Foliar Spraying Suitable Dosage of Silicon Fertilizer on Wheat Suppressed Inoculated Aphid Reproduction by Activating Plant Induced Defense Response

Background: Although the ability of silicon to induce plant resistance against some insect is known, it is still unknown that the effect of the different concentrations of silicon on activates the secondary defense system in wheat, and then inuence the life cycle table of wheat aphids. In this experiment, different levels of silicon (Si) fertilizer was applied to change the secondary defense system of wheat, to study the impact on the life table parameters of aphid (Sitobion avenae F.) (Hemiptera: Aphididae), and to explore the mechanism of Si application in enhancing wheat resistance to aphids. Wheat seedlings were cultured in a hydroponic experiment with the seven different levels of Si (0, 0.3, 1, 2, 3, 4, 5 and 9 mmol/L). Results: Our ﬁ ndings indicated that the application of Si can suppress the net reproductive rate (R 0 ), intrinsic rate of increase (r m ), mean generation time (T), nite rate of increase (λ), and prolong the population doubling time (t) of aphid. Besides, aphid infection elicited immune responses in wheat, which was enhanced by Si: Between the treatments, a foliar application of Si improved the activity of four defense enzymes (PAL, PPO, CAT, LOX), and increased the content of signal transduction substances (JA, SA) and secondary metabolites (tannin, alkaloids, lignin, total phenolics and ﬂ avonoids). Moreover, the results of the correlation analysis showed that the concentration of Si in wheat was positively associated with the activities of defense enzymes and the concentrations of secondary metabolites and signal transduction substances. There was a signicant positive correlation between the activities of four defense enzymes and the concentrations of secondary metabolites and signal transduction substances in wheat. Furthermore, the activities of defense enzymes, the concentrations of secondary metabolites and signal transduction substances and Si in wheat were negatively associated with the R 0 , T, r m , (cid:0) and positively related to the t of the life table parameters from the aphid. Conclusions: In conclusion, the current result suggested that the application of Si can increase the signal transduction pathway of wheat and regulate the secondary metabolism by


Background
Aphids are piercing and sucking by feeding on the phloem of wheat (Dedryver et al., 2010). Aphids can build extraordinary populations under the right conditions due to high reproductive potential (parthenogenesis), shorter generation times, higher population intrinsic growth rate, and a high proportion of viviparous females in the population (Chapman et al., 2018;Kaitlin et al., 2018). Therefore, the occurrence of aphids is extremely harmful to agricultural production. For a long time, aphids are primarily controlled by insecticides. But on the one hand, overdependence on insecticides can lead to the emergence of insecticide-resistant aphid populations (Foster et al., 2014), reduce the control effect against aphids. On the other hand, the application of pesticides may also adversely affect the growth of non-targeted insects, and result in unintended environmental or nontarget impacts. Therefore, the use of integrated pest control has always received great attention.
Plants are not always in a passive position when facing the occurrence of pests in nature. In the process of long-term co-evolution with pests, plants have developed complex resistance mechanisms to resist the invasion and damage of various pests. The mechanisms of defense against the aphids include constitutive resistance and induced resistance, the defense response of plants to aphids reveals the defense response of wheat to aphids (Wu and Baldwin, 2010;Zhou et al., 2015). Among them, the former refers to the resistance of the plant itself when it is not invaded by aphids, and the latter is the resistance induced by aphids. Research shown that aphid infection increases in ROS levels, and induces the up expression of genes involved in signaling pathways and some defensive enzymes (Lu et  Studies have shown that the application of Si can reduce fecundity, reproductive period, longevity, r m , and R 0 of aphid, and alter survival, reproduction, and host plant preferences of sucking insects which were found in wheat (Dias et al., 2014;Gomes et al., 2008;Korndorfer et al., 2011). Its internal mechanism is considered to involve the following aspects. First, as a bene cial element, Si is accumulated for subsequent deposition as amorphous hydrated silica', forming a cuticle-Si double layer in the leaf blade of the plant (Luyckx 2017; Debona 2017), or form a polysilicon acid complex with low solubility and strong stability, and increase the intensive cell silici cation in the frequency of silici ed cells per unit area of leaf epidermal cells, improve the abrasion resistance of plant tissues, and reduce the palatability and digestibility of tissues (Hao et al., 2008;Matoh, 1986;Law, 2011;Cai, 2008). Cell silici cation can play a pivotal role in the quality of a wide variety of monocot and dicot plant species and help them, whether directly or indirectly, counteract abiotic and/or biotic stresses (Tripathi et al., 2017;Luyckx et al., 2017;Adil et al., 2020). Secondly, the study found the application of Si can increase the activities of some defense enzymes, and the accumulation of lignins, phenolic compounds, phytoalexins in plants (Ma et al., 2016;Abbas et al., 2015;Ye et al., 2013;Cai, 2008;Rahman et al., 2015;Tripathi et al., 2017). And Si can also induce the release of insect-resistant chemicals from the epidermis or mesophyll of wheat, reduce the reproductive capacity of aphids, and improve the insect resistance of plants (Ye et al., 2013;Van et al., 2013;Sivanesan et al., 2014). However, it is rarely reported about the systematic research on the effects of different concentrations of Si on the signal transduction, the activity of defense enzymes, and various secondary defense substances, and then affects the secondary defense system in wheat, and to explore the impact on the life cycle table of grain aphid.
The grain aphid (Sitobion avenae F.) is the dominant species among natural aphid populations (Xu et al., 2017), especially in the Huanghuai wheat region of China, it is the main pest affecting wheat production.
In the current study, a culture experiment was used to set different Si supply levels to study the parameters of the life cycle of inoculated aphids, and affect the level of Signal conducting substance (JA, SA), defensive enzyme (PAL, PPO, CAT, LOX) activity and secondary metabolites (tannin, alkaloids, lignin, total phenolics and flavonoids) in wheat under the conditions of aphid infecting. Systematically explore the effect of different concentrations of Si on the defense response of wheat induced by aphid feeding, to further clarify the in uence of silicon on wheat aphid resistance and its internal mechanism.

Effect of Si on life table parameters of aphid
Performance parameters in the growth and development of the wheat aphid varied among treatments but were generally better on T4 treatment compared to that for other treatments (Table 1). Compared with control, all treatments decreased the net reproductive rate (R 0 ), the mean generation time (T), the intrinsic rate (r m ), the nite rate of increase (λ) and prolonged the population doubling time (t) of the wheat aphid.

Uptake of Si by wheat
The concentration of Si in the wheat varied among treatments (Fig.1). Compared with control, other treatments increased the concentration of Si in the wheat. In particular, a comparison of Si levels among Si treatments revealed that it was signi cantly higher (P<0.01) in the T4 treatment as compared to that of the remaining Si treatments (Fig. 1).

Effect of Si on the activities of the enzyme activity of wheat
The PAL, PPO, CAT, and LOX are major defense enzymes of secondary metabolism. In Si-sprayed (+Si) wheat, 1, 2, 3, 4, 5mmol/L of Si improved the PAL activity in leaves of wheat, were signi cantly higher than those in control, 0.3, 9mmol/L of Si-sprayed (+Si) wheat, becoming highest at 3mmol/L ( Fig. 2A).
Activities of PPO increased with Si foliar spray treatments. Maximum PPO activity was noted with the treatment at T4, and signi cantly higher than other treatments. However, in other treatments, the activities of PPO were not different between +Si and −Si plants (Fig. 2B).
In the CAT, the rst increase in enzyme activity was found during T1, followed by the next increase starting from T2, becoming highest at T4 treatment, and then decreased, and the increased range was 10.51%-100.20% but was generally higher than control.
Analyses of LOX activity in leaves of wheat seedlings showed markedly higher activity of this enzyme at the different Si treatments than in the control (Fig. 2D). In particular, a comparison of the LOX activity in wheat among Si treatments revealed that it was remarkably higher (P<0.01) in the T4 treatment as compared to that of the remaining Si treatments.

Effect of Si on the concentrations of secondary metabolites of wheat
Analysis of variance indicated that the levels of secondary metabolites were increased by Si application. The levels of secondary metabolites in the wheat varied among Si treatments (Fig. 3). In general, as the concentration of Si application increases, the content of secondary biomass rst increases and then decreases, the maximum values of these parameters are seen with the application of Si at T4 treatment, followed by T3 treatment, which was signi cantly higher than other Si treatments (Fig. 3A, B, C, E).
However, the concentration of total phenolic (D) was observed that no dramatic difference among the T1, T2, T3, T5. Up-regulation of tannin (  3E) recorded in wheat were a slow increased, but, T2-T6 treatments were higher than control and T1. In these secondary metabolites, a comparison of Si levels among Si treatments revealed that it was increased range was 1.50%-74.96%, but were generally higher than control (Fig. 3).
Effect of Si on the concentrations of signal transduction substances of wheat We analyzed endogenous SA and JA levels after Si application. JA levels were increased with Si application (Fig. 4). In particular, a comparison of JA levels among Si treatments revealed that it was signi cantly higher (P<0.01) in the T4 treatment as compared to that of the remaining Si treatments (Fig.   3). Endogenous hormone SA also exhibited a similar pattern as JA. Endogenous JA levels were signi cantly increased at all concentrations of Si application as compared to that of the control, and this tendency was consistently observed in all indicators (Fig. 4).
Correlation between the activities of the defense enzymes, secondary metabolites and signal transduction substances in wheat at the different concentrations of Si in aphidinfection wheat PCA analysis was conducted to report the impact of the levels of Si on the dispersion of the defense enzymes activity, secondary metabolites and signal transduction substances of wheat. PCA score plot was given in Fig. 5A showed that PC1 and PC2 contributing 36.3% and 19.2 % respectively to the total variance. The loading plot explained that PAL, PPO, Si, phenolic, lignin, JA contributes to PC1, whereas tannin, alkaloid, SA contribute to PC2. However, avonoids, CAT contributed to both PC1 and PC2 (Fig.  5B). The elements such as the defense enzymes activity, secondary metabolites and signal transduction substances of wheat were analyzed by plotting the correlation matrix (Fig. 6A). The concentration of the activities of defense enzymes (include PAL, PPO, LOX, and CAT) was signi cantly positively associated with the concentrations of secondary metabolites (include tannin, phenolic, flavonoids, lignin, and alkaloid) and JA and Si in wheat. Moreover, the activities of PAL and PPO presented a positive correlation with SA. There was a signi cant positive correlation between the levels of secondary metabolites and signal transduction substances and Si in wheat. Additionally, Fig. 6A found a positive correlation between the concentration of Si from wheat and signal transduction substances in wheat at the different concentrations of Si after aphid infection. Our data was further con rmed by the analysis of the heatmap (Fig. 6B).
Correlation between the life table parameters of aphid and biochemical indexes in wheat at the different concentrations of Si in aphid-infection wheat As shown in (Fig. 7), R 0 and T were dramatically negatively associated with the levels of secondary metabolites and signal transduction substances and Si in wheat. The r m and of the life table parameters from aphid were dramatically negatively associated with the activities of PPO, the concentrations of secondary metabolites (include phenolic, flavonoids, lignin, and alkaloid) and signal transduction substances and Si in wheat. Additionally, the Fig. 7 found a signi cant positive correlation between the t of the life table parameters from aphid and the activities of the enzymes (include PPO, LOX), the concentrations of secondary metabolites (include tannin, phenolic, flavonoids and alkaloid), signal transduction substances and Si in wheat. This study showed that the application of Si increased the accumulation of Si in wheat and decreased the R 0 , T, r m and , prolonged the t of aphid, so that the resistance of wheat to aphid was improved. The resistance level of the genotype to the pest attack was thought to be involved in the T and t, and the higher the resistance of the genotype to the pest attack, the higher were the values for T and t (Kennedy and Abou 1979; Machacha et al., 2012). This study has shown that Si shortness T, maybe the insect's lifecycle was slowed down in Si-treated plants (James, 2003;Connick, 2011), and shortened the time (T) from birth to the rst reproduction of aphids and accelerated the death of aphids. It shows that the application of silicon may increase the resistance of wheat to aphids. Besides, Han et al. (2015) have shown that the r m , λ and R 0 of the rice leaf folder population were all reduced at both the low and high Si addition rates. But we found that the Si was 3 mmol/L, the aphid resistance was the best. Because when the Si concentration is too high, it may be produced Si poison, changed the permeability of the cell membrane, and affected the normal defense of the leaves. This correlation study has shown that the accumulation of Si was signi cantly negatively correlated with the R 0 , T, r m , of aphid, and was  Debona et al., 2017). In this study, different levels of Si were applied to in uence the Si content in wheat, which in turn affected the secondary defense mechanism in wheat. The results of this study showed that Si increases in the activities of defense enzymes, the accumulation of signal transduction substances and secondary metabolites.

Discussion
Moreover, the content of JA was higher than that of SA under the different Si levels, which may be SA and JA act antagonistically, where SA inhibits the activity of JA and vice versa (Maffei et al., 2007). This study showed that a positive correlation has been observed among the accumulation of Si in wheat and the activities of defense enzymes, and the accumulation of signal transduction substances and secondary metabolites. Si may be increased the phloem defense, participated in the secondary metabolism and other physiological processes of wheat, increased the biochemical mechanisms of defense against the aphids, as a result of this the fecundity of aphids are decreased.
Most of the previous studies have separately analyzed some antioxidant enzymes and certain secondary metabolites in plants, to explore the internal mechanism of the decrease in density of piercing insects or the change of some indicators of the lifetable parameters. For example, Yang and Teixeira et al. (2020) shown that the application of Si hindered the feeding of brown planthopper (Nilaparvata lugens, BPH) by changing the enzyme activity and the content of some substances in the antioxidant system in plants, and improving silici cation of leaf sheaths that BPH feed on, as well as reduced the R 0 and r m of the sapsucking cabbage aphid, Brevicoryne brassica L. (Hemiptera: Aphididae) by changing the Si content of collard leaves, total glucosinolates, and leaf cell wall thickness. This test systematically linked the defensive signal substances, defensive enzyme activities, and defensive secondary substances under the feeding condition of aphids with the aphid life cycle table, and explored the internal mechanism of different silicon levels affecting its internal mechanism. This correlation study has shown that the activities of defense enzymes and the accumulation of signal transduction substances and secondary metabolites were remarkably negatively correlated with the R 0 , T, r m , of aphid, and was signi cantly  Barbehenn and Peter, 2011). The presence of secondary compounds such as alkaloids, limonoids, and cucurbitacins tends to reduce palatability, thereby making plants a less preferred host food source for insects (Aoyama and Labinas, 2012), thus reducing its survival rate and reproduction rate (Zhang et al., 2017). Furthermore, SA signals the release of plant volatiles that attract the natural enemies of insect pests (de Boer et al., 2004), and enhances wheat defense mechanisms against the aphid. However, the Si application method used in most studies is rhizosphere Si application (Teixeira et al., 2020;Ma et al., 2015), but this experiment was foliar spraying, and the Si content of leaf was higher than rhizosphere Si application. Since Si is easily deposited in plants, and once deposited, Si is not remobilized (Raven, 1983).

Conclusions
In conclusion, in the present study, we revealed that the perception of aphid infestation by wheat seedlings and the application of Si induces a whole sequence of potential defensive reactions. It is proposed that the application of Si can enhance secondary metabolism, and reduce the R 0 , T, r m , , prolong the t, thus enhances wheat defense mechanisms against the aphid. Additionally, the content of TEOS was 3 mmol/L, the aphid resistance was the best.

Test design
Four germinated seeds were sown in each polyethylene plastic pot that was lled 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℃.

Plant culture
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% H 2 O 2 for 30 min and rinsed thoroughly with distilled water four times, then soaked 24 h with distilled water and germinated on moist lter paper for 2 days in petri dishes, and germinated at 25℃. The germinating seeds were sown on moistened gauze xed 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 (

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 (R 0 ) 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: R 0 =∑l x m x 2) The mean generation time (T) is the time of each aphid from birth to the rst reproduction, is calculated as: T=∑xl x m x / R 0 3) The intrinsic rate (r m ): The maximum instantaneous growth rate of a population in an ideal state, and it re ects the population's expansion ability in an ideal state, which can be used to measure the population's tness, is calculated as: r m =lnR 0 / T 4) The nite 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: =e rm 5) The population doubling time (t): Population doubling time in a certain period time, is calculated as: t= ln2/ R 0 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 ltered. The ltrate was evaporated to 1/4 volume in vacuo. The MeOH extract was suspended in 0.1mol/L HCl (20 ml), vacuum ltration and adjust pH = 9 with concentrated MeOH, and then After 30 minutes, the ltrate 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. Endogenous free SA was measured by high-performance liquid chromatography (HPLC) (Jang, 2018).

Signal transduction substances and Si assays
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 puri ed 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).

Statistical Analysis
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. funding agency did not participate in the design of study and collection, analysis, and interpretation of data and in writing the manuscript. Tables Table 1 Effect of Si on life table parameters  Note: Silicon is represented by "Si". Life table parameters include net reproduction rate (R 0 ), intrinsic growth rate (r m ), nite growth rate (λ), average childbearing time (T) and population doubling time (t). CK, T1-T7 represent different treatments, which are spraying different levels 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. Numbers followed by different letters within a row are signi cantly different according to the t-test (P < 0.05).