1. Materials and methods

1.1. Materials

The commercially available potato variety Qingshu 9 was purchased after 1 month of harvest. The fresh potato samples were washed and dried in sunlight for several weeks to turn the potato skin green and allow germination. Subsequently, the green potato skin and buds were dried in a vacuum blast drying oven and pulverized into a powder using a plant pulverizer through an 80-mesh sieve and stored at 0–4°C until further use.
The test strain of F. solani was isolated from Lycium barbarum root rot in Gansu Province, China (Fang, 1998), and its pathogenicity was confirmed based on Koch’s postulates. After identification, the strain was stored at 0–4°C until further use. Potato dextrose broth (PDB) and potato dextrose agar (PDA) were employed for fungal cultivation. The PDB comprised 200 g of peeled potato, 20 g of glucose, and 1000 mL of distilled water (neutral pH). The PDA was prepared by adding 17–20 g of agar to the constituents of PDB. All reagents used were of domestic analytical grade and purchased from Gansu Zhongrui Chemical Co. Ltd., China.

1.2. Methods

1.2.1. Extraction of potato glycoalkaloids

Potato glycoalkaloids were extracted using an acetic acid extraction–ammonia precipitation method with slight modifications (Bo et al., 2012). In brief, 100 g of the potato sample was mixed with 400 mL of 5% acetic acid, stirred for 60 min (JB-1 magnetic stirrer, Shanghai Leici Xinjing Instrument Co. Ltd., China), and filtered (SHZ-D III circulating water vacuum pump, Gongyi Yuhua Instrument Co. Ltd., China). The residue was extracted twice with 200 mL of 5% acetic acid, and the filtrate was combined and its pH adjusted to 11 with ammonia. After extracting three times with 200 mL of water-saturated n-butanol, the extracts were combined and dried on a rotary evaporator (RE-3000, Shanghai Yarong Biochemical Instrument Factory, China), and the residue mixed with 20 mL of methanol to obtain total glycoalkaloid extract. The mass concentration of the glycoalkaloid extract was 5 g∙mL−1.

1.2.2. Effect of potato glycoalkaloids onF. solaniultrastructure

The F. solani was inoculated onto PDA at a concentration of 0.3036 g∙mL−1 (EC50), along with 2 g∙mL−1 potato glycoalkaloid extract, and incubated at 25°C for 48 h. Subsequently, sterile filter paper strips (0.7 cm × 5 cm) were placed around the colony (covering an area of 5 cm × 5 cm) and incubated at 25°C. After 72 h, the edge of the colony was sampled. The collected sample was fixed by double fixation with glutaraldehyde and citric acid (Zeng, 2012), and observed and photographed under a transmission electron microscope (JEM2000EX; JEOL, Japan).

1.2.3. Effect of potato glycoalkaloids onF. solani cell membrane permeability

The mycelia of F. solani were cultured in PDB for 4 days and washed four times with ultrapure sterile water. Then, the washed mycelia were freeze-dried to a constant weight (Labconco freeze drier, USA) and 1 g of the mycelia was transferred into 5 mL of potato glycoalkaloid (EC50) extract and incubated at 25°C under constant shaking at 120 r∙min−1. Subsequently, conductivity of the culture broth was measured (DDB-303A digital conductivity meter, Shanghai Yidian Scientific Instrument Co. Ltd., China) hourly during 0–9 h. Finally, the culture broth was boiled in a water bath (HH-S6 digital display thermostat water bath, Jintan Medical Instrument Factory, China) for 10 min and conductivity was determined. The experiment was repeated thrice, with sterile water and methanol as controls, The permeability of cell membrane was expressed as relative permeability (%) = (relative time conductivity value − initial conductivity value)/(kill conductivity value − initial conductivity value) × 100% (Shen, 2014).

1.2.4. Effect of potato glycoalkaloids on soluble mycoprotein in F. solani

The effect of potato glycoalkaloids on soluble mycoprotein was determined by Coomassie Brilliant Blue G-250 staining (Song, 2010). In brief, mycelia of F. solani were cultured in PDB for 4 days, and washed four times with ultrapure sterile water. Then, the washed mycelia were freeze-dried (Labconco) to a constant weight and 1 g of the mycelia was added to 5 mL of potato glycoalkaloid (EC50) extract, and sampled at 0, 2, 4, 6, and 8 h. The collected samples were centrifuged (D-37520 centrifuge, Heraeus Biofuge, Germany), and the absorbance of samples was recorded at 595 nm (Jenway 6505 UV/Vis UV Spectrophotometer; Gaonan Instrument (Shenzhen) Co. Ltd. China). The protein concentration was calculated according to the protein standard curve, and the experiment was repeated thrice, with sterile water and methanol as controls.

1.2.5. Effect of potato glycoalkaloids on soluble sugar in F. solani

The effect of potato glycoalkaloids on soluble sugar in F. solaniwas determined by anthrone colorimetry (Yao et al., 1992). In brief,F. solani mycelia were cultured in PDB for 4 days, and then washed four times with ultrapure sterile water. Then, the washed mycelia were freeze-dried (Labconco) to a constant weight and 1 g of mycelia mixed with 5 mL of potato glycoalkaloid (EC50) extract, and sampled at 0, 1, 2, 4, 6, 8, 10, and 12 h. Subsequently, collected samples were subjected to centrifugation (6000 rpm; 5 min), heated with anthrone reagent, and cooled to room temperature, and the absorbance of the samples measured at 620 nm. The soluble sugar content was calculated according to the glucose standard curve, and the experiment was repeated thrice, with sterile water and methanol as controls.

1.2.6. Effect of potato glycoalkaloids on reducing sugar in F. solani

The effect of potato glycoalkaloids on reducing sugar in F. solani was determined by 3,5-dinitrosalicylic acid (DNS) method (Chen, 2002). In brief, F. solani mycelia were cultured in PDB for 4 days, and washed four times with ultrapure sterile water. Then, the washed mycelia were freeze-dried (Labconco) to a constant weight and 1 g of mycelia was added to 5 mL of potato glycoalkaloid (EC50) extract, and sampled hourly during 0–8 h. Subsequently, 1 mL of the collected samples was respectively subjected to centrifugation (6000 rpm; 5 min), and 0.5 mL of the supernatant was mixed with 1.5 mL of distilled water and 1.5 mL of DNS reagent to determine the absorbance at 520 nm. The reducing sugar content was calculated according to the standard curve, and the experiment was repeated thrice, with sterile water and methanol as controls.

1.2.7. Effect of potato glycoalkaloids on fat content in F. solani

The oil weight method was employed for sample processing and fat content determination (Li, 1987). The F. solani mycelia were cultured in PDB for 4 days and washed four times with ultrapure sterile water. Then, the washed mycelia were freeze-dried (Labconco) to a constant weight and 1 g of the mycelia was added to 5 mL of potato glycoalkaloid (EC50) extract, and sampled at 24, 48, and 72 h. Subsequently, the collected samples were filtered (SHZ-D III circulating water vacuum pump) and rinsed with redistilled water four times; the obtained wet hyphae were dried at 60–80°C for 4 h, smashed with a mortar and pestle, and filtered through a mesh sieve to obtain dried powder. The procedure was repeated thrice, and sterile water and methanol were used as controls. For fat content determination, the Soxhlet extractor was cleaned, heated in a blast drying oven at 105°C for 20 min, cooled to room temperature, and weighed (m). Then, 12-cm quantitative filter paper was weighed (m1) and made into a bucket, and 2 g of the dried sample powder were added to the filter paper bucket and weighed (m2). The difference between the two masses indicated the quality of the dried sample powder (m3 = m2 − m1). Subsequently, the dried sample was soaked in petroleum ether overnight, and heated in a thermostat water bath for 65°C. Reflux extraction was performed for 12 h using a Soxhlet fat extractor, and then the extract was heated at 100°C for 8 h, cooled to room temperature, and weighed (m′). Crude fat was calculated as follows: crude fat (%) = (m′ − m)/(m2 − m1) × 100%.

1.3. Statistical analysis

All data were analyzed using Excel 2007. The variance was examined using SPSS 19.0 and the difference investigated by employing Duncan’s new complex range method (Zhou et al., 2014).