Fusarium graminearum and F. culmorum are phytopathogens, which cause destructive diseases in cereals. Epidemics of these phytopathogens are caused by mycotoxin contamination and the reduction of crop quality. In this study, the alteration due to in vitro camphor treatment on F. culmorum 9F and F. graminearum H11 isolates was investigated in terms of epigenetic, cellular, and transcription levels. Camphor with different concentrations (0.2, 0.4, 0.8, 1, 2, and 4 µg/µL) was applied to potato dextrose agar (PDA) growth media. The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC50) were calculated as 2 and 1 µg/µL, respectively. hog1, mst20, CAT, POD, mgv1, stuA, and tri5 genes, which are related to various cellular processes and pathogenesis, were examined by qPCR assay. qPCR analysis showed that camphor treatment leads to the downregulation of tri5 expression but the upregulation of the remaining genes. Apoptosis and oxidative stress were confirmed via acridine orange/ethidium bromide (AO/EB) and dichlorofluorescin diacetate (DCF-DA) staining, respectively. Moreover, coupled restriction enzyme digestion-random amplification (CRED-RA) assay, used for DNA methylation analysis, was carried out to evaluate epigenetic alterations. The decrease in genomic template stability (GTS) values, which resulted due to the alterations in random amplified polymorphic DNA (RAPD) profiles caused by camphor treatment, were detected as 97.60% in F. culmorum 9F and 66.27% in F. graminearum H-11. The outer and inner methylated cytosine profiles are determined by CRED-RA assay as type I–IV epigenetic alterations. The outcomes indicated that camphor could lead to alterations at several molecular levels of F. graminearum and F. culmorum.

To characterize the isolates of F. graminearum and F. culmorum fungi from Turkey and Iran, we performed ISSR analysis with 18 non-anchored and 23 anchored (including ten novel) primers. Amplification product sizes were 0.2-3.5 kb. In total, 405 bands were scored, 24 of which (5.92%) were polymorphic. The similarities among F. graminearum isolates were calculated as 62.3-99%, and among F. culmorum as 65.7-94.3%. Moderate genetic variation at intra- and inter-specific levels was determined, and the average intraspecific genetic diversity values were 80.65% for F. graminearum, and 80% for F. culmorum. Cluster analysis separated the isolates into two main clades. Group I consisted of F. culmorum isolates from Turkey that produced DON mycotoxin. Group II contained all F. graminearum isolates that were deoxynivalenol (DON) and nivalenol (NIV) chemotypes from Turkey and Iran. Both groups I and II were divided into two subgroups including their divisions. Phenons in group II included isolates distributed in the same geographic region. ISSR markers clustered isolates within a definite order according to their species. Isolates from the same agro-ecological locations were also kept together in subdivisions. The novel ISSR markers developed for the first time in this study contribute to differentiating between Fusarium isolates according to their species and geographic regions.

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Intraspecific and interspecific diversity between Phoenix theophrasti individuals (92 from Turkey and 70 from Crete, Greece) and P. dactylifera specimens (28 from Turkey) were investigated by inter-simple sequence repeat (ISSR) analysis. A total of 45 polymorphic fragments, 360-3454 bps long, were produced. Intraspecific diversity for P. dactylifera was 26.63% and similarities ranged between 0.5 and 1. In the constructed dendrogram, P. dactylifera specimens clustered together in the first main group, outside branches consisting of P. theophrasti samples that generated the second main group. The intraspecific diversity for Turkish P. theophrasti populations was found to be 18.60% and for Cretan populations 13.45%. Antalya-Kumluca-Karaöz samples were grouped outside the branches of the remaining P. theophrasti samples. All three Cretan populations formed their own, separate branch. Datça-Eksera Stream samples together with two Datça-Hurmalıbük specimens constituted a group excluding Datça-Hurmalıbük and Bodrum-Gölköy specimens. Five Bodrum-Gölköy genotypes were clustered separately. Gene flow (N m ) values among populations were estimated from 0.157 to 59.615. AMOVA analysis revealed the percentages of variance among and within Phoenix populations: 73% and 27%, respectively. The first three principal coordinate components accounted for 37.60, 29.32 and 20.04%, respectively, thus the total variance obtained from the first three principal coordinate components was 86.96%. A positive correlation between geographic and genetic distances of populations was detected by Mantel tests (Rx,y = 0.44, p = 0.04). The populations were classified into four clusters by STRUCTURE analysis, supported the PCoA data. To conclude, ISSR results support that P. dactylifera and P. theophrasti are different species. Moreover, the findings not only revealed relationships between natural Phoenix theophrasti populations but also supported the identification of the P. theophrasti individuals that are phenotypically differentiated in the divided Bodrum-Gölköy population (P. theophrasti subsp. golkoyana).ISSR genotyping of Phoenix theophrasti natural populations in Turkey and Crete (Greece) and P. dactylifera

Fusarium culmorum produces polyketide-structured aurofusarin and its precursor metabolites. Aurofusarin biosynthetic pathway is catalysed by the products of eleven genes located in gene cluster. In this study, the effects of the expression levels of Pks12, Gip6, and Gip7, responsible genes for the intracellular steps of the pathway, on pigmentation were investigated in an F. culmorum field isolate. Presence of three genes were determined via PCR and sequencing. Their expressions were investigated by qPCR at 48 th , 72 nd , 120 th and 168 th hours. Aurofusarin and rubrofusarin production were verified by HPLC at the same time periods. Mycelial pigmentation, which was initially white, turned yellow, then carmine red during the sevenday culture. Pks12, Gip6, Gip7 genes with 1282, 449 and 1278 bps had sequence similarities (86,6%, 99,4% and 80,5% respectively) with the genes of reference (FcUK99). The ΣCp values of all genes ranged from 29th to 32nd cycles, whereas the ΣΔΔCt values were calculated between 9,59 E-02 and 1,30 E-02. Relative quantification revealed these genes were controlled by down-regulation compared to the β-tubulin expression. Aurofusarin quantities ranged from 2,355 ppm to 27,350 ppm between the 72 nd and 168 th hours whereas the yield of rubrofusarin decreased from 0,098 ppm to 0,063 ppm. ( 4) Conclusion: This study becomes first report for both investigating the expression levels of the genes responsible for the catalysis of intracellular steps of aurofusarin biosynthesis in F. culmorum, and for examining the relationship between gene expression and mycelium pigmentation. HIGHLIGHTS• Molecular analysis of aurofusarin biosynthesis in F. culmorum.• Mycelial pigmentation depending on gene expression and aurofusarin accumulation.• Pigmentation variations depending on growth phase.

Fusarium isolates are divided into three chemotypes according to produce of class B trichothecenes; 3-acetyldeoxynivalenol (3ADON) 15-acetyldeoxynivalenol (15ADON) and nivalenol (NIV) and 4- acetyldeoxynivalenol (NIV) chemotypes. In this study, chemotyping of seventeen isolates from Turkey and Iran belonging to F. graminearum, F. culmorum, F. poae and F. pseudograminearum species were carried out by polymerase chain reaction (PCR) analysis. While all F. culmorum and F. poae isolates determined as 3ADON, remaining F. graminearum and F. pseudograminearum isolates were either 3ADON or 15ADON chemotypes. A common band of 583 bp long DNA fragment was amplified in all of F. culmorum and F. poae, one F. pseudograminearum (21F) and four F. graminearum (14F, sh14, sh15, sh7) isolates with 3ADON chemotype. However, remaining two F. pseudograminearum and four F. graminearum isolates with 15ADON chemotype, yielded amplicons that of 863 bp. It was shown that 3ADON was more predominant chemotype from other class B trichothecenes. This is the first report on chemotyping of F. poae and F. pseudograminearum isolates and also to show presence of 3ADON chemotype in F. graminearum isolate from Turkey.

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