4 DISCUSSION
The infestation of aphids in plants can cause huge damage to crop production. Currently, exogenous interventions such as chemical pesticides are the most effective approach to control insect pests and achieve high crop yields (Y.-F. Li et al., 2018). However, the long-term use of pesticides could lead to insects developing resistance and cause the ineffectiveness of these chemicals. Activation of the plant itself anti-aphid system is considered a green protocol in plant protection and sustainable agricultural production. In this study, we demonstrated that the application of nano-Se could effectively reduce aphid populations in wheat seedlings (Table 1). This could be due to the mechanism that nano-Se could promote the synthesis of flavones, phenolic acids, plant hormones and the expression of the related genes, which could further influence the phenylpropane pathway and the metabolism of indole alkaloids. The application of nano-Se also increased antioxidants and VOCs concentrations, which promotes the resistance of wheat seedlings to aphids (Figure 8).
Plant secondary metabolites play an important role in the resistance to insect feedings. These chemicals commonly include phenolics (phenolic acids and flavonoids) and terpenoids (diterpenes and monoterpenes), which could attract specific insects and their natural enemies (Jamieson et al., 2017). A previous study showed that Se hyperaccumulators could promote plant growth by protecting Artemisia ludoviciana andSymphyotrichum ericoides against grasshoppers (El Mehdawi, Quinn, & Pilon-Smits, 2011). The effect of Se on secondary metabolites was mainly based on abiotic stress, including salt, heavy metal, and drought (Hawrylak-Nowak, Hasanuzzaman, & Matraszek-Gawron, 2018). However, few studies were conducted to elucidate the effects of Se on the formation of secondary metabolites (phenylpropane and indole alkaloid metabolism) and their impact on insects. Our previous study reported that foliar application of nano-Se (10 mg/L) could alleviate pesticide stress via regulating the ascorbate-glutathione cycle in tea trees (D. Li et al., 2021). In pepper plants, nano-Se (20 mg/L) could enhance phenylpropane and fatty acid metabolism (D. Li et al., 2020), causing a significant increase in the concentration of flavonoids (by 80%) and phenolic acids (by 65%). In this study, the increased concentration of myricetin, rutin, apigenin, kaempferol, quercetin, caffeic acid, hydroxy-3, 5-dimethoxycinnamic acid, 4-hydroxybenzoic acid, 4-hydroxycinnamic acid, and chlorogenic acid was observed after the application of nano-Se (5mg/L) to wheat seedlings with aphid infection (Figure 4). These metabolites of apigenin and caffeic acid were then selected to evaluate the growth and metabolism of wheat under aphid stress. This practice could enhance wheat growth including plant height, length, weight, flavonoids (luteolin and quercetin), and phenolic acids (caffeic acid, 4-hydroxybenzoic acid, and 4-hydroxycinnamic acid) in the seedlings (Figures S1, S2, and S3). The application of plant strengthener 4-fluorophenoxyacetic acid was found to suppress pest populations (Wang et al., 2020). The enhanced resistance to insects could be due to the deposition of flavonoid polymers in plant cells. Czerniewicz’s et al. found that the formation of luteolin, apigenin, caffeic acid, and 4-hydroxybenzoic acid compounds could impair insect feeding, thus protecting wheat from aphid infection (Czerniewicz et al., 2017).
In general, plants can establish a dynamic equilibrium of ROS via formation or removal processes depending on the growth conditions. Plant secondary metabolism and antioxidant system can synergistically improve plant resistance to biotic and abiotic stresses. In the present study, the antioxidant enzymes (CAT, POD, SOD, LOX, GSH-Px, and APX) and non-enzymes (VE, MDA, GSH, Pro, and LPO) system were found to change to varying degrees with nano-Se treatment (Figure.3). Selenite present in soil (0.5 mg/kg) enhanced the antioxidant enzymes (CAT, POD, and PPO) and reduced the MDA concentration, forming a favorable situation in oilseed against Sclerotinia sclerotiorum stress (J. Xu et al., 2020). Li et al. used 200 µM of Se treatment to inhibit silkworms via significantly decreasing their growth and reproduction (Jiang et al., 2020). In addition, sodium selenate (6.5 µM) foliar application can effectively protect rice against N. lugens infestation (Scheys, De Schutter, Subramanyam, Van Damme, & Smagghe, 2020). Sodium selenate has been shown to effectively inhibit the growth of insect pests in crops, but without elucidating the underneath mechanism (Hanson, Lindblom, Loeffler, & Pilon‐Smits, 2004; Z. Xu et al., 2021). Our study suggests that nano-Se application could alleviate the effects of herbivores by regulating the secondary metabolism and antioxidative ability.
Phytohormone was considered as the first line to defense against the pests (Erb, Meldau, & Howe, 2012). The JA, SA, and MT signaling was intensively involved in herbivore defense in plants. In the present study, the application of nano-Se (5, 10, and 20 mg/L) could lead to the increase in JA, MT, and DIMBOA concentration, while the SA concentration was reduced (Figure.2). The remarkable effects were observed on MT and SA concentration changes, these two phytohormones were selected to apply directly on wheat seedlings to evaluate the plant metabolism. The growth parameters (height, length, and weight) and phytohormones (JA, SA, DIMOBA and MT) were measured in wheat seedlings (Figure. S1 and S4). JA and other signaling pathways interact to regular and enhance plant resistance to insects (Lortzing & Steppuhn, 2016). It has been demonstrated that the ROS and hormonal signaling were integrated to respond to insect herbivores (Kerchev, Fenton, Foyer, & Hancock, 2012). Silicon could also regulate the biosynthesis of phytohormones (JA, SA and IAA) and reduce the concentration of ROS; these two processes together enhance plant resistance to herbivorous insects (Tripathi et al., 2021). In our study, nano-Se could activate the indole alkaloid pathway and the related genes, which correspondingly increased the MT and DIMBOA contents. Extensive research demonstrated that MT is involved in regulating circadian rhythms in aphids (Barbera et al., 2020). The signal alternatively activates the molecular pathway that leads to the reproduction and migration responses. MT application (50µM) could induce the antioxidant enzymes activities (PPO, CHI, and PAL), thus regulating the ROS concentration in plants (Liu et al., 2019). Furthermore, MT treatment increased MeJA concentration and up-regulated the JA biosynthesis genes, as a result, it enhanced the tomato defense against disease (Arnao & Hernandez-Ruiz, 2019). MT is found to regulate flavonoids and carotenoid biosynthesis as well, promote the synthesis of amino acids, organic acids and sugars, and enhance the plant resilience under biotic (fungi, bacteria, and insects) and abiotic (heat, cold, drought, and salt) stresses (Tiwari et al., 2020). DIMBOA is an allelochemical and can react with glucose to enhance resistance to aphids by promoting the callose deposition in maize (Meihls et al., 2013). DIMBOA (0.1 mM) could reduce the Sitobion avenae F numbers to levels below the economic damage threshold (HANSEN, 2006). However, the anti-aphid mechanism related to nano-Se and DIMBOA was not elucidated. In this study, the combination of nano-Se and MT application increased DIMBOA and MT, as well as the levels of the related genes which could be associated with the indole alkaloid pathway, thereby could decrease the aphid numbers in the wheat seedlings.
In present study, the release number of VOCs increased in the wheat seedlings treated by nano-Se. GC-IMS analysis revealed the increased concentration of ethanol, 1-popanol, Isopentyl alcohol, 2-butanone, 2-heptanone, acetoin, acetone, benzaldehyde, ethyl 2-methylpropanoate, and ethyl acetate dimer in the presence of nano-Se bio-fortification. However, 2-pentenal (E) dimer, hexanal monomer, ethyl hexanoate, and phenylacetaldehyde concentrations were reduced. Plants can produce a wide array of volatile metabolites, including terpenes, fatty acid derivatives, aromatic compounds, and amino acid derivatives (Clavijo McCormick, Unsicker, & Gershenzon, 2012). The increased concentration of benzaldehyde, acetoin, and 1-propanol facilitated the repelling response to herbivorous insects in sorghum seedlings (Park et al., 2020). Zhuang et al. found that the volatile indoles increased with insect feeding; these chemicals could improve the resistance in rice plants (Zhuang et al., 2012). Zhang et al. showed that VOCs including acetone, hexanal, and pentalamide play a vital role in impacting insect behaviors (Y. Zhang, Teng, Wang, & Jiang, 2021). For example, VOCs could function as either attractants or antifeedants, and effectively control insect activities. It is commonly accepted that VOCs and insect pheromones could be combined to regulate insect behaviors. Our present study found that the combinations of nano-Se with MT can markedly reduce the insect numbers most likely due to the increasing VOCs in wheat seedlings.
In summary, foliar application of nano-Se could effectively decrease the aphid number compared with the control. The corresponding insect-resistant mechanism could be associated with the increasing levels of secondary metabolites (API and caffeic acid) and plant hormones (MT and DIMBOA) in the indole alkaloid and phenylpropane metabolisms in wheat seedlings. Different ratios of nano-Se and these metabolite combinations were tested the defense-related compounds toSitobion avenae in wheat plants. The combination of nano-Se and MT achieved the best controls in reducing the S. avenaenumber. The concentrations of antioxidants, phenolic acids, flavonoids, and plant hormones were found to be markedly enhanced by acting synergistically of nano-Se and MT combination. The VOCs could function as insect attractants and repellents, which included ethanol, 1-popanol, isopentyl alcohol, 2-butanone, 2-heptanone, acetoin, acetone, benzaldehyde, ethyl-2-methylpropanoate, and ethyl acetate dimer. Therefore, the application of the optimal ratio of nano-Se and MT could be a promising strategy to protect wheat seedlings from S. avenaeinfection by collective actions from the secondary metabolic pathways and contributions of the increasing antioxidants and VOCs levels.