Plant-to-plant defence induction in cotton is mediated by delayed release of volatiles upon herbivory

Introduction

Plants produce a wide range of secondary metabolites that enable them to defend themselves against antagonists, such as herbivores and pathogens. These compounds can function as toxins that directly reduce herbivore survival or reproductive success (e.g. quinones, alkaloids, anthocyanins, and terpenoids), or, as in the case of volatile organic compounds (VOCs), serve as indirect defence signals (Pichersky & Lewinsohn, 2011; Mithöfer & Boland, 2012; Kessler & Kalske, 2018; Pichersky & Raguso, 2018). These VOCs can be stored and emitted constitutively (Gershenzon, 1994, 2000; Clancy et al., 2016), or induced and synthesised de novo following herbivory (Paré & Tumlinson, 1997). Importantly, these herbivore-induced changes include shifts in the composition and relative ratios of compounds within a volatile blend released by a plant (Turlings & Erb, 2018), which contain ecologically relevant cues of risk of attack. Herbivore-induced plant volatiles (HIPVs) may repel herbivores and attract their enemies; they can also serve as signals between different parts of an individual plant (within-plant signalling) to activate preventive systemic defences (Heil & Silva Bueno, 2007; Meents & Mithöfer, 2020), and may be used by neighbouring plants to prepare for future attacks (Morrell & Kessler, 2017; Schuman, 2023).

Initial discoveries demonstrating volatile-mediated interactions between plants in response to herbivore attack (Baldwin & Schultz, 1983; Farmer & Ryan, 1990; Bruin et al., 1992) were met with some scepticism but are now widely accepted as being both common and ecologically relevant (Heil & Karban, 2010; Ninkovic et al., 2019; Kessler et al., 2023). Numerous studies have reported on the role of signalling between plants mediated by HIPVs (Baldwin & Schultz, 1983; Dolch & Tscharntke, 2000; Karban et al., 2003; Heil & Silva Bueno, 2007), with field studies revealing specificity in the volatile cues involved (Karban et al., 2004; Moreira et al., 2016; Kalske et al., 2019). Herbivore-induced plant volatiles reported to act as potential signalling cues include jasmonates (Farmer & Ryan, 1990), green leaf volatiles (Engelberth & Engelberth, 2019), and aromatic compounds (Erb et al., 2015). These HIPVs from a damaged plant can reach an undamaged neighbouring plant, which can then enter a so-called ‘primed’ state (Ton et al., 2007; Mauch-Mani et al., 2017). Although defences in primed plants are sometimes not expressed or only at low levels, these plants exhibit greatly enhanced induction of defence compounds after being attacked (Conrath et al., 2006; Martinez-Medina et al., 2016). In addition, undamaged plants exposed to HIPVs may also immediately upregulate their defences without the need of a direct contact with the attacker; these induced defences will be present before herbivore attack (Karban et al., 2003; Waterman et al., 2024).

Gossypium hirsutum L. (Malvaceae), known as upland cotton, is cultivated world-wide primarily for the production of textile fibres. It is heavily attacked by pests and requires high amounts of pesticide application, accounting for a substantial portion of world-wide pesticide use (Coupe & Capel, 2016; Huang et al., 2021). While the use of these chemicals has resulted in increased crop yields, it has also had extremely negative impacts on the environment (Van Der Werf, 1996; Aktar et al., 2009), particularly in soil and water pollution. More benign pest control strategies are sought, including the enhancement of the plants' natural defences (Llandres et al., 2018). Gossypium hirsutum is known to respond to insect herbivory by altering its volatile emission profile both quantitatively and qualitatively, as well as increasing its content of nonvolatile defensive terpenoid aldehydes, such as gossypol and heliocides (Loughrin et al., 1994; McCall et al., 1994; Röse et al., 1996; McAuslane et al., 1997; Arce et al., 2021). Interestingly, the volatile blends emitted by damaged plants also change over time from herbivory onset, with stored volatile compounds being released immediately after damage (such as the terpenes α-pinene and caryophyllene), and de novo synthesised compounds being emitted in high quantities after at least 24 h of attack onset (Loughrin et al., 1994; Paré & Tumlinson, 1997). The latter compounds include terpenes such as β-ocimene and β-farnesene and the aromatic indole, emitted in very low amounts or not at all from undamaged or freshly damaged plants. Thus, two pools of volatiles are released after herbivory, hereafter named fresh damage volatiles and old damage volatiles.

It is known that cotton plants attacked by herbivorous mites are more resistant to new colonisation by mites than undamaged plants in both laboratory and field conditions (Karban, 1985, 1986). Similarly, cotton is more resistant to Spodoptera caterpillars when damaged by mites (Karban, 1988), and it has also been found that Spodoptera caterpillars are deterred from feeding when plants have been previously damaged by caterpillars (Alborn et al., 1996). These findings indicate that induction by herbivores is a broad response that protects cotton against future attacks, although its importance for plant fitness has been difficult to test given that cotton is perennial (Karban, 1993). Plant–plant signalling by cotton volatiles was first studied by Bruin et al. (1992), who found that cotton seedlings infested with herbivorous mites produced VOCs that caused a decrease in oviposition by herbivorous mites on neighbouring plants, which were also more attractive to predatory mites. More recently, Zakir et al. (2013) showed, both in laboratory and field, a significant reduction in oviposition by Spodoptera littoralis (Lepidoptera: Noctuidae) moths on undamaged cotton plants previously exposed to damaged neighbouring cotton plants. In addition, using wild cotton plants, Briones-May et al. (2023) found that exposure to HIPVs from neighbouring plants primes the induction of extrafloral nectar of receiver plants under glasshouse conditions. Field studies performed in Mali afford additional evidence for VOC-mediated signalling, by showing that topping (i.e. manual removal of the apical part of flowering cotton plants) resulted in reduced infestation by the cotton bollworm (Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)) on the topped plant, as well as on intact neighbouring plants (Llandres et al., 2018). Similar signalling effects have been recently found for infestation by Aphis gossypii Glover (Llandres et al., 2023). Despite progress made thus far, there is no precise information about how and which direct defences are triggered by HIPV exposure in cotton (Quijano-Medina et al., 2024). The signalling effects of different pools of volatile compounds that are released at distinct time points after damage onset have so far been ignored, although it is likely that they do not convey the same information about herbivory risk. Cotton is ideal for addressing this question and to test the functional role and adaptive significance of volatiles in plant signalling. As de novo synthesised volatiles released after herbivory can be expected to carry the most reliable information, we hypothesised that specifically this pool of volatiles would trigger responses in neighbouring plants.

In this study, we investigated the effects of exposure to HIPVs from emitter plants on undamaged receiver plants by measuring chemical profiles (including volatiles and direct defence metabolites, namely gossypol and heliocides, as well as phytohormones), associated gene expression (to get at subtler responses associated with priming), and caterpillar feeding preference as proxy of downstream consequences for plant resistance. To do this, we exposed undamaged G. hirsutum plants to airborne VOCs emitted by plants infested with Spodoptera caterpillars. We also assessed the impact of the timing of herbivory by exposing receiver plants to HIPVs from plants at two contrasting time points since herbivory onset, namely 0–24 h since damage onset vs 24–48 h since damage onset. This allowed us to determine whether the chronological changes in volatile emissions are relevant for plant signalling between cotton plants. By taking into account the temporal dynamics of volatile emissions, we were able to separate the effects of the two different pools of volatiles released by damaged plants. This separation helped to elucidate how each pool activates defensive cascades in neighbouring plants that prepares them for incoming attacks. The results presented here indicate that the volatiles released after 24 h after damage, which most reliably indicate an attack by caterpillars, are the most relevant for plant-to-plant information conveyance.