Emre Yörük

Genetic characterization of Fusarium graminearum and F. culmorum isolates from Turkey by using random-amplified polymorphic DNA.

Five Fusarium graminearum and 12 F. culmorum isolates, primarily pathogenic species of Fusarium head blight, were obtained from naturally infected wheat from various agro-ecological regions of Turkey. Genotyping of the isolates was carried out using random-amplified polymorphic DNA (RAPD). Sixty-five 10-mer oligonucleotide primers were used to amplify the RAPD markers. Among them, 50 primers produced strong and reproducible DNA amplicons. The remaining primers generated either insufficient or no amplification patterns. In total, 1200 fragments were scored, 311 of which were determined to be polymorphic and unique to the isolates. The produced RAPD markers ranged from 0.2 to 5 kb. The mean genetic similarity values of the F. graminearum and F. culmorum isolates were 61.5 and 65%, respectively. The similarity coefficient was 43 to 76.1% among F. graminearum isolates and 49 to 81.1% among F. culmorum isolates. Genetically, the most similar F. graminearum isolates were F6 and F7 (76.1%), which originated from the same agro-ecological region (Sakarya). The most similar F. culmorum isolates were F20 and F21 (81.1%), which were from different geographic regions (Bilecik and UÅŸak, respectively). Moreover, interspecific variation between the two species was determined to be 86.3 to 93.3%. Cluster analysis generated two branched groups, each containing isolates of one species, except F13 of F. culmorum. The sequencing of stable and reproducible monomorphic and polymorphic RAPD markers indicated that the Fusarium genome shared high similarity (105-625 bit scores) with the genomes of other organisms as well as with the F. graminearum reference genome.

Geneticin (G418) resistance and electroporation-mediated transformation of Fusarium graminearum and F. culmorum

Fusarium graminearum and F. culmorum are phytopathogenic species causing scab and root rot diseases in all small grain cereals worldwide including Turkey. In this study, resistance levels to geneticin (G418) of 14 F. graminearum and 24 F. culmorum isolates collected from cereals were determined. Fungal cultures were grown on potato dextrose agar medium supplemented with 0, 25, 50, 75 and 100 µg/mL of G418. Minimum inhibitory concentration was determined as 25 µg/mL. As a result, it was concluded that all isolates were highly sensitive to G418. Plasmid pFA6-kanmx4 containing geneticin resistance gene (kanmx) was introduced singly or co-electroporated with pEGFP75 plasmid, containing GFP gene, into fungal protoplast cultures obtained with lytic enzyme. Transformants were grown in media including 25 µg/mL G418. Transformation frequencies were 2.8 and 1.8 transformant per µg plasmid for F. graminearum and F. culmorum isolates, respectively. Transformation process was also confirmed by spectrofluorimetric assay. Relative fluorescence unit values in co-transformants were calculated as 1.87 ± 0.04 for F. graminearum and 2.26 ± 0.08 for F. culmorum. The results obtained from the study gave information about antibiotic resistance levels of two Fusarium species in Turkey. Moreover, it was shown that pFA6-kanmx4 plasmid was a suitable vector, which can be used in genetic manipulation studies of these two fungal species in particular suppression of endogenous and/or the expression of exogenous genes.

Expression Analysis of PKS13, FG08079.1 and PKS10 Genes in Fusarium graminearum and Fusarium culmorum

BACKGROUND Identification and quantification of mycotoxins produced by Fusarium species are important in controlling fungal diseases. OBJECTIVES Potential of zearalenone, butenolide and fusarin C production was investigated in five Fusarium graminearum and five F. culmorum isolates at molecular level. MATERIALS AND METHODS Presence of PKS13, FG08079.1 and PKS10 genes, associated with production of zearalenone, butenolide and fusarin C, respectively, were confirmed by PCR. In addition, expression levels of them together with housekeeping gene (β-tubulin) were detected by real time PCR. RESULTS PKS13 and FG08079.1 transcripts were determined in all isolates, while PKS10 specific primers failed to amplify any product, indicative of no expression. ΔΔCTCT of PKS13 was ranged between 1.79E-03-3.97E-03 and for FG08079.1 was between 0.25E-03 and 6.02E-03. The highest PKS13 expressions were 3.86E-03 in F. graminearum F9 and 3.97E-03 in F. culmorum F16. Maximum FG08079.1 expressions were calculated as 6.02E-03 and 3.81E-03 in F. graminearum 2F and F. culmorum F2, respectively. CONCLUSIONS We revealed that ten Fusarium isolates produced zearalenone and butenolide under culture conditions. However, fusarin C was not generated by them in these conditions.