Identification of Seedborne Fungi on Soybean (Glycine max L.) Seeds Grown in Mediterranean Region of Turkey

Abstract

Soybean (Glycine max L.) is one of the most valuable oilseed crops in the world. It is not only an oil seed crop and feed for livestock, but also valuable mineral and vitamins sources for the human diet. The soybean yield is affected by various biotic and abiotic stress factors in all growing seasons. Diseases are one of the most significant biotic factors that reduce soybean growth and yield. Fungi are important pathogens affecting yield and quality by attacking plants during the growth period and after harvest. This study was conducted to detect and identify the seed-borne fungi associated with the soybean seed. From this context, 150 soybean seeds were randomly chosen from the experimental fields of Akdeniz University in Antalya province of Turkey. These seeds were sterilized with 70% ethanol for 1 min, followed by 10% sodium hypochlorite for 1 min and then rinsed with sterile water and then placed in Petri plates by using the agar plate method. A total of four seed-borne fungi species namely Aspergillus spp., Penicillium spp., Cladosporium spp. and Fusarium spp. were isolated from the soybean seeds. Additionally, Genomic DNAs of these fungal species were extracted and the internal transcribed spacer (ITS) region of ribosomal DNA was amplified with the ITS-1 and ITS-4 primers using a thermal cycler. After sequencing of amplified products, the sequences were aligned. BLASTn analysis of each sequence showed that the sequences of the fungi had the similarity (99%) to the fungal isolates deposited in the GenBank.

Keywords

• Soybean
• seedborne fungi
• seed decay
• molecular analysis
• ITS region

References

1. Ahammed, S.K., Anandam, R.J., Babu, P.G., Munikrishnaiah, M., Gopal, K., 2006. Studies on seed mycoflora of soybean and its effect on seed and seedling quality characters. Legume Research, 29(3): 186-190.
2. Ahmad, A., Hayat, I., Arif, S., Masud, T., Khalid, N., Ahmed, A., 2014. Mechanisms involved in the therapeutic effects of soybean (Glycine max). International Journal of Food Properties, 17(6): 1332-1354.
3. Ahmed, O., Balogun, O.S., Fawole, O.B., Fabiyi, O.A., Hussein, A.T., Kassoum, K.O., 2016. Seed-borne fungi of soybeans (Glycine max) in the guinea savannah agroecology of Nigeria. Journal of Agricultural Sciences Belgrade, 61(1): 57-68.
4. Alemu, K., 2014. Seedborne fungal pathogen associated with soybean (Glycine max L.) and their management in Jimma, Southwestern Ethiopia. Journal of Biology, Agriculture and Healthcare, 25(4): 14-19.
5. Anonymous, 2021a. Statistic Databases. American Soybean Association SoyStats. (http://soystats.com/ international-world-soybean-production/), (Accessed date: 19.10.2021).
6. Anonymous, 2021b. Crop Production Statistics. Turkish Statistical Institute, (https://biruni.tuik.gov.tr/medas), (Accessed date: 19.10.2021).
7. Barnett, H.L., Hunter, B.B., 1987. Illustrated Genera of Imperfect Fungi. 4th Ed., MacMillian, NewYork.
8. Catal, M., Adams, G.C., Fulbrigt, D.W., 2010. Evaluation of resistance to rhabdocline needlecast in douglas fir variety shuswap, with quantitative polymerase chain reaction. Phytopathology, 100(4): 337-344.

9. Chang, X., Li, H., Naeem, M., Wu, X., Yong, T., Song, C., Liu, T., Chen, W., Yang, W., 2020. Diversity of the seedborne fungi and pathogenicity of fusarium species associated with intercropped soybean. Pathogens, 9(7): 531.
10. Chiotta, M.L., Alaniz Zanon, M.S., Palazzini, J.M., Scandiani, M.M., Formento, A.N., Barros, G.G., Chulze, S.N., 2016. Pathogenicity of Fusarium graminearum and F. meridionale on soybean pod blight and trichothecene accumulation. Plant Pathology, 65(9): 1492-1497.
11. Escamilla, D., Rosso, M.L., Zhang, B., 2019. Identification of fungi associated with soybeans and effective seed disinfection treatments. Food Science and Nutrition, 7(10): 3194-3205.
12. Gutleb, A.C., Caloni, F., Giraud, F., Cortinovis, C., Pizzo, F., Hoffmann, L., Bohn, T., Pasquali, M., 2015. Detection of multiple mycotoxin occurrences in soy animal feed by traditional mycological identification combined with molecular species identification. Toxicology Reports, 2: 275-279.
13. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41(1): 95-98.
14. Killebrew, J.F., Roy, K.W., Abney, T.S., 1993. Fusaria and other fungi on soybean seedlings arid roots of older plants and interrelationships among fungi, symptoms, and soil characteristics. Canadian Journal of Plant Pathology, 15(3): 139-146.
15. Kim, D.H., Kim, S.H., Kwon, S.W., Lee, J.K., Hong, S.B., 2013. Mycoflora of soybeans used for Meju fermentation. Mycobiology, 41(2): 100-107.
16. Kim, I.S., Kim, C.H., Yang, W.S., 2021. Physiologically active molecules and functional properties of soybeans in human health a current perspective. International Journal of Molecular Sciences, 22(8): 4054.
17. Kövics, G.J., Sandor, E., Rai, M.K., Irinyi, L., 2014. Phoma-like fungi on soybeans. Critical Reviews in Microbiology, 40(1): 49-62.
18. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7): 1870-1874.
19. Levic, J., Stankоvić, S., Krnjaja, V., Bočarov-Stančić, A., Ivanović, D., 2012. Distribution and frequency and incidence of seed-borne pathogen of some cereals and industrial crops in Serbia. Journal Pesticides and Phytomedicine, 27(1): 33-40.
20. Li, S., Darwish, O., Alkharouf, N.W., Musungu, B., Matthews, B.F., 2017. Analysis of the genome sequence of Phomopsis longicolla: a fungal pathogen causing phomopsis seed decay in soybean. BMC Genomics, 18(1): 688.
21. Liu, J., Deng, J., Zhang, K., Wu, H., Yang, C., Zhang, X., Du, J., Shu, K., Yang, W., 2016. Pod mildew on soybeans can mitigate the damage to the seed arising from field mold at harvest time. Journal of Agricultural and Food Chemistry, 64(48): 9135-9142.
22. Maleki, A., Naderi, A., Naseri, R., Fathi, A., Bahamin, S., Maleki, R., 2013. Physiological performance of soybean cultivars under drought stress. Bulletin of Environment, Pharmacology and Life Sciences, 2(6): 38-44.
23. Munkvold, G.P., 2009. Seed pathology progress in academia and industry. Annual Review of Phytopathology, 47: 285-311.
24. Mutava, R.N., Prince, S.J.K., Syed, N.H., Song, L., Valliyodan, B., Chen, W., Nguyen, H.T., 2015. Understanding abiotic stress tolerance mechanisms in soybean: A comparative evaluation of soybean response to drought and flooding stress. Plant Physiology and Biochemistry, 86: 109-120.
25. Pedrozo, R., Fenoglio, J.J., Little, C.R., 2015. First report of seedborne Fusarium fujikuroi and its potential to cause pre and post emergent damping off on soybean (Glycine max) in the United States. Plant Disease, 99(12): 1865-1865.
26. Pedrozo, R., Little, C.R., 2017. Fusarium verticillioides inoculum potential influences soybean seed quality. European Journal of Plant Pathology, 148(3): 749-754.
27. Pitt, J.L., Hocking, A.D., Bhudhasamai, K., Miscamble, B.F., Wheeler, K.A., Tanboon-Ek, P., 1994. The normal mycoflora of commodities from Thailand. 2. Beans, rice, small grains and other commodities. International Journal of Food Microbiology, 23(1): 35-53.
28. Porter, L.D., Pasche, J.S., Chen, W., Harveson, R.M., 2015. Isolation, identification, storage, pathogenicity tests, hosts, and geographic range of Fusarium solani f. sp. pisi causing fusarium root rot of pea. Plant Health Progress, 16(3): 136-145.
29. Roy, K., Baird, R., Abney, T., 2001. A review of soybean (Glycine max) seed, pod, and flower mycofloras in North America, with methods and a key for identification of selected fungi. Mycopathologia, 150(1): 15-27.
30. Saitou, N., Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4): 406-425.
31. Ustun, R., Cat, A., Çatal, M., Uzun, B., 2018. First report of Fusarium proliferatum causing seed rot on soybean (Glycine max) in Turkey. Journal of Biotechnology, 280: 28-29.
32. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In PCR Protocols a Guide to Methods and Applications, pp. 315-322.
33. Wicklow, D.T., Bennett, G.A., Shotwell, O.L., 1987. Secondary invasion of soybeans by Fusarium graminearum and resulting mycotoxin contamination. Plant Disease, 71(12): 1146-1146.