Fusariotoxin-Induced Toxicity in Mesenchymal Stem Cells and Fibroblasts: A Comparison Between Differentiated and Undifferentiated Cells.

Objectives: Humans are unknowingly exposed to mycotoxins through the consumption of plant-derived foods and processed products contaminated with these toxic compounds. In addition to agricultural losses, Fusarium toxins pose a threat to human health. However, the effects of fusariotoxins on the viability and proliferation of stem cells have not been fully explored. We investigated the cytotoxic effects of deoxynivalenol (DON) and B-trichothecene mix (MIX) on mesenchymal stem cells (MSCs) and the L929 fibroblast cell line. Materials and Methods: MSCs were isolated from the dental pulp tissue. The doubling time and viability of dental pulp stem cells (DPSCs) and L929 cells were determined using the MTT assay. The following doses of B-trichothecenes (0.25-16 µg/mL; 24 hours and 48 hours) were used to evaluate cytotoxicity. In addition, changes in the confluency-dependent response of DPSCs to DON toxicity were determined. Moreover, we investigated the effect of DON on cell death via acridine orange/ethidium bromide (AO/EB) double staining. Results: A DON and MIX showed a dose- and time-dependent inhibitory effect on the proliferation of both cells. DPSCs exposed to DON for 48 hours (IC50 = 0.5 µg/mL) were found to be 16-fold more sensitive than L929 cells (IC50 = 8 µg/mL). Compared with a culture with 80% confluency, DPSCs from a 50% confluent culture were more sensitive to varying doses of DON (0.25-4 µg/mL, 24-48 hours). Moreover, AO/EB staining showed that treatment of DPSCs with DON led to a significant increase in cell death (17% for 2.4 µg/mL; 50% for 4.8 µg/mL). Conclusion: This study reveals that undifferentiated MSCs are significantly more sensitive to DON than differentiated somatic cells (L929). Given that humans are frequently exposed to these mycotoxins, our findings imply that prolonged exposure to them may also have harmful effects on cellular differentiation and embryonic development.

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.

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.

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 (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.

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.

Regeneration capacity of mature embryo-derived callus in barley ( Hordeum vulgare L.).

In this study, induction of regenerable callus from mature embryos in eight Turkish barley varieties was analysed by using different plant growth regulators (PGRs). Varying concentrations (0.5-4 mg l -1 ) of 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba (3,6-dichloro-o-anisic acid) were tested for callus induction from mature embryos. Highest percent of callus induction was observed in Bornova 92 variety (98.3%) on MS medium supplemented with 4 mg l -1 dicamba. Calli were transferred to regeneration media with 0.5 mg l -1 dicamba, 0.5 mg l -1 zeatin riboside (ZR) and 2 mg l -1 thidiazuron (TDZ). Low concentrations of dicamba induced multiple shoots during callus regeneration. When the effect of precultivation with 2,4-D or dicamba on the shoot induction were evaluated, lower concentrations (< 4 mg l -1 ) of auxins have been found optimal. On the regeneration medium with 0.5 mg l -1 dicamba, shoots were able to elongate up to 20 cm and shoot numbers were between 1-23 per callus. The use of ZR led to formation of short shoot buds and somatic embryos in 2 weeks period. The effect of TDZ was different from other PGRs by inducing green solid sectors on calli surfaces (Total 51 sectors/20 callus/Akhisar variety). Five plantlets have been grown from these solid cell clumps and transferred to specific media for root formation. As a result, five varieties (Süleyman Bey, Bornova 92, Vamyk Hoca, Kaya and Akhisar) tested in our study showed the potential to produce regenerable callus by using low amounts of dicamba or TDZ. The optimization process starts from culturing embryos to plantlet formation took nearly 4 weeks.

Calli cultures from Abies equi-trojani (Aschers et Sinten) and changes in antioxidant defense system enzymes.

This study was conducted to explain difficulties of indirect regeneration of forest trees in tissue culture conditions. For this purpose, changes of antioxidant defense system enzymes; superoxide dismutase (SOD), peroxidase (POD) activities were determined during calli formation on young apical shoots of Abies equi-trojani (Aschers et Sinten). Young apical shoots were collected from naturally growing trees and cultured on two different media; Murashige and Skoog (MS) and McCown Woody plant medium (WPM) supplemented with growth regulators benzyl amino purine (BAP), 2,4-dichloro phenoxy acetic acid (2.4-D), kinetin (Kn) in various concentrations for callus induction. WPM media containing 1 mg ml(-1) BAP and 1 mg ml(-1) 2,4-D gave the best calli induction ratio (74%) between tested combinations. POD and SOD enzyme activities were measured both on young shoot explants and 10 day-old calli derived from these explants. POD and SOD enzyme activities were higher being 81.02% and 74.82%, respectively on calli when compared to shoots. The results showed that culture stress tolerated with increased antioxidant enzyme activities could be considered as protective physiological responses in calli cells.