Materials and methods
Four germinated seeds were sown in each polyethylene plastic pot that was filled with 700 ml of Hoagland’s nutrient solution. In the hydroponic culture experiments, the nutrient solutions were renewed every 3 days. Wheat seedlings were grown in a grown chamber at 70% relative humidity, a photoperiod of 14:10 (L: D), a light intensity of 3000lx. The whole wheat plants were sprayed with the different concentration of Si that was 0 (CK), 0.3 (T1), 1 (T2), 2 (T3), 3 (T4), 4 (T5), 5 (T6), 9 (T7) mmol/L on wheat with the second expanded leaves and trifoliate heart respectively. When the 4 leaf of wheat seedlings started to unfold, the nymphs were transferred onto the plants. In this experiment, each seedling grew a nymph on the entire plant, and 4 wheat plants were planted in each pot, each treatment consisted of 15 individuals. Per plant were checked and nymphs born were removed daily. Adult longevity, nymphal production, and mortality were recorded daily until the aphids died, and missing adult aphids resulting from escape or predation were counted as censored observations on the day of their disappearance. To prevent the aphids from escaping, each plant was covered with a cellophane cover with an opening at the upper end, and a cover made of 80 mesh (0.178 mm. nylon net is covered at the opening). After the aphid died, the wheat was sampled and stored at -80℃.
The wheat seed used in the experiment was Zhengmai 9023, which were provided by Qiule Seed Co., Ltd., Zhengzhou China. Wheat seeds surface were sterilized with 10% H2O2 for 30 min and rinsed thoroughly with distilled water four times, then soaked 24 h with distilled water and germinated on moist filter paper for 2 days in petri dishes, and germinated at 25℃. The germinating seeds were sown on moistened gauze fixed to the plastic tray containing distilled water at 25℃. When the seedlings grow to two leaves, wheat seeds with consistent germination was selected and transplanted into a plastic pot to be cultured in a light culture room for later use. The nutrient solution was Hoagland’s nutrient solution (Arditti and Dunn 1969): Ca(NO3)2·4H2O 0.945g/L, KNO3 0.607g/L, MgSO4·7H2O 0.493g/L, NaH2PO4·2H2O 0.156g/L, H3BO3 2.86mg/L, MnCl2·4H2O 1.81mg/L, ZnSO4·7H2O 0.22mg/L, CuSO4·5H2O 0.08mg/L, Na2MoO4·H2O 0.02mg/L, EDTA-NaFe 10.525g/L.
Aphids were the dominant species of Sitobion avenae in Huanghuai region, China. Sitobion avenae F used in our study were provided by Henan Academy of Agricultural Sciences. The aphid was then reared on wheat in a greenhouse at 20℃, 70% relative humidity, and a photoperiod of 14:10 (L: D), and the light intensity of 3000lx for being used in experiments.
TEOS: First, 4 ml of absolute ethyl alcohol and 190 ml of distilled water are fully stirred for 0.5h, then the mixed solution of 0, 0.0134, 0.0448, 0.0896, 0.1344, 0.1792, 0.224, 0.4032 ml distilled water and TEOS, respectively, 4 ml of absolute ethyl alcohol and 2 ml of Tween 80 is slowly dropped and fully stirred for 2h.
Measurement indexes and methods
Determination of the life table parameters of aphid
The parameters are calculated as follows (Birch, 1948; Hsin and Hsi, 1985; Chi, 1988):
1) The net reproductive rate (R0) is recorded as the number of offspring that females produce in a period equivalent to the time from birth to adult death, is calculated as: R0=∑lxmx
2) The mean generation time (T) is the time of each aphid from birth to the first reproduction, is calculated as: T=∑xlxmx/ R0
3) The intrinsic rate (rm): The maximum instantaneous growth rate of a population in an ideal state, and it reflects the population's expansion ability in an ideal state, which can be used to measure the population's fitness, is calculated as: rm=lnR0/ T
4) The finite rate of increase (λ) refers to the total growth rate of the population in a certain period time, and refers to the population growth trend in a long period time, is calculated as: ƛ =e rm
5) The population doubling time (t): Population doubling time in a certain period time, is calculated as: t= ln2/ R0
Where x is the age interval (days), lx is the agespeciﬁc survival, mx is the total number of nymphs produced by each aphid for a period equal to their corresponding x.
Enzyme activity assays
The activities of PAL, CAT, LOX, and PPO were determined using commercial assay kits (Suzhou Keming Biotechnology Co., Ltd, Jiangsu, China) according to the manufacturer’s instructions.
Secondary metabolites assays
Measurement of lignin content (Lin and Kao, 2001)
Wheat leaves were homogenized with a pestle in a mortar in 95% ethanol. The homogenate was centrifuged at 13860 g for 5 min. The pellet was washed three times with 95% ethanol and twice with a mixture of ethanol and hexane (1:2, v/v). The dried sample was washed once with 2 ml acetyl bromide in acetic acid (1:3, v/v). Then 1 ml acetyl bromide in acetic acid (1:3, v/v) was added to the pellet and incubated at 70℃ for 30 min. After cool down, 0.9 ml of 2 mol/L NaOH and 0.1 ml 7.5 mol/L hydroxylamine hydrochloride were added, and the volume was made up to 10 ml with acetic acid. After centrifugation at13860g for 5 min, the absorbance of the supernatant was measured at 280 nm (A280).
Total Phenolic Content (Tawaha et al., 2007)
100 mg dried leaves were weighed into a PE and extracted with 5 ml of distilled water at 100℃ for 30 min in a shaking water bath. After cooling, the extract was centrifuged at 3500 g for 10 min, and the supernatant was recovered and stored at 4℃ until used for the total phenolic content assay. The total phenolic content was estimated by using the Folin–Ciocalteu colorimetric method (Folin and Ciocalteu, 1927), using gallic acid as a standard phenolic compound.
Determination of total ﬂavonoid content (Uarrota et al., 2014)
For extraction, the wheat dried in a hot oven (60℃) for 1 h. Next, 100 mg dried leaves were weighed into a PE and extracted with 95% ethanol (1:60, v/v) at ultrasound in shaking for 12 h. For that, 1 ml of extract solution was mixed with 0.3 ml 5% sodium nitrite (v/v). After 6 min, 0.3 ml l0% aluminum nitrate. After 6 min, 4 ml 4% sodium hydroxide, and 50% ethanol to a total volume of 10 ml. The mixture was well mixed and incubated at room temperature for 15 min versus reagent blank containing water instead of the sample. The absorbance of the resulting solution was measured at 700 nm by using a microplate reader. Rutin was used as the standard for the quantiﬁcation of the total amount of ﬂavonoids. Results were expressed as milligrams of rutin equivalent per gram of dry weight (mg/g).
Determination of tannin (Hagerman and Butler, 1978)
Tannin was extracted from 100 mg dried leaves with 10 ml of distilled water at 60°C for 12 h. After centrifugation at 5000 g for 5 min, the supernatant was collected and used for tannin measurements. The tannin content was estimated by using the F–D reagent colorimetric method, and then absorbance was read at 680 nm. Tannic acid was used as a standard tannin compound.
Determination of alkaloids (Ehmann, 1977)
The dried leaves powders (100 mg) were extracted with 80% aqueous methanol (MeOH) (0.2 L×2) at room temperature for 1.5 hours through sonication and were then filtered. The filtrate was evaporated to 1/4 volume in vacuo. The MeOH extract was suspended in 0.1mol/L HCl (20 ml), vacuum filtration and adjust pH = 9 with concentrated MeOH, and then After 30 minutes, the filtrate was extracted three times with an equal volume of chloroform, and the organic phase was evaporated to dryness, and 70% constant volume of 25 ml. A sample of the extract was added with Ehmann reagent, and then absorbance was read at 555 nm. Quantitative measurements were performed, based on a standard calibration curve of gramine in distilled water.
Signal transduction substances and Si assays
Endogenous JA was measured by GC-MS-SIM (McCloud, 1997; Jang, 2018)
A total of 0.5 g dried sample was extracted with a solution of acetone and 50 mM citric acid (70:30 v/v), and [9, 10-2H2] 9, 10 dihydro-JA (20 ng) was added as an internal standard. The aqueous solution was then filtered and extracted with 10 ml of diethyl ether three times. Both JAs (endogenous JA and the internal standard) were eluted with 10 ml of diethyl ether and acetic acid (98:2 v/v). The sample was adjusted to 50 μL with dichloromethane after the evaporation of solvents and etherification of the residue with excess diazomethane. Then, the extracts were analyzed by GC-MS-SIM.
Endogenous free SA was measured by high-performance liquid chromatography (HPLC) (Jang, 2018).
A 0.2 g freeze-dried sample was sequentially extracted with 90% and 100% methanol in a centrifuge (10,000×g). Both extracts were dried in a vacuum. The dry pellets were resuspended in 2.5 ml of 5% trichloroacetic acid and the supernatant was partitioned with ethyl acetate/cyclopentane/isopropanol (49.5:49.5:1, v/v). The top layer was transferred to a 4 ml vial and dried with purified N gas. The SA was again suspended in 1 ml of 70% methanol and analyzed by HPLC.
The Si content of rice leaves was determined by the colorimetric molybdenum blue method described by van der Vorm (1987).
The mean life table parameters of aphids and the content of nutritional reserves of wheat in treatments were analyzed using SPSS and EXCEL and were drawing using Origin 2016. PCA and heat-map were analyzed using meta analyst. Differences were compared using a t-test at signiﬁcance levels of P < =0.05 or P < =0.01.