A molecular method for identification of aflatoxigenic fungi in pistachio of Khorasan region (Gonabad and Feyzabad)

مجله پژوهشهای علوم و صنایع غذایی ایران

Article 16, Volume 12, Issue 2 - Serial Number 38, May and June 2016, Pages 318-329   

Document Type: Research Article

DOI: 10.22067/ifstrj.v1395i2.42364

Authors

Nasim Pourebrahim^{* 1}; Masoud Yavarmanesh²

¹Islamic Azad University of Quchan

²Ferdowsi University of Mashhad

Abstract

Introduction:Pistachio nut is one of the popular tree nuts. Among the different species of the genus Pistacia, only the fruits of Pistacia vera attain optimal size to be acceptable to consumers as edible nuts. Contamination of pistachio by Aspergillus species and their mycotoxins is the most important problem for consumption and export of this product. Aflatoxins are potent toxic, carcinogenic and mutagenic secondary metabolites primarily produced by two fungal species, Aspergillus flavus and Aspergillus parasiticus. Aspergillus flavus produces AFB1 and AFB2, while Aspergillus parasiticus produces AFB1, AFB2, AFG1 and AFG2. Among four main groups of aflatoxins, AFB1 is the most potent carcinogenic compound. Therefore, identification of toxigenic fungi is necessary for evaluating the foods quality and the presence of mycotoxins. The current methods being used for assessing fungi presence in foods based on cultivation methods and microscopic characteristics are time-consuming and labor-intensive. Recently, molecular techniques such as polymerase chain reaction (PCR) due to high sensitivity, specificity and rapidity has been introduced as powerful tools for detecting toxigenic fungi. Many genes involved in the biosynthesis of these mycotoxins have been identified and their DNA sequences have been published. PCR methods can be used to detect of aflatoxigenic Aspergilli based on structural genes (nor1, ver1 and omtA) encoding key enzymes in aflatoxin biosynthesis pathway and the regulatory gene aflR.
Materials and method: Pistachio samples were collected from different cultivation regions of two towns including Gonabad and Feyzabad. Samples were packed in sterile plastic bags and immediately transferred to the laboratory. The moisture content of samples was determined using thermal method and drying in at 95-100°C. Among fungal isolates 30 Aspergillus genus were detected and purified by cultural-based methods using PDA (potato dextrose agar) medium. Colonies of the fungus were transferred to PDB (potato dextrose broth) medium and incubated for 5 days at 28°C with shaking at 150 rpm. The mycelium was frozen in liquid nitrogen and ground to a powder for later DNA isolation. DNA was extracted with CTAB (cetyl trimethyl ammonium bromide) extraction buffer, then was purified with organic solvents such as chloroform/isoamyl alcohol and finaly was precipitated by isopropanol. Aspergillus genus were detected using polymerase chain reaction by specific primer pair Asp1/Asp2 for amplification of 18S rRNA region. Furthermore, aflatoxigenic genes were detected by three sets of primers (APA-450/APA-1482, ver1/ver2 and OMT-208/OMT-1232). PCR was performed in a volume of 25 µl containing 0.5 µl of each primer, 12.5 µl of Taq DNA polymerase master mix red, 10.5 µl of sterile distilled water and 1 µl of genomic DNA as template. A PCR consisted of an initial denaturing step of 5 min at 94°C followed by 35 cycles (30 s at 94°C, 35 s at 65°C and 40 s at 72°C) finished by a final extension step at 72°C for 10 min. The PCR products were analyzed by electrophoresis on a 1% agarose gel in TBE.
Results and Discussion: Among fungal isolates 30 Aspergillus genus were detected using microscopic characterstics and colony color. Under the microscope, conidia were one-celled, spherical, hyaline or pigmented and they formed long chains. 12 and 4 out of 30 samples had omtA and ver1 genes respectively. No observation was found for aflR regulatory gene in the fungal isolates. The results showed that although some isolates had one or two structural genes in the aflatoxin biosynthetic pathway, they could not produce aflatoxin due to not having any aflR gene. Coefficient of correlation was calculated to find the relationship between the existence of Aspergillus molds and aflatoxigenic genes in pistachio. The statistical results indicated that there is a significant correlation between the enumeration of Aspergillus molds and the existence of genes (omtA and ver1) in different moisture domains (p> 0.05) while no significant correlation was identified between the enumeration of Aspergillus molds and the existence of genes in different domains of enumeration of mesophilic bacteria, yeasts and molds. Contamination of nut seeds by fungi occurs during growth, harvesting, transport and storage. The production of aflatoxin is affected by different factors, such as genetic properties of the producing fungi, temperature, moisture content, the chemical composition of food and antimicrobial agents produced by other microorganisms. Water stress and temperature are the most relevant environmental factors which influence fungal growth and mycotoxin production. Other studies showed that there was a good correlation between the expression of an early structural gene (aflD) and aflatoxin B1 production in peanut seeds. Also previous studies have shown that there was a significant relationship between A.flavus contamination in the peanuts and pistachio with high humidity (p> 0.05). Since other factors such as temperature, pH and chemical composition of pistachio can affect the existence of Aspergillus molds and expression of aflatoxigenic genes, the influence of these factors on existence of Aspergillus molds and genes involved in aflatoxin biosynthesis pathway need to be investigated.

Keywords

Aspergillus; Aflatoxin; Pistachio; Aflatoxigenic genes; Polymerase chain reaction

References

ارمی، م.، صفاری، م.، پوربخش، ع و هاشمی، ج.، 1390، ردیابی ژن‌های مشارکت کننده در تولید آفلاتوکسین در تخم مرغ‌های مشکوک به آلودگی جهت غربالگری اولیه آن‌ها. مجله علمی پژوهشی دانشگاه علوم پزشکی اراک، دوره 14(2)، 9-1.

رحیمی، پ.، شریف نبی، ب. و بهار، م.، 1386، گونه‌های آسپرژیلوس جدا شده از میوه‌های پسته و بررسی تولید آفلاتوکسین در آن‌ها. رستنیها، دوره 8 (1)، 42-30.

رزاقی ابیانه، م.، پیله ور سلطان آبادی، ی.، شمس قهفرخی، م. و علی نژاد، س.، 1390، آفلاتوکسین‌ها و اهمیت آن‌ها در بهداشت عمومی و کشاورزی. انتشارات موسسه آموزش عالی علمی کاربردی جهاد کشاورزی، تهران، 31-18.

علامه، الف. و رزاقی ابیانه، م.، 1380، مایکوتوکسین‌ها. انتشارات دانشگاه امام حسین، تهران، چاپ اول.

مؤسسه استاندارد و تحقیقات صنعتی ایران، 1386، مغز پسته- ویژگی‌ها و روش‌های آزمون. تجدید نظر چهارم، شماره 218.

مؤسسه استاندارد و تحقیقات صنعتی ایران، 1386، میکروبیولوژی مواد غذایی و خوراک دام- روش جامع برای شمارش کلی میکروارگانیسم‌ها در 30 درجه سلیسیوس. تجدید نظر اول، شماره 5272.

مؤسسه استاندارد و تحقیقات صنعتی ایران، 1387، میکروبیولوژی مواد غذایی و خوراک دام- روش جامع برای شمارش کپک‌ها و مخمرها- قسمت دوم: روش شمارش کلنی در فراورده‌های با فعالیت آبی (aw) مساوی یا کمتر از 95/0. چاپ اول، شماره 2-10899.

مؤسسه استاندارد و تحقیقات صنعتی ایران، 1389، مواد غذایی- نمونه برداری از مغزهای درختی، بادام زمینی، سایر دانه‌های روغنی و مغز هسته زردآلو و مشتقات آن‌ها برای آزمون آفلاتوکسین- روش آزمون. چاپ اول، شماره 13534.

نجفی کهکی، الف.، 1388، ردیابی گونه‌های آسپرژیلوس فلاووس و آسپرژیلوس پارازیتیکوس تولید‌کننده آفلاتوکسین به روش مولکولی در پسته. پایان نامه کارشناسی ارشد، کتابخانه دیجیتال دانشگاه تهران.

Abdel-Hadi, A., Carter, D., Magan, N., 2010, Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different environmental conditions. Journal of Applied Microbiology, 109, 1914-1922.

Calvo, A. M., Wilson, R. A., Bok, J. W., Keller, N. P., 2002, Relationship between secondary metabolism and fungal development. Microbiology and Molecular Biology Reviews, 66(3), 447-459.

Charmley, L. L., Rosenber, A., Trenholm, H. L., 1994, Factocrs responsible for economic losses due to Fusarium mycotoxin contamination of grains, foods and feedstuffs. In: Miller, J. D., Trenholm, H. L., eds, Mycotoxins in grain: compounds other than aflatoxin, St. Paul,MN: Eagan Press, 471-456.

Chen, R.S., Tsay, J.G., Hung, Y.-F., Chion, R.Y.-Y, 2002, Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction. Journal of Food Protection, 65, 840-844.

Criseo, G., Bagnara, A., Bisignano, G., 2001, Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group. Letters in Applied Microbiology, 33, 291-295.

Diener, U. L. and Davis, N. D., 1967, Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids by Aspergillus flavus in sterile peanuts. Journal of the American Oil Chemists’ Society, 44(1967), 259-263.

Doyle, J.J., Doyle, J.L., 1990, Isolation of plant DNA from fresh tissue. Focus, 12, 13-15.

European Commission (EC)., 2010, amending regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Official Journal of the European Union, L50, 8-12.

FAOSTAT., 2012, FAO Production, Consumption, Resources Statistics [on-line]. FAO.

Geisen, R., 1996, Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. Systematic and Applied Microbiology, 19, 388-392.

Gourama, H. and Bullerman, L., 1995, Aspergillus flavus and Aspergillus parasiticus: Aflatoxigenic fungi of concern in foods and feeds: A review. Journal of Food Protection, 58, 1395-1404.

Kabirian H.R., Afshari H., Mohammadi Moghadam M. and Hokmabadi H., 2011, Evaluation of pistachio contamination to Aspergillus flavus in Semnan provinc. International Journal of Nuts and Related Sciences, 2(3), 1-6.

Khodavaisy, S., Maleki, A., Hossainzade, B., Rezai, S., Ahmadi, F., Validi, A., Rashidi, A., Ghahramani, E., 2012, Occurrence of fungal contamination in pistachio and peanut samples from retail shops in Sanandaj province, Iran. African Journal of Microbiology Research, 6(39), 6781-6784.

Konietzny, U. and Greiner, R., 2003, The application of PCR in the detection of mycotoxigenic fungi in foods. Brazilian Journal of Microbiology, 34, 283-300.

Melchers, W. J., verweij, P. E., Van den Hurk, P., Van Belkum, A., De Pauw, B. E., Hoogkamp-korstanje, J. A., Meis, J. F., 1994, General primer-mediated PCR for detection of Aspergillus species. Journal of Clinical Microbiology, 32(7), 1710-1717.

Moghaddam, M. M., Goltapeh, E. M., Hokmabadi, H., Haghdel, M., & Mortazavi, A. M., 2006, Evaluation of susceptibility of pistachio cultivars to aflatoxigenic Aspergillus flavus and aflatoxin B1 production. Acta Hort. (ISHS), 726, 655-658.

Mojtahedi, H., Rabie, C. J., Lubben, A., Steyn, M. & Danesh, D., 1979, Toxic Aspergillus from pistachio nuts. Mycopathologia, 67, 123-127.

OBrian, G. R., Georgianna, D. R., Wilkinson, J. R., Yu, J., Abbas, H. K., Bhatnagar, D., Cleveland, T. E., Nierman, W. C., Payne, G. A., 2007, The effect of elevated temperature on gene transcription and aflatoxin biosynthesis. Mycologia, 99, 232-239.

Rahimi, P., Sharifnabi, B., & Bahar, M., 2008, Detection of aflatoxin in Aspergillus species isolated from pistachio in iran. Journal of Phytopathology, 156, 15-20.

Razzaghi-Abyaneh, M., Shams-Ghahfarokhi, M. and Chang, P.-K., 2011, Aflatoxins: Mechanisms of inhibition by antagonistic plants and microorganisms. In: Guevara-Gonzalez, Ramon G. (Ed.), Aflatoxins: Biochemistry and Molecular Biology. INTECH Open Access Publisher, 285-304.

Schmidt-Heydt, M., Magan, N., Geisen, R., 2008, Stress induction of mycotoxin biosynthesis genes by abiotic factors. FEMS Microbiology Letters, 284, 142-149.

Schmidt-Heydt, M., Abdel-Hadi, A., Magan, N., Geisen, R., 2009, Complex regulation of aflatoxin biosynthesis gene cluster of A. flavus in relation to various combinations of water activity and temperature. International Journal of Food Microbiology, 135, 231-237.

Schmidt-Heydt, M., Rufer, C. E., Abdel-Hadi, A., Magan, N., Geisen, R, 2010, The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression. Mycotoxin Res. 26, 241-246.

Schnerr, H., Vogel, R. F., Niessen, L., 2002, Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat-samples. Letters in Applied Microbiology, 35, 121-125.

Sedaghat, R., 2011, Constraints in production and marketing of iran’s pistachio and the policies concerned: An application of the garret ranking technique. International Journal of Nuts and Related Sciences, 2(1), 27-30.

Shapira R., Paster N., Eyal O., Menasherov M., Mett A. and Salomon R., 1996, Detection of aflatoxigenic molds in grains by PCR. Applied and Enviromental Microbiology, 62, 3270-3273.

Shokraii, E. H. and Esen, A, 1988, Composition, solubility, and electrophoretic patterns of proteins isolated from kerman pistachio nuts (Pistacia vera L.). Journal of Agricultural and Food Chemistry, 36, 425-429.

Simsek, O., Arici, M. and Demir, C., 2002, Mycoflora of hazelnut (Corylus avellana L.) and aflatoxin content in hazelnut kernels artificially infected with Aspergillus parasiticus. Nahrung 46(3), 194-196.

Suanthie, Y., Cousin, M. A., Woloshuk, C. P., 2009, Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penecillium and Fusarium. Journal of stored products research, 45, 139-145.

Trial, F., Mahanti, N. and Linz, J., 1995, Molecular biology of aflatoxin biosynthesis. Microbiology, 141, 755-765.

Vyzantiadis, T-A. A., Johnson, E. M., Kibbler, C. C., 2012, From the patient to the clinical mycology laboraty; how can we optimize microscopy and culture methods for mould identification. Journal of Clinical Pathology, 65, 475-483.

Wen, Y., Hatabayashi, H., Arai, H., Kitamoto, H. and Yabe, K., 2004, Function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 6, 3192-3198.

Xu, H. X., Annis, S., Linz, J. and Trail, F., 2000, Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene, nor-1, in infection hyphae. Physiological and Molecular Plant Pathology, 56(5), 185-196.

Yu, J., Chang, P.-K., Ehrlich, K. C., Cary, J. W., Bhatnagar, D., Cleveland, T. E., Payne, G. A., Linz, J. E., Woloshuk, C. P. and Bennett, J. W., 2004, Clustered pathway genes in aflatoxin biosynthesis. Applied and Environmental Microbiology, 1253-1262.