Inhibitory action of ginkgolic acid against pathogenic fungi and characterisation of its inhibitory activities on Nigrospora oryzae

N. oryzae, Alternaria alternata and Penicillium expansum were stored in the Microbiological Culture Collection Centre of Dalian Polytechnic University, China. These pathogenic fungi were maintained on potato dextrose agar (PDA: extract of boiled potatoes, 5 g; anhydrous glucose, 20 g; agar, 20 g; and distilled water, 1,000 mL) stored at 4 °C and activated by inoculating on a PDA plate and cultured at 28 °C for 4 d before the experiment.

The chemicals used in the culture medium were of reagent grade; other chemicals were of analytical grade. The kits (superoxide dismutase [SOD], A001-3; catalase [CAT], A007-1) used to measure SOD and CAT were purchased from the Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China. Ginkgolic acid standard was purchased from Shanghai Tauto Biotech Co., Shanghai, China.

Ginkgo fruits (G. biloba L. cv. ‘Luanguofoshou’) were harvested at a mature stage from a special garden base in Dalian, Liaoning, China (longitude: 121°57′, latitude: 38°95′). All rotten or visibly damaged fruits were removed to obtain seeds; then, the testae were air-dried at room temperature. We then washed the G. biloba testa, dried it to a constant weight, crushed it and sifted it. Crude ginkgolic acid was extracted according to the method reported by Hausen (1998). Briefly, the powder was reflux-extracted with 80% ethanol (solid–liquid ratio of 1:20) at 80 °C for 3 h. Then, the mixture was centrifuged; the supernatant was concentrated and freeze-dried to obtain crude ginkgolic acid.

Crude ginkgolic acid was purified using AB-8 macroporous resin and silica gel column chromatography, followed by analysis using high-performance liquid chromatography (HPLC). In brief, ginkgolic acid standard and purified ginkgolic acid (5 mg) were dissolved separately in anhydrous methanol (1 mL) and filtered through a 0.2 μm syringe filter. The ginkgolic acid was analysed by HPLC (2695E; Waters, Massachusetts, USA) using a C18 chromatographic column (4.6 mm × 250 mm, 5 μm) (Agilent, California, USA), a method described by Lei et al. (2014) with slight modifications. Various concentrations of ginkgolic acid were prepared before the following experiments.

The purified ginkgolic acid sample was mixed with KBr under vacuum, ground and pressed into tablets, and the infrared spectrum was scanned in the range of 4,000–400 cm^-1.

Fungistatic rate was measured by radial growth assay with slight modifications (Tan et al., 2017). A medium containing mycelium with a diameter of 5 mm was taken from the same circumference of the edge of the pre-cultured colony by a puncher. Various concentrations of ginkgolic acid were added to the medium (2 mL · 100 mL^-1 medium). Then, the colony was cultured in PDA medium with ginkgolic acid (0 mg · mL^-1, 2.5 mg · mL^-1, 5 mg · mL^-1, 10 mg · mL^-1) at 30 °C; ginkgolic acid showed an inhibitory effect in this range. Ginkgolic acid was dissolved in 80% ethanol; so the trial with 80% ethanol instead of ginkgolic acid was used as a control. When the colony in the control group grew close to the plate wall, the diameter of a single colony was measured, and the fungistatic rate was calculated according to the following equation: 1 Fungistaticrate(%)=[ (D1−D2)/D2 ]×100%, $${\rm{Fungistatic}}\,{\rm{rate}}\,(\% ) = [({{\rm{D}}_1} - {{\rm{D}}_2})/{{\rm{D}}_2}] \times 100\% ,$$ where D₁ was the increment of colony diameter in the control group; D₂ was the increment of colony diameter in the treatment group.

The effect of ginkgolic acid on the cell membrane of N. oryzae was investigated using an optical microscope. N. oryzae was inoculated in potato dextrose liquid medium with or without ginkgolic acid treatment. It was then placed under an optical microscope (10 × 40) for observation.

N. oryzae was inoculated in potato dextrose liquid medium and cultured at 28 °C for 3 d on a rotary shaker at 150×g. Mycelium (3 g; wet weight) was obtained from the above mixture using a sterile inoculator, dried with sterile filter paper and transferred into 100 mL potato dextrose liquid medium. After sub-culturing at 28 °C with shaking at 150×g for 3 d, ginkgolic acid was added and N. oryzae was cultured in the presence of ginkgolic acid (5 mg · mL^-1) for subsequent experiments. Ethanol (80%) was used in the trial as a control instead of ginkgolic acid.

The mycelium of N. oryzae was inoculated into 100 mL liquid medium, to which 5 mg · mL^-1 ginkgolic acid solution was added. N. oryzae was cultured in shake flasks (150×g) at 28 °C for 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h, 48 h and 55 h in the media supplemented with ginkgolic acid. The conductivity of the medium was measured using a conductivity meter (DDS-307A; Yoke Instrument Co., Shanghai, China) to analyse the changing trend of cell membrane permeability (Xu et al., 2003).

At 0 h, 3 h, 6 h, 9 h, 12 h, 24 h and 48 h, respectively, 1 g of mycelium was obtained from the mixed culture in which ginkgolic acid was added. The mycelium was thoroughly ground in a mortar containing a small amount of quartz sand and phosphate buffer saline (PBS), and the mixture was collected and put into a centrifuge tube. After centrifugation at a speed of 4,000×g for 10 min, the supernatant was transferred into a colorimetric tube whose specification was 10 mL, diluted with PBS to the specified scale and mixed well.

We then put 1 mL of the diluted solution into a test tube and added 4 mL of Coomassie brilliant blue G-250 reagent. The mixture was allowed to react for 2 min and then the absorbance was measured at 595 nm (Asryants et al., 1985).

Mycelium (1 g) was obtained from the liquid medium of N. oryzae treated with ginkgolic acid for 0 h, 3 h, 6 h, 9 h, 12 h, 24 h and 48 h. After the mycelium was thoroughly ground, the activities of SOD and CAT were measured using the enzyme-linked immunosorbent assay (ELISA) test kit.

The enzyme activity of SOD is calculated as the amount of enzyme that inhibits the auto-oxidation rate of hydroquinone up to 50% per minute in 1 mL of the reaction liquid. The unit enzyme activity of CAT is defined as the amount of 1 μmol H₂O₂ decomposed per gram of sample per second.

E. aureum (Family: Araceae) with green leaves (>6 cm in diameter) was cultivated as an ornamental plant, which was planted in the plantation of the Dalian Polytechnic University for >3 years.

Spore suspensions were prepared by scraping spores from PDA cultures of N. oryzae using a sterile inoculator and suspending them in sterile distilled water. Spore concentrations were adjusted to 10⁶ colony-forming units (CFU) · mL^-1 assisted by the haemocytometer.

Leaves of similar size and no obvious mechanical damage were selected from healthy E. aureum and disinfected with 0.1% sodium hypochlorite for 1 min, washed with sterile water and cultured at room temperature (20 °C).

A sterilised scalpel was used to make a uniform wound (1 mm × 1 mm) in the same position on each blade. The wound site was inoculated with 10 μL sterile distilled water or N. oryzae spore suspension (10⁶ CFU · mL^-1). After 3 d of incubation at room temperature, 20 μL sterile distilled water or ginkgolic acid (5 mg · mL^-1) was inoculated at the wound site. Leaves were cultured at room temperature for 20 d after being treated as described.

In this paper, all experiments were performed in triplicate. The obtained data were subjected to one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS, version 16.0, Chicago, USA); p < 0.05 was regarded as statistically significant.

The HPLC spectrum of ginkgolic acid was analysed. The results revealed that the purity of ginkgolic acid was 69.18% and it was composed of five ginkgolic acids, that is, C13:0, C15:1, C17:2, C15:0 and C17:1, and their derivatives. The peak area of purified ginkgolic acid was 710.74 mAU*s. The content of C15:1 was the highest, accounting for 38.53% of the purified substance and 55.70% of the total purified ginkgolic acid (Figure 1).

Figure 1.

The infrared spectrum of ginkgolic acid was determined by the KBr tablet method; the results are shown in Figure 2, and the main characteristic peaks are assigned in Table 1. It can be seen from the spectrum that there were multiple absorption peaks at 3,426.36 cm^-1, 2,925.45 cm^-1, 1,718.90 cm^-1, 1,626.12 cm^-1, 1,484.59 cm^-1, 1,077.92 cm^-1, 818.20 cm^-1 and 778.24 cm^-1.

Figure 2.

The experiment results showed that various concentrations of ginkgolic acid had different inhibitory effects on the pathogens. With an increase in concentration, ginkgolic acid showed a similar inhibitory trend to N. oryzae and A. alternata; the inhibition was the strongest at the concentration of 5 mg · mL^-1, and the inhibition rates against N. oryzae and A. alternata reached 65.82% and 56.96%, respectively. In addition, it exhibited the highest inhibitory activity against N. oryzae and no significant inhibitory effect on P. expansum (Table 2). So, further research on the effect of ginkgolic acid against N. oryzae was conducted in the study.

Therefore, according to the results from Figure 3, the inhibitory action of ginkgolic acid at a concentration of 5 mg · mL^-1 against N. oryzae was studied.

Figure 3.

The untreated N. oryzae showed an intact cell membrane structure when observed under an optical microscope (Figure 4A). However, after being treated with ginkgolic acid, a blurred cell membrane and intracellular material leakage were observed under the optical microscope (Figure 4B).

Figure 4.

The extracellular conductivity was measured to evaluate the cell membrane permeability of N. oryzae. The effect of ginkgolic acid on the electrical conductivity of N. oryzae is shown in Figure 5A. After adding ginkgolic acid, the conductivity of the sample increased significantly between 0 h and 2 h, decreased slightly after 6 h and then stabilised at a high level. Electrical conductivity of the control group showed a general downward trend; although it rose during the period of 0–4 h and reached the peak at the fourth hour, it never exceeded that of the treatment group.

Figure 5.

For the treatment group, the protein content in the mycelia fell significantly between 0 h and 12 h and then remained stable at an extremely low level. However, in the control group, the protein content in N. oryzae increased slightly within 6 h of co-culture, and there was no significant change after 6 h. In addition, the protein content in the treatment group was much lower than that in the control group (Figure 5B).

As shown in Figure 6A, the activity of SOD in the treatment group first rose sharply from 0 h to 3 h, then declined rapidly until 10 h and finally, it remained stable. In the control group, the overall trend of enzyme activity was up slightly. After treatment for 6 h, SOD activity in the treatment group was significantly lower than that in the control group.

Figure 6.

Effect of ginkgolic acid on the activity of CAT in N. oryzae is shown in Figure 6B. CAT activity increased significantly from 0 h to 3 h and peaked at the sixth hour, then decreased rapidly between 6 h and 9 h and finally dropped slowly. In the control group, CAT almost remained at the same level after rising, and CAT activity was significantly higher than that in the treatment group after 6 h.

Leaves of E. aureum were infected by N. oryzae and cultured at room temperature for 20 d (Figure 7). The results showed that ginkgolic acid slowed down the disease symptoms. The degree of disease on the leaves is shown in Figure 8. After infection with N. oryzae spore suspension, leaves treated with sterile distilled water had sub-circular brown spots around the wounds with black dotted grains around the spot margins, while leaves treated with ginkgolic acid showed significantly less morbidity and no black dotted grains around the spot margins. Similarly, after mechanical damage, leaves treated with sterile distilled water showed slight lesions around the wound. On the other hand, the wound healed well after treatment with ginkgolic acid.

Figure 7.

Figure 8.