1 INTRODUCTION
Plant hosts have defenses to counter attacks from antagonists such as herbivores and pathogens (Pandey, Ramegowda, & Senthil-Kumar, 2015; Miller, Costa Alves, & Van Sluys, 2017). For example, the jasmonic acid pathway often regulates plant defenses against herbivores, while the salicylic acid pathway often regulates defenses against pathogens (Koornneef & Pieterse, 2008; Thaler, Humphrey, & Whiteman, 2012). While many studies have assessed plant responses to particular antagonists, plants are often challenged by many stressors concurrently, and plant defenses can depend on the order in which antagonists arrive on plants (Thaler et al., 2012; Nejat & Mantri, 2017). For example, herbivores often limit plant defenses against pathogens when they arrive first on plants, but herbivores often have few impacts on plant defenses against pathogens when they arrive after pathogens on plants (Okada, Abe, & Arimura, 2015; Lin et al. , 2019). In other contexts, certain organisms ‘prime’ pathways, promoting defense against subsequent organisms activating the same pathway, such that attack order may not matter (Mauch-Mani, Baccelli, Luna, & Flors, 2017; Ramírez-Carrasco, Martínez-Aguilar, & Alvarez-Venegas, 2017).
Biotic stressors may also alter the nutritional quality of plants by regulating amino acid metabolism (Casteel et al., 2014; Zhou, Lou, Tzin, & Jander, 2015), which can alter the feeding behavior and nutrient uptake by subsequent herbivores (Behmer, 2009; Zhu, Poelman, & Dicke, 2014). For example, the composition of free amino acids constitutively changes in leaves of soybean plants in response to soybean aphids (Chiozza, O’Neal, & MacIntosh, 2010). Tomato yellow leaf curl virus also alters the nutritional quality of tomato plants by affecting free amino acid levels in phloem, which alters the amino acid composition of whitefly (Bemisia tabaci ) honeydew (Guo et al., 2019). However, few studies have explored how the diversity and identity of attacking organisms, and variation in the order of attack, affect nutritional traits of plants.
While there has been considerable research on the jasmonic and salicylic acid pathways, to understand complexities of plant defense it is necessary to assess how biotic antagonists mediate other signaling pathways (e.g., Lacerda, Vasconcelos, Pelegrini, & Grossi de Sa, 2014; Suzuki, 2016). Moreover, it is key to assess how changes in plant defense correlate with plant nutrients. For example, plants in low-nitrogen soil often adopt carbon-based defenses, while plants grown with fertilizer often accumulate more nitrogenous toxins (Cipollini, Walters, & Voelckel, 2017). Nitrogen in plants may also affect both pathogens and herbivores through synthesis of defensive metabolite, nitric oxide, and by nitrogen mobilization (War et al., 2012; Mur, Simpson, Kumari, Gupta, & Gupta, 2017). However, few studies have correlated effects of multiple biotic stressors on both plant chemical signaling and nutritional properties (Petek et al., 2014; Su et al., 2016).
We addressed these knowledge gaps by assessing the response ofPisum sativum plants to attack from a piercing-sucking vector herbivore, the pea aphid (Acrythosiphon pisum ), a chewing non-vector herbivore, the pea leaf weevil (Sitona lineatus ), and an aphid-borne pathogen, Pea-enation mosaic virus (PEMV). These organisms co-occur in ecosystems of eastern Washington and northern Idaho, USA, and interactions between them can affect plant traits and signaling pathways affecting insects and pathogens (Chisholm, Eigenbrode, Clark, Basu, & Crowder, 2019; Bera, Blundell, Liang, Crowder, & Casteel, 2020). However, the order in which herbivores and pathogens arrive on hosts, which varies across sites (Chisholm et al., 2019), may impact plant traits and defenses. To address this, we varied the diversity, identity, and order of attack among this community of biotic antagonists and assessed resulting changes in gene expression and phytohormones related to plant defense and nutrition. Our study revealed how plant responses to diverse stressors can mediate complex species interactions within a pathosystem.