Targeted genotype analysis of fusarium isolates in 3-ADON chemotype

Within the scope of this master's thesis, it was aimed to perform molecular level chemotyping of Fusarium isolates, to investigate a mycotoxin (NX-2) with unknown distribution yet via targeted genotype analysis and also to reveal the mycotoxin production capacity of isolates. In this context, a total of 78 Fusarium isolates which are isolated from various regions of our country and Iran were studied. In 30 of these isolates, fungal identification was performed by amplification of 28S rDNA region (620 bp). The isolates were identified at the species level by scanning OPT18, which is specific to F. culmorum and UBC85, which is specific to F. graminearum, SCAR markers. 478 bp fragments, which are amplified from 9 isolates, revealed the isolates as F.culmorum; 332 bp fragments amplified from 6 isolates revealed that the isolates as F. graminearum. TEF1α (Translated Elongation Factor 1α) gene was amplified for species identification of 14 samples. Amplification products (700 bp) were sequenced and aligned with the sequences in the reference Fusarium genomes in database by using bioinformatic tools. The impedence of 13 isolates with F. proliferatum (97.60-99.55%) and one with F. oxysporum (99.55%) was determined. For identification of deoxynivalenol (DON) and nivalenol (NIV) chemotypes, amplification of tri13 in the Tri5 gene cluster was targeted. 282 bp amplified product provided the selection of DON chemotype and the 1075 bp fragment allowed selection of NIV chemotype. Interestingly, both amplified products were observed in 17-16 coded isolate. 15 isolates carrying the tri3 gene as 583 bp were identified as 3-acetyldeoxynivalenol (3-ADON) and 3 isolates carrying the tri3 gene as 863 bp were identified as 15-acetyldeoxynivalenol (15-ADON). For targeted genotype analysis, 821 bp regions belongs to tri1 gene were amplified from 30 out of 53 isolates. As the amplification products were not cut with ApoI restriction endonuclease (RE) to 346 and 475 bp fragments, NX-2 chemotype could not be determined experimentally from these isolates. However, it was shown that F. graminearum 17-11 amplification site carries the ApoI RE cut site by the using bioinformatic tools based on sequencing. Additionally, it is revealed that 17-11 has 120 bp insertion via multiple alignment with gene region of reference isolate and the sequences belongs to other isolates. 78 isolates were screened in terms of DON mycotoxin production capacity by targeted genotype analysis of tri5-tri6 intergenic region. It was revealed that 45 isolates become large amount producers and one is small amount producer with of 200 bp and 650 bp fragments, respectively. Findings from molecular genotyping and targeted genotyping studies will contribute to the mapping of potential toxicological risks in the agro-ecological region where phytopathogen is effective. Data from such studies can be used to develop diagnostic kits. In this case, infectious agents and toxins can be detected in the field in an early period and it can be contributed to the protection of public health by preventing the food chain contamination.