium were found to contaminate low number of rice samples (15.68% and 21.56%) participating in fungal population with 2.45% and 3.98% respectively.

On DG18

A total of 47 species of fungi belonging to 16 genera were isolated. Aspergillus was the most dominant genus (49 samples matching 96.07% of rice samples) representing 65.51% of total fungal counts. It was represented by 19 species, of which A. candidus and A. niger prevaled in 52.94% and 50.98% of samples matching 8.28% and 6.93% of total fungal counts respectively as shown in Table (2).

Table 2: Total count (TC), percentage total count (%TC), Incidence (I) out of 51 samples and percentage incidence (%I) of fungal species recovered from rice samples on DRBC and DG18 Media.

High incidence= 50-100%.

Moderate =25-< 50%.

Low=13-< 25%.

Rare=1-12.

Six species of Aspergillus appeared in moderate incidence and these were A. chevalieri, A. flavus, A. flavus var. columnaris, A. montevidensis, A. rubrum and A. versicolor (31.37%, 37.25%, 25.49%, 45.09%, 33.33% and 31.37% respectively). The following 5 species of Aspergillus (A. clavatus, A. fumigatus, A. ochraceus, A. oryzae and A. terreus) were found to contaminate low number of rice samples (15.68%, 15.68%, 15.68%, 15.68% and 17.64%) participating in fungal population with 1.55%, 1.65%, 1.55%, 0.93% and 1.65% respectively.

The genus Penicillium appeared in high incidence (62.74% of samples) giving rise to 18.42% of total fungi. Eleven species of Penicillium were recovered, among which, P. aurantiogriseum, P. chrysogenum, P. citrinum, P. duclauxii, P. glabrum, P. islandicum and P. oxalicum were of low incidence (13.72%, 17.64%, 21.56%, 15.68%, 13.72%, 15.68% and 15.68% respectively).

Each of Alternaria and Cladosporium occurred in moderate incidence (25.49 % and 27.45%) participating in fungal population with 3.51% and 4.03% respectively. Lichtheimia corymbifera was found to contaminate low number of rice samples (15.68%) accounting for 1.86% of total fungal count.

The majority of fungi isolated on DRBC were also recovered on DG18 but the following differences were observed:

Each of A. chevalieri, A. montevidensis and A. rubrum were isolated in moderate frequency on DG18 but they were of rare incidence on DRBC. This is often due to the osmophilic character of these species.

Aspergillus niger which occurred in high frequency (50.95%) on DG18 , was found to be of moderate incidence on DRBC (45.09%). On the other hand A. flavus occurred in high incidence on DRBC (62.74%) but was moderately found on DG18 (37.25%).

Each of A. clavatus, A. fumigatus and A. ochraceus were of low frequency on DG18 but they were rare on DRBC. Aspergillus oryzae occurred in low incidence on DG18 (15.68%), while it was absent on DRBC.

Cladosporium genus occurred in low incidence on DRBC (21.56%) but was moderately found on DG18 (27.45%).

Each of Penicillium aurantiogriseum, P. citrinum, P. duclauxii, P. glabrum and P. oxalicum, were isolated in low frequency on DG18 but they were of rare incidence on DRBC.

Fusarium genus and F. semitectum which occurred in low incidence on DG18 (15.68%) was found to be of rare frequency on DRBC (9.80% and 5.88%).

Lichtheimia corymbifera appeared in low frequency on DG18 (15.68%) but was rarely found on DRBC (7.84%), Alternaria alternata was isolated in moderate frequency on DG18 (25.49%) but it was of low incidence on DRBC (15.68%).

Natural Occurrence of Mycotoxins 

With reference to Table (3), twenty four samples  of  rice  were randomly selected and analyzed for natural  occurrence of  mycotoxins, only 3 samples were found contaminated with 3 different types of mycotoxins namely streigmatocystin (10-20 µg/ kg), ochratoxin-A (50-100 µg/ kg ) and aflatoxin B1(100-200 µg/ kg) ranging from  10 to 200 µg/ kg. These samples were collected from El- Minia City (NO.33), Mattay (NO.19) and Samalott (NO.22), respectively as shown in Table 3. Other tested rice samples were free from any mycotoxin contamination.  All of the toxin contaminated samples were of grade 3 where more than 10% of grains were broken. Moreover, samples 19 and 22 were infested with rice weevil. The presence of A. flavus is of main concern because the fungus is a potent aflatoxin producer.

Table 3: Natural occurrence of mycotoxins in rice grain samples.

Mycotoxins Produced by Fungal Strains

Twenty eight fungal isolates belonging to A. flavus (21 isolates), A. ochraceus (3), A. parasiticus (1), A. fumigatus (1) and A. terreus (2) were analyzed for mycotoxin production using TLC technique. as general these  strains  were  able  to  produce  aflatoxins, Ochratoxin-A, fumagillin,  Gliotoxin  and  terrein,  respectively (Table 4).

All isolates of A. flavus produced aflatoxins in variable degrees as shown in Table 4.  Levels of aflatoxins (B1, B2, G1, and G2) at levels ranging from 10 to 300 ug/L. A. parasiticus produced B1 and G1 (10-20 ug/L). A. terreus (2 isolates) were able to elaborate terrein (10 -300 ug/L).  Ochratoxin-A (20 -200 ug/L) was produced by 3 isolates of A. ochraceus. Finally, A. fumigatus produced Gliotoxin, fumagillin (10-20 ug/L for each).

Table 4: Mycotoxins produced by fungal strains (positive strains).

AUMC,  Assiut university mycological centre.

Discussions

These results showed that the total fungal counts which were prduced on two medium types were revealed the most predominant genus were Aspergillus, Penicillium, Alternaria, cladosporium and Fusarium respectively.

This finding corresponding with previous  studies recorded by^26,27 who investigated the mycobiota of rice which were grown on two isolation media including DRBC (dichloran rose- bengal chloramphenicol agar) and DG18 (dichloran 18% glycerol agar) media and he reported that a total of sixty two species related to 34 genera. The broadest species spectrum were from the genera  Aspergillus, Penicillium, Eurotium followed by Fusarium, Cladosporium and Cochliobolus. Rice grains were mostly contaminated by Aspergillus candidus, A. flavus, A. niger, A. amstelodami, A. rubrum, Penicillium citrinum, P. oxalicum and Talaromyces spp.

In Swed, Fredlund et al.²⁸ collected 99 rice samples from the Swedish retail market. Aspergillus was the most common fungal genus identified but also Penicillium, Eurotium, Cladosporium, Wallemia, Alternaria, Epicoccum and Trichotecium were isolated. A. flavus presented in 21% of the samples.

Abdel-Hafez et al.²⁹ sampled Egyptian paddy rice and isolated Aspergillus flavus, A. sydowii, A. terreus, A. fumigatus and A. ochraceus) and Penicillium species (P. chrysogenum and P. corylophilum) along with Fusarium oxysporum, Alternaria alternata, Cladosporium cladosporioides, Trichoderma viride and Mucor racemosus. Abd-Allah & Ezzat¹¹ Sampled Paddy rice from EI-Sharkia, E1-Dakahlia , EI-Gharbia, and Kafr E1-Shekh governorates and recorded an average of 6.79 x 10⁴ fungal spores per gram rice. The fungal isolates were 47 species belonging to twenty eight genera. Aspergillus, Cladosporium and Penicillium were the most predominant genera. Aspergilli were represented by 22 species, Aspergillus niger and A. flavus had the highest occurrence. Previous studies on Egyptian rice, reported by El-Shanshoury et al.¹² who surveyed the incidence and load of fungi and aflatoxins in maize, wheat and peanut, collected from some markets in central Delta provinces, Egypt.

Other studies on the contamination of storage rice in many regions as compared to this study have been reported.^{30,31,32,33,2,28,34,35,36,37} Sales and Yoshizawa^38,39 studied occurrence of Aspergillus section Flavi in rice from Philippines. Toman et al.⁴⁰ collected 60 samples of white and  parboiled  rice purchased  from the  Czech  food   market.  It was reported that Ochratoxin-A (OTA) analysis showed that 58 samples (96.7%) were found to be positive. OTA levels in white and parboiled rice fluctuated from 0.05 to 0.17 ug/g. These all finding are almost in agreement with our result in this part of the study.

The natural occurrence of mycotoxins in rice has been studied in different countries of the world: In Nigeria,² studied fungi and some mycotoxins contaminating rice., high levels of AFB1 contamination in rice have also been reported at 200.19± 320.98µg/ kg² and  37.2±14.0µg/ kg.⁴¹ Also,⁴² determined the mycobiota associated with rice (Oryza sativa), maize (Zea mays), and millet (Pennisetum typhoiodes) in storage.

While⁴³ screened samples of rice, maize, cocoa and cocoa-based powder beverage collected from different markets and stores in south-western Nigeria.

In China, different studies on rice and other cereal contamination with mycotoxins have been reported;^{44,45,6,46,47} Other studies included those of India;^48,49,50,51 Korea;^52,53 Spanish;^54,55,10 Turkey;^56,57 Canada,³ South America.⁵⁸ and Pakistan.⁵⁹

In South Vietnam, Trung et al.³³ screened twenty five samples of Vietnamese rice coming from the Mekong Delta for fungal contamination. Ergosterol content measurement and total fungal load determination indicated that moulds contamination was quite weak. Identification of fungal species revealed that Aspergillus was the most common genus (43.75 % of isolates) followed by Fusarium (21.8 %) and Penicillium (10.9 %). The presence of toxigenic strains such as A. flavus, A. ochraceus and P. citrinum was confirmed by cultures. Fungal strains were tested for their toxinogenic potential and the results showed that 80 % of A. flavus strains were able to synthesized cyclopiazonic acid (levels up to 32.3 ppm), all strains of A. ochraceus produced ochratoxin A (one at 178 ppm) and the studied strain of P. citrinum was moderately toxigenic for citrinin. Also two rice samples were found contaminated with high level of ochratoxin A (21.3 and 26.2 ppb). This contamination can probably be linked to unfavorable post harvest storage and climatic conditions.

Also Sani and Sheikhzadeh discussed the different methods of aflatoxin (AFT) degradation in rice. Mycotoxins are mainly present in cereal grains such as rice and are not completely destroyed during their processing and cooking.⁶⁰

Detection of aflatoxins as aprimary mycotoxin in stored rice was also reported by demonstrated time to time by.^61,62,48 These mycotoxins were detected on the basis of fluorescence and retention factors (R.F.) values. Presence of them was confirmed by long wave UV light.

The retention factors of all the mycotoxins produced were determined, this was done by thin layer chromatography. Konishi et al.^44,63 studied aflatoxins and other mycotoxins in rice from China and Japan, respectively. Subsequently, Tanaka et al.⁶⁴ who find the variation in mycotoxin contamination in rice from different regions may be due to differences in toxigenic microflora influenced by different agricultural practices and the differences in climate and also their storage conditions, Taligoola et al.^65,27 morover reported toxigenic fungi and mycotoxins in rice.

Earlier, Madsen & Rasmussen⁶⁶ reported the presence of AFB1 contamination in milled rice. Prasad et al.⁶⁷ screened sixty five samples of stored rice and twelve were positive for aflatoxin. Levels of aflatoxins ranged between 84 to 2830 µg/ kg mycelium.

Recently, Kushiro; Toman et al.; Xiang et al.; Sani & Sheikh zadeh; Urooj et al.; Mukhtar et al.; Aingkharat et al.; Majeed et al.,^{68,40,69,60,70-73} were studies contamination of storage rice and other cereals by different fungal toxins.

Conclusion

Our results suggest that there is a need for proper storage of rice seed to minimize the fungal contamination and their mycotoxin production. Aflatoxins and ochratoxin are among the five most significant and abundant mycotoxins contaminating foods and food stuffs in the world⁷⁴ and have also been shown in this work to be major contaminants of rice in El-Minia Government.

Acknowledgements

Authors thanks, staff members of Assiut university mycological centre (AUMC), Egypt for their support in this research.

Conflict of Interest

There is no conflict of interest.

Funding Source

There is no funding source.

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