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).