Our results showed that defense responses to FAW were increased in maize plants by Si fertilization, which partially interacted with the effects of herbivory and were more specific to the landrace variety. Greater root growth was observed in Si-fertilized landrace plants in the absence of herbivory, while in the hybrid greater root growth occurred in the absence of both Si and herbivory, which was correlated with higher chlorophyll index. In turn, landrace maize plants fertilized with Si and subjected to herbivory showed lower weight gain of FAW larvae and less injury, in exchange for a lower plant height. Such effects indicate that there was a specific priming effect in the landrace variety by Si application followed by induction of resistance with FAW herbivory. In addition, the landrace variety is naturally more resistant and tolerant to FAW than the hybrid of maize, as evidenced by the lower injury and higher shoot biomass and plant height after larval infestation.
The lowest weight gain of FAW larvae occurred in plants of the landrace variety only when herbivory took place after Si application. The lower larval weight gain may have occurred because the larvae fed less on the landrace variety due to resistance induced by attacked plants under Si fertilization, which resulted in reduced injury to the plants. These results suggest that the effects primed by Si fertilization were triggered by subsequent herbivory, slowing the growth of infesting FAW larvae and reducing the injury to the landrace variety due to increased deterrence. Based on these results, we hypothesize that the landrace variety has specific resistance mechanisms, such as genes that encode Si transporter proteins in the roots, since the effects were observed only in this maize genotype that has broader genetic variability due to less intensified breeding for agronomic traits. Lsi1 and Lsi2 transporters of Si have been found in the roots of rice, maize, wheat, and pumpkin. Lsi6 is an intervascular transport protein that plays a role in Si discharge from the xylem to leaf tissues. No external transport proteins have been reported in the leaves (Yamaji and M, 2009; Reynolds et al. 2016).
Analysis of leaf Si concentrations indicated that Si applied in the soil led to greater accumulation in the shoots of maize plants, with no significant difference of herbivory in Si leaf concentration; in the absence of Si fertilization though, maize plants accumulated more Si when FAW herbivory occurred than when there was no herbivory. This suggests that Si applied to soil was absorbed by maize roots, transported to the aerial part of plants, and deposited in the leaves; under the herbivory stress condition, even without Si supplementation the plants absorbed Si to aid in induced resistance to insect attack, since the element is naturally present in soil (Epstein 1994). Previous studies have shown an increase in leaf Si concentration in maize plants fertilized with Si (Gossain et al. 2002; Alvarenga et al. 2017; Pereira 2018). Studies with other grass species have also highlighted the higher Si accumulation in the leaves of fertilized plants. For instance, Nascimento et al. (2017) and Vilela et al. (2014) found higher levels of Si in rice and sugarcane leaves fertilized with Si, respectively.
Other studies have demonstrated the Si-induced resistance to FAW in different grass species other than maize. Nascimento et al. (2017) found lower larval weight of FAW fed rice leaves fertilized with Si via the soil. Nogueira et al. (2018) also found a lower weight of FAW larvae fed leaves of rice plants treated with Si. In contrast to effects of Si-induced resistance to insects, information is lacking on Si interaction with insect herbivory to increase tolerance in plants. Jhonson et al. (2019) showed an increase in the shoot biomass of wheat plants fertilized with Si under herbivory compared to those that were fertilized but did not undergo herbivory, suggesting that insect attack could not reduce the shoot biomass in Si-fertilized plants. In the present study, the highest shoot dry mass was observed in the landrace variety when there was herbivory, which suggests that landrace plants activated tolerance mechanisms to compensate for injury caused by FAW, resulting in greater vegetative growth of the plants. The study of Jhonson et al. (2019) was pioneering in showing that Si can also elicit induction of plant tolerance to insect herbivory in grass species.
The jasmonic acid (JA) signaling pathway plays an important role in mediating the defense responses of plants against chewing herbivorous insects. After being attacked, the plant recognizes the molecular patterns associated with the herbivores and mounts a defense response. Ye et al. (2013) showed that there is a strong interaction between Si and the JA signaling pathway as Si-fertilized rice plants increased the defense levels mediated by this phytohormone and acted as a priming agent against larvae herbivory. The authors also found increased activities of defense enzymes and proteins, greater induction of transcripts encoding proteins involved in JA signaling, and greater phtytohormone accumulation after insect attack (Ye et al. 2013). According to Hall et al. (2019), when plants are fertilized with Si and later attacked by herbivores, the induction of JA production occurs more rapidly due to the priming effect triggered by Si application.
Based on the results obtained herein, Si application itself increased H2O2 concentrations in the leaves of fertilized maize plants, signaling a stress condition (Fester and Hause 2005). Hydrogen peroxide is one of the main ROS produced by plants under stress conditions that stimulates reactions leading to the expression of defense genes, which protect the plants from future attacks by pathogens and insects (Torres 2010). At low concentrations, H2O2 acts as a signaling molecule of plant defenses (Maffei et al. 2007). However, it is difficult to estimate the threshold between a high and low H2O2 concentration that can be compared with the values obtained in our study that could help understand the correlation between its concentrations and the plant defense responses. Future studies are warranted on this topic to give insight on the plant signaling processes mediated by H2O2 following Si fertilization and the interaction with insect herbivory. The present study is the second in the literature to demonstrate an increase in H2O2 concentrations upon Si application. Yang et al. (2017) first reported an increase in H2O2 concentrations in rice plants fertilized with Si.
The results allowed us to conclude that Si did not act per se as an inducer of resistance in maize to FAW. Such effects can be inferred from the results observed in the insect performance, where only under the condition of Si fertilization followed by insect herbivory was there a reduction in FAW larval weight gain in plants of the landrace variety. This was probably due to greater deterrence induced by landrace maize to infesting larvae in the plants previously primed by Si fertilization, since there was also less injury caused by FAW on the plants under this condition, indicating lower larval weight gain due to reduced feeding on plants. This is the first study dissecting the effects of priming from induction of resistance promoted by Si fertilization to insect herbivory.
Previous studies conducted with Si and FAW in maize plants only evaluated the effects of the fertilization, without testing the interactive effect with herbivory (Goussain et al. 2002; Alvarenga et al. 2017; Oliveira et al. 2017). Although in these studies the authors claimed that Si successfully induced resistance to FAW, the negative effects recorded on experimental insects were only slight, without affecting the immature development of FAW, which is the biological phase that causes injury to host plants. This suggests that the beneficial effects promoted by Si fertilization depend on the interaction with insect herbivory. This finding agrees with Coskun et al. (2018) that compiled information from the literature and concluded that the majority of positive effects with Si application were observed in stressed plants, whereas the fertilization in unstressed plants has shown to provide no additive effects. In recent studies by Oliveira et al. (2020) and Sampaio et al. (2020) that evaluated Si application to wheat and sorghum plants on the performance of aphids species, respectively, the negative effects of the fertilization per se on the insects were only modest.
Plants can respond to the presence of insect herbivores in a complex way, where priming and induced resistance can occur together, minimizing the energy costs of mounting a defense to the biotic stress (Frost et al. 2008). Thus, it is more likely that Si acts as a primer than an inducer of resistance itself, leading to the expression of induced resistance only when the plant is under substantial stress, in this case insect herbivory (Dixon et al. 1994; Fawe et al. 2001; Reynolds et al. 2009; Alhousari and Greger 2018). In the present study, H2O2 production in the leaves by Si fertilization played an important role in priming defense in the landrace maize against FAW herbivory. Molecular, biochemical, and physiological traits associated with the phenotype can be used to evaluate defense priming in plants (Balmer et al. 2015; Martinez-Medina et al. 2016; Mauch-Mani et al. 2017), and measuring the concentrations of H2O2 produced upon application of resistance elicitors seems to be a reliable biochemical marker to dissect the effects of priming and induced resistance.
The landrace variety of maize showed higher APX activity when Si was applied in the absence of FAW herbivory, which indicates that Si played an important role in ROS scavenging by increasing the activities of antioxidant metabolism. The highest CAT and SOD activities in the landrace variety occurred in contrasting conditions where only one type of treatment was applied, i.e. when there was no Si application and presence of herbivory or when there was Si application without herbivory. The greater enzyme activities may have resulted in higher plant shoot biomass, so the increased activities in response to FAW herbivory may have contributed to ROS scavenging, thus resulting in greater tolerance in plants of the landrace variety.
In the study by Yang et al. (2017), there was an increase in CAT and SOD activities in Si-fertilized rice plants under insect herbivory, so that Si delayed the decrease in the enzyme activities under infestation condition. Contrasting results were found by Torabi et al. (2005) that observed that SOD activity increased when Si was applied to rice plants, but CAT and APX activities were decreased. In addition, Si was reported to increase plant tolerance to abiotic stress through improvement of antioxidant metabolism, as demonstrated by Gong et al. (2005) and Shi et al. (2014; 2016), in which Si fertilization increased tolerance in tomato and wheat plants subjected to water stress by increasing the enzymatic activities of SOD and CAT. Jhonson et al. (2019) first demonstrated that Si induced tolerance in wheat by promoting overcompensation in plants growth under insect herbivory. Therefore, there is evidence that Si fertilization can increase tolerance to both biotic and abiotic stress conditions by upregulating the activities of antioxidant metabolism in plant species classified as Si-accumulators.
The highest enzymatic activities of CAT and APX in the maize hybrid were observed in the conditions without Si application and without herbivory. Higher concentrations of MDA and greater larval weights of FAW were also observed under these conditions. Because the effects depended on the maize genotype, higher activities of the antioxidant enzymes served to remove excess ROS from the cells to reduce oxidative stress in the hybrid plants due to possible abiotic stress provided by the experimental conditions in the greenhouse. For example, the lesser availability of nutrients to the plants grown in pots may have been an influential factor, since maize hybrids are usually bred to respond to higher levels of macronutrient fertilization for high yields (Amorim and Souza 2005).
The increase in lipid peroxidation by ROS results in increased concentrations of MDA, one of the main products of lipid peroxidation of cell membranes, which indicates oxidative damage to these structures (Corbineau et al. 2002). MDA is used as one of the main proxies of oxidative stress in plants (Yang et al. 2017). Therefore, the results of the present study indicate that Si fertilization reduces MDA concentrations in maize plants under herbivory stress condition, reducing oxidative damage to plant cells. Ma et al. (2016) also concluded that Si decreased lipid peroxidation in fertilized plants. The results of our study along with data in the literature agree with the hypothesis that Si fertilization improves plants antioxidant metabolism, especially the increased activities of antioxidant enzymes, contributing to reducing ROS levels that would ultimately cause oxidative damage to stressed plants.
The antioxidant enzymes SOD, CAT, and APX are the main enzymes involved in plant tolerance to oxidative stress. Plants that express high antioxidant enzyme activities can more efficiently eliminate excess ROS, which protects the cellular components from the toxic effects of ROS produced under stress situations, and consequently the plants experience less oxidative damage, allowing them to tolerate the stress (Caverzan et al. 2016). In general, the results of the present study showed that increase in the activities of antioxidant enzymes occurred independently among plants fertilized with Si or subjected to herbivory, and that the responses of antioxidant enzyme activities were probably due to the sequence of events in methodology used here, i.e. first application of Si and then larval infestation, and time at which samples were collected for chemical analysis.
In the larval performance bioassay with FAW, the same effects as in the greenhouse experiment were not observed, and there were no significant differences in leaf consumption and larvae biomass for the landrace variety in the laboratory. In the hybrid there was lower leaf consumption rate and lower larval weight gain in the laboratory and greenhouse experiments, respectively. Differences in the effects between experiments were due to the methods used. The presence of FAW larvae feeding on the plants in the greenhouse was essential for the induced defense responses to occur in a more dynamic and effective way; in turn, in the lab bioassay we most likely observed the predetermined effects of the Si fertilization. These effects may be due to the mechanical barrier formed in the leaves by deposition of Si and polymerization as SiO2 (Ma and Yamaji 2006). Thus, the methods used in the greenhouse are more appropriate for experiments aimed at evaluating the Si-induced defense effects in plants to insect herbivory.
Although Si is not classified as an essential nutrient for plants development, this element can interact with plant defense signaling pathways by regulating various physiological activities under biotic and abiotic stress conditions, attenuating oxidative stress caused by increased ROS production (Ye et al. 2013). The results of the present study demonstrate that the defense effects provided by Si probably cannot be characterized as induction of resistance, since the application of Si per se did not induce resistance in maize plants. Thus, we suggest a more appropriate term to be used in scientific literature of integrated pest management for Si in function of the negative effects it promotes on insects feeding and development via plants fertilization as “resistance elicitor” (Souza and Boiça Júnior 2019), which broadly encompasses the defense effects of both priming and induced resistance.
Another interesting result was the biostimulant effect of Si in the landrace variety under fertilization in the absence of herbivory, which resulted in higher root growth of the plants. Biostimulatory effects of Si have been reported (Van Oosten et al. 2017). “Biostimulant” is a term recently adopted by the scientific community and industry to characterize chemical and microbial compounds that improve the nutritional efficiency, tolerance to abiotic stresses, and crop quality traits, regardless of nutrient content (Du Jardin 2015; Yakhin et al. 2017). Thus, biostimulants are compounds that act in the metabolic and defense signaling pathways of plants. In the present study, the biostimulant effect provided by Si was genotype-dependent and occurred only when there was no concomitant herbivory. Vargas-Hernandez et al. (2017) suggest that some compounds play the roles of both resistance elicitor and biostimulant, depending on the doses used, which resembles an hormesis-like effect, a subject that deserves future in-depth investigation for Si (Abreu et al. 2021).
Molecular analyses will also be needed to better answer important questions on Si interactions with plant metabolic and defense signaling pathways, such as which genes are up- and down-regulated upon Si fertilization, and which transporters are present in the roots of different plant genotypes, so that desired effects can be potentiated in integrated pest management by using plants that were previously bred to better respond to Si fertilization and induce higher levels of resistance or tolerance to insect herbivory. Another interesting issue to be addressed is whether priming in Si-fertilized plants can be passed on to the produced seeds, i.e., whether the activation effect of epigenetic defenses are transgenerational. The results obtained in this study are important for characterizing the induced defense effects provided by Si fertilization and encouraging its use in a more appropriate way in integrated pest management. The implementation of Si fertilization to maize plants could contribute to the management of FAW both in countries where it is native and in the regions of the world the pest was recently established.