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Effects of Kaempferol on Growth and Toxin Production of Head Blight Agent Fusarium culmorum

Year 2017, Volume: 12 Issue: 1, 24 - 33, 30.06.2017

Abstract

Fusarium species cause root rot and head blight diseases on wheat and barley. In this study, quantitative spore production, expression of tri4 gene which is one of genes responsible for DON production, and qualitative DON production were examined in F. culmorum F24 isolate, agent of head blight. It was determined that spore production was decreased by increasing concentrations of kaempferol (0, 10 ve 20 µg mL-1 kaempferol) and these differences were significant (p<0.001). Total RNA molecules isolated from samples subjected or non-subjected to kaempferol were used in Reverse Transcription Polymerase Chain Reaction (RT-PCR) in order to examine the tri4 expression after they were converted to cDNA. While gene expression was determined in control group, it was not determined in experimental groups. Similarly, spot of 0.27 Rf value was detected on control group on thin layer chromatography, but no spot belonging to DON was detected on experimental groups. Findings obtained from this study showed that kaempferol even at low concentrations decreased the production of spores and repressed the biosynthesis of DON in F. culmorum species. Results of the study is original since that the antifungal effects of kaempferol on this species was not investigated before; output data have importance in terms of plant pathology research.

References

  • Al-Burtamani, S.K., Fatope, M.O., Marwah, R.G., Onifade, A.K. and Al-Saidi S.H. 2005. Chemical Composition, Antibacterial and Antifungal Activities of the Essential Oil of Haplophyllum Tuberculatum From Oman. Journal of Ethnopharmacology, 2005, 4;96 (1-2): 107-112.
  • Anand, A., Zhou, T., Trick, H.N., Gill, B.S., Bockus, W.W. and Muthukrishnanl S. 2003. Greenhouse and Field Testing of Transgenic Wheat Plants Stably Expressing Genes for Thaumatin-Like Protein, Chitinase and Glucanase Against Fusarium graminearum. Journal of Experimental Biology, 2003, 54 (384): 1101-1111.
  • Arıcı, Ş.E. ve Koç, N.K. 2008. in vitro Seleksiyon Tekniği ile Buğday (Triticum aestivum)'da Fusarium (Fusarium spp)'a Dayanıklı Hücre Hatlarının Elde Edilmesi ve Bitki Regenerasyonu. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 2008, 21 (1): 55-64.
  • Arslan, Ü. ve Baykal, N. 2002. Kök ve Kökboğazı Fungal Patojenlerine Karşı Bazı Buğday Çeşitlerinin Reaksiyonları ve Tohum Koruyucu Fungusitlerin Fusarium culmorum (w.g.sm.) sacc.’a Etkisi. Uludağ Üniviversitesi Ziraat Fakültesi Dergisi, 2002, 16: 69-76.
  • Bai, G., Desjardins, A.E. and Plattner, R.D. 2001. Deoxynivalenol-Nonproducing Fusarium graminearum Causes İnitial İnfection, But Does Not Cause Disease Spread İnwheat Spikes. Mycopathologia, 2001, 153: 914-98.
  • Bai, G. and Shaner, G. 2004. Management and Resistance in Wheat and Barley to Fusarium Head Blight. Annual Review of Phytopathology, 2004, 42: 135-161.
  • Bernardo, A., Bai, G., Guo, P., Xiao, K., Guenzi, A.C. and Ayoubi, P. 2007. Fusarium graminearum-Induced Changes in Gene Expression Between Fusarium Head Blight-Resistant and Susceptible Wheat Cultivar. Functional and Integrative Genomics, 2007, 7: 69-77.
  • Cabral, L.C., Pinto, V.F. and Patriarca, A. 2013. Application of Plant Derived Compounds to Control Fungal Spoilage and Mycotoxin Production in Foods. International Journal of Food Microbiology, 2013, 166: 1–14.
  • Calderón-Montaño, J.M., Burgos-Morón, E., Pérez-Guerrero, C. and López-Lázaro, M. 2011. A Review on the Dietary Flavonoid Kaempferol. Mini-Reviews in Medicinal Chemistry, 11: 298-344.
  • Castillo, F., Hernández, D., Gallegos, G., Rodriguez, R. and Aguilar, C.N. 2012. Antifungal Properties of Bioactive Compounds from Plants. (Ed. Dhanasekaran, D.), Fungicides for Plant and Animal Diseases, InTech, Mexico, pp. 81-106.
  • Desjardins, A.E. and Proctor, R.H. 2007. Molecular Biology of Fusarium Mycotoxins. International Journal of Food Microbiology, 2007, 119: 47-50.
  • Dyakov, T.Y., Dzhavakhlya, V.G. and Korpela, T. 2007. Comprehensive and Molecular Phytopathology, (1th edition), Elsevier, Amsterdam.
  • FAO, 2013. Prodstat Crops. http://faostat.fao.org (erişim tarihi: 28. 08. 2016)
  • Gutleb, A.C., Morrison, E. and Murk. A.J. 2002. Cytotoxicity Assay for Mycotoxins Produced by Fusarium Strains. Enviromental Toxicology and Pharmocology, 2002, 11: 309-320.
  • Haratian, M., Sharifnabi, B., Alizadeh, A. and Safaie, N. 2008. PCR Analysis of the tri13 Gene to Determine the Genetic Potential of Fusarium graminearum İsolates from Iran to Produce Nivalenol And Deoxynivalenol. Mycopathologia, 2008, 166:109–116.
  • Hendra, R., Ahmad, S., Sukari, A., Shukor, M.Y. and Oskoueian, E. 2011. Flavonoid Analyses and Antimicrobial Activity of Various Parts of Phaleria Macrocarpa (Scheff.) Boerl Fruit. International Journal of Molecular Sciences, 2011, 12: 3422-3431.
  • Hogg, S. 2005. Essential Microbiology, (1th edition), John Wiley and Sons, West Sussex.
  • Kimura, M., Tokai, T., Takahashi-Ando, N., Ohsato, S. and Fujimura, M. 2007. Molecular and Genetic Studies of Fusarium Trichothecen Pathways Gene and Evolution. Bioscience, Biotechnology and Biochemistry, 2007, 71: 2105-2123.
  • Kowalski, J., Samojedny, A., Paul, M., Pietsz, G. and Wilczok, T. 2005. Effect of kaempferol on the production and gene expression of monocyte chemoattractant protein-1 in J774.2 macrophages. Pharmacological Reports, 2005, 57: 107-112.
  • Llorens, A., Hinojo, M.J., Mateo, R., Medina, A., Valle-Algarre, F.M., Gonzalez-Jaen, M.T. and Jimenez, M. 2006. Variability and Characterization of Mycotoxin Producing Fusarium spp. Isolates by PCR-RFLP Analysis of the IGS-rDNA Region. Antonie van Leeuwenhoek, 2006, 89: 465-478
  • McDonald, T., Brown, D., Keller, N.P. and Hammond, T.M. 2005. RNA Silencing of Mycotoxin Production in Aspergillus and Fusarium Species. Molecular Plant-Microbe Interactions, 2005, 18 (6): 539-545.
  • Mert-Türk, F. ve Gencer, G. 2013. Distribution of the 3-AcDON, 15-AcDON, and NIV Chemotypes of Fusarium culmorum in the North-West of Turkey. Plant Protection Science, 2013, 49 (2): 57-64.
  • Miedaner, T., Cumagun, C.J.R. and Chakraborty, S. 2008. Population Genetics of Three İmportant Head Blight Pathogens Fusarium graminearum, F. pseudograminearum and F. culmorum. Journal of Phytopathology, 2008, 156: 129-139.
  • Miedaner, T., Caixeta, F. and Talas, F. 2013. Head-blighting Populations of Fusarium culmorum from Germany, Russia and Syria Analyzed by Microsatellite Markers Show a Recombining Structure. European Journal of Plant Pathology, 2008, 137: 743-752.
  • NCBI, 2016. Genome. http://www.ncbi.nlm.nih.gov (erişim tarihi: 28. 08. 2016)
  • Özer, N. ve Soran, H. 1991. Fusarium Species of Turkey. Hacettepe Üniversitesi Eğitim Fakültesi Dergisi, 1991, 6: 259-271.
  • Parry, D.W., Jenkinson, P. and McLeod, L. 1995. Fusarium Ear Blight (scab) in Small Grain Cereals-A Review. Plant Pathology, 1995, 44: 207-238.
  • Scherm, B., Orru, M., Balmas, V., Spanu, F., Azara, E., Delogu, G., Hammond, T.M., Keller, N.P. and Migheli, Q. 2011. Altered Trichothecene Biosynthesis in tri6-Silenced Transformants of Fusarium culmorum İnfluences the Severity of Crown and Foot Rot on Durum Wheat Seedlings. Molecular Plant Pathology, 2011, 12 (8): 759-771.
  • Scherm, B., Balmas, V., Spanu, F., Pani, G., Delogu, G., Pasquali, G. and Migheli, Q. 2013. Fusarium culmorum: Causal Agent of Foot and Root Rot and Head Blight on Wheat. Molecular Plant Pathology, 2013, 14 (4): 323-341.
  • Shao, J., Zhang, M.X., Wang, T.M., Li, Y. And Wang, C.Z. 2016. The roles of CDR1, CDR2, and MDR1 in kaempferolinduced suppression with fluconazole-resistant Candida albicans. Pharmaceutical Biology, 2016, 54 (6): 984-992.
  • Tok, F.M. ve Arslan, M. 2016. Distribution and Genetic Chemotyping of Fusarium graminearum and Fusarium culmorum Populations in Wheat Fields in the Eastern Mediterranean Region of Turkey. Biotechnology and Biotechnological Equipment, 2016, 30 (2): 254-260.
  • Tunali, B., Özseven, İ., Büyük, O., Erdurmuş, D. ve Demirci, A. 2006. Fusarium Head Blight and Deoxynivalenol Accumulation of Wheat in Marmara Region and Reactions of Wheat Cultivars and Lines to F. graminearum and Fusarium culmorum. Plant Pathology Journal, 2006, 5 (2): 150-156.
  • Wang, J.H., Ndoye, M., Zhang, J.B., Li, H.P. and Liao, Y.C. 2011. Population Structure and Genetic Diversity of the Fusarium graminearum Species Complex. Toxins, 2011, 3 (8): 1020-1037.
  • Windels, C.R. 2000. Economic and Social Impacts of Fusarium Head Blight: Changing Farms and Rural Communities in the Northern Great Plains. Phytopathology, 2000, 90 (1): 17-21.
  • Yli-Mattila, T., Rämö, S., Hietaniemi, V., Hussien, T., Carlobos-Lopez, A.L. and Cumagun, C.J.R. 2013. Molecular Quantification and Genetic Diversity of Toxigenic Fusarium Species in Northern Europe as Compared to Those in Southern Europe. Microorganisms, 2013, 1: 162-174.
  • Yordanov, M., Dimitrova, P., Patkar, S., Saso, L. and Ivanovska, N. 2008. Inhibition of Candida albicans extracellular enzyme activity by selected natural substances and their application in Candida infection. Canadian Journal of Microbiology, 2008, 54: 435-440.
  • Yörük, E., Gazdağlı, A. ve Albayrak, A. 2014. Class B Trichothecene Chemotyping in Fusarium Species by PCR Assay. Genetika-Belgrade, 2014, 46 (3): 661-669.
  • Yörük, E., Tunali, B., Kansu, B., Ölmez, F., Uz, G., Zümrüt, I.M., Sarıkaya, A. ve Meyva, G. 2016. Characterization of High-Level Deoxynivalenol Producer Fusarium graminearum and F. culmorum İsolates Caused Head Blightand Crown Rot Diseases in Turkey.  Journal of Plant Diseases and Protection, 2016, 123:177–186.

Kaempferol’ün Başak Yanıklığı Etmeni Fusarium culmorum’un Üremesi ve Toksin Üretimi Üzerine Olan Etkileri

Year 2017, Volume: 12 Issue: 1, 24 - 33, 30.06.2017

Abstract

Fusarium türleri buğday ve arpada, kök çürüklüğü ve
başak yanıklığı hastalıklarına neden olmaktadır. Bu çalışmada kaempferol uygulaması sonucunda başak yanıklığı
etmeni F. culmorum F24 izolatında niceliksel
olarak spor üretimi, DON üretiminde sorumlu genlerden tri4 geninin anlatımı ve kalitatif olarak DON üretimi
incelenmiştir. Spor üretiminin artan kaempferol konsantrasyonlarında (0, 10 ve
20 µg
mL-1 kaempferol)
düştüğü ve bu farkın anlamlı olduğu (p<0.001) saptanmıştır. Kaempferol
uygulanmış ve uygulanmamış örneklerden izole edilen total RNA molekülleri
cDNA’ya çevrildikten sonra Ters Transkripsiyon Polimeraz Zincir Reaksiyonu’nda (RT-PZR)
tri4 anlatımının incelenmesinde kullanılmıştır.
Kontrol grubunda gen anlatımı belirlenirken, deney gruplarında gen anlatımı
saptanmamıştır. Benzer şekilde ince tabaka kromatografisinde de kontrol
grubunda 0.27 Rf (ing., ‘Retention
factor’) değerine sahip spot görülürken, deney gruplarında DON’a ait spot
saptanmamıştır. Elde edilen veriler kaemferol’ün düşük konsantrasyonlarda bile F. culmorum türünde spor üretimini azalttığını
ve DON üretimini baskılayabildiğini göstermiştir. Elde edilen veriler kaempferol’ün
daha önce bu türde antifungal etkisinin araştırılmaması sebebiyle özgünlük
içermekte; veriler bitki patolojisi araştırmaları açısından önem arz
etmektedir.

References

  • Al-Burtamani, S.K., Fatope, M.O., Marwah, R.G., Onifade, A.K. and Al-Saidi S.H. 2005. Chemical Composition, Antibacterial and Antifungal Activities of the Essential Oil of Haplophyllum Tuberculatum From Oman. Journal of Ethnopharmacology, 2005, 4;96 (1-2): 107-112.
  • Anand, A., Zhou, T., Trick, H.N., Gill, B.S., Bockus, W.W. and Muthukrishnanl S. 2003. Greenhouse and Field Testing of Transgenic Wheat Plants Stably Expressing Genes for Thaumatin-Like Protein, Chitinase and Glucanase Against Fusarium graminearum. Journal of Experimental Biology, 2003, 54 (384): 1101-1111.
  • Arıcı, Ş.E. ve Koç, N.K. 2008. in vitro Seleksiyon Tekniği ile Buğday (Triticum aestivum)'da Fusarium (Fusarium spp)'a Dayanıklı Hücre Hatlarının Elde Edilmesi ve Bitki Regenerasyonu. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 2008, 21 (1): 55-64.
  • Arslan, Ü. ve Baykal, N. 2002. Kök ve Kökboğazı Fungal Patojenlerine Karşı Bazı Buğday Çeşitlerinin Reaksiyonları ve Tohum Koruyucu Fungusitlerin Fusarium culmorum (w.g.sm.) sacc.’a Etkisi. Uludağ Üniviversitesi Ziraat Fakültesi Dergisi, 2002, 16: 69-76.
  • Bai, G., Desjardins, A.E. and Plattner, R.D. 2001. Deoxynivalenol-Nonproducing Fusarium graminearum Causes İnitial İnfection, But Does Not Cause Disease Spread İnwheat Spikes. Mycopathologia, 2001, 153: 914-98.
  • Bai, G. and Shaner, G. 2004. Management and Resistance in Wheat and Barley to Fusarium Head Blight. Annual Review of Phytopathology, 2004, 42: 135-161.
  • Bernardo, A., Bai, G., Guo, P., Xiao, K., Guenzi, A.C. and Ayoubi, P. 2007. Fusarium graminearum-Induced Changes in Gene Expression Between Fusarium Head Blight-Resistant and Susceptible Wheat Cultivar. Functional and Integrative Genomics, 2007, 7: 69-77.
  • Cabral, L.C., Pinto, V.F. and Patriarca, A. 2013. Application of Plant Derived Compounds to Control Fungal Spoilage and Mycotoxin Production in Foods. International Journal of Food Microbiology, 2013, 166: 1–14.
  • Calderón-Montaño, J.M., Burgos-Morón, E., Pérez-Guerrero, C. and López-Lázaro, M. 2011. A Review on the Dietary Flavonoid Kaempferol. Mini-Reviews in Medicinal Chemistry, 11: 298-344.
  • Castillo, F., Hernández, D., Gallegos, G., Rodriguez, R. and Aguilar, C.N. 2012. Antifungal Properties of Bioactive Compounds from Plants. (Ed. Dhanasekaran, D.), Fungicides for Plant and Animal Diseases, InTech, Mexico, pp. 81-106.
  • Desjardins, A.E. and Proctor, R.H. 2007. Molecular Biology of Fusarium Mycotoxins. International Journal of Food Microbiology, 2007, 119: 47-50.
  • Dyakov, T.Y., Dzhavakhlya, V.G. and Korpela, T. 2007. Comprehensive and Molecular Phytopathology, (1th edition), Elsevier, Amsterdam.
  • FAO, 2013. Prodstat Crops. http://faostat.fao.org (erişim tarihi: 28. 08. 2016)
  • Gutleb, A.C., Morrison, E. and Murk. A.J. 2002. Cytotoxicity Assay for Mycotoxins Produced by Fusarium Strains. Enviromental Toxicology and Pharmocology, 2002, 11: 309-320.
  • Haratian, M., Sharifnabi, B., Alizadeh, A. and Safaie, N. 2008. PCR Analysis of the tri13 Gene to Determine the Genetic Potential of Fusarium graminearum İsolates from Iran to Produce Nivalenol And Deoxynivalenol. Mycopathologia, 2008, 166:109–116.
  • Hendra, R., Ahmad, S., Sukari, A., Shukor, M.Y. and Oskoueian, E. 2011. Flavonoid Analyses and Antimicrobial Activity of Various Parts of Phaleria Macrocarpa (Scheff.) Boerl Fruit. International Journal of Molecular Sciences, 2011, 12: 3422-3431.
  • Hogg, S. 2005. Essential Microbiology, (1th edition), John Wiley and Sons, West Sussex.
  • Kimura, M., Tokai, T., Takahashi-Ando, N., Ohsato, S. and Fujimura, M. 2007. Molecular and Genetic Studies of Fusarium Trichothecen Pathways Gene and Evolution. Bioscience, Biotechnology and Biochemistry, 2007, 71: 2105-2123.
  • Kowalski, J., Samojedny, A., Paul, M., Pietsz, G. and Wilczok, T. 2005. Effect of kaempferol on the production and gene expression of monocyte chemoattractant protein-1 in J774.2 macrophages. Pharmacological Reports, 2005, 57: 107-112.
  • Llorens, A., Hinojo, M.J., Mateo, R., Medina, A., Valle-Algarre, F.M., Gonzalez-Jaen, M.T. and Jimenez, M. 2006. Variability and Characterization of Mycotoxin Producing Fusarium spp. Isolates by PCR-RFLP Analysis of the IGS-rDNA Region. Antonie van Leeuwenhoek, 2006, 89: 465-478
  • McDonald, T., Brown, D., Keller, N.P. and Hammond, T.M. 2005. RNA Silencing of Mycotoxin Production in Aspergillus and Fusarium Species. Molecular Plant-Microbe Interactions, 2005, 18 (6): 539-545.
  • Mert-Türk, F. ve Gencer, G. 2013. Distribution of the 3-AcDON, 15-AcDON, and NIV Chemotypes of Fusarium culmorum in the North-West of Turkey. Plant Protection Science, 2013, 49 (2): 57-64.
  • Miedaner, T., Cumagun, C.J.R. and Chakraborty, S. 2008. Population Genetics of Three İmportant Head Blight Pathogens Fusarium graminearum, F. pseudograminearum and F. culmorum. Journal of Phytopathology, 2008, 156: 129-139.
  • Miedaner, T., Caixeta, F. and Talas, F. 2013. Head-blighting Populations of Fusarium culmorum from Germany, Russia and Syria Analyzed by Microsatellite Markers Show a Recombining Structure. European Journal of Plant Pathology, 2008, 137: 743-752.
  • NCBI, 2016. Genome. http://www.ncbi.nlm.nih.gov (erişim tarihi: 28. 08. 2016)
  • Özer, N. ve Soran, H. 1991. Fusarium Species of Turkey. Hacettepe Üniversitesi Eğitim Fakültesi Dergisi, 1991, 6: 259-271.
  • Parry, D.W., Jenkinson, P. and McLeod, L. 1995. Fusarium Ear Blight (scab) in Small Grain Cereals-A Review. Plant Pathology, 1995, 44: 207-238.
  • Scherm, B., Orru, M., Balmas, V., Spanu, F., Azara, E., Delogu, G., Hammond, T.M., Keller, N.P. and Migheli, Q. 2011. Altered Trichothecene Biosynthesis in tri6-Silenced Transformants of Fusarium culmorum İnfluences the Severity of Crown and Foot Rot on Durum Wheat Seedlings. Molecular Plant Pathology, 2011, 12 (8): 759-771.
  • Scherm, B., Balmas, V., Spanu, F., Pani, G., Delogu, G., Pasquali, G. and Migheli, Q. 2013. Fusarium culmorum: Causal Agent of Foot and Root Rot and Head Blight on Wheat. Molecular Plant Pathology, 2013, 14 (4): 323-341.
  • Shao, J., Zhang, M.X., Wang, T.M., Li, Y. And Wang, C.Z. 2016. The roles of CDR1, CDR2, and MDR1 in kaempferolinduced suppression with fluconazole-resistant Candida albicans. Pharmaceutical Biology, 2016, 54 (6): 984-992.
  • Tok, F.M. ve Arslan, M. 2016. Distribution and Genetic Chemotyping of Fusarium graminearum and Fusarium culmorum Populations in Wheat Fields in the Eastern Mediterranean Region of Turkey. Biotechnology and Biotechnological Equipment, 2016, 30 (2): 254-260.
  • Tunali, B., Özseven, İ., Büyük, O., Erdurmuş, D. ve Demirci, A. 2006. Fusarium Head Blight and Deoxynivalenol Accumulation of Wheat in Marmara Region and Reactions of Wheat Cultivars and Lines to F. graminearum and Fusarium culmorum. Plant Pathology Journal, 2006, 5 (2): 150-156.
  • Wang, J.H., Ndoye, M., Zhang, J.B., Li, H.P. and Liao, Y.C. 2011. Population Structure and Genetic Diversity of the Fusarium graminearum Species Complex. Toxins, 2011, 3 (8): 1020-1037.
  • Windels, C.R. 2000. Economic and Social Impacts of Fusarium Head Blight: Changing Farms and Rural Communities in the Northern Great Plains. Phytopathology, 2000, 90 (1): 17-21.
  • Yli-Mattila, T., Rämö, S., Hietaniemi, V., Hussien, T., Carlobos-Lopez, A.L. and Cumagun, C.J.R. 2013. Molecular Quantification and Genetic Diversity of Toxigenic Fusarium Species in Northern Europe as Compared to Those in Southern Europe. Microorganisms, 2013, 1: 162-174.
  • Yordanov, M., Dimitrova, P., Patkar, S., Saso, L. and Ivanovska, N. 2008. Inhibition of Candida albicans extracellular enzyme activity by selected natural substances and their application in Candida infection. Canadian Journal of Microbiology, 2008, 54: 435-440.
  • Yörük, E., Gazdağlı, A. ve Albayrak, A. 2014. Class B Trichothecene Chemotyping in Fusarium Species by PCR Assay. Genetika-Belgrade, 2014, 46 (3): 661-669.
  • Yörük, E., Tunali, B., Kansu, B., Ölmez, F., Uz, G., Zümrüt, I.M., Sarıkaya, A. ve Meyva, G. 2016. Characterization of High-Level Deoxynivalenol Producer Fusarium graminearum and F. culmorum İsolates Caused Head Blightand Crown Rot Diseases in Turkey.  Journal of Plant Diseases and Protection, 2016, 123:177–186.
There are 38 citations in total.

Details

Primary Language Turkish
Subjects Agricultural Engineering
Journal Section Research
Authors

Özlem Sefer This is me

Emre Yörük

Elif Sedef Develi This is me

İşıl Melis Zümrüt This is me

Ayşe Server Sezer This is me

Zeynep Konukçu This is me

Publication Date June 30, 2017
Submission Date September 2, 2016
Acceptance Date August 8, 2018
Published in Issue Year 2017 Volume: 12 Issue: 1

Cite

APA Sefer, Ö., Yörük, E., Develi, E. S., Zümrüt, İ. M., et al. (2017). Kaempferol’ün Başak Yanıklığı Etmeni Fusarium culmorum’un Üremesi ve Toksin Üretimi Üzerine Olan Etkileri. Ziraat Fakültesi Dergisi, 12(1), 24-33.

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