PCR-RFLP analyses of Aspergillus spp. Isolated from Pistachio in Turkey

Yıl 2020, Cilt: 49 Sayı: 2, 25 - 33, 12.11.2020

Ali Alaayadi Safa Ali Selçuk Başbuğa Omar Atik Canan Can

Öz

Pistachio (Pistacia vera L.) is a strategic crop plant and Turkey is one of the major pistachio producing county together with Iran, Syria and the United States of America. Pistachio trees are affected by many fungal diseases. The Aspergillus spp. are one of the most important agent that produce mycotoxins and also known as human and animal pathogens. This study was conducted to determine the genomic DNA polymorphisms from Aspergillus spp. which were isolated from pistachio fruits collected from different climatic zones in Turkey. Total of 27 Aspergillus spp. isolates were used and specific primers for ITS, Actin and β-tubulin genes were subjected to RFLP analysis by using Hind III, Pst I, Taq I and Eco RI restriction enzymes. Three different groups were defined with the enzyme Taq I in the β-tubulin region. The results revealed low polymorphism and genetic diversity in Aspergillus spp. with PCR-RFLP of the ITS and actin regions.

Anahtar Kelimeler

Pistachio, Aspergillus spp, PCR-RFLP

Türkiye'de Antep Fıstığı'ndan İzole Edilen Aspergillus spp.'nin PCR-RFLP Analizleri

Öz

Antep fıstığı (Pistacia vera L.) stratejik öneme sahip bir ürün ve Türkiye, İran, Suriye ve Amerika Birleşik Devletleri ile birlikte önemli bir üretici ülke konumundadır. Antep fıstığı ağaçlarında hastalık yapan birçok fungus türü vardır. Bunlardan Aspergillus spp. aflatoksin üretimi yanında insan ve hayvan patojeni olması dolayısı ile Antep fıstığı için en önemli tehditlerden birisi olarak kabul edilmektedir. Bu çalışma ülkemizin farklı bölgelerinden toplanmış meyve örneklerinden izole edilmiş Aspergillus spp.’de genomik DNA polimorfizmini belirlemek amacı ile yapılmıştır. Toplam 27 Aspergillus spp. izolatında ITS, Aktin ve β-tubulin genlerinde Hind III, Pst I, Taq I ve Eco RI kesim enzimleri kullanılarak RFLP analizi yapılmıştır. β -tubulin bölgesinde Taq I enzimi ile üç farklı grup saptanmıştır. Sonuçlar, ITS ve Aktin bölgelerinde Aspergillus spp.’nin PCR-RFP ile düşük polimorfizm ve genetik çeşitlilik sergilediğini ortaya koymuştur.

Anahtar Kelimeler

Antep Fıstığı, Aspergillus spp, PCR-RFLP

Kaynakça

• Abraca, M.L., Bragulat, G. and Cabanes, F.J. 1994. Ochratoxin A Production by Strains of Aspergillus flavus var. niger. App. Environ. Microbiol. 60: 2650-2652.
• Afsah-Hejri, L., Jinap, S., Hajeb, P., Radu, S. and Shakibazadeh, S. 2013. A Review on Mycotoxins in Food and Feed: Malaysia Case Study. Comp. Rev. Food Sci. Food Saf. 12:629-651.
• Atoui, A. and El Khoury, A. 2017. PCR-RFLP for Aspergillus Species. Pages 313-320 in Mycotoxigenic Fungi. Methods in Molecular Biology. Moretti, A. and Susca, A. eds. Humana Press, New York.
• Balajee, S.A., Borman, A.M., Brandt, M.E., Cano, J., Cuenca-Estrella, M., Dannaoui, E., Guarro, J., Haase, G., Kibbler, C.C., Meyer, W., O’Donnell, K., Petti, C.A., Rodriguez-Tudela, J.L., Sutton, D., Velegraki, A. and Wickes, B.L. 2009. Sequence-based Identification of Aspergillus, Fusarium, and Mucorales Species in the Clinical Mycology Laboratory: Where are We and Where Should We Go from Here? J. Clin. Microbiol. 47:877–884.
• Balajee, S.A., Gribskov, J.L., Hanley, E., Nickle, D. and Marr, K.A. 2005. Aspergillus lentulus sp. nov., A New Sibling Species of A. fumigatus. Eukaryotic Cell. 4: 625–632.
• Balajee, S.A., Nickle, D., Varga, J. and Marr, K.A. 2006. Molecular Studies Reveal Frequent Misidentification of Aspergillus fumigatus by Morphotyping. Eukaryotic Cell. 5: 1705-1712.
• Bruce, C., Campbell, H., Schatzk, F., Russell, J. and Monlyneux, T. 2003. Current Research On Reducing Pre and Post-Harvest Aflatoxin Contamination of USA Almond, Pistachio and Walnut. Toxin Rev. 22:225-260.
• Carbone, I. and Kohn, L.M. 1999. A Method for Designing Primer Sets for Speciation Studies in Filamentous Ascomycetes. Mycologia 91: 553-556.
• Chang, J. M., Oyaizu, H. and Sugiyama, J. 1991. Phylogenetic-Relationships Among Eleven Select Species of Aspergillus and Associated Teleomorphic Genera Estimated from 18S Ribosomal-RNA Partial Sequences. J. Gen. Appl. Microbiol. 37: 289-308.
• Cooley, R.N. and Caten, C.E. 1993. Molecular Analysis of the Septoria nodorum Beta Tubulin Gene and Characterization of a Benomyl-Resistance Mutation. Mol. Gen. Genet. 237: 58-64.
• Crous, P.W., Quaedvlieg, W., Sarpkaya, K., Can, C. and Erkılıç, A. 2013. Septoria-Like Pathogens Causing Leaf and Fruit Spot of Pistachio, IMA Fungus, 4(2): 187–199.
• Daniela, H.M. and Meyera, W.C. 2003. Evaluation of Ribosomal RNA and Actin Gene Sequences for the Identification of Ascomycetous Yeasts. Int. J. Food Microbiol. 86: 61–78.
• Dao, H.P., Mathieu F. and Lebrihi, A. 2005. Two Primer Pairs to Detect OTA Producers by PCR Method. Int. J. Food. Microbiol., 104: 61–67.
• Davidson, R. M., Hanson, L. E., Franc, G. D. and Panella, L. 2006. Analysis of β-tubulin Gene Fragments from Benzimidazole-Sensitive and Tolerant Cercospora beticola. J. Phytopathol., 154: 321–328.
• Denizel, T., Jarvis, B. and Rolf, E.J. 2006. A field Survey of Pistachio (Pistacia vera) Nut Production and Storage in Turkey with Particular Reference to Aflatoxin Contamination. J. Sci. Food Agric. 27 (11):1021-1026.
• Dekoe, W.J., Sanson, R.A., Egmond, P.V., Gilbert, J. and Sabino, M. 2000. Proceedings of The International IUPAC Symposium on Mycotoxins and Phcotoxins, Guarja Brazil. pp. 21-25.
• Doster, M.A. and Michailides, T.J. 1994. Aspergillus Molds and Aflatoxins in Pistachio Nuts in California. Phytopathology. 84 583-590.
• Edwin, R., Palencia, I., Hinton, D.M. and Bacon, C.W. 2010. The Black Aspergillus Species of Maize and Peanuts and Their Potential for Mycotoxin Production. Toxins. 2: 399-416.
• Eskalen, A., Küsek, M., Danisti, L. and Karadag, S. 2001. Fungal Diseases in Pistachio Trees in East Mediterranean and Southeast Anatolian Regions. In: Ak B.E. (ed.). XI GREMPA Seminar on Pistachios and Almonds, Zaragoza: CIHEAM, 2001, 261-264.
• FAOSTAT 2018. Food and Agriculture Organization Production Resource Statistics. (Web page: http://faostat.org.) Ferdaws, B., Sobita, S. and Abhilasha, A. 2016. Effect of Selected Spices Against Aspergillus flavus of Pistachio (Pistachia vera L.) in vitro Condition. Int. J. Multidiscip. Res. Dev. 7: 185-187.
• Geiser, D.M., Frisvad, J.C. and Taylor, J.W. 1998. Evolutionary Relationships in Aspergillus Section Fumigati Inferred from Partial Beta-Tubulin and Hydrophobin DNA Sequences. Mycologia. 90: 831–845.
• Glass, N.L. and Donaldson, G.C. 1995. Development of Primer Sets Designed for Use with The PCR to Amplify Conserved Genes from Filamentous Ascomycetes. App. Environ. Microbiol. 61: 1323–1330.
• Goldman, G.H., Temmerman, W., Jacobs, D., Contreras, R., van Montagu, M. and Herrera-Estrella, A. 1993. A Nucleotide Substitution in One of the Beta-Tubulin Genes of Trichoderma Viride Confers Resistance to the Antimitotic Drug Methyl Benzimidazole-2-Yl-Carbamate. Mol. Gen. Genet. 240: 73–80.
• Hiraoka, Y., Toda, T. and Yanagida, M. 1984. The NDA3 Gene of Fission Yeast Encodes Beta-Tubulin: A Cold Sensitive Nda3 Mutation Reversibly Blocks Spindle Formation and Chromosome Movement in Mitosis. Cell. 39: 349–358. Jalali, L. and Avagyan, G. 2016. Evaluation of Contamination of Aspergillus flavus and Aflatoxin Production in Pistachio Cultivars and Investigation of a Chemical Controlling Method. J. Nuts. 7: 51–58.
• Jones, A.L., Shabi, E. and Ehret, G.R. 1987. Genetics of Negatively Correlated Cross Resistance to a N-Phenylcarbamate in Benomyl-Resistant Venturia inaqualis. Can. J. Plant Pathol. 9: 195-199. Kaska, N. 2005. Pistachio nut growing in Turkey. Acta Hortic. 419: 161-164.
• Kesawat, M.S. and Kumar, B.D. 2009. Molecular Markers: Its Application in Crop Improvement. J. Crop. Sci. Biotechnol. 4: 169–181.
• Klich, M.A. 2002. Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Utrecht. The Netherlands, 128 pp.
• Klich, M.A. and Pitt, J.I. 1988. Differentiation of Aspergillus flavus from Aspergillus parasiticus and Other Closely Related Species. T. Brit. Mycol. Soc. 91:99-108.
• Koenraadt, H., Somerville, S.C. and Jones, A.L. 1992. Characterization of Mutations in the Beta-Tubulin Gene of Benomyl-Resistant Yield Strains Of Venturia inaequalis and Other Plant Pathogenic Fungi. Phytopathology. 82: 1348-1354. Konukoğlu, F., Kusek, M., Sarpkaya, K., Karadag, S. and Can, C. 2011. Discoloration of Pistachio Nuts Grown in the Gaziantep Province of Turkey. Acta Horticulturae, 912:761-763.
• Kurtzman, C.P., S, Smiley, M.J., Robnett, C.J. and Wicklow, D.T. 1986. DNA Relatedness Among Wild and Domesticated Species in Aspergillus flavus Group. Mycologia. 78: 955-959.
• Maskan, M. and Karatas, S. 1998. Fatty Acid Oxidation of Pistachio Nuts Stored Under Various Atmospheric Conditions and Different Temperatures. J. Sci. Food Agric. 77: 334-340.
• Mirhendi, S.H., Diba, K. and Kordbacheh, P. 2007. Identification of Pathogenis Aspergillus Species by a PCR-Restriction Enzyme Method. J. Med. Microbiol. 56:1568-1570.
• Moody, S.F. and Tyler, B.M. 1990. Use of Nuclear DNA Restriction Fragment Length Polymorphism to Analyze the Diversity of the Aspergillus flavus group; A. flavus, A. parasiticus and A. nomius. J. App. Environ. Microbiol. 56: 2453-2461. Muranty, H., Jorge, V., Bastien, C., Lepoittevin, C., Bouffier, L. and Sanchez, L. 2014. Potential for Marker-Assisted Selection for Forest Tree Breeding: Lessons from 20 Years of MAS in Crops. Tree Genet. Genom. 6: 1491–1510.
• Nasri, T., Hedayati, M., Abastabar, M., Pasqualotto, A., Armaki, M., Hosseinnejad, A. and Nabili, M. 2015. PCR-RFLP on β-Tubulin Gene for Rapid Identification of the Most Clinically Important Species of Aspergillus. J. Microbiol. Meth. 117. 10.1016/j.mimet.2015.08.007.
• Nikkuni, S., Kosaka, N., Suzuki, C. and Mori, K. 1996. Comparative Sequence Analysis on the 18S rRNA Gene of Aspergillus oryzae, A. sojae, A. flavus, A. parasiticus, A. niger, A. awamori and A. tamarii. J. Gen. App. Microbiol. 42:181-187.
• Nikkuni, S., Nakajima, H., Hoshina, S., Ohno, M., Suzuki, C., Kashiwagi, Y. and Mori, K. 1998. Evolutionary Relationships Among Aspergillus Oryzae and Related Species Based on the Sequences of 18S rRNA Genes and Internal Transcribed Spacers. J. Gen. App. Microbiol. 44: 225-230.
• Noonimabc, P., Mahakarnchanakulb, W., Nielsend, K.F., Frisvadd, J.C. and Samsona, R.A. 2009. Fumonisin B2 Production by Aspergillus niger in Thai Coffee Beans. Food Addit. Cont. 26: 94-100.
• Ozkilinc, H., Sarpkaya, K., Kur,t S., Can, C., Polatbilek, H., Yasar, A., Sevinc, U., Uysal, A. and Konukoglu, F. 2017. Pathogenicity, Morpho-Species and Mating Types of Alternaria spp. Causing Alternaria Blight in Pistacia spp. in Turkey, Phytoparasitica. Doi: 10.1007/s12600-017-0624-8.
• Özgün, E. 2013. Antep fıstığında (Pistacia vera L.) Aflatoksin Oluşturan Aspergillus Türlerinin Belirlenmesi. Gaziantep Üniversitesi, Yüksek Lisans Tezi, Türkiye, 52 pp.
• Peever, T. L., Canihos, Y., Olsen, L., Ibanez, A., Liu, Y.C. and Timmer, L.W. 1999. Population Genetic Structure and Host Specificity of Alternaria spp. Causing Brown Spot of Minneola tangelo and Rough Lemon in Florida. Phytopathology. 89: 851-860.
• Peterson, S.W. 2008. Phylogenetic Analysis of Aspergillus Species Using DNA Sequences from Four Loci. Mycologia. 100: 205-26.
• Samson, R., Hong, S., Peterson, S.W., Frisvad, J.C. and Varga, J. 2007. Polyphasic Taxonomy Of Aspergillus Section Fumigati and Its Teleomorph Neosartorya. Stud. Mycol. 59: 147–203.
• Samson, R.A. and Pitt, J. I. 2000. Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification. Harwood Academic Publishers, Singapore, 510 pp.
• Samson, R.A., Peterson, S.W., Frisvad, J.C. and Varga, J. 2011. New Species in Aspergillus Section Terrei. Stud. Mycol. 69: 39-55. Samson, R.A., Seifert, K.A., Kuijpers, A.F.A. and Frisvad, J.C. 2004. Phylogenetic Analysis of Penicillium Subgenus Penicillium Using Partial β-Tubulin Sequences. Stud. Mycol. 49:175–200.
• Sarpkaya, K., 2014. Antep Fıstığında Karazenk Hastalığına Neden Olan Pseudocercospora Pistacina’nın Biyolojisi, Epidemiyolojisi ve Mücadelesine Yönelik Çalışmalar, Ç.Ü. Doktora Tezi, 120 sayfa.
• Schoch, CL, Seifert, KA, Huhndorf, S., Robert, V., Spouge J.L., Levesque, C.A. and Chen, W. 2012. Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi. Proc Natl Acad Sci U S A. 2012;109(16):6241-6246. doi:10.1073/pnas.1117018109.
• Schuster, E., Dunn-Coleman, N., Frisvad, J.C. and Van Dijck, P.W.M. 2002. On the Safety of Aspergillus niger - A Review. App. Microbiol. and Biotechnol. 59: 426-435.
• Seip, E.R., Woloshuk, C.P., Payne, G.A. and Curti, S.E. 1990. Isolation and Sequence Analysis of a β-Tubulin Gene from Aspergillus flavus and Its use as a Selectable Marker. App. Environ. Microbiol. 56: 3686–3692.
• Shokraii, E.H. 1977. Chemical Composition of the Pistachio Nuts (Pistacia vera L.) of Kerman, Iran. J. Food Sci. 42: 244-245. Somashekar, D., Ratl, E.R. and Chandrashekar, A. 2004. PCR-Restriction Fragment Length Analysis of afIR Gene for Differentiation and Detection of Aspergillus flavus and Aspergillus parasiticus in Maize. Int. J. Food Microbiol. 93:101-107.
• Tsantili, E., Takidelli, C., Christopoulos, M.V., Lambrinea, E., Rouskas, D. and Roussos, P.A. 2010. Physical, Compositional and Sensory Differences in Nuts Among Pistachio (Pistachia vera L.) varieties. Sci. Hortic. 125: 562- 568.
• White, T.J., Bruns, T., Lee, S. and Taylor. J. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. Pages 315-322 in PCR protocols: a guide to methods and applications PCR protocols: a guide to methods and applications. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., eds. Academic Press, New York.
• Zarrin, M., Rashidnia, Z., Faramarzi, S. and Harooni, L. 2017. Rapid Identification of Aspergillus fumigatus Using Βeta-tubulin and RodletA Genes. Mac. J. Med. Sci. 5. 10.3889/oamjms.2017.203.
• Zheng, Z., Saghaian, S. and Reedc, M. 2012. Factors Affecting the Export Demand for U.S. Pistachios. Int. Food Agribus. Man. 15: 139-154.