Investigation of antifungal activities of myrcene on Fusarium reference strains.

Antifungal effects of myrcene, the plant-based naturel compound, were investigated on Fusarium graminearum PH-1 and Fusarium culmorum FcUK99 references, for the first time. Minimum inhibitory concentration (MIC) and half of MIC (MIC50) of both Fusarium strains against myrcene were found as 25 µg/µl and 12.5 µg/µl, respectively. MIC50 application decreased the cell viabilities in the ratios of 34.90% and 33.91% in PH-1 and FcUK99, respectively (p < 0.01). The significantly increased catalase (CAT) activity was recorded in MIC50 treated strains (p < 0.01). Apoptosis-like process and cellular oxidative stress were also monitored with acridine orange/ethidium bromide (Ao/Eb) dual staining and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) staining. The genomic template stability (GTS) percentages were calculated as 79% for PH-1 and 71% for FcUK99 via random amplified polymorphic DNA (RAPD). Methylation polymorphism values were calculated as 53.8% and 50.6% in PH-1 and 40.4% and 39% in FcUK99 for HapII and MspI, respectively by coupled restriction enzyme digestion-random amplification (CRED-RA). Methylation-sensitive amplified polymorphism (MSAP) revealed that myrcene caused both type I and type III epigenetic modifications in both genomes. MIC50 dose caused up to 13.86 ± 0.42-fold changes in the expressions of cat, mst20, and stuA, whereas downregulation in tri5 was recorded. Myrcene application did not change the retrotransposon movement in both species by the amplifying of idiomorphic retrotransposon patterns through inter-retrotransposon polymorphism-polymerase chain reaction (IRAP-PCR). This study demonstrated that myrcene is an effective compound in the management of phytopathogenic Fusarium species by causing morphological, genetic, epigenetic, and cellular alterations, and has a potential to utilize as an antifungal agent.

The roles of hormones in the modulation of growth and virulence genes' expressions in UPEC strains.

BACKGROUND: Host factors such as hormones are known to modulate growth, virulence and antibiotic susceptibility of bacteria. In the present study, the effect of norepinephrine (NE) and estradiol (Est) on growth and expression levels of virulence genes (usp, sfa/foc, cnf1, aer) of uropathogenic E. coli (UPEC) strains C7 and C149 were investigated. METHODS: E. coli C7 and C149 were grown in serum based SAPI broth with and without three different concentrations of norepinephrine and estradiol. Growths were determined via optical density measurement in a spectrophotometer. Real-time polymerase chain reaction was used to determine gene expression levels. Statistical analyses were performed by one way Anova Tukey's post hoc-test. RESULTS: According to our results it has been shown that, growths of bacteria could be affected in the presence of hormones which are variable according to incubation period and hormones' concentrations. Up regulation of usp, sfa/foc, cnf1 were shown to be statistically significant (p < 0.05) in the presence of low, medium levels NE and all concentrations of Est. The expression of aer was down regulated significantly in the presence of low (p < 0.001) and medium level of Est; but all levels of NE was shown to be increased the expression of aer significantly (p < 0.05). CONCLUSIONS: The results of the present study has shown once more that host factors (norepinephrine and estradiol) could influence the growth of a bacterium as well as gene expressions.

The Effects of Insulin and Glucose on Different Characteristics of a UPEC: Alterations in Growth Rate and Expression Levels of some Virulence Genes.

BACKGROUND: Host factors are known to modulate virulence, antibiotic susceptibility, and growth rate of bacteria. The effect of human insulin and glucose on growth rate and expression of virulence genes (usp, sfa/foc, cnf1) of a uropathogenic E. coli (UPEC) strain were investigated in this study. METHODS: E. coli C7 was grown in tryptic soy broth (TSB-control) and TSB containing 20 µU/mL insulin, 200 µU/mL insulin, 0.1% glucose, and 200 µU/mL insulin + 0.1% glucose. Growth rates were determined via optical density measurement in a spectrophotometer. Real-time polymerase chain reaction was used to determine the gene expression levels. Statistical analyses were performed via Tukey's post hoc-test. RESULTS: Differences were found to be not statistically significant for bacterial growth rate in TSB and TSB with insulin and/or glucose. The expression levels of all three virulence genes were shown to be reduced significantly in the presence of insulin and/or glucose. The highest degree of repression was observed in 200 µU/mL insulin added to TSB. Also, the repression level of the gene expression was revealed to be reduced in 0.1% glucose supplemented TSB. CONCLUSIONS: In the present study, it was shown that insulin and glucose can modulate UPEC's gene expression while the growth rate was not affected.

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.

Cumin Seed Oil Induces Oxidative Stress-Based Antifungal Activities on Fusarium graminearum.

In this study, the antifungal activity of cumin seed oil (CSO) was tested on Fusarium graminearum. (i) Minimum inhibitory concentrations (MICs) and related concentrations (IC75, IC50, and IC25) were detected; (ii) toxicity was evaluated by a water-soluble tetrazolium salt-1 (WST-1) assay; (iii) genomic/epigenomic alterations were evaluated by the coupled restriction enzyme digestion-random amplification (CRED-RA) method; (iv) oxidative stress was investigated by CAT expression, catalase activity, and DCF-DA staining; (v) deoxynivalenol biosynthesis was evaluated by tri6 expression; (vi) and potential effects of CSO on wheat were tested by a water loss rate (WLR) assay. MIC, IC75, IC50 and IC25 values were detected at 0.5, 0.375, 0.25, and 0.125 mg mL-1. In WST-1 assays, significant decreases (p < 0.001) were detected. Genomic template stability (GTS) related to methylation differences ranged from 94.60% to 96.30%. Percentage polymorphism for HapII/MspI values were as 9.1%/15.8%. CAT (oxidative stress-related catalase) and tri6 (zinc finger motif transcription factor) gene expressions were recorded between 5.29 ± 0.74 and 0.46 ± 0.10 (p < 0.05). Increased catalase activity was detected (p < 0.05) by spectrophotometric assays. DCF-DA-stained (oxidative stressed) cells were increased in response to increased concentrations, and there were no significant changes in WLR values. It was concluded that CSO showed strong antifungal activity on F. graminearum via different physiological levels.

Determining the biocontrol capacities of Trichoderma spp. originating from Turkey on Fusarium culmorum by transcriptional and antagonistic analyses.

In this study aiming to investigate potential fungal biocontrol agents for Fusarium culmorum, several isolates of Trichoderma spp. were evaluated for their antagonistic effects by means of transcriptional analyses. At first, 21 monosporic Trichoderma spp. isolates were obtained from natural wood debris and wood area soils in Manisa, Turkey. Trichoderma spp. Isolates were identified as belonging to four different species (T. atroviride, T. harzianum, T. koningii, and T. brevicompactum) by tef1-α sequencing. Then, the linear growth rate (LGR) of each species was calculated and determined to be in a range between 13.22 ± 0.71 mm/day (T. atroviride TR2) and 25.06 ± 1.45 mm/day (T. harzianum K30). Inter-simple sequence repeat (ISSR) genotyping validated the tef1-α sequencing results by presenting two sub-clusters in the dendrogram. We determined the genetically most similar (TR1 & TR2; 97.77%) and dissimilar (K9 & K17; 40.40%) individuals belonging to the same and different species, respectively. Dual sandwich culture tests (which are useful for antagonism studies) revealed that T. harzianum K21 (the least suppressive) and T. brevicompactum K26 (the most suppressive) isolates suppressed F. culmorum with growth rates of 3% and 46%, respectively. Expressions of genes previously associated with mycoparasitism-plant protection-secondary metabolism (nag1, tgf-1, and tmk-1) were tested by quantitative real-time polymerase chain reaction (qRT-PCR) in both those isolates. While there were no significant differences (p>0.05) in expression that were present in the K21 isolate, those three genes were upregulated with fold change values of 2.69 ± 0.26 (p<0.001), 2.23 ± 0.16 (p<0.001), and 5.38 ± 2.01 (p<0.05) in K26, meaning that the presence of significant alteration in the physiological processes of the fungus. Also, its mycoparasitism potential was tested on Triticum aestivum L. cv Basribey in planta, which was infected with the F. culmorum FcUK99 strain. Results of the trials, including specific plant growth parameters (weight or length of plantlets), confirmed the mycoparasitic potential of the isolate. It can be concluded that (i) nag1, tgf-1, and tmk-1 genes could be approved as reliable markers for evaluation of BCA capacities of Trichoderma spp. and (ii) the T. brevicompactum K26 strain can be suggested as a promising candidate for combating in F. culmorum diseases following the necessary procedures to ensure it is non-hazardous and safe.

Chemotyping of Fusarium graminearum and F. culmorum isolates from Turkey by PCR assay.

Fusarium graminearum and F. culmorum are the major causal agents of Fusarium head blight in Turkey. They produce trichothecenes such as deoxynivalenol (DON), nivalenol (NIV) and their several acetylated derivatives, 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). In this study, a total of thirty-three isolates of F. graminearum and F. culmorum were collected from various regions and three different hosts. They were identified by amplification of tri5 gene cluster. Totally 32 isolates, 21 of F. culmorum and 11 of F. graminearum, were determined as DON chemotype, while only one F. graminearum isolate (1F) was detected as a NIV. A 282 base pair (bp) band for tri13 gene and also ranging from 458 to 535 bp bands for tri7 gene were amplified in all DON producers' genomes. Further analysis of DON chemotype based on tri3 gene amplification showed that all isolates of F. graminearum displayed 15-ADON sub-chemotype. They yielded a 863 bp amplicon. Similarly, 3-ADON sub-chemotype was identified in F. culmorum' isolates except F13. As a result of tri3 gene assay, it was produced a 583 bp fragment in these twenty isolates. It is the first report that a F. graminearum isolate depicts NIV chemotype in agricultural regions of Turkey. According to our findings, DON chemotype is predominating in our country. Also, it is presented that most of the F. graminearum isolates have 15-ADON sub-chemotype, while all F. culmorum's belong to 3-ADON which possess full length amplicon of tri7 gene.

Investigation of Camphor Effects on Fusarium graminearum and F. culmorum at Different Molecular Levels.

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.

Alterations of growth rate and gene expression levels of UPEC by antibiotics at sub-MIC.

The host is the main environment for bacteria, and they also expose to many antibiotics during the treatment of infectious diseases in host body. In this study, it was aimed to investigate possible changes in growth rate and expression levels of three virulence genes (foc/foc, cnf1, and usp) in a uropathogenic E. coli standard strain within the presence of ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. The UPEC C7 strain was grown on tryptic soy broth-TSB (control), TSB + ciprofloxacin, TSB + nitrofurantoin, and TSB + trimethoprim-sulfamethoxazole for determination of both growth rate and gene expression level. Antibiotics were added according to their sub-minimal inhibition concentrations. E-test was used to determine MIC values of antibiotics. Growth changes were measured in absorbance 600 nm during 24-h period. Total RNA isolations were performed after incubation for 24 h at 37 °C. Gene expression levels were determined by quantitative PCR. Tukey's post hoc test was used for statistical analysis. According to absorbance values, it has been shown that only ciprofloxacin and trimethoprim-sulfamethoxazole have lead significant decrease on growth rate. We also detected statistically significant differences in each gene expression levels for all antibiotics via relative quantification analysis. Fold changes in gene expression was found 0.65, 1.42, 0.23 for foc/foc gene; 0.01, 0.01, 2.84 for cnf1 gene; and 0.1, 0.01, 0.01 for usp gene in the presence of ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole, respectively. This investigation has shown that antibiotics can play a role as an environmental factor which may determine the pathogenicity of bacteria in vivo.

Multiplex Detection of Fusarium Species.

Multiplex PCR is a powerful method to detect, identify, and quantify the mycotoxigenic fungus by targeting the amplification of genes associated with mycotoxin production and detection, identification, and quantification of Fusarium species. As compared with uniplex PCR, it has several advantages such as low cost, shortened time, and simultaneous amplification of more than two genes (in only one reaction tube). Here, we describe multiplex PCR-based detection and identification of trichothecene-, zearalenone-, fumonisin-, and enniatin-producing Fusarium species, the use of multiplex PCR in multiplex genotype assay and the use of multiplex TaqMan real-time qPCR.

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 (ß-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.