Identification of Aspergillus section Flavi Species from Peanuts and their Mycotoxigenic Properties

Year 2019, Volume: 17 Issue: 4, 450 - 457, 31.12.2019

İşılay Lavkor

https://doi.org/10.24323/akademik-gida.667256

Abstract

This study is aimed to identify 50 A. section Flavi isolates from peanut samples collected in the Adana and Osmaniye provinces of Turkey by using morphological and polymerase chain reaction (PCR), and to determine the in vitro aflatoxin and cyclopyazonic acid formation properties of the isolates. DNA was extracted for the identification of Aspergillus spp., and β-tubulin gene region on DNA was amplified by PCR. Identification of species was determined by comparing the result of sequences which belong to partial β-tubulin of the sequences with known ribosomal sequences using Basic Local Alignment Search Tool (BLAST). In the study, out of a total 50 fungal isolates, 17 were identified as A. parasiticus, and 33 were identified as A. flavus. As a result of Blast analysis of β-tubulin gene sequence comparisons A. flavus and A. parasiticus showed a major similarity in ranges 89-100% and 96-100%, respectively. Furthermore, all of the A. flavus and A. parasiticus isolates were determined as aflatoxin producers in the range from 0.45 to 4521.64 µg/L and 0.22 to 2562.87 µg/L with high-performance liquid chromatography (HPLC) under in vitro conditions, respectively. Total 32 A. flavus isolates produced cyclopiazonic acid in the range between 0.35-8.21 μg/g with thin layer chromatography (TLC). The present study provides a new method on molecular characterization of A. section Flavi on peanut samples collected from the Adana and Osmaniye provinces of Turkey. This research also demonstrated that A. flavus and A. parasiticus were responsible for mycotoxin contamination on peanut samples collected from Adana and Osmaniye provinces.

Keywords

Aspergillus flavus, Aspergillus parasiticus, PCR, Mycotoxin, Peanut

Yerfıstığından İzole Edilen Aspergillus section Flavi Türlerinin Tanımlanması ve Mikotoksijenik Özelliklerinin Belirlenmesi

Abstract

Bu çalışmada, Adana ve Osmaniye illerinden toplanan yerfıstığı örneklerinden izole edilmiş 50 Aspergillus section Flavi üyesi izolatın morfolojik ve polimeraz zincir reaksiyonu (PZR) yöntemiyle tanımlamaları yapılmıştır. Ayrıca, izolatların in vitro koşullarında aflatoksin ve siklopiazonik asit oluşturma özellikleri belirlenmiştir. Aspergillus spp. tanımlanmasında DNA ekstraksiyonları yapılmış, β-tubulin gen bölgesi ile PZR çoğaltılmıştır. Türlerin tanımlamasında β-tubulin gen bölgesine ait sekans sonuçları Blast (Basic Local Alignment Search Tool)’da bilinen ribozamal sekanslar ile karşılaştırılmıştır. Çalışmanın sonucunda, fungal izolatların 33’ü Aspergillus flavus, 17’si Aspergillus parasiticus olarak tanımlanmıştır. Blast analizi sonucunda ise A. flavus ve A. parasiticus türüne ait β-tubulin gen sekansı karşılaştırmaları sırası ile %89-100 ve %96-100 oranları arasında benzerlik göstermiştir. Ayrıca, A. flavus ve A. parasiticus izolatlarının tümü in vitro koşullarında yüksek performanslı sıvı kromatografisi (HPLC) ile sırasıyla 0.45 ile 4521.64 µg/L ve 0.22 ile 2562.87 µg/L aralığında aflatoksin üreticisi olduğu belirlenmiştir. İnce tabaka kromatografisiyle (TLC) 32 A flavus izolatı 0.35-8.21 μg/g aralığında siklopiazonik asit üretmiştir. Bu çalışma, Adana ve Osmaniye illerinden toplanan yerfıstığı örneklerinden izole edilen A. section Flavi'nin moleküler karakterizasyonu için yeni bir yöntem sunmaktadır. Ayrıca bu araştırma Adana ve Osmaniye illerinden toplanan yerfıstığı örneklerinden izole edilen A. flavus ve A. parasiticus izolatlarının mikotoksin oluşumundan sorumlu türler olduğunu göstermiştir.

Keywords

Aspergillus flavus, Aspergillus parasiticus, PZR, Mikotoksin, Yerfıstığı

References

• [1] Duran, R.M., Cary, J.W., Calvo, A.M. (2009). The role of veA in Aspergillus flavus infection of peanut, corn and cotton. The Open Mycology Journal, 3(1), 27-36.
• [2] Kuiper-Goodman, T. (2004). Risk Assessment and Risk Management of Mycotoxins in Food. In: Mycotoxins in Food: Detection and Control, Edited by N. Magan, M. Olsen, Woodhead Publishing Limited, Cambridge, UK, 3-31p.
• [3] Rao, S.B.N., Chopra, R.C. (2001). Influence of sodium bentonite and activated charcoal on aflatoxin M1 excretion in milk of goats. Small Ruminant Research, 41(3), 203-213.
• [4] Frisvad, J., Thrane, U., Samson, R., Pitt, J. (2006). Important mycotoxins and the fungi which produce them. Advances in Experimental Medicine and Biology, 571, 3-31.
• [5] Pushvinder, R., Desai, S. (2006). Variability among isolates of Aspergillus flavus from groundnut for aflatoxin and cyclopiazonic acid production. Journal of Environmental, Agricultural and Food Chemistry, 5(4), 1458-1463.
• [6] Vaamonde, G., Patriarca, A., Fernandez Pinto, V., Comerio, R., Degrossi, C. (2003). Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section Flavi from different substrates in Argentina. International Journal of Food Microbiology, 88(1), 79-84.
• [7] Rodrigues, P., Soares, C., Kozakiewicz, Z., Paterson, R.R.M., Lima, N., Venancio, A. (2007). Identification and Characterization of Aspergillus flavus and Aflatoxins. In: Communicating Current Research and Educational Topics and Trends in Applied Microbiology, Edited by A. Mendez-Vilas, Formatex Research Center, Badajoz, Spain, 527-534p.
• [8] Pildain, M.B., Frisvad, J.C., Vaamonde, G., Cabral, D., Varga, J., Samson, R.A. (2008). Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. International Journal of Systematic and Evolutionary, 58(Pt 3), 725-735.
• [9] Godet, M., Munaut, F. (2010). Molecular strategy for identification in Aspergillus section Flavi. FEMS Microbiology Letters, 304(2), 157-168.
• [10] Nelson, A.J., Elias, K.S., Arévalo, E.G., Darlington, L.C., Bailey, B.A. (1997). Genetic characterization by RAPD analysis of isolates of Fusarium oxysporum f. sp. associated with an emerging epidemic in Peru. Phytopathology, 87(12), 1220-1225.
• [11] Oktay, H.I. (2010). Aspergillus İzolatlarının Tanısı, Bazı Mikotoksijenik Özelliklerinin Belirlenmesi, Sıcaklık ve sürenin İncirde Aflatoksin ve Siklopiazonik Asit Oluşumuna Etkisi. Doktora Tezi, İstanbul Teknik Üniversitesi Fen Bilimleri Enstitüsü, İstanbul, 154s.
• [12] Gonzalez-Salgado, A., Gonzales-Jaen, T., Vazquez, C., Patino, B. (2008). Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour. Food Additives & Contaminants: Part A: Chemistry, Analysis Control Exposure Risk Assessment, 25(6), 758-764.
• [13] Niessen, L. (2007). PCR-based diagnosis and quantification of mycotoxin producing fungi. International Journal of Food Microbiology, 119(1-2), 38-46.
• [14] Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A., Salomon, R. (1996). Detection of aflatoxigenic molds in grains by PCR. Applied Environmental Microbiology, 62(9), 3270-3273.
• [15] Somashekar, D., Rati, E.R., Anand, S., Chandrashekar, A. (2004). Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India. Food Microbiology, 21(6), 809-813.
• [16] Manonmani, H.K., Anand, S., Chandrashekar, A., Rati, E.R. (2005). Detection of aflatoxigenic fungi in selected food commodities by PCR. Process Biochemistry, 40(8), 2859-2864.
• [17] Lavkor, I. (2013). Yerfıstığı tarımında Uygun Kültürel İşlemler ve Hastalık Yönetim Pratikleri ile Hastalık ve Aflatoksin Oluşumunun Önlenmesi. Doktora Tezi, Çukurova Üniversitesi Fen Bilimleri Enstitüsü, Bitki Koruma Anabilim Dalı, Adana, 300s.
• [18] Raper, B.K., Fennel, D.I. (1977). The Genus Aspergillus. New York: Robert, E. Krieger Publishing Company.
• [19] Samson, R.A., Pitt, I.J. (1990). Modern Consepts in Penicillium and Aspergillus Classification. NATO ASI Series, Plenum Pres, New York and London.
• [20] Doyle, J.J., Dickson, E.E. (1987). Preservation of plant samples for DNA restriction endonuclease analysis. Taxon, 36(4), 715-722.
• [21] Ehrlich, K.C., Montalbano, B.G., Cotty, P.J. (2003). Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production. Fungal Genetics and Biology, 38(1), 63-74.
• [22] Ehrlich, K.C., Kobbeman, K., Montalbano, B.G., Cotty, P.J. (2007). Aflatoxin-producing Aspergillus species from Thailand. International Journal of Food Microbiology, 114(2), 153-159.
• [23] Thompson, J.D., Higgins, D.G., Gibson, T.J. (1994). Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680.
• [24] Kumar, S., Stecher, G., Peterson, D., Tamura, K. (2012). Mega cc: computing core of molecular evolutionary genetics analysis program for automated and iterative data analysis. Bioinformatics, 28(20), 2685-2686.
• [25] Abbas, H.K., Zablotowicz, R.M., Horn, B.W., Phillips, N.A., Johnson, B.J., Jin, X., Abel, C.A. (2011). Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize. Food Additives & Contaminants: Part A: Chemistry, Analysis Control Exposure Risk Assessment, 28(2), 198-208.
• [26] AOAC (2002). Official Method 991.31. Aflatoxins in corn, raw peanuts, and peanut butter immunoaffinity column (AflaTest) method, AOAC International, 2000, 42, 2-18.
• [27] Sosa, A., Kobashigawa, E., Galindo, J., Bocourt, R., Corassin, H., Oliverira, C.A.F. (2013). Mycotoxicological studies of an Aspergillus oryzae strain. Journal of Yeast and Fungal Research, 4(3), 26-32.
• [28] Somuncuoğlu, Ş. (2007). Kuru İncirlerde Siklopiazonik Asit Varlığının ve Miktarının Belirlenmesi. Yüksek Lisans Tezi, İstanbul Teknik Üniversitesi Fen Bilimleri Enstitüsü, Bitki Koruma Anabilim Dalı, İstanbul, 60s.
• [29] Abdullahzadeh, S.S., Sangtarash, M.H., Dehkordi, M. (2015). Rapid identification of aflatoxigenic fungals isolated from environments of Chaharmahal and Bakhtiari, Iran. Current Medical Mycology, 16(1), 38.
• [30] Gallo, A., Stea, G., Battilani, P., Logrieco, A.F., Perrone, G. (2012). Molecular characterization of an Aspergillus flavus population ısolated from maize during the first outbreak of aflatoxin contamination in Italy. Phytopathologia Mediterranea, 51(1), 198−206.
• [31] Baquião, A.C., Oliveira, M.M.M., Reis, T.A., Zorzete, P., Atayde, D.D., Correa, B. (2013). polyphasic approach to the identification of Aspergillus section Flavi isolated from Brazil nuts. Food Chemistry, 139(1-4), 1127-1132.
• [32] Eshetu, L. (2010). Aflatoxin Content of Peanut (Arachis hypogaea L.) in Relation to Shelling and Storage Practice of Ethiopian Farmers. M.Sc. Thesis. Addis Ababa University, Ethiopia, 68p.
• [33] Pildain, M.B., Vaamonde, G., Cabral, D. (2004). Analysis of population structure of Aspergillus flavus from peanut based on vegetative compatibility, geographic origin, mycotoxin and sclerotia production. International Journal of Food Microbiology, 93(1), 31-40.
• [34] Mphande, F.A., Siame, B.A., Taylor, J.E. (2004). Fungi, aflatoxins and cyclopiazonic acid associated with peanut retailing in Botswana. Journal of Food Protection, 67(1), 96-102.
• [35] Cisarová, M., Tančinová, D., Rapčanová, J. (2015). Incidence of aflatoxigenic fungi in peanuts (Arachis hypogea L.) from markets in Slovakia. Scientific Papers. Animal Science and Biotechnologies, 48(1), 118-122.
• [36] Klich, M.A., Pitt, J.I. (1988). A Laboratory guide to common Aspergillus species and their teleomorphs. North Ryde: Commonwealth Scientific and Industrial Research Organization.
• [37] Clevstrom, G., Ljunggren, H. (1985). Anatoxin formation and the dual phenomenon in Aspergillus flavus Link. Mycopathologia, 92(3), 129–139.
• [38] Kumeda, Y., Asao, T., Takahashi, H., Ichinoe, M. (2003). High prevalence of B and G aflatoxin-producing fungi in sugarcane field soil in Japan: heteroduplex panel analysis identifies a new genotype within Aspergillus section Flavi and Aspergillus nomius. FEMS Microbiology Ecology, 45(3), 229-238.
• [39] Rodrigues, P., Venâncio, A., Kozakiewicz, Z., Lima, N. (2009). A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds. International Journal of Food Microbiology, 129(2), 187-193.
• [40] Razzaghi-Abyaneh, M., Shams-Ghahfarokhi, M., Allameh, A., Kazeroon-Shiri, A., Ranjbar-Bahadori, S., Mirzahoseini, H., Rezaee, M. (2006). A survey on distribution of Aspergillus section Flavi in corn field soils in Iran: populations patterns based on aflatoxins, cyclopiazonic acid and sclerotia production. Mycophatologia, 161(3), 183-192.
• [41] Chang, P.K., Horn, B.W., Dorner, J.W. (2009). Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus. Fungal Genetics and Biology, 46(2), 176-182.
• [42] Astoreca, A.L., Dalcero. A.M., Pinto, V.F., Vaamonde, G.A. (2011). A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds. International Journal of Food Microbiology, 146(1), 38-43.
• [43] Türköz Bakırcı, G. (2014). Tahıl ve tahıl ürünlerinin aflatoksin, okratoksin a, zearalenon, fumonisin ve deoksinivalenol mikotoksinleri yönünden incelenmesi. Akademik Gıda, 12(2), 46-56.