Phylogenetic Diversity Analysis and Molecular Identification of Endofitic Fusarium spp. Isolated From Salep (Orchid)

Öz

The endophytic fungi species isolated from Salep (Orchid) roots and tubers, endemic plants of Turkey, their identification using the molecular techniques is utmost important. The aim of the present study was to identify the species of endophytic Fusarium Link isolated from Salep (Orchid). For this purpose, beta-tubulin (β-tubulin: benA), Internal Transcribed Spacer (ITS) and Translation Elongation Factor (EF) gene regions were amplified by Polymerase Chain Reaction using specific primers. Then, all PCR products were sequenced and compared with the other related sequences in GenBank. The Fusarium isolates were identified to species level as Fusarium redolens Wollenw. (n=7) and Fusarium oxysporum Schlecht. (n=3). The phylogenetic analysis was carried out based on the β-tubulin, ITS and EF gene sequences. In the present study, Fusarium spp. isolated from Saleps (Orchids) in Turkey were firstly identified by molecular identification methods including amplification of the three different gene region.

Anahtar Kelimeler

• Salep
• Orchid
• Endophytic fungi
• Polymerase Chain Reaction
• Fusarium redolens
• Fusarium oxysporum

Saleplerden (Orkide) İzole Edilen Endofitik Fusarium spp.’nin Moleküler Tanımlanması ve Filogenetik Çeşitlilik Analizleri

Öz

Türkiyenin endemik bitkisi Salep (orkide) kök ve yumrulardan izole edilmiş endofitik fungus türlerinin moleküler teknikler kullanılarak tanımlanması son derece önemlidir. Bu çalışmada Salep (Orchid) bitkisinden izole edilen endofitik Fusarium Link türlerinin tanımlanması amaçlanmıştır. Bu amaçla beta-tubulin (β-tubulin: benA), Internal Transcribed Spacer (ITS) ve Translation Elongation Factor (EF) gen bölgeleri Polimeraz Zincir Reaksiyonu (PZR) ile özel primerler kullanılarak çoğaltılmıştır. Daha sonra tüm PZR ürünlerinin dizi analizi yapılmış ve GenBank’daki diğer ilgili diziler ile karşılaştırılmıştır. Fusarium izolatları tür düzeyinde Fusarium redolens Wollenw. (n=7) ve Fusarium oxysporum Schlecht. (n=3) olarak tanımlanmışlardır. Filogenetik analizler the β-tubulin, ITS ve EF gen dizilerini temel alarak gerçekleştirilmiştir. Bu çalışmada, ilk olarak üç farklı gen bölgesinin çoğaltılması içeren moleküler tanımlama yöntemleri kullanılarak Türkiye’deki saleplerden (orkidelerden) izole edilen endofitik Fusarium spp. izolatları tür düzeyinde tanımlanmıştır.

Anahtar Kelimeler

• Salep
• Orkide
• Endofitik fungus
• Polimeraz Zincir Reaksiyonu
• Fusarium redolens
• Fusarium oxysporum

Kaynakça

1. Balmas, V., Migheli, Q., Scherm, B., Garau, P., O’Donnell, K., Ceccherelli, G., Kang, S. and Geiser, D.M., (2010). Multilocus phylogenetics show high levels of endemic fusaria inhabiting Sardinian soils (Tyrrhenian Islands). Mycologia, 102(4), pp.803-812.
2. Deshmukh, S.K., Verekar, S.A. and Bhave, S.V., (2015). Endophytic fungi: a reservoir of antibacterials. Frontiers in microbiology, 5, p.715.
3. Gargouri, S., Balmas, V., Burgess, L., Paulitz, T., Laraba, I., Kim, H.S., Proctor, R.H., Busman, M., Felker, F.C., Murray, T. and O’Donnell, K., (2020). An endophyte of Macrochloa tenacissima (esparto or needle grass) from Tunisia is a novel species in the Fusarium redolens species complex. Mycologia, 112(4), pp.792-807.
4. Geiser, D.M., del Mar Jiménez-Gasco, M., Kang, S., Makalowska, I., Veeraraghavan, N., Ward, T.J., Zhang, N., Kuldau, G.A. and O'donnell, K., (2004). FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. European Journal of Plant Pathology, 110(5), pp.473-479.
5. Gezgin, Y. and Eltem, R., (2009). Diversity of endophytic fungi from various Aegean and Mediterranean orchids (saleps). Turkish journal of Botany, 33(6), pp.439-445.
6. 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. Applied and environmental microbiology, 61(4), pp.1323-1330.
7. Herron, D.A., Wingfield, M.J., Wingfield, B.D., Rodas, C.A., Marincowitz, S. and Steenkamp, E.T., (2015). Novel taxa in the Fusarium fujikuroi species complex from Pinus spp. Studies in Mycology, 80, pp.131-150.
8. Jiang, J., Zhang, K., Cheng, S., Nie, Q., Zhou, S.X., Chen, Q., Zhou, J., Zhen, X., ting Li, X., wen Zhen, T. and Xu, M., 2019. Fusarium oxysporum KB-3 from Bletilla striata: an orchid mycorrhizal fungus. Mycorrhiza, 29(5), pp.531-540.

9. Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35:1547-1549.
10. Lee, H.J. and Min, B.R. (2005). RFLP and partial DNA sequence analysis of beta-tubulin gene in the genus Fusarium. Direct Submission, GenBank. Unpublished.
11. Lombard, L., Van der Merwe, N.A., Groenewald, J.Z. and Crous, P.W., (2015). Generic concepts in Nectriaceae. Studies in Mycology, 80, pp.189-245.
12. Lupien,S.L., Dugan,F.M., Ward,K. and O'Donnell,K.(2016). Wilt, crown and root rot of common rose mallow (Hibiscus moscheutos) caused by a novel Fusarium sp. Direct Submission, GenBank. Unpublished.
13. Meghatli, M., Krimi, Z., Tunali, B. and Kansu, B. (2020). Fusarium oxysporum isolate Foa 72 translation elongation factor 1-alpha (EF 1-alpha) gene, partial cds, Direct Submission, GenBank. Unpublished.
14. Melo, I.S., Santos, S.N., Rosa, L.H., Parma, M.M., Silva, L.J., Queiroz, S.C. and Pellizari, V.H., (2014). Isolation and biological activities of an endophytic Mortierella alpina strain from the Antarctic moss Schistidium antarctici. Extremophiles, 18(1), pp.15-23.
15. O'Donnell, K., Cigelnik, E. and Nirenberg, H.I., (1998). Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia, 90(3), pp.465-493. Osbourn, A., (2010). Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation. Trends in Genetics, 26(10), pp.449-457.
16. Santos, A.G., da Rocha, G.O. and de Andrade, J.B., (2019). Occurrence of the potent mutagens 2-nitrobenzanthrone and 3-nitrobenzanthrone in fine airborne particles. Scientific reports, 9(1), pp.1-13.
17. Tamura, K. and Nei, M., (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution, 10(3), pp.512-526.
18. Tan, R.X. and Zou, W.X., (2001). Endophytes: a rich source of functional metabolites. Natural product reports, 18(4), pp.448-459.
19. Thompson, J.D., Higgins, D.G. and 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), pp.4673-4680.
20. Van Den Ende, A.G. and De Hoog, G.S., 1999. Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol, 43, pp.151-162.
21. Vu, D., Groenewald, M., De Vries, M., Gehrmann, T., Stielow, B., Eberhardt, U., Al-Hatmi, A., Groenewald, J.Z., Cardinali, G., Houbraken, J. and Boekhout, T., (2019). Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in mycology, 92, pp.135-154.
22. Yang, M., Zhang, H., van der Lee, T.A., Waalwijk, C., van Diepeningen, A.D., Feng, J., Brankovics, B. and Chen, W., (2020). Population genomic analysis reveals a highly conserved mitochondrial genome in Fusarium asiaticum. Frontiers in Microbiology, 11, p.839.
23. http://www.blast.ncbi.nlm.nih.gov (Son erişim tarihi: Haziran 2021)