Bozcaada çavuş ve karalahna üzümlerinden izole edilen okratoksijenik Aspergillus türlerinin biyoçeşitliliği

Abstract

Bu çalışmada, Bozcaada Çavuş ve Karalahna üzümlerinden izole edilen okratoksijenik Aspergillus türlerinin varlığı ve OTA üretimi rapor edilmiştir. Çalışma, 2015 ve 2016 yıllarında olgunlaşma sırasında toplanan üzüm örneklerinden izole edilen Aspergillus izolatları ile gerçekleştirilmiştir. İlk olarak 290 Aspergillus izolatının ikincil metabolit profilleri İnce Tabaka Kromatografisi kullanılarak belirlenmiştir. İzolatlardan 122 tanesinin ise muhtemel OTA üreticisi olduğu tespit edilmiştir. 122 izolatın farklı kültür ortamlarındaki koloni morfolojilerine göre seçilen 43 izolat, calmodulin gen dizileme analizi kullanılarak tanımlanmıştır. Tanımlanan izolatların A. tubingensis, A. carbonarius, A. niger/welwitschia/awamori, A. welwitschia, A. spelaeus ve A. fructus olduğu belirlenmiştir. OTA üretimi ise altı izolatta araştırılmıştır. HPLC-FLD kullanılarak bu izolatların 3,550 ± 0,240 ile 92,346 ± 0,818 ppb arasında değişen miktarlarda OTA ürettiği bulunmuştur. Sonuç olarak üzümlerden OTA üreten Aspergillus türleri izole edildi. A. spelaeus ve A. fructus türlerinin varlığı üzümlerde ilk kez rapor edilmiştir. A. fructus Türkiye için yenit kayıt olduğu tespit edilmiştir.

Keywords

• Aspergillus
• calmodulin
• HPLC
• Moleküler identifikasyon
• TLC.

Project Number

FBA-2019-3028

Biodiversity of ochratoxigenic Aspergillus species isolated from çavuş and karalahna grapes in Bozcaada, Türkiye

Abstract

This study reported the presence and OTA production of ochratoxigenic Aspergillus species isolated from Bozcaada Çavuş and Karalahna grapes. The study was conducted with Aspergillus isolates isolated from grape samples collected during ripening in 2015 and 2016. Thin-layer chromatography was used to determine the secondary metabolite profiles of 290 Aspergillus isolates. Out of these, 122 isolates were found to be possible OTA producers. 43 isolates, selected based on their colony morphology of 122 isolates in different culture media, were identified using calmodulin gene sequencing analysis. The identified isolates were determined to be A. tubingensis, A. carbonarius, A. niger/welwitschia/awamori, A. welwitschia, A. spelaeus, and A. fructus. OTA production was investigated in six isolates. Using HPLC-FLD, these isolates were found to produce OTA in quantities ranging from 3.550 ± 0.240 to 92.346 ± 0.818 ppb. Consequently, OTA-producing Aspergillus species were isolated from grapes. The presence of A. spelaeus and A. fructus was reported for the first time in grapes. A. fructus has been found to be a new record for Türkiye.

Keywords

• Aspergillus
• Calmodulin
• HPLC
• Molecular Identification
• TLC.

Supporting Institution

This study was supported by Çanakkale Onsekiz Mart University Scientific Research Projects Coordination Unit with project number FBA-2019-3028.

Project Number

FBA-2019-3028

Thanks

We would like to thank Erhan AKDAĞ, Deputy Director of the Çanakkale Food Control Laboratory Directorate of the Ministry of Agriculture and Forestry, and the staff of the Mycotoxin Laboratory Unit, who helped with the ochratoxin analysis within the scope of the study. We thank Assoc. Prof. Dr. Alper Tunga AKARSUBAŞI for her support for the article.

References

1. Anonymous, (2023). http://www.bozcaada.bel.tr/bozcaada/ekonomi-sosyal/ [accessed 09 March 2023]
2. Asan A, Selçuk F, Giray G, Aydoğdu H, Ulukapı M, Ceylan MF (2022). Türkiye Mantarları Listesi’ne İlaveler-1. Bağbahçe Bilim Dergisi 9(3): 65-89. https://doi.org/10.35163/bagbahce.1130135
3. Aşkun T, Eltem R, Taşkin E (2007). Comparison of rose-bengal chloramphenicol agar and dichloran glycerol agar (DG18) for enumeration and isolation of moulds from raisins. Journal of Applied Biological Sciences 1(2): 71-74. https://dergipark.org.tr/en/pub/jabs/issue/34886/386751
4. Ayan EG, Asan A, Şen B, Okten S (2018). Edirne ili Söğütlük Ormanı Toprağından izole edilen Aspergillus türlerinin biyoçeşitliliği. Mantar Dergisi 9(2): 126-141. https://doi.org/10.30708/mantar.424172
5. Balajee SA, Houbraken J, Verweij PE, Hong SB, Yaghuchi T, Varga J, Samson RA (2007). Aspergillus species identification in the clinical setting. Study in Mycology 59(1): 39-46. https://doi.org/10.3114/sim.2007.59.05
6. Battilani P, Pietri A, Bertuzzi T, Languasco L, Giorni P, Kozakiewicz Z (2003). Occurrence of ochratoxin A–producing fungi in grapes grown in Italy. Journal of Food Protection 66(4): 633-636. https://doi.org/10.4315/0362-028X-66.4.633
7. Chiotta ML, Ponsone ML, Combina M, Torres AM, Chulze SN (2009). Aspergillus section Nigri species isolated from different wine-grape growing regions in Argentina. International Journal of Food Microbiology 136(1): 137-141. https://doi.org/10.1016/j.ijfoodmicro.2009.08.013
8. Dardeniz A, Bahar E, Şimşek L (2007). Bozcaada bağcılığındaki gelişmeler, sorunlar ve öneriler. Çanakkale Araştırmaları Türk Yıllığı 5(5): 147-61.

9. Dyer SK, McCammon S (1994). Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar. Journal of Applied Bacteriology 76(1): 75-78. https://doi.org/10.1111/j.1365-2672.1994.tb04418.x
10. Eltem R, Aşkun T, Sarigül N, Özkale E, Efendiler H (2004). Colonial and morphological characteristics of some Aspergillus Fr.: Fr. species isolated from vineyards in Manisa and Izmir provinces (Turkey). Turkish Journal of Botany 28(3): 287-298. https://journals.tubitak.gov.tr/botany/vol28/iss3/3
11. Eltem R, Taskin E, Pazarbasi S. (2009). Biodiversity and flora of microfungi from Sultana-Type vineyard soil in Turkey. Fresenius Environmental Bulletin 18(1): 82-86. https://www.prt-parlar.de/download_feb_2009/
12. García‐Cela E, Crespo‐Sempere A, Gil‐Serna J, Porqueres A, Marin S (2015). Fungal diversity, incidence and mycotoxin contamination in grapes from two agro‐climatic Spanish regions with emphasis on Aspergillus species. Journal of the Science of Food and Agriculture 95(8): 1716-1729. https://doi.org/10.1002/jsfa.6876
13. Garmendia G, Vero S (2016). Occurrence and biodiversity of Aspergillus section Nigri on ‘Tannat’grapes in Uruguay. International Journal of Food Microbiology 216: 31-39. https://doi.org/10.1016/j.ijfoodmicro.2015.08.020
14. Gulsunoglu Z, Kilic-Akyilmaz M, Karbancioglu-Guler F, Katleen, R (2019). Production of multiple hydrolytic enzymes by black aspergilli isolated from date and grape. Journal of Agricultural Sciences 25(4): 459-466. https://doi.org/10.15832/ankutbd.448264
15. Guzev L, Danshin A, Ziv S, Lichter A (2006). Occurrence of ochratoxin A producing fungi in wine and table grapes in Israel. International Journal of Food Microbiology 111: 67-71. https://doi.org/10.1016/j.ijfoodmicro.2006.03.003
16. Hall TA (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41(41): 95-98.
17. Index Fungorum. https://www.speciesfungorum.org/Names/SynSpecies.asp?RecordID=800600 [accessed 12 December 2023].
18. Khoury AE, Rizk T, Lteif R, Azouri H, Delia ML, Lebrihi A (2008). Fungal contamination and Aflatoxin B1 and Ochratoxin A in Lebanese wine–grapes and musts. Food and Chemical Toxicology 46(6): 2244-2250. https://doi.org/10.1016/j.fct.2008.02.026
19. Krijgsheld P, Bleichrodt RV, Van Veluw GJ, Wang F, Müller WH, Dijksterhuis J, Wösten HAB (2013). Development in Aspergillus. Study in Mycology 74(1): 1-29. https://doi.org/10.3114/sim0006
20. Lasram S, Oueslati S, Mliki A, Ghorbel A, Silar P, Chebil S (2012). Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas. Food Control, 25(1): 75-80. https://doi.org/10.1016/j.foodcont.2011.10.006
21. Magnoli C, Violante M, Combina M, Palacio G, Dalcero A (2003). Mycoflora and ochratoxin‐producing strains of Aspergillus section Nigri in wine grapes in Argentina. Letters in Applied Microbiology 37(2): 179-184. https://doi.org/10.1046/j.1472-765X.2003.01376.x
22. Martinez-Culebras PV, Ramon D (2007). An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine. International Journal of Food Microbiology 113(2): 147-153. https://doi.org/10.1016/j.ijfoodmicro.2006.06.023
23. Oliveri C, Bella P, Tessitori M, Catara V, La Rosa R (2017). Grape and environmental mycoflora monitoring in old, traditionally cultivated vineyards on Mount Etna, southern Italy. Journal of the Science of Food and Agriculture 97(1): 65-73. https://doi.org/10.1002/jsfa.7683
24. Özcan Ateş G, Zorba NND (2021). Determination of mycobiota and microbial loads of grapes grown in Bozcaada, Turkey. Journal of Microbiology, Biotechnology and Food Sciences 9(5): 899-906. https://doi.org/10.15414/jmbfs.2020.9.5.899-906
25. Özdener Kömpe Y, Akin Mutlu V, Özkoç İ, Demiray S, Serhat B (2022). Fungal diversity and ex vitro symbiotic germination of Serapias vomeracea (Orchidaceae). Acta Botanica Croatica 81(1): 108-116. https://doi.org/10.37427/botcro-2022-008
26. Palumbo JD, O'Keeffe TL (2015). Detection and discrimination of four Aspergillus section Nigri species by PCR. Letters in Applied Microbiology 60(2):188-195. https://doi.org/10.1111/lam.12358
27. Pantelides IS, Aristeidou E, Lazari M, Tsolakidou M, Tsaltas D, Christofidou M, Kafouris D, Christou E, Ioannou N (2017). Biodiversity and ochratoxin A profile of Aspergillus section Nigri populations isolated from wine grapes in Cyprus vineyards. Food Microbiology 67: 106-115. https://doi.org/10.1016/j.fm.2017.06.010
28. Perrone G, Stea G, Epifani F, Varga J, Frisvad JC, Samson R A (2011). Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biology 115(11): 1138-1150. https://doi.org/10.1016/j.funbio.2011.07.008
29. Ponsone ML, Combina M, Dalcero A, Chulze S (2007). Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems. International Journal of Food Microbiology 114(2): 131-135. https://doi.org/10.1016/j.ijfoodmicro.2006.07.001
30. Samson RA, Houbraken J, Thrane U, Frishad JC, Andersen B (2010). Food and Indoor Fungi. Utrecht: CBS-Knaw Fungal Biodiversity Center.
31. Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CH, Perrone G, Seifert KA, Susca A, Tanney JB, Varga J, Kocsubé S, Szigeti G, Yaguchi T, Frisvad J C (2014). Phylogeny, identification and nomenclature of the genus Aspergillus. Study in Mycology 78: 141-173. https://doi.org/10.1016/j.simyco.2014.07.004
32. Serra R, Abrunhosa L, Kozakiewicz Z, Venâncio A (2003). Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes. International Journal of Food Microbiology 88(1): 63-68. https://doi.org/10.1016/S0168-1605(03)00085-0
33. Sesli E, Asan A, Selçuk F, (edlr.) Abacı Günyar Ö, Akata I, Akgül H, Aktaş S, Alkan S, Allı H, Aydoğdu H, Berikten D, Demirel K, Demirel R, Doğan HH, Erdoğdu M, Ergül CC, Eroğlu G, Giray G, Halikî Uztan A, Kabaktepe Ş, Kadaifçiler D, Kalyoncu F, Karaltı İ, Kaşık G, Kaya A, Keleş A, Kırbağ S, Kıvanç M, Ocak İ, Ökten S, Özkale E, Öztürk C, Sevindik M, Şen B, Şen İ, Türkekul İ, Ulukapı M, Uzun Ya, Uzun Yu,Yoltaş A (2020). Türkiye Mantarları Listesi (The Checklist of the Fungi of Turkey) (XVII + 1177 pp). İstanbul: Ali Nihat Gökyiğit Vakfı Yayını.
34. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725-2729. https://doi.org/10.1093/molbev/mst197
35. Taskin E, Eltem R, da Silva ES, de Souza, JVB (2008). Screening of Aspergillus strains isolated from vineyards for pectinase production. Journal of Food, Agriculture and Environment 6: 412-414. https://doi.org/10.1234/4.2008.1364