МИКРОБИОЛОГИЯ, 2011, том 80, № 5, с. 707-713
= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
MOLECULAR IDENTIFICATION OF ASPERGILLUS AND EUROTIUM SPECIES ISOLATED FROM RICE AND THEIR TOXIN-PRODUCING ABILITY
© 2011 n D. Y&zdani", b, M. A. Zainal Abidin", *, Y. H. Tan", and S. Kamaruzaman"
aDepartment of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia bDepartment of Biotechnology, Institute of Medicinal Plants, ACECR, 131451446, Tehran, Iran
Received December 19, 2010
Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holo-morphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.
Keywords: rice, Aspergillus, Eurotium, DNA sequencing, aflatoxin.
The genus Aspergillus is a group of filamentous fungi consist of more than 250 species and the number will continue to grow as scientists discover new species over time. At present, Aspergillus is among the most economically important of the fungal genera. They are responsible for production of several toxins, including aflatoxin and ochratoxin A [1].
Identification of the Aspergillus species based on the morphological characteristics is not stable because some morphological features do not present in all isolates of a species and their presence can vary among cultures of the same isolate. Besides, physiological characters of As-pergillus vary as some metabolites may absent totally in some isolates. Meanwhile, DNA sequence data obtained from molecular approaches are very useful in identification of the Aspergillus species. However, there is no strict criterion on the line drawing between phylogenetic species. Therefore, none of the morphological, physiological or molecular methods works perfectly in identification of the Aspergillus species [2]. To date, several Aspergillus species have been reported in rice. Among them are E. amstelodami, E. chevalieri [3—5], A. flavus, A. fumigatus [6—8], and A. tamarii [3]. In Malaysia, A. niger, A. Candidus, A. flavus, A. fumigatus and A. versicolor have been reported in rice [8]. Besides, some studies have reported A. parasiticus and A. ochraceus as the producers of aflatoxin and ochratoxin A, respectively in rice [9, 10]. Rice (Oryza sativa L.) is one of the most important staple food crops in Malaysia and approximately 668000 hectares rice is grown in Peninsular Malaysia [11]. The chem-
* Corresponding author, e-mail: zainal@agri.upm.edu.my Tel: +603-89466986; Fax: +603-86560698;
ical composition of rice grain make it an ideal substrate for the establishment and growth of some fungal species, especially toxigenic fungi including Aspergillus [3]. Moisture content, relative humidity, temperature, period of storage, initial levels of contamination, toxigenic potential of fungal strains influence the production of my-cotoxins [12].
Considering the economic and nutritional importance of rice, this research was conducted to determine the Aspergillus species contaminating rice under natural conditions in Peninsular Malaysia using nucleotide sequences ofthe internal transcribed spacers 1 & 2 region of rDNA and determination of their toxin producing ability using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), the most common used methods for the detection of mycotoxins [13].
MATERIALS AND METHODS
Collection of rice samples. A total of 29 milled rice samples were collected from retailers in four states (Selangor, Perak, Penang and Kedah) of Peninsular Malaysia in October and November of 2008. All samples were stored in nylon bag and kept at 4°C before use.
Isolation of Aspergillus spp. from rice samples. The As-
pergillus and Eurotium species were isolated from rice grains using agar plating method on malt extract salt agar (MESA: malt extract 20 g, NaCl 75 g, agar 15 g in 1 L distilled water) without surface disinfection. Four hundred seeds of each sample were subjected to MESA plates and incubated at 28°C for 7 days [14].
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Extraction of toxins. The isolates of Aspergillus were grown on yeast extract sucrose agar (YES; 2% yeast extract, 15% sucrose, 1.5% agar) as a single colony in the centre ofplates and incubated at 28°C for 3 days. Extraction was conducted as described [15, 16] with some modifications. Briefly, colony margins together with adjacent surrounding zones of cultures were scraped into a large test tube (32 x 200 mm) containing 10 ml ofchloro-form : acetone (85 : 15 v/v). The suspension was left at room temperature (25°C) for 15—20 min and agitated every 5 min using a vortex stirrer. The extract was then filtered through Whatman No. 1 filter paper. The filtrate was evaporated to dryness under 40°C in an air circulated oven dryer. The residue was resuspended in 500 ^l of methanol and filtered using a 0.2 ^m syringe filter (Whatman, GD/x 13 mm). Samples were stored at 4°C before using for TLC and HPLC tests.
TLC test. TLC was carried out on a silica gel 60 plate 20 x 20 cm (Merck) using toluene : chloroform : acetone (15 : 75 : 10 v/v) for aflatoxin and acetonitrile : acetic acid : methanol (90 : 5 : 5 v/v) as mobile phases for OTA detection. 5 ^l ofB1, B2, G1 and G2 aflatoxin ("Supelco", USA) and OTA standards ("Sigma-Aldrich", Germany) with concentration of 1 ^g ml-1 each and 20 ^l of test samples were spotted on TLC plates and was run for 45 min in a TLC tank at room temperature. Plates were observed under UV light at 254 and 365 nm.
HPLC test. Confirmation of TLC results were performed using a simultaneous program for determination of aflatoxin and OTA as described by [17, 18]. The toxins were filtered and 20 ^l of each extracted toxin were injected into a Shimadzu Liquid Chromatograph LC-20AT fitted with a fluorescence detector (Shimadzu RF-10AXL) and with a column C18 Wakosil II (SGE Analytical Science, Australia), 250 x 4.6 mm. The HPLC was run using a mobile phase with a flow rate of 1 ml min-1, consisted of three solvents (A: 100% methanol, B: 100% acetonitrile and C: water with 0.1% acetic acid). The gradient was 0-12 min isocratic 25% A, 15% B, 60% C, 1214 min linear gradient to 10% A, 50% B, 40% C, 1424 min held at 10% A, 50% B, 40% C and immediately returned to 25% A, 15% B, 60% C at minute 24 and followed by a 2 min delay for equilibration. The excitation/emission wavelengths for aflatoxin from 0-14 min and ochratoxin from 21-25 min were 365/455 nm and 330/460 nm, respectively.
Identification of isolates using nucleotide sequences of ITS1 and ITS2
1. Culture preparation and DNA extraction. All Aspergillus colonies were prepared on CYA at 28°C for 5 days and DNAwas extracted according to Liu etal. [19] with some modifications. Fungal mycelium (200300 mg) except for A. niger and A. fumigatus were directly collected from culture plates using a sterile toothpick and added to 1.5 mL Eppendorftube containing 500 ml ofly-sis buffer (400 mM Tris-HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate).
For A. niger and A. fumigatus, the cultures were prepared in 50 ml tubes containing 20 ml of Potato Dextrose Broth (Difco) with incubation for 48 h in an orbital shaker (300 rpm) at 28°C. Mycelium was then filtered using Whatman No. 1 filter paper and washed with sterile distilled water, frozen in liquid nitrogen and ground to a fine powder using mortar. The powder was added to 1.5 ml Eppendorf tube containing 500 ml of lysis buffer.
The tube was left at room temperature for 10 min. After adding 150 ml ofpotassium acetate (pH 4.8), the tube was vortexed briefly and spun at 10000 g for 1 min. Then, the supernatant was transferred to a new 1.5 ml Eppendorf tube and an equal volume of phenol : chloroform : isoamyl alcohol (25 : 24 : 1) was added. The tube was vortexed briefly and spun at 10000 g for 1 min. This method was repeated before transferring the supernatant to another 1.5 ml Eppendorf tube. It was followed by addition of an equal volume of isopropyl alcohol and the suspension was mixed by inversion briefly. The tube was spun at 10000 g for 3 min and the supernatant was discarded. The DNA pellet was washed in 300 ml of 70% ice-cold etha-nol. After the pellet was spun at 10000 rpm for 3 min, the supernatant was discarded and this step was repeated. Lastly, the DNA pellet was air dried and dissolved in 50 ^l of deionized water. The quality of DNA was determined by agarose gel electrophoresis.
2. PCR amplification and DNA sequencing. The amplification of ITS1-5.8S-ITS2 regions of rDNA was performed in 50 ^l PCR Master Mix ("Fermentas", Canada) consisting of 200 nM oligonucleotide primers and 1 ^l of DNA template. Oligonucleotide primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') were used [20]. The amplification was performed in a thermal cycler (Biometra® T3, Syngene, UK) programmed for pre-denaturation of5 min at 95°C, 35 cycles of1 min at 95°C, 1 min at 55°C and 2 min at 72°C. After a final extension of 5 min at 72°C, the samples were cooled to 4°C. The PCR products were analyzed in 1% (w/v) agarose gel in 1x TBE buffer (Tris-Borate-EDTA, "Sigma"). The gel was stained in 0.5 ^g ml-1 ethidium bromide solution and the bands visualized and photo
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