Silicon (Si) has not historically been considered an essential plant nutrient though it has been termed ‘quasi-essential’ (Epstein, 1994; Luyckx et al., 2017). Evidence has mounted in the last decade that Si plays important roles in plant defense against biotic and abiotic stress (Ma et al., 2001; Ma, 2004; Ahmed et al., 2013; Balakhnina and Borkowska, 2013; Pontigo et al., 2015; Cooke and Leishman, 2016) including against insect herbivores and pathogens in agriculture (Ye et al., 2013; Wang et al., 2017). Among studies of the effects of Si on plant defense against herbivores, two primary modes of action have emerged as important. The first of these is the mechanical mode afforded by the deposition of inorganic amorphous oxide (SiO₂) phytoliths in the epidermis of foliage and in spines and trichomes (Ma, 2004; Hartley et al., 2015). These defenses are often constitutive (‘always on’) but can also be induced by damage such that the plant is responding to herbivory with greater deposition of silicon in defense structures (Hartley et al., 2015). Silicon deposition provides structural rigidity to plants and the resulting physical toughness also makes the plant surface less vulnerable to penetration and colonization by fungal pathogens (Wang et al., 2017) and tougher for herbivores to masticate and digest (Kvedaras and Keeping, 2007; Reynolds et al., 2016). The second mode of action of silicon in plant defense is enhancement of the induced production of defense chemicals (Wu and Baldwin, 2010) including enzymes such as polyphenol oxidase and inhibitors such as trypsin protease inhibitor. For both of the foregoing mechanisms, there is strong evidence that the influence of Si is mediated by the jasmonate signaling pathway (Ye et al., 2013). The lipid-derived plant hormone, jasmonate, plays several important roles in plant metabolism including prioritization of defense over plant growth when a plant is under biotic stress (Yang et al., 2012). Ye et al. (2013) demonstrated Si elevated rice plant defense against rice leaffolder (Cnaphalocrocis medinalis) by promoting phytolith accumulation in leaves as well as polyphenol oxidase, peroxidase, trypsin protease inhibitor, and Bowman–Birk protease inhibitor activity. The significance of the jasmonate pathway in these effects was established by use of RNAi to silence expression of the CORONATINE INSENSITIVE1 gene which is involved in jasmonate perception. For the RNAi rice plants, the positive effects of Si on plant defenses were negated, leading to normal development of the herbivore. By logical extension, it is expected that the influence of Si on plant defense will also apply to mechanisms other than the two broad types described above, provided that these mechanisms too are mediated by the jasmonate pathway.

Plant defenses are not always direct in nature; they can also operate indirectly via the third trophic level, the natural enemies of herbivores. Work in recent decades has amply demonstrated that plants are able to manipulate the composition of the volatiles they emit, such that they serve as synomones, semiochemicals that benefit both the emitter and receiver (Becker et al., 2015). In this mechanism, a plant responds to herbivore attack by producing a particular herbivore induced plant volatiles (HIPVs) blend to which natural enemies such as predators and parasitoids are attracted, leading them to attack the herbivores (Mumm and Dicke, 2010; Schuman et al., 2012). Crucially, the jasmonate pathway is the most important of the three known signal-transduction pathways that underlie the induction of HIPVs (Ament et al., 2004; Wei et al., 2011; Zhang et al., 2013). Accordingly, the positive effects of Si on direct plant defenses described above may apply to indirect plant defense based on HIPV production.

To date, no work has been published on the effects of Si on HIPVs in any plant system. There is, however, indirect evidence that HIPVs may be affected by Si treatment to plants. Using a cucumber system, Kvedaras et al. (2010) demonstrated that Si enhanced the attraction of the predator, Dicranolaius bellulus, to plants infested by Helicoverpa armigera. An associated field study showed that Si-treated plants were more attractive to ‘wild’ predators than Si deficient control plants (Kvedaras et al., 2010). Whilst those results are consistent with Si altering the HIPV blend of pest-infested plants, that paper did not include any results for the volatiles emitted by the plants. This leads to the hypothesis that Si alters the HIPV blend thus enhancing the attraction of natural enemies. To test our hypothesis, we studied the volatiles of rice plants grown under Si deficient conditions versus with Si supplementation, and determined the effects on volatiles when infested by a major economic insect pest. We then complemented volatile studies with behavioral assays to determine whether attraction of biologically relevant parasitoid species was affected by changes in volatiles. Finally, RNAi plants were used to explore whether silenced perception affected the influence of the Si regime on plant volatiles.

Silicon is the second most abundant element in the earth’s crust, yet the vast majority of this is not available to plants (Epstein, 1994; Savant et al., 1997; Ma and Yamaji, 2006), existing predominantly as feldspars and quartz minerals. Plants take up Si from the soil solution as silicic acid [Si(OH)₄], or Si(OH)₃O^- at high pH, so although the Si content of soils can be as high as 45% (Currie and Perry, 2007), availability can be low. Deficiencies are especially important for plants such as rice that require this element in large amounts (Horuz et al., 2013). Evidence of this is the fact that plants have evolved influx, efflux and channel-type transporters to actively uptake Si (Ma and Yamaji, 2006, 2015; Trembath-Reichert et al., 2015; Vivancos et al., 2016) as well as that yield increases when plant-available Si is added to the growing medium (Tavakkoli et al., 2011). The beneficial roles of Si to various plants have been the subject of extensive research and include alleviation of abiotic stresses such as drought (Marques et al., 2016), metal toxicity, and micronutrient deficiency (Hernandez-Apaolaza, 2014); and biotic stress from pathogens (Winslow, 1992; Vivancos et al., 2015) and herbivores (Reynolds et al., 2009, 2016; Ye et al., 2013). To date, there is increasing evidence of Si priming plants for defense against herbivore attack (Reynolds et al., 2009; Hartley and DeGabriel, 2016). But, a conspicuous gap in knowledge about the effects of Si on plant biology is for the volatiles that are produced by plants and that are often key semiochemicals that drive ecological interactions including induced, indirect plant defense against herbivores enemies (Pare and Tumlinson, 1999; Dicke and Loon, 2000; Degenhardt et al., 2003).

The present results support our hypothesis and show, for the first time in any plant species, that plants grown with available Si have a different HIPV profile compared to plants grown under Si deficiency. Earlier work by Kvedaras et al. (2010) showed that +Si plants were more attractive than -Si plants to a generalist predator in olfactometer tests and to wild predators (of unknown identity) in a field experiment. That study did not, however, include work on plant volatiles so the mechanism underpinning the apparent effects on predators remained unclear. In the present study, several of the volatile compounds that were influenced by herbivore infested rice plants were shown to be HIPVs, since they were not produced by uninfested plants, irrespective of the Si status of the plant. These were β-linalool, α-bergamotene, and β-sesquiohellandrene, each of which have been identified as HIPVs in other studies (Hare, 2011; De Backer et al., 2016; Pinto-Zevallos et al., 2016). Whilst it is possible to record antennal responses for parasitoids to individual HIPVs (Takemoto and Takabayashi, 2015), it is known that blends of HIPVs and the relative quantity of different compounds is more important than any single compound (Van Wijk et al., 2010, 2011). Accordingly, in addition to the three compounds that were produced only by infested plants, several others were produced in greater quantities by infested rather than healthy plants: hexanal 2-ethyl, 1-hexanol 2-ethyl, methyl salicylate, γ-elemene and β-caryophyllene. Most crucially, the differences in blends between plants with a different Si regime (within herbivore treatment) involved a significant change in the composition of volatiles produced, with lower production of two of the HIPVs (α-bergamotene and β-sesquiohellandrene) and of hexanal 2-ethyl and cedrol. Hexanal 2-ethyl (synonym, 2-ethyl hexanol) has been reported as a volatile compound released by Rutaceae spp. foliage (Robbins et al., 2012) and is produced by insect-damaged roots of carrot (Daucus carota) (Weissteiner and Schütz, 2006). Cedrol has been reported as a foliar and fruit volatile produced by Ficus carica L. (Soltana et al., 2017) and, though it has not been reported to be a HIPV, exhibits bioactivity against arthropod pests (Eller et al., 2014).

The foregoing differences in volatile blends between experimental treatments were demonstrated to have ecological relevance by the behavior of two parasitoid wasp species. Both T. flavo-orbitalis and M. mediator responded more strongly to the volatile blend from +Herbivore plants than -Herbivore plants within each of the Si regimes, demonstrating that the experimental conditions were suitable for these parasitoids to exhibit biologically appropriate behavior consistent with HIPVs guiding them to infested plants. Crucially, both parasitoids also exhibited a significant preference to the odor blend from +Si over -Si when both of these treatments were herbivore-infested.

The nature of the change in volatile blend brought about by Si pre-treatment (Figures 1, 2) was not a clear cut switching-on, or -off, for the release of a given compound. Rather, the results are consistent with earlier studies showing that it is the ratio of compounds in blends that is crucial for attraction to natural enemies (Pare and Tumlinson, 1999; Dicke and Loon, 2000; Degenhardt et al., 2003). Specifically, our results are consistent with the heuristic that both parasitoids were attracted to a blend (Figures 3, 4) in which the major HIPVs, especially linalool, were present but in which more minor volatiles (hexanal-2-ethyl and cedrol) were at background levels, equivalent to those emitted by herbivore-free plants.

The present study establishes a stronger evidence base for exploring scope to promote biological control of pest herbivores by ensuring that crops have an optimal supply of Si so that they are able to mount a strong induced, indirect defense based on HIPV production. Fully exploiting the apparent priming effects of Si on plant defense demands a more comprehensive understanding of the underlying metabolic pathways. Work by Ye et al. (2013) demonstrated the importance of the jasmonate signaling pathway for positive effects of Si on rice defenses. The present study demonstrates that Si can enhance the attractiveness of the HIPV blends produced by WT rice when attacked by a herbivore but that this effect of Si does not occur among RNAi plants with silenced jasmonate perception. The emission of HIPVs is known to be influenced by the jasmonate pathway, particularly in the case of chewing herbivores such as those used in the present study (Dicke and Baldwin, 2010). Accordingly, the present results support the finding of Ye et al. (2013) that Si promotes plant defenses via the jasmonate pathway, but extends the range of effects to include HIPVs production. Further studies are required to determine whether Si also interacts with the salicylic acid and ethylene defense pathways of plants (Ruther and Kleier, 2005; Zarate et al., 2007; Koornneef et al., 2008; Dicke et al., 2009). Each of these, as well as the jasmonate pathway, can lead to the induction of HIPVs including by feeding of sucking pests such as aphids (Ozawa et al., 2000; van Poecke and Dicke, 2002). It will, therefore, be important to determine the extent of interactions between Si and varying plant defense mechanisms in order to fully exploit the potential of this element in novel plant protection approaches.

JL and GG designed the experiments. JL, JZ, LH, JW, and YS conducted the experiment. JL, PZ, and GG analyzed and interpreted the data. JL, OR, MY, and GG drafted and revised the paper. All authors read and approved the final manuscript.

GG and OR hold research funding from Australian Steel Mill Services (a potential source of silicacious material) and this supports a project being conducted independently of the present work. Australian Steel Mill Services had no role in the present study, interpretation of results or decision to publish. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.