Mycobiota and Mycotoxins Contaminating Rice Grains in El Minia, Governorate, Egypt

The mycological analysis of 51 samples of rice grains collected from different localities in El-Minia Governorate revealed the isolation of 54 species of fungi belonging to 21 genera. Most common mycobiota (geneus) were Aspergillus and Penicillium being isolated from 96.07% and 54.9% of samples contributing 63.08 % and 21.89% of total fungal count. The prevalent species were represented by Aspergillus flavus, A. candidus, A. niger, Penicillium chrysogenum, P. islandicum especially on dichloran rose bengal chloramphenicol agar medium (DRBC). These species in addition to some osmophilic fungi including A. chevalieri, A. montevidensis, A. rubrum were also common when Dichloran Glycerol agar (DG18) was used for culturing of rice samples. About 12.5% of samples analysed for natural occurrence of mycotoxins were contaminated either with Aflatoxin – B1 (100-200 μg/ kg), ochratoxin –A (50-100 μg/ kg) or sterigmatocystin (10-20 μg/ kg). These samples were of grade 3 (with more than 10% broken grains) and showed infestation with rice weevil. The majority of fungal strains tested for their mycotoxin production in liquid cultures were able to produce variable levels of aflatoxin B1, Aflatoxin G1 (A. flavus and A. parasiticus), Ochratoxin –A (A. ochraceus), terrein (A. terreus), gliotoxin and fumagillin (A. fumigatus).

Rice is one of the most famous and important cereals worldwide. According to 1 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 Sathyanarayana 7 listed 143 fungal species from rice. Reddy et al. 8 published a review contain the important groups of mycotoxins including aflatoxins (AFS), fumonisins , ochratoxin A(OTA), deoxynivlenol, and zearalenone (ZEN) , which contaminated rice in different countries. Sempere & Santamarina 9 confirmed that the majority of mycotoxins were produced by Aspergillus, penicillium, and Fusarium, Ferre 10 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 fungi contaminating rice grains as well as for evaluation of mycotoxin levels in the tested samples and in cultures of toxinogenic fungi.

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 were transported immediately to laboratory and kept in plastic bags at 5-7°C till mycological and mycotoxin analysis.

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 .
All plates were inoculated with rice grains (5 grains/plate). Cultures in quadriplicates were incubated for 7-8 days at 30 o 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][15][16][17][18][19][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 medium were autoclaved, inoculated with fungi, and incubated for 7 days at 30 o C . After incubation period, the extraction of mycotoxins was carried out according to [21][22][23] . Then 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 whatman 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 funnell 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 Whatman 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.
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: a-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. b-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%).
c-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.
e-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.
f-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%).

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.

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 rosebengal 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  Grade 1= less than 5%, grade 2 = from 5% to 10% and grade 3 = more than 10%.  Also some studies about the contamination of storage rice in many regions as compared to this study 30,31,32,33,2,28,34,35,36,37 .While 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 on the Czech food market. 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 ng/g.
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/ kg 2 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.
While 43 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 were showed about rice and other cereal contamination with mycotoxins; 44,45,6,46,47 . In India; 48,49,50,51 . In Korea; 52,53 . In Spanish; 54,55,10 . In Turkey; 56,57 . In Canada, 3 . In South America, 58 . In 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. 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. Liu et al, 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 68,40,69,60,[70][71][72][73] , were studies contamination of storage rice and other cereals by different fungal toxins.

ConClusions
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 world 74 , and have also been shown in this work to be major contaminants of rice in El-Minia Government.
aCknowledGMents Authors thanks, staff members of Assiut university mycological centre(AUMC) ,Egypt for their support in this research.