Role of Large Cabbage White butterfly male-derived compounds in elicitation of direct and indirect egg-killing defenses in the black mustard

Abstract

To successfully exert defenses against herbivores and pathogens plants need to recognize reliable cues produced by their attackers. Up to now, few elicitors associated with herbivorous insects have been identified. We have previously shown that accessory reproductive gland secretions associated with eggs of Cabbage White butterflies (Pieris spp.) induce chemical changes in Brussels sprouts plants recruiting egg-killing parasitoids. Only secretions of mated female butterflies contain minute amounts of male-derived anti-aphrodisiac compounds that elicit this indirect plant defense. Here, we used the black mustard (Brassica nigra) to investigate how eggs of the Large Cabbage White butterfly (Pieris brassicae) induce, either an egg-killing direct [i.e., hypersensitive response (HR)-like necrosis] or indirect defense (i.e., oviposition-induced plant volatiles attracting Trichogramma egg parasitoids). Plants induced by P. brassicae egg-associated secretions expressed both traits and previous mating enhanced elicitation. Treatment with the anti-aphrodisiac compound of P. brassicae, benzyl cyanide (BC), induced stronger HR when compared to controls. Expression of the salicylic (SA) pathway- and HR-marker PATHOGENESIS-RELATED GENE1 was induced only in plants showing an HR-like necrosis. Trichogramma wasps were attracted to volatiles induced by secretion of mated P. brassicae females but application of BC did not elicit the parasitoid-attracting volatiles. We conclude that egg-associated secretions of Pieris butterflies contain specific elicitors of the different plant defense traits against eggs in Brassica plants. While in Brussels sprouts plants anti-aphrodisiac compounds in Pieris egg-associated secretions were clearly shown to elicit indirect defense, the wild relative B. nigra, recognizes different herbivore cues that mediate the defensive responses. These results add another level of specificity to the mechanisms by which plants recognize their attackers.

Keywords: Brassicaceae; PR-1; Pierisaccessory reproductive glands; egg parasitoid; hypersensitive response; induced plant defenses; oviposition-induced plant volatiles.

FIGURE 1

Hypersensitive response (HR)-like necrosis of Brassica nigra plants induced by Pieris eggs and female reproductive tract. (A) Pieris brassicae egg clutch on a leaf showing no necrosis (HR-; credits: E. Griese), (B) P. brassicae egg clutch inducing strong necrosis (HR+; credits: E. Griese), (C) reproductive tract of a virgin P. brassicae female, and (D) reproductive tract of a mated P. brassicae female. Ovi, oviduct; ARG, accessory reproductive gland; Ovar, ovariole; Bc, bursa copulatrix; Sp, spermatheca.

FIGURE 2

Different severities of HR-like necrosis after application of ARG homogenate or anti-aphrodisiac compounds. Surface area of necrotic tissue and intensity of necrosis was assessed using a relative scale: (A) weak (HR+): no or little necrotic tissue on upper leaf side, (B) medium (HR++): larger parts of treated area with brownish necrotic tissue on upper leaf side, and (C) strong necrosis (HR+++): almost whole treated area necrotic on both sides.

FIGURE 3

Percentage of B. nigra plants treated with ARG secretion from P. brassicae females or anti-aphrodisiac compound expressing HR-like necrosis. (A) treatments with ARG secretions from P. brassicae females (n = 8 plants/treatment, all treatments on one plant), (B) treatments with anti-aphrodisiac compound from P. brassicae females, benzyl cyanide (BC) at three different concentrations 0.5–1.5 ng/100 μl ethanol (EtOH) or EtOH only (n = 9 plants/ treatment. HR-: no necrosis observed, HR+ to +++: necrosis, which was assessed using a relative scale as shown in Figures 1A and 2A–C.

FIGURE 4

Proportion (mean ± SE) of female Trichogramma evanescens wasps choosing volatiles of differently treated B. nigra plants when tested against untreated or solvent-treated control plants in a Y-tube olfactometer. Each treatment, (1) P. brassicae eggs 24 h after egg deposition, (2) ARG (of virgin P. brassicae females, (3) ARG of mated P. brassicae females 2 h after mating, (4) ARG of mated P. brassicae females 48 h after mating, (5–6) BC in two concentrations (1 and 10 ng/100 μl ethanol) was replicated with four to seven plant pairs and 20 wasps per plant pair (n = 80–140 wasps per treatment). ^∗p < 0.05, ns, not significant (p > 0.05); one-sample t-test. The dashed line indicates 0.5 = no preference. For statistical analysis, data were first arcsine transformed and then back-transformed for presentation.

FIGURE 5

Gene expression of (A)PR1 and (B)LOX2 genes in B. nigra plants exhibiting a HR-like necrosis (HR+) or not (HR-) infested by P. brassicae eggs (4 and 24 h after oviposition) or in plants treated with BC (1 ng). Bars represent mean expression levels normalized (relative to the reference gene GAPDH) as 2^-ΔΔCt with standard error bars (n = 3–4 biological replicates). Asterisk indicates significant difference (ANOVA P < 0.05; Dunnett post hoc test).