Osthol displayed a broad-spectrum antifungal activity against phytopathogens [10]. Bioassay demonstrated that osthol showed a high activity against F. graminearum (Figures 1–3) and the in vitro assays in this study also indicated that osthol could inhibit hyphal growth of F. graminearum. Treated with 100 μg·mL⁻¹osthol for 24 h, the hyphae of F. graminearum growing in aqueous culture broke into fragments and the fragments could develop to colonies after inoculation (Figure 3C). This indicated that the treatment inhibited the growth of the fungi, but was not lethal to them.

The morphological observations showed cytoplasmic vacuolation and blurring of organelles and cell walls after the treatment with osthol (Figure 4). The fungal cell wall consists basically of a complex network of glucan and chitin, playing important roles in several biological processes such as cell shape, morphogenesis, reproduction, cell-cell and cell-matrix interaction, and osmotic, physical protection.

The fungal cell wall is critical for cell viability and pathogenicity. Chitin and β-1,6-glucanase in the cell wall of F. graminearum both confer a high mechanical resistance to the cell wall. In previous study, Chaffin and Lopez-Ribot [11] found that chitin was important although only a low content (0.6%–9%) could be found in the wall of Candida albicans (mainly in the septum of the hyphae). When the fragments appeared after treated with osthol, the components of cell wall of hyphae and hydrolysis-relative enzymes were measured. The results showed that the chitinase activity increased after the treatment with osthol compared with the control and that N-GlcNAc, the chitin unit, remained to a high content in the hyphae. However, β-1,6-glucanase activity did not obviously change (Figure 7–9), which indicated that osthol treatment could not activate the up-regulated expression of glucanase genes in hyphae of F. graminearum. So glucan, the important component within cell wall, was not hydrolyzed due to the stabilization of glucanolytic activities. In agreement with this, the results of the TEM experiments (Figure 4) showed clearly that the blurring of cell wall after treated with osthol was mainly caused by the hydrolyzation of chitin in the wall. Hence, the broken hyphae appeared due to the loosing of cell wall together with the water flow pressure in liquid medium (Figure 3B).

As a necessary component of cell wall, chitin could be hydrolyzed when sugar starvation occurred [12]. A previous report had demonstrated that the coumarin compounds could inhibit the glucose absorption by N. frontalis [13]. In the present study, decreasing glucose content led to changes to V-shaped form in osthol-treated hyphae of F. graminearum suggesting a starvation and a recovery of nutrient (Figure 5). From assay of morphology observation, the large vesicles in osthol-treated hyphae indicated that the treatment of osthol might originate glucose starvation [14]. The origin of the starvation might be the inability of nutrient absorption caused by osthol. The results of the trial about ¹⁴C-labeled glucose absorption agree with this conclusion (Figure 7). When cells of fungi encounter stress or unfavorable nutrient conditions, the changes of cell were physiologically and morphologically different from the cells growing exponentially. Especially sugar starvation initiates changes in substantial physiological and biochemical processes with the goal of sustaining respiration and other essential metabolic processes [15].

The results also showed that plenty of vacuoles appeared in the osthol-treated hyphae (Figure 4). Autophagy was the major route which cells require enhanced structural compounds degradation and remodeling of components for the maintenance of viability of fungi under starvation conditions [16, 17].

Although basic activity of osthol against the phytopathogen and some changes of physiological activity were reported herein, further detailed research should be focused on direct targets in the phytopathogenic cells.

Osthol (97.5%, w/v) was extracted by Plant Protection Institute of Jiangsu Academy of Agricultural Sciences (PPIJA); DMAB (p-dimethylaminobenzaldehyde) was purchased from ShangHai SSS Reagent CO., LTD; chitin was purchased from Shanghai Chemical Reagent Corporation; ¹⁴C-labeled glucose was purchased from China Isotope Corporation; DNS (3,5-dinitrosalicylic acid), ATP (adenosine 5′-triphosphate), β-1,6-glucan, GlcNAc (N-acetyl-D-glucosamine), DMSO (dimethyl sulfoxide), Triton X-100, PPO (2,5-diphenyloxazole), POPOP (1,4-bis(5-phenyloxazol-2-yl)benzene) and all other chemicals were obtained from Sigma. Osthol was dissolved in DMSO and prepared as osthol stock solution (20,000 μg·mL⁻¹) which was used in the trials. F. graminearum was presented by PPIJA and maintained on potato dextrose agar (PDA) medium at 4 °C.

Antifungal activity of osthol against F. graminearum was determined by inhibition of radial growth and biomass of the fungus in vitro, respectively. The disc diffusion assay was performed in radiation sterilized (¹⁴C-labeled glucose) petri plates of 90 mm in diameter. Osthol stock solution was added to autoclaved PDA mediums to the final concentrations of 0, 6.25, 12.5, 25, 50 and 100 μg·mL⁻¹ just before the curdle of mediums, and then 5-mm discs of the growing fungus were inoculated at the center of 90 mm-diameter Petri plates, respectively. The plates were incubated at 25 °C and the radial growths were observed in 48 h intervals.

Osthol was added to Czapek liquor media (50 mL) in flasks to the final concentrations of 0, 6.25, 12.5, 25, 50 and 100 μg·mL⁻¹, respectively. Each flask were inoculated with a 5-mm disc of F. graminearum and incubated at 25 °C in a rotary shaker of 140 rpm. Mycelium was harvested from each medium after 72 h, oven-dried, and the biomass was recorded.

At the same time, equal volumes of DMSO without osthol were also used as solvent controls in the same treatment. All experiments (including the following experiments) were repeated three times and each one experiment was done in triplicates.

The effects of osthol on the hyphal structure of F. graminearum were observed by light and transmission electron microscope (TEM). Osthol was added to incubation shaking flasks of F. graminearum to the final concentration of 100 μg·mL⁻¹ three days after inoculation. Mycelium was harvested for morphology observation 24 hours after the treatment. For transmission electron microscopy (TEM), the samples were infiltrated, embedded with Epon-Araldite and polymerized at 60 °C for 24h. Ultrathin sections of the samples were cut with a diamond knife and collected on 200-mesh copper grids. After contrasting with uranyl acetate and lead citrate, the grids were examined with a HITACHI H-600 electron microscope at 75KV [18].

The pathogen F. graminearum was grown on potato dextrose agar (PDA) at 28 °C for 5 days and the 10 mm mycelial plates were inoculated into 500 mL flasks containing sterilized Czapek culture medium (250 mL). F. graminearum were cultured in flasks at 25 °C, 140 rpm for 72 h, and then were treated with osthol at a final concentration of 100 μg·mL⁻¹. After 1, 3, 6, 12 and 24 h treatment, mycelial modality was observed separately. The harvested hyphal materials were stored in a refrigerator at −80 °C for the following trials.

Hyphae plates of F. graminearum grown on PDA medium were inoculated in Czapek culture medium in shake flasks and incubated at 25 °C, 140 rpm for 72 h and 15 μL 303.00 m Ci/mmol ¹⁴C-labelled glucose were added thereafter. Then the hyphae in the culture medium were treated with osthol at a final concentration of 100 μg·mL⁻¹. After incubation for 1, 3, 6, 12 and 24 h, the hyphae were sampled and filtered through filter paper and stored at −20 °C. Hyphae from osthol-free mediums were used as control.

Scintillator solution: 5 g PPO (2,5-diphenyloxazole), 0.5 g POPOP (1,4-bis(5-phenyl-2-oxazolyl) benzene) were dissolved in toluene respectively mixed with 300 mL Triton X-100 and made up with toluene to a final volume of 1000 mL.

Accurately weighed 0.1 g freeze dried mycelial samples were homogenized by hand with Tris-HCl (0.05 M, pH=7.5, 1 mL), then the homogenate was centrifuged at 10000 rpm for 10 min at 4 °C. Supernatant (0.1 mL) was were mixed with scintillator solution (6 mL) and measured by scintillation counter. [19].

For each treatment, mycelial material (0.4 g dry weight) was homogenized in extraction solution (0.05 M Tris-HCl, pH 7.5, 2 mL). The homogenate was centrifuged at 15000 rpm for 10 min at 4 °C, and the supernatant was used for the following trials. Supernatant (0.1 mL) was carefully transferred into an Eppendorf tube, and then DNS reagent (2.0 mL) was added to the ED tube. The tubes were heated at 100 °C in a water bath for 5 minutes, cooled down to room temperature and filled up to a predetermined volume of 25 mL. At the same time, distilled water (0.1 mL) plus DNS reagent (2.0 mL) were treated as described above and served as blank sample for zeroing of the spectrophotomater. Three repeats were carried out in each treatment and a glucose standard curve was prepared at a wavelength of 595 nm for calibration purposes [20].

Supernatant liquid (0.2 mL) was treated with potassium borate solution (0.8 M, 0.1 mL) and the mixture was placed in a boiling water bath for 3 min. After cooling down, 1% DMAB (3 mL) was added and the samples were kept at 36 °C for 20 min. The absorbencies of the samples were detected at 544 nm using a spectrophotometer. Three repeats were carried out in each treatment and N-acetyl-D-glucosamine was used to make the standard curve [21].

Gelatiniform chitin (N-acetylation product of chitosan, 0.2 mL) was added to supernatant liquid (0.3 mL from 2.5) and the mixture were kept at 37 °C for 1 h and inactivated in boiling water for 5 min. After centrifugation at 5000 rpm for 10 min, supernatant liquid (0.2 mL) was kept in a boiling water bath for 3 min. After cooling down, potassium borate solution (0.8 M, 0.1 mL) and 1% DMAB (3 mL) were added. After incubation at 36 °C for 20 min, the values of absorbency for each treated sample were detected at 544 nm using a spectrophotometer. Three repeats were carried out in each treatment and N-acetyl-D-glucosamine was used to make the standard curve [22].

To supernatant liquid (0.3 mL from 2.5) 1% β-1,6-glucanase (0.4 mL) was added and the mixture was kept at 40 °C for 5 min, and then DNS (2 mL) was added. After incubation for 5 min in a boiling water bath and cooling down to the room temperature, the absorbance values for each treated sample were detected at 595 nm using a spectrophotometer. Three repeats were carried out in each treatment and glucose was used to make the standard curve [23].