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Research Article *Corresponding author Wellington G. Campos, Departament of Bio systems Engineering, Federal University of São João del Rei, Stress affects Brazil, E-mail: Submitted: 30 May 2017 Accepted: 06 June 2017 Constitutive but not - Published: 08 June 2017 ISSN: 2333-6668 Induced Glucosinolates in Copyright © 2017 Campos et al. Collard OPEN ACCESS Keywords 1 1 Janete Oliveira S. Valim , Natália C. Teixeira , Maria Goreti A. • Brassica oleracea Oliveira2,3, and Wellington G. Campos1,3* • Brevicorynebrassicae • Plant defense 1 Departament of Bio systems Engineering, Federal University of São João del Rei, • Plutellaxylostella Brazil • Water stress 2Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Brazil 3National Institute of Science and Technology in Pest-Plant Interactions, Brazil

Abstract The presence of glucosinolates (GLS) in Brassicaceaeis thought to be the first line of plant defense against a variety of insects and pathogens. Influence of drought stress on constitutive GLS has been documented; however, its effect on herbivory-induced GLS is poorly known, especially with respect to different feeding guilds of insects. Collard (Brassica oleracea L. cv. acephala) plants were grown in well-watered, slightly water-stressed and severely water-stressed conditions. Then, the plants were subjected to infestations by two host-specific , the leaf-chewingPlutellaxylostella L. (Lepidoptera: Plutellidae) or the phloem-feeding Brevicorynebrassicae L. (Hemiptera: Aphididae). Concentration of total leaf GLS decreased in severely drought-stressed plants; however, in no water regime collards had their total GLS increased due to damage by both specialist insects. Thus, we did not found evidence for total GLS induction by insect damage, irrespective of insect feeding or level of plant water-stress. Naturally occurring or deliberate submission of collard crops to water limitation may not affect herbivore-induced GLS, but constitutive resistance against insect attack may be reduced.

INTRODUCTION The presence of glucosinolates (GLS) in Brassicaceae and

Induced responses are changes in plant chemistry and defense against a variety of invading organisms [11]. Even though structure following damage or stress. These traits may negatively related plant families [10] is thought to serve as a first line of abiotic and biotic factors [12]. Brassicaceous plants usually face when herbivores are present (induced defense) [1]. Thus, in damageGLS are by constitutive multiple insect defenses, herbivore their species, levels which are influenced in most cases by affect herbivores (induced resistance) and benefit the plant addition to defense mechanisms that function independently of belong to different feeding guilds and different degrees of feeding damage (constitutive defenses), plants can be protected against specialization [13]. Thus, such plants may adjust their defense herbivores by defenses previously induced by biotic and abiotic strategies in response to herbivore feeding mode. In fact, studies stresses. Many of such stress conditions operate simultaneously, have shown that induced responses and GLS accumulation by in a way that the response of plants to combinations of two or more conditions cannot be directly extrapolated from the response to each of the different stresses applied individually [2]. some plants depend on the herbivore species and on the insect- Among the abiotic stresses, drought affects plants at various herbivore,feeding guild but [14-16]. induced It responseshas been suggested depend mainly that plant on insect responses taxa levels of their organization [3]. However, knowledge remains (i.e.,are notaphid influenced vs. lepidopteran by the species), degree oflikely specialization due to their ofdifferent insect feeding modes [14]. in plant resistance or plant defense compounds [4,5]. On the L. scant on the specific expression of drought-mediated changes Plutellaxylostella other hand, biotic stress caused by herbivores can induce, (Lepidoptera: Plutellidae) and the phloem feeding cabbage aphid besides slower changes in morphological traits, rapid increases BrevicorynebrassicaeThe leaf-chewing diamond L. (Hemiptera: back moth Aphididae) (DBM) are among the major insect pests that attack brassicaceous plants worldwide. secondary metabolites [6,7]. Induction of secondary metabolites in concentrations of anti-nutritional enzymes and proteins, and by biotic stresses is well documented, but less information is individually led to a decreased level of total GLS in collard available on how drought and other abiotic conditions affect BrassicaoleraceaBoth of them are L.highly cv. acephala host-specific(Brassicaoleracea [13]. Drought L) stress [5], while applied GLS induced plant responses to herbivory [8,9].

Cite this article: Janete Oliveira SV, Teixeira NC, Maria Goreti AO, Campos WG (2017) Drought Stress affects Constitutive but not Herbivore-Induced Gluco- sinolates in Collard. Int J Plant Biol Res 5(3): 1069. Campos et al. (2017) Email:

Central Bringing Excellence in Open Access induction by herbivore damage has been documented in many other plants [17]. However, few studies have investigated the interaction of drought stress with herbivory in inducing changes analysistreatment of (nGLS = on4 x the 8 x same 3). The day groups that the were treatment as follows: groups (1) received Control day-zero: undamaged plants, whose leaves were harvested for of insects. We addressed this issue using collards and herbivores, were harvested for analysis of GLS in the seventh day after the of GLS [18-20], especially with respect to differentB. feedingbrassicae guilds. We insects; (2) Control day-seven: undamaged plants, whose leaves assessed whether drought stress decreases both, constitutive the leaf-chewing DBM and the phloem-feeding treatment groups received insects; (3) DBM damaged: plants damaged by DBM, whose leaves were harvested for analysis of alsoand tested herbivore-induced whether caterpillars GLS. induce Considering greater that production leaf-chewing of GLS B.GLS brassicae in the seventh, whose day leaves after werethey received harvested 20 for third-instar analysis oflarvae GLS thanherbivores aphids. perceptibly cause leaf-damage of a greater extent, we inper the plant; seventh and (4) day Cabbage after they aphid received damaged: 20 plants nymphs damaged per plant. by MATERIAL AND METHODS Each plant, including control groups, was caged individually in a bag of transparent organza. We established seven days of B. oleracea cv. acephala, hybrid Georgia with glossy plant exposure to insects because earlier studies have indicated and dark green leaves, were grown in a greenhouse (9 m x 25 systemic increase in GLS levels as quickly as four days after m) Collard, covered with transparent water proof plastic and laterally days period for GLS induction [16,18]. The cabbage aphid for the experimentherbivore infestation was collected [6]. fromOther commercial authors have B. oleraceaalso used crops, a seven- and coated with anti-aphid netting. The collards were grown in 10 L soil, without organic matter and humus addition. Firstly, soil . fertilityplastic pots, was which analyzed were and filled improvements with standardized in nutrients homogeneous were carried out at the beginning and during plant growth. We used it was reared in a greenhouse on potted-collards The DBM was a standard fertilization recommended for growing potted plants. ofobtained cabbage from (B. oleraceaea breedingcv. stockcapitata that) wasbefore kept the for trial ≈14 period. generations Macronutrients (N, P, K and Mg) were added via NPK fertilizer in a climate-controlled room, where they fed on detached leaves 4:14:8, urea, potassium chloride and magnesium sulfate. We harvested all the leaves (assuming systemic effect) from the eight plants of each water treatment per group of damage (n = sulfate, manganese chloride and ammonium molybdate. Physical 8 x 3 x 4), to determine systemic changes in GLS. The leaves were soilMicronutrients analysis indicated (B, Cu, Mnsandy and texture. Mo) by means of boric acid, copper frozen and ground to a powder in liquid nitrogen and maintained

followed Jezeket al. [22], and Gallaher et al. [23], with protocol were grown in a greenhouse in 10 L plastic pots, which were at -20ºC [21] for further analysis. The determination of total GLS Collard, hybrid Georgia with glossy and dark green leaves, details in Valim et al. [5], The analytical method is based on alkalin matter and humus addition. Firstly, soil fertility was analyzed and improvementsfilled with standardized in nutrients homogeneouswere carried out soil, at the without beginning organic and ferricyanideet al., hydrolysis can be of assessed GLS to spectrophotometrically yield 1-thioglucose. Ferricyanide and used to during plant growth. We used a standard fertilization for growing determineoxidizes 1-thioglucosetotal GLS content and [22]. the loss of the chromogenic potted plants [5]. Physical soil analysis indicated sandy texture. Statistical analyzes were performed using the Graph The amount of water delivery required to achieve different water conditions (treatments) in the soil for 10 L pots followed homogeneityPad Prism 5.0 of software, variances Prism was assessed Graph Inc., by the San Bartlett’s Diego. Data test. normality was verified using the Kolmogorov-Smirnov test, and Valim et al. [5], (I) Well-watered: 2000 ml of water every 48 h, wasthe soil kept was moderately kept well-watered watered and and leaf leaf wilting wilting usually never occurred;occurred a function of water regime and insect damage, followed by the (II) Slightly water-stressed: 700 ml of water every 48 h, the soil The GLS concentrationP < 0.05). was subjected to a two-way ANOVA as ml of water every 48 h, the minimum amount of water to prevent RESULTSTukey’s HSD AND test ( DISCUSSION plantin the deathhottest by hours dehydration. of the day; The (III) pots Severely were placed water-stressed: inside plastic 170 GLS concentration in collards differed due to irrigation after watering. regime (F2,95= 42.8, p< 0.0001), but did not differ due to insect dishes (38 cm-diam. x 4.5 cm-deep) to retain the water drained

B. brassicae (F2,95= 0.4, p= 0.74). The biotic and L plastic pots, where they were maintained under same watering damage, whether caused by the leaf-chewing DBM or the 6, conditionThirty-day for 15seedlings days. The of collard pots were were placedtransplanted on a greenhouse into the 10 phloem-feeding 95= 1.4, p= bench 1 m high. After this period, the seedlings were subjected abiotic independent variables did not influence on each other (F to the three watering treatments that were described above. The 0.22). Thus, severely-stressed collards had less total water treatments were arranged on the greenhouse benches GLStotal than GLS well-wateredvariation induced and slightlyby both water-stressed specialist insects plants (Figure (Figure 2). in a uniform distribution. Each row consisted of three pots 1); however, regardless of soil water regime, collards showed no (plants), one for each treatment, which were changed in position Increasing water scarcity decreases photosynthetic and along the rows. Thus, treatments were distributed in a way that effects of sun and temperatures were evenly distributed over all collards maximize mechanisms to retain and save water, treatments, so each plant was an experimental unit. The plants in probablydevelopmental to increase performance survival of incollards. such condition Severely water-stressed [5]. Therefore, each treatment were allowed to grow and to adapt for 45 days. in a condition of limited and , there After 45 days of water treatments, we distributed 96 plants growth, reproduction, energy storage and chemical defenses in four groups, each group with eight plants from each water againstis a possible herbivores trade-off and p athogens. in An assumption allocation betweeninherent in plant the

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Brassicaceae [4,5,27-29] support both hypotheses. According GDBH and SCH, an increased level of GLS induced collardby herbivores did not should show be any less additional likely to investmentoccur in severely in GLS drought- against bothstressed specialist plants. insects. However, Additionally, even in sufficientour results water do not condition, support the hypothesis that the type of feeding guild affects induction of

aphids,total GLS. caterpillars Phloem-feeding cause ruptures insects are in plant thought tissues to cause and remove a weak induction of GLS when compared to leaf-chewing [14,30]. Unlike

productionleaf fragments of GLS. in aIn predictable any case, an way; increase however, in the despiteGLS level this, would the leaf damage caused by DBM in collards did notB. brassicae stimulate attacks,further because both are highly adapted to overcome these compounds be useless to prevent further DBM and

thatWe are alternativelytoxic to non-specialized propose that insects GLS [12,13,30,31]. inducibility in collards depends on the level of feeding specialization by herbivores, Figure 1 Effect of irrigation regime on total glucosinolate rather than feeding guilds. Firstly, collards may have evolved concentration in leaves of Brassica oleraceacv. acephala. Bar and in line show mean ± B. brassicae P relation to generalist herbivores. It has been hypothesized SD, and different letters between columns indicate thatsome generalistability to discriminate herbivores damages may trigger by DBM higher and induction of significant differences ( < 0.05) by the Tukey’s HSD test. plant defenses compared to specialist ones [32,33]. Secondly, specialists have evolved strategies to minimize the activation of

B. brassicae may have evolved some ability to hinder GLSdefense-related induction. A geneslarger [34];number therefore, of taxa should the highly be used host-specific in further testsDBM forand these hypotheses, including species with varying degrees of specialization in feeding on brassicaceous plants. There is other possibility to be considered beyond the differential relation of collards with their specialist and generalist herbivores. Although GLS induction by herbivory has been documented for a number of plant species, there are substantial differences in GLS inducibility among plant species or even among cultivars of B. oleracea [17]. Variations in responses to environmental stress between plant species and cultivars are common, and need to be considered when generalizing results for Brassicaceae [4]. Therefore, the domesticated collard may naturally have a low GLS inducibility, regardless of feeding guilds and degrees of feeding specialization. Finally, individual Figure 2 compounds of GLS in plants that were damaged by insects may differ among drought conditions (20). Thus, in addition to total of damageEffect by Plutellaxylostella of damage condition (C-0 = control day-zero, without Brevicorynebrassicaedamage; C-7 = control) onseven-days, total glucosinolate without damage; concentration Px = seven in leaves days of Brassica oleraceac v. acephala; andplants Bb = seventhat were days ofgrown damage under by GLS,CONCLUSION further studies should consider GLS compound profiles. three different irrigation regimes. There was no difference (p >0.05) between damage conditions in the same water regime by the Tukey’s This study shows that collard under severe and continuous

metabolism, with decreased constitutive GLS. On the other hand, HSD test. nowater-stress evidence was experienced found for GLS adjustments induction inby insect its secondary damages, decreased photosynthetic rate imposes limits on the production growth–differentiation balance hypothesis (GDBH) [24] is that a Thus, naturally occurring droughts or deliberate submission of defense compounds. Further than in defense, GLS may has a irrespective of insect feeding guildor level of plant water-stress. special role in minimizing drought stress, because they can be relocated and hydrolyzed to assist in stomatal control [25,26]. induced GLS, but constitutive resistance against insect attack mayof collard be reduced. crops to water limitation may not affect herbivore- drought stress increases hydrolysis and reduces concentration of ACKNOWLEDGMENTS GLSThus, in plant the stomatal tissues. Similar control to hypothesisour results, reports (SCH) predictson decreased that concentration of GLS in drought-stressed plants of the family The financial supportprovided by Conselho Nacional de Int J Plant Biol Res 5(3): 1069 (2017) 3/4 Campos et al. (2017) Email:

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