Assessment of Genetic Diversity Among Fusarium Species Isolated from Wheat Using SCoT Markers

Öz

Fusarium species pose significant threats to wheat cultivation by inducing root rot, crown rot, and head blight. Understanding the genetic variations within these species is pivotal for effective disease management strategies. This investigation aimed to evaluate the genetic variation among isolates of Fusarium culmorum, F. graminearum, and F. pseudograminearum following their identification via cultural and species-specific PCR assays. The application of species-specific PCR facilitated the precise identification of these species by generating bands of distinct sizes (F. culmorum: 570 bp, F. pseudograminearum: 520 bp; F. graminearum: 420 bp), aligning with morphological classifications. A total of 36 Start Codon Targeted (SCoT) markers were employed to characterize the genetic variability among Fusarium isolates. Among the selected SCoT primers, 181 bands were amplified, with 131 exhibiting polymorphism (72.37%). The percentages of polymorphic bands in primers varied between 61.54 and 89.29. The highest polymorphism locus was observed in Fusarium culmorum with 12.71%, followed by F. pseudograminearum and F. graminearum. The average polymorphism information content and resolving power for the SCoT markers were calculated as 0.26 and 10.04, respectively. Utilizing the unweighted pair group method with arithmetic mean (UPGMA) clustering analysis employing Dice's method, the SCoT data delineated the Fusarium isolates into three distinct clusters corresponding to species. The study which focused on specifically evaluating the genetic diversity of Fusarium culmorum, F. graminearum and F. pseudograminearum, recognized as primary threats to global wheat cultivation, with SCoT markers, substantial genetic variations were revealed among Fusarium isolates at both inter- and intra-species levels. This investigation represents the initial endeavor to elucidate the genetic diversity of Fusarium isolates obtained from wheat utilizing SCoT markers.

Anahtar Kelimeler

• Fusarium spp.
• Genetic diversity
• Root rot
• SCoT markers
• Wheat

Buğdaydan İzole Edilen Fusarium Türlerinde SCoT Markörleri Kullanılarak Genetik Çeşitliliğin Değerlendirilmesi

Öz

Fusarium türleri kök çürüklüğü, kökboğazı çürüklüğü ve başak yanıklığına neden olarak buğday yetiştiriciliğinde ciddi zararlara neden olmaktadır. Bu türler içerisindeki genetik varyasyonun anlaşılması, etkili hastalık yönetimi stratejileri için oldukça önemli bir yere sahiptir. Bu çalışmanın amacı, kültürel yöntemler ve tür özgü PCR testleri aracılığıyla Fusarium culmorum, F. graminearum ve F. pseudograminearum izolatlarının tanımlanmasının ardından, bu türler arasındaki genetik çeşitliliği değerlendirmektir. Türe özgü PCR uygulaması, morfolojik sınıflandırmalarla uyumlu olarak çeşitli boyutlarda bantlar (F. culmorum: 570 bp, F. pseudograminearum: 520 bp; F. graminearum: 420 bp) oluşturmuştur. Fusarium izolatları içindeki genetik değişkenliği karakterize etmek için toplam 36 adet başlangıç kodonunu hedefleyen (SCoT) markör kullanılmıştır. Seçilen SCoT primerlerinden 181 bant amplifiye edilmiş ve bunların 131'i (%72.37) polimorfizm göstermiştir. Primerler arasındaki polimorfik band yüzdeleri 61.54 ve 89.29 arasında değişim göstermiştir. En yüksek polimorfizm lokusu %12.71 ile Fusarium culmorum izolatlarında görülmüş, bunu F. pseudograminearum ve F. graminearum izolatları takip etmiştir. SCoT markörleri için ortalama polimorfizm bilgi içeriği ve çözünürlük gücü sırasıyla 0.26 ve 10.04 olarak hesaplanmıştır. Dice'in yöntemine göre aritmetik ortalamalı ağırlıksız çift grup yöntemi (UPGMA) kümeleme analiz kullanılarak, SCoT verileri Fusarium izolatlarını türlerle uyumlu bir şekilde üç ayrı kümeye ayırmıştır. Küresel buğday yetiştiriciliğine yönelik birincil tehditler olarak kabul edilen F. culmorum, F. graminearum ve F. pseudograminearum'un genetik çeşitliliğinin SCoT markörleri ile değerlendirilmesine özel olarak odaklanan bu çalışmada, Fusarium izolatları arasında hem türler arası hem de tür içi seviyelerde önemli genetik varyasyonlar ortaya konulmuştur. Bu araştırma, başlangıç kodonunu hedefleyen belirteçlerin kullanılarak buğdaydan elde edilen Fusarium izolatlarının genetik çeşitliliğini belirlemek amacıyla SCoT markörlerinin kullanıldığı ilk çalışma niteliğindedir.

Anahtar Kelimeler

• Buğday
• Fusarium spp.
• Genetik çeşitlilik
• Kök çürüklüğü
• SCoT markörleri

Kaynakça

1. Agustí-Brisach, C., Raya-Ortega, M. C., Trapero, C., Roca, L. F., Luque, F., López-Moral, A. and Trapero, A. (2018). First report of Fusarium pseudograminearum causing crown rot of wheat in Europe. Plant Disease, 102(8): 1670–1670. https://doi.org/10.1094/PDIS-11-17-1840-PDN
2. Albayrak, G., Yörük, E., Gazdağli, A. and Sharifnabi, B. (2016). Genetic diversity among Fusarium graminearum and F. culmorum isolates based on ISSR markers. Archives of Biological Sciences, 68(2): 333–343. https://doi.org/10.13140/RG.2.1.4666.7125
3. Alkan, M., Göre, M. E., Bayraktar, H. and Özer, G. (2019). Genetic variation of Fusarium spp. isolates associated with root and crown rot of winter wheat using retrotransposon-based iPBS assays. International Journal of Agricultural and Wildlife Sciences, 5(2): 250–259. https://doi.org/10.24180/ijaws.537423
4. Anonymous (2024). Food and Agriculture Organization Statistical Database (FAO). http://www.fao.org/faostat/en/#data/QC (Accessed Date: 23.02.2024)
5. Aoki, T. and O'Donnell, K. (1999). Morphological and molecular characterization of Fusarium pseudograminearum sp. nov., formerly recognized as the Group 1 population of F. graminearum. Mycologia, 91(4): 597–609. https://doi.org/10.1080/00275514.1999.12061058
6. Aydın, F., Özer, G., Alkan, M. and Çakır, İ. (2022). Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough. Food Microbiology, 107: 104081.
7. Bayraktar, H. and Dolar, F. (2009). Genetic diversity of wilt and root rot pathogens of chickpea, as assessed by RAPD and ISSR. Turkish Journal of Agriculture and Forestry, 33(1): 1–10. https://doi.org/10.3906/tar-0709-37
8. Bentley, A. R., Tunali, B., Nicol, J. M., Burgess, L. W. and Summerell, B. A. (2004). A Preliminary Survey of Fusarium Species Associated with Wheat Stem Bases in Northern Turkey. 3. Ind Australasian Soilborne Diseases Symposium, February, Sydney, Australia.

9. Bockus, W. W., Bowden, R. L., Hunger, R. M., Morrill, W. L., Murray, T. D. and Smiley, R. W. (2010). Compendium of Wheat Diseases and Pests. 3rd ed. American Phytopathological Society, Minnesota, U.S.A.
10. Bozoğlu, T., Derviş, S., Imren, M., Amer, M., Özdemir, F., Paulitz, T. C. and Özer, G. (2022). Fungal pathogens associated with crown and root rot of wheat in central, eastern, and southeastern Kazakhstan. Journal of Fungi, 8(5): 417. https://doi.org/10.3390/jof8050417
11. Çakar, D., Özer, G., Akıllı Şimşek, S. and Maden, S. (2023). Determination of vc and mating types of Cryphonectria parasitica isolates by multiplex PCR and their genetic diversity in 13 chestnut‐growing provinces of Turkey. Forest Pathology, 53(3): e12813.
12. Chakraborty, S., Liu, C. J. and Mitter, V. (2006). Pathogen population structure and epidemiology are keys to wheat crown rot and Fusarium head blight management. Australasian Plant Pathology, 35: 643–655. https://doi.org/10.1071/AP06068
13. Collard, B. C. and Mackill, D. J. (2009). Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Molecular Biology Reporter, 27: 86–93. https://doi.org/10.1007/s11105-008-0060-5
14. Consolo, V. F., Ortega, L. M., Salerno, G., Astoreca, A. L. and Alconada, T. M. (2015). Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina. Revista Argentina de Microbiología, 47(3): 245–250. https://doi.org/10.1016/j.ram.2015.05.004
15. Cook, R. J. (2010). Fusarium Root, Crown, and Foot Rots and Associated Seedling Diseases. In: Compendium of Wheat Diseases and Pests, Eds: APS Press, St. Paul, Minnesota, USA.
16. Demirel, R., Ilhan, S., Asan, A., Kinaci, E. and Oner, S. (2005). Microfungi in cultivated fields in Eskişehir provience (Turkey). Journal of Basic Microbiology, 45(4): 279–293. https://doi.org/10.1002/jobm.200410526
17. Dyer, A. T., Johnston, R. H., Hogg, A. C. and Johnston, J. A. (2009). Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (F. culmorum, F. pseudograminearum and F. graminearum) on hard red spring and durum wheat. European Journal Plant Pathology, 125: 387–395.
18. Gholami, S., Vafaee, Y., Nazari, F. and Ghorbani, A. (2021). Exploring genetic variations in threatened medicinal orchids using start codon targeted (SCoT) polymorphism and marker-association with seed morphometric traits. Physiology and Molecular Biology of Plants, 27: 769–785. https://doi.org/10.1007/s12298-021-00978-4
19. Güney, İ. G., Tekin, F., Günen, T. U., Özer, G. and Derviş, S. (2023). Alternaria alternata causing inner black rot of lemon (Citrus limon) fruits in Turkey: Genetic diversity and characterisation. Physiological and Molecular Plant Pathology, 125: 101998. https://doi.org/10.1016/j.pmpp.2023.101998
20. Gupta, V., Jatav, P. K., Haq, S. U., Verma, K. S., Kaul, V. K., Kothari, S. L. and Kachhwaha, S. (2019). Translation initiation codon (ATG) or SCoT markers-based polymorphism study within and across various Capsicum accessions: insight from their amplification, cross-transferability and genetic diversity. Journal of Genetics, 98: 1–12. https://doi.org/10.1007/s12041-019-1095-0
21. Hornok, L., Waalwijk, C. and Leslie, J. F. (2007). Genetic factors affecting sexual reproduction in toxigenic Fusarium species. International Journal of Food Microbiology, 119(1-2): 54–58. https://doi.org/10.1016/j.ijfoodmicro.2007.07.026
22. Igwe, D. O., Afiukwa, C. A., Ubi, B. E., Ogbu, K. I., Ojuederie, O. B. and Ude, G. N. (2017). Assessment of genetic diversity in Vigna unguiculata L. (Walp) accessions using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphic markers. BMC Genetics, 18(1): 1–13. https://doi.org/10.1186/s12863-017-0567-6
23. Köycü, N. D. and Sukut, F. (2018). Effect of unregistreted fungicides to Fusarium culmorum on wheat. Journal of Tekirdag Agricultural Faculty, 15(2): 26-35.
24. Leslie, J. F. and Summerell, B. A. (2006). The Fusarium Laboratory Manual. Blackwell Professional, Iowa, U.S.A. Machenahalli, S., Jingade, P., Giri, M. S., Huded, A. K. C., Sudha, M., Daivasikamani, S. and Mishra, M. K. (2021). Cross infection and molecular characterization of Colletotrichum spp. infecting coffee and black pepper. Physiological and Molecular Plant Pathology, 113: 101600. https://doi.org/10.1016/j.pmpp.2021.101600
25. Matny, O. N., Shamsallah, S. A. and Haas, M. (2018). Genetic diversity study of Fusarium culmorum: causal agent of wheat crown rot in Iraq. BioRxiv, 341909. https://doi.org/10.1101/341909
26. Mehri, Z., Khodaparast, S. A., Aalami, A., and Mousanejad, S. (2020). Genetic diversity of Athelia rolfsii populations in the north of Iran. Rostaniha, 21(1): 14-26. 10.22092/BOTANY.2020.127939.1175
27. Mueller, B. D., Groves, C. L., Holtz, D., Deutsch, A. and Smith, D. L. (2018). First report of Fusarium culmorum causing Fusarium head blight of wheat in Wisconsin. Plant Disease, 102(5): 1028–1028. https://doi.org/10.1094/PDIS-08-17-1220-PDN
28. Nath, V. S., Hegde, V. M., Jeeva, M. L., Misra, R. S., Veena, S. S., Raj, M. and Nair, S. S. D. (2013). Genetic diversity of Phytophthora colocasiae causing taro leaf blight: analysis using start codon targeted (SCoT) polymorphism. Journal of Root Crops, 39(2): 168–177.
29. Nicholson, P., Simpson, D. R., Weston, G., Rezanoor, H. N., Lees, A. K., Parry, D. W. and Joyce, D. (1998). Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology, 53(1): 17–37. https://doi.org/10.1006/pmpp.1998.0170
30. Ölmez, F. and Tunali, B. (2019). Fusarium species isolated from wheat samples showing root and crown rot. Plant Protection Bulletin, 59(3): 31–37. https://doi.org/10.16955/bitkorb.515066
31. Özdemir, F. G. G., Arici, Ş. E. and Elekçioğlu, İ. H. (2023). Interaction of different Fusarium culmorum isolates and Pratylenchus thornei on wheat. Journal of Tekirdag Agricultural Faculty, 20(1): 1-11.
32. Özer, G., Erper, İ., Yıldız, Ş., Bozoğlu, T., Zholdoshbekova, S., Alkan, M. and Derviş, S. (2023). Fungal pathogens associated with crown and root rot in wheat-growing areas of Northern Kyrgyzstan. Journal of Fungi, 9(1): 124. https://doi.org/10.3390/jof9010124
33. Özer, G., Paulitz, T. C., Imren, M., Alkan, M., Muminjanov, H. andDababat, A. A. (2020). Identity and pathogenicity of fungi associated with crown and root rot of dryland winter wheat in Azerbaijan. Plant Disease, 104(8): 2149–2157. https://doi.org/10.1094/PDIS-08-19-1799-RE
34. Palacıoğlu, G., Alkan, M., Derviş, S. Bayraktar H. and Özer G. (2023). Molecular phylogeny of plant pathogenic fungi based on start codon targeted (SCoT) polymorphism. Molecular Biology Reports, 50: 8271–8279 https://doi.org/10.1007/s11033-023-08735-4
35. Pouzeshimiab, B., Razavi, M., Zamanizadeh, H. R., Zare, R. and ReZaee, S. (2014). Assessment of genotypic diversity among Fusarium culmorum populations on wheat in Iran. Phytopathologia Mediterranea, 53(2): 300–310. http://www.jstor.org/stable/43871782
36. Prevost, A. and Wilkinson, M. J. (1999). A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics, 98: 107–112. https://doi.org/10.1007/s001220051046
37. Roldan-Ruiz, I., Dendauw, J., Van Bockstaele, E., Depicker, A. and De Loose, M. A. F. L. P. (2000). AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.). Molecular Breeding, 6: 125–134. https://doi.org/10.1023/A:1009680614564
38. Tunali, B., Nicol, J. M., Hodson, D., Uçkun, Z., Büyük, O., Erdurmuş, D. and Bağci, S. A. (2008). Root and crown rot fungi associated with spring, facultative, and winter wheat in Turkey. Plant Disease, 92(9): 1299–1306. https://doi.org/10.1094/PDIS-92-9-1299
39. Tunali, B., Nicol, J., Erol, F. Y. and Altiparmak, G. (2006a). Pathogenicity of Turkish crown and head scab isolates on stem bases on winter wheat under greenhouse conditions. Plant Pathology Journal, 5(2): 143-149. https://doi.org/10.3923/ppj.2006.143.149
40. Tunali, B., Ozseven, I., Buyuk, O., Erdurmus, D. and Demirci, A. (2006b). Fusarium head blight and deoxynivalenol accumulation of wheat in Marmara region and reactions of wheat cultivars and lines to F. graminearum and F. culmorum. Plant Pathology Journal, 5(2): 150-156. https://doi.org/10.3923/ppj.2006.150.156
41. Ünal, F., Dolar, F. S., Tekiner, N. and Yeğin, N. Z. (2017). Molecular identification, virulens and genetic diversity of Fusarium species on wheat. Eurasian Journal of Agricultural Research, 1(2): 13–22.
42. Xu, F., Song, Y. L., Yang, G. Q., Wang, J. M., Liu, L. L. and Li, Y. H. (2015). First report of Fusarium pseudograminearum from wheat heads with fusarium head blight in North China Plain. Plant Disease, 99(1): 156–156. https://doi.org/10.1094/PDIS-05-14-0543-PDN
43. Yeken, M. Z., Emiralioğlu, O., Çiftçi, V., Bayraktar, H., Palacıoğlu, G. and Özer, G. (2022). Analysis of genetic diversity among common bean germplasm by start codon targeted (SCoT) markers. Molecular Biology Reports, 49(5): 3839–3847. https://doi.org/10.1007/s11033-022-07229-z
44. Yue, Q., Zhang, C., Wang, Q., Wang, W., Wang, J. and Wu, Y. (2019). Analysis on genetic diversity of 51 Grape germplasm resources. Ciência Rural, 49(11): e20190247. http://www.10.1590/0103-8478cr20190247