Arthropod-Plant Interactions (2017) 11:71–78 DOI 10.1007/s11829-016-9467-3

ORIGINAL PAPER

Behavioral responses of obsoletus to host-plant volatiles cues

1 1 2 1 Paola Riolo • Roxana L. Minuz • Ezio Peri • Nunzio Isidoro

Received: 6 May 2016 / Accepted: 7 October 2016 / Published online: 25 November 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract The polyphagous Hyalesthes obso- identified suggest the possibility of using them in moni- letus Signoret is considered to be the principal vector of toring and management of H. obsoletus. stolbur phytoplasma, which is associated with yellow dis- eases of grapevine. To explore the possibility of developing Keywords Phytoplasma vector Á Plant volatiles Á novel control strategies, the behavioral responses to six Semiochemical Á Y-tube olfactometer synthetic mixtures and nine single compounds, previously identified from the headspace of Vitex agnus-castus L. (chaste tree) and Urtica dioica L. (nettle), were investi- Introduction gated in Y-tube bioassays. Choice tests revealed differ- ences in the behavioral responses of males and females to Bois noir (BN) is a grapevine (Vitis vinifera L.) disease that the volatiles that they were exposed to. Males were is associated with ‘Candidatus Phytoplasma solani’ attracted to a mixture containing (E)-b-caryophyllene, 1,8- (16SrXII-A subgroup) (Quaglino et al. 2013). Infected cineole, (E,E)-a-farnesene, (E)-b-farnesene, and methyl grapevine symptoms include chlorosis and downward salicylate (mixture 2). The addition of methyl benzoate to rolling of leaves, stunted shoots, and shriveling of berries this five-compound mixture (mixture 3) did not attract (Belli et al. 2010). In recent years, rapid spreading of BN males but elicited positive responses in females. Further- has been seen frequently, and this can be a limiting factor more, females were attracted to a mixture containing (E)-b- in grapevine production (Maixner 2011; Zahavi et al. caryophyllene, (E,E)-a-farnesene, (Z)-3-hexenyl acetate, 2013). (Z)-3-hexen-1-ol, and benzothiazole (mixture 4), but here The epidemiology of vector-borne plant pathogens is addition of methyl salicylate (mixture 5) did not attract directly dependent on the vector species and its life females. Neither males nor females showed attractivity or strategy, which include its host-plant specificity, feeding repellency toward the singly tested compounds. This study preferences, dispersal capacity, and sex behavioral differ- enhances knowledge on the interaction of insect behav- ences (Hoy et al. 1999; Weintraub and Beanland 2006; iorally effective constituents in complex plant volatile Maixner 2010). mixtures. The attractive mixtures of plant volatiles The planthopper Signoret (Hemi- ptera: ) is considered to be the principal vector of the BN causal agent in both southern and central European Handling Editor: Jarmo Holopainen. vineyards (Maixner et al. 1994; Sforza and Boudon-Padieu 1998). Postembryonic development occurs on the roots of & Paola Riolo host plants, on which the young stages feed and overwinter [email protected] (Sforza et al. 1999). Adults live above ground and transmit 1 Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, the phytoplasma on healthy plants by feeding (Bressan Universita` Politecnica delle Marche, 60131 Ancona, Italy et al. 2007). 2 Dipartimento di Scienze Agrarie e Forestali, Universita` degli The occurrence and spread of BN are associated also Studi di Palermo, 90128 Palermo, Italy with the presence in the vineyard agroecosystem of wild 123 72 P. Riolo et al. vegetation that can host both the insect vector and the single compounds that can be used as cues by H. obsoletus. phytoplasma (Murolo et al. 2010; Landi et al. 2013, 2015; Such key mixtures or single compounds could be part of a Kosovac et al. 2015). Adults of H. obsoletus can feed on sustainable and environmentally friendly strategy to con- various plants, although only a few species are generally trol H. obsoletus adults in vineyard agroecosystems. selected to complete their whole life cycle (primary hosts). Its host-plant range is still under investigation, as it includes very different plant families and it changes Materials and methods depending on geographic area (Hoch and Remane 1985; Riolo et al. 2012, and references therein). Grapevines represent only an occasional host plant for adults, whereby they can transmit the phytoplasma during their feeding Adults of H. obsoletus were collected from nettle plants in probing (Maixner 2010). the Ancona area (43°320 N; 13°230 E) of the Marche region The life history of H. obsoletus, its affiliation (prefer- in central-eastern Italy. The insects were transferred to the ence of adult for the plant species on which it developed) to laboratory, separated according to sex, and caged with wild host plants, and its erratic feeding behavior on fresh shoots of nettle. Twenty-four hours before the grapevine impede the effective control of this planthopper beginning of the bioassays, adult H. obsoletus were trans- (Maixner 2010). In H. obsoletus both sexes produce ferred to clean cages and fed with aqueous sucrose solution vibratory signals: the females have a major role in the (5% sucrose, 0.5 % sorbitol), to avoid any influence on the stimulation of the male search process, whereas the males compounds used in the subsequent experiments. The adults are more vibrationally active in the courtship and precopula were kept at 26 ± 1 °C and 60 ± 10 % relative humidity, stages. There is no evidence of the presence of sex pher- under a natural photoperiod (L16:D8). omones in this species (Mazzoni et al. 2010). Generally, synthetic herbicides and insecticides have Volatiles sources and experiments been proposed for the control of H. obsoletus on vineyard ground cover and the surrounding vegetation (Maixner Experiment 1 2010; Mori et al. 2015). Among possible alternatives to synthetic pesticides, the use of plant volatiles to manipulate This experiment was designed to determine the olfactory insect behavior has become a suitable tool for management responses of H. obsoletus males and females to relatively of a number of insect pests, including vectors of plant complex synthetic mixtures that contained 4–6 chaste tree- pathogens (Szendrei and Rodriguez-Saona 2010; Eben and derived compounds (Table 1, Mixtures 1–3) and 5–7 net- Gross 2013). Volatiles provide important cues for insects in tle-derived compounds (Table 1, Mixtures 4–6), versus a their search for host plants on which to feed or lay eggs and blank. The first mixtures evaluated included the major in the avoidance of plants that might be unsuitable as hosts antennally active volatiles (in terms of relative abundance) (Visser 1986; Szendrei and Rodriguez-Saona 2010). Also, of the chaste tree [i.e., 1,8-cineole, (E,E)-a-farnesene, (E)- volatile compounds in mixtures can act antagonistically, b-farnesene, (E)-b-caryophyllene] and nettle [i.e., (Z)-3- additively, or synergistically, depending on whether the hexenyl acetate, benzothiazole, (Z)-3-hexen-1-ol, (E,E)-a- insect response to a mixture of such volatiles is less than, farnesene, (E)-b-caryophyllene]. In the subsequent mix- equal to, or more intense than the sum of responses to the tures tested were added some antennally active compounds individual components, respectively (Bruce and Pickett that are present in small amounts in shoot blends from the 2011). natural chaste tree (i.e., methyl salicylate, methyl benzoate) In our previous studies, where the attractiveness to and nettle [i.e., methyl salicylate, (Z)-jasmone] (Riolo et al. H. obsoletus females and males of volatiles from nettle, 2012; Table 1). The aim of the mixtures was to mimic the field bindweed, hedge bindweed, chaste tree, and grapevine natural ratios of antennally active compounds emitted by was tested, we showed significant attraction of males to the shoots of chaste tree and nettle (Riolo et al. 2012; chaste tree volatiles, and of females to nettle volatiles. Table 1). For every mixture, a hexane ([99 % pure; Furthermore, volatiles from these two host plants were Sigma-Aldrich, Italy) stock solution was prepared at the collected and analyzed, and compounds that elicited elec- concentration of 10 lg/ll. The concentration specified trophysiological responses in planthopper antennae were refers to the most abundant compound, which was 1,8- identified (Riolo et al. 2012). cineole for mixtures 1–3, and (Z)-3-hexenyl acetate for Hence, the objective of the current study was to examine mixtures 4–6 (Riolo et al. 2012). The mixtures were pre- the olfactory responses of males and females to synthetic pared from the stock solutions each day and diluted to volatiles and to identify key mixtures of compounds or 0.1 lg/ll before the bioassays.

123 Behavioral responses of Hyalesthes obsoletus to host-plant volatiles cues 73

Table 1 Composition of the Compound Sourcea Chaste tree mixturesb Nettle mixturesb synthetic mixtures tested in the bioassays, according to the 123 456 ratios of the natural blend of compounds emitted by fresh Alcohol shoots of the chaste tree and (Z)-3-hexen-1-ol SA – – – 5 5 5 nettle (Riolo et al. 2012) Ester (Z)-3-hexenyl acetate SA – – – 100 100 100 Aromatics Methyl salicylate SA – 0.5 0.5 – 0.2 0.2 Methyl benzoate SA – – 0.05 – – – Benzothiazole SA – – – 10 10 10 Monoterpene 1,8-cineole SA 100 100 100 – – – Sesquiterpenes (E,E)-a-farnesene C 40 40 40 10 10 10 (E)-b-farnesene F 10 10 10 – – – (E)-b-caryophyllene SA 20 20 20 1 1 1 Other compound (Z)-jasmone SA – – – – – 0.05 a Standards obtained from Sigma-Aldrich, Italy (SA), Contech Inc., Canada (C), Fluka Chemie, Switzerland (F) b Numbers represent doses of each compound, expressed in ng/ll

Experiment 2 square of filter paper (1 cm2; Whatman, grade 5), with the blank represented by a square of filter paper with 10 ll Each single synthetic compound present in volatile mix- hexane applied. Airflow of 0.8 l/min was maintained tures that attracted males and/or females (see Results, Ex- through each cylinder using an air pump, with the airflow periment 1) was bioassayed. We evaluated the olfactory passing through the olfactometer arms. The incoming air responses of males to five single synthetic compounds [(E)- was purified through activated charcoal and humidified b-caryophyllene, 1,8-cineole, (E,E)-a-farnesene, (E)-b- with double-distilled, deionized water. The glass Y-tube farnesene, and methyl salicylate] and the responses of was positioned with an upward slope toward the blank and females to nine single synthetic compounds [(E)-b- test samples, of 10° from the horizontal plane. Two fluo- caryophyllene, 1,8-cineole, benzothiazole, (E,E)-a-farne- rescent lamps (TL-D, 18 W, Philips, France) were posi- sene, (E)-b-farnesene, (Z)-3-hexen-1-ol, (Z)-3-hexenyl tioned centrally behind the Y-tube olfactometer. acetate, methyl benzoate, and methyl salicylate] versus the The H. obsoletus adults were inserted individually into blank. For every single compound, a hexane stock solution the olfactometer at the entrance of the stem, using a release was prepared at the concentration of 10 lg/ll. The single vial, and they were observed until they had walked at least compounds were prepared from the stock solutions each 13 cm up one of the arms. Each individual was tested only day and diluted to 0.1 lg/ll before the bioassays. once. Specimens that did not choose a side arm within 5 min were recorded as ‘no choice’. For each individual Y-tube olfactometer bioassays planthopper, their activation time (from the beginning of bioassay to the exit from the release vial) and choice time The responses of H. obsoletus adults to these synthetic (from the beginning of the bioassay to the crossing of the mixtures and single compounds were investigated using a 13 cm arm line) were recorded using a manual dual-choice Y-tube olfactometer (glass; stem length, chronometer. The stimuli (mixtures and single compounds) 25 cm; arm length, 20 cm; arm angle, 75°; internal diam- were randomly assigned to one arm at the beginning of eter, 4 cm). Each arm of the Y-tube was connected by each bioassay, and each stimulus was reapplied after each Teflon tubing (Sigma-Aldrich, Italy) to a glass cylinder insect tested. The position of the Y-tube was reversed after (9 9 18 cm): one served as the control (blank), and the every 10 min, to avoid positional bias. After each treat- other included the stimulus. Ten microliters of the solution ment, the Y-tube was changed for a clean one, rinsed in of mixtures or single compounds was applied onto a small acetone (Sigma-Aldrich, Italy), and baked for 10 min at

123 74 P. Riolo et al.

200 °C. At the end of the day, the olfactometers were blank (Fig. 2a). However, no differences in H. obsoletus washed with detergent, rinsed with distilled water and preferences were recorded among the mixtures themselves acetone, and baked overnight at 200 °C. The experiments (males: v2 = 4.950, df = 5, P = 0.422; females: v2 = 6.628, were conducted from *12 p.m. to 5 p.m., during the period df = 5, P = 0.250). In terms of the responses to the various of maximum flight activity of H. obsoletus (Bressan et al. mixtures, the activation times were not influenced by H. ob- 2007), at a temperature of 26 ± 1 °C and a relative soletus sex (F = 0.751,P = 0.386) and mixture tested(males: humidity of 60 ± 10 %. In all, 136 (64 males, 72 females) F = 0.579, P = 0.716; females: F = 0.952, P = 0.448) H. obsoletus were tested for each mixture (Experiment 1), (Table 2). Conversely, males showed significantly shorter and 120 (60 males and 60 females) were tested for each choice times than females (F = 13.82, P \ 0.001; Table 2), single compound (Experiment 2). but no differences were detected among mixtures (males: F = 0.989, P = 0.424; females: F = 1.783, P = 0.116) Statistical analysis (Table 2). The percentage of females that made a choice between the arms of the olfactometer was significantly lower The Y-tube olfactometer data were analyzed using one-tailed than that of the males, as 66 versus 79 %, respectively

Chi-square tests with Yates correction (H0: the insects are not (F = 15.63, P = 0.003). attracted by the mixture/single compound, compared to the blank). The differences among ‘treatments’ (mixtures/single Experiment 2 compounds) were evaluated by contingency table analysis, based on Chi-square (Zar 1999). One-way ANOVA tests The olfactometer assays showed that H. obsoletus males were used to compare the percentages (arcsine transformed) and females were not attracted by any of the single com- of H. obsoletus males and females that made a choice between pounds, compared to the blank (Figs. 1b, 2b), and there the arms. Two-way ANOVA tests were used to compare the were no differences among the single compounds (males: activation and choice times between the sexes and among the v2 = 1.509, df = 4, P = 0.825; females: v2 = 6.678, ‘treatments’ of Experiments 1 and 2. The significance level of df = 8, P = 0.572). In terms of these responses to the all of the statistical tests was set at a \ 0.05. The analyses single compounds, the total activation time was signifi- were performed using Systat 11 (Systat Software Inc.). The cantly influenced by the H. obsoletus sex, whereby females specimens that did not make a choice were not included in the exited the release vial significantly faster than males analysis. (7.89 ± 2.19 s vs. 13.36 ± 3.34; F = 5.70, P = 0.017). Conversely, males showed a shorter choice time than females (72.07 ± 8.78 s vs. 86.86 ± 9.81 s; F = 10.17, Results P = 0.006). No differences were detected in the activation (males: F = 1.017, P = 0.399; females: F = 1.308, Experiment 1 P = 0.238) and choice (males: F = 1.696, P = 0.151; females: F = 0.668, P = 0.720) times among the single The olfactometer assays showed that H. obsoletus males were compounds within the sexes. Similar to the bioassays with significantly attracted by mixture 2, compared to the blank the mixtures, the percentage of H. obsoletus females that (v2 = 2.88, P = 0.045) (Fig. 1a), while H. obsoletus females made a choice between the arms of the olfactometer was were significantly attracted by mixtures 3 (v2 = 3.43, significantly lower than that of the males, as 75 versus P = 0.032) and 4 (v2 = 3.50, P = 0.031), compared to the 91 %, respectively (F = 13.696, P = 0.006).

Fig. 1 Behavioral responses of Hyalesthes obsoletus males to the synthetic mixtures tested (a) and to the single synthetic compounds tested (b). Level of significance is indicated on the left (one-tailed Chi-square test: ns nonsignificant); NC, number of insects that did not make a choice 123 Behavioral responses of Hyalesthes obsoletus to host-plant volatiles cues 75

Fig. 2 Behavioral responses of Hyalesthes obsoletus females to the synthetic mixtures tested (a) and to the single synthetic compounds tested (b). See legend to Fig. 1

Table 2 Activation and choice Mixtures Activation time (s) Choice time (s) times of Hyalesthes obsoletus males and females in the Males Females Males Females mixtures, compared to the blank bioassays Chaste tree 1 18.1 ± 3.6 26.5 ± 5.2 84.7 ± 10.8 115.3 ± 13.1 2 22.5 ± 4.6 20.1 ± 5.5 99.5 ± 11.7 98.1 ± 13.3 3 22.6 ± 4.1 33.4 ± 7.2 79.9 ± 11.0 151.6 ± 14.4 Nettle 4 27.9 ± 4.9 27.6 ± 4.3 96.3 ± 12.2 114.8 ± 11.4 5 21.6 ± 4.4 22.6 ± 3.9 81.1 ± 11.3 107.8 ± 12.2 6 24.4 ± 4.3 20.1 ± 4.5 108.3 ± 11.9 121.7 ± 14.0 Total (mean) 22.9 ± 4.3 25.0 ± 5.1 91.6 ± 11.5*** 118.2 ± 13.1 Data are mean ± SE (n = 60 males or females for each comparison) Significance in male versus female comparisons: *** P \ 0.001 (one-way ANOVA tests)

Discussion bioactive terpenes, green-leaf volatiles and aromatics [(E)- b-caryophyllene, (E,E)-a-farnesene, (Z)-3-hexenyl acetate, These findings provide an insight into the potential roles (Z)-3-hexen-1-ol, and benzothiazole (mixture 4)], but in that particular combinations of chemical constituents from this case the addition of methyl salicylate (mixture 5) chaste tree and nettle shoot volatiles play in the behavior of reduced the attractivity. Synergistic effects among different H. obsoletus males and females. The choice tests revealed chemical classes of compounds have been suggested to that the presence of several compounds is necessary to explain the attraction of other insects to their host plants induce significant attraction of H. obsoletus males and/or (Bruce and Pickett 2011). females, with an important role for the aromatic com- Emission of volatile compounds can change depending pounds. Methyl salicylate elicited a positive behavioral on the plant organ, the developmental stage, and the pho- response in males as a mixture with four other major toperiod (Dudareva et al. 2004). Moreover, airborne vola- bioactive chaste tree-derived terpenes [(E)-b-caryophyl- tiles are thought to be involved in defense reactions elicited lene, 1,8-cineole, (E,E)-a-farnesene, (E)-b-farnesene by phytophagous species (Arimura et al. 2005), pathogens (mixture 2)], while the addition of methyl benzoate (mix- (Jansen et al. 2011), and even against abiotic stress factors, ture 3) reduced the attractivity of this mixture to males. On such as mechanical injury, climatic factors, or pollution the other hand, addition of methyl benzoate to this five- (Loreto and Schnitzler 2010). The benzenoid esters compound chaste tree-derived mixture (mixture 3) signifi- investigated here, as methyl salicylate and methyl ben- cantly attracted H. obsoletus females. Females were also zoate, have been detected as released from insect-damaged significantly attracted by a nettle-derived mixture of plants (Zhao et al. 2010). In particular, methyl salicylate

123 76 P. Riolo et al. has been reported as the most distinctive indicator of insect might be a sign that their host-plant affiliation is not only sap feeding in induced volatile mixtures of different plant based on associative learning of chemical and/or physical species (Zhu and Park 2005; Pareja et al. 2009; Blande cues, but is also determined by genetic factors (Bernays et al. 2010). Methyl salicylate has also been frequently 2001; Johannesen et al. 2008). Indeed, chaste tree is not reported after disease-induced changes in the emission of found in the central Italy agroecosystems (the geographic plant volatiles (Jansen et al. 2011). For H. obsoletus area of the investigated in this study) although females, an already damaged/infested host plant that thus it has been recorded as a H. obsoletus host plant in Israel increases the release of methyl salicylate might indicate a (Sharon et al. 2005) and recently also in Montenegro resource for which competition is high or that is charac- (Kosovac et al. 2015). terized by a sub-optimal nutritional content, as has been As all of these investigated volatiles are also produced reported also for Cicadulina storeyi China (Oluwafemi by plant species other than the chaste tree and nettle et al. 2011) and Diaphorina citri Kuwayama (: (Knudsen et al. 2006), they might also function as non-host Psyllidae) (Martini et al. 2014). Furthermore, the stolbur cues when encountered individually. In many insect spe- phytoplasma might manipulate movement of H. obsoletus cies, volatile mixtures are more behaviorally effective than within the agroecosystem, which is a mechanism that might single odorants, so the ratios and the key odorants in a have evolved to promote their own spread, as observed, for given blend can be critical for host-plant recognition example, in Rhopalosiphum padi (L.) (Hemiptera: Aphi- (Bruce and Pickett 2011). Our findings provide striking didae) (Ingwell et al. 2012) and D. citri (Mann et al. 2012; examples of context-dependent behavioral activity of Martini et al. 2014, 2015). In particular, Mann et al. (2012) H. obsoletus based on these volatile compounds, whereby hypothesized that ‘Candidatus Liberibacter asiaticus’ can none of these individual compounds served as a chemical induce a plant to increase the rate of methyl salicylate signature for host-plant recognition. emission to enhance the attractivity of the infected plant to Field applications of host-plant volatile compounds, the vector D. citri; then after feeding on an infected plant, alone or in combination with other stimuli, have proven the psyllid tends to feed on non-infected plants. The same successful for monitoring and management of several phy- situation might also be hypothesized for H. obsoletus males tophagous species (Rodriguez-Saona and Stelinski 2009), in the vector/chaste tree/‘Ca. Phytoplasma solani’ e.g., for Leptinotarsa decemlineata (Say) (Coleoptera: pathosystem. Chrysomelidae) (Martel et al. 2005), Rhynchophorus fer- Our data from mixture preferences have also revealed rugineus Olivier (Coleoptera: Curculionidae) (Vacas et al. differences in the behavioral responses of H. obsoletus males 2016), and Cydia pomonella L. (Lepidoptera Tortricidae) and females to the volatiles that they were exposed to. This (Knight and Light 2005). Moreover, the use of plant volatiles may have a role in H. obsoletus ‘assessment’ of a suit- to manipulate the behavior of a vector species has been able host plant (e.g., discrimination between host and non- recently investigated in Cacopsylla picta (Foerster) (Hemi- host plants, discrimination between primary and non-pri- ptera: Psyllidae): traps baited with (E)-b-caryophyllene mary host plants, and/or as an indicator of host-plant status) dispensers represent a promising innovative technique for (Gripenberg et al. 2010; Bruce and Pickett 2011). Moreover, monitoring or mass trapping (Eben and Gross 2013). in our trials, males progressed upwind in the olfactometer Our novel findings can be used as baseline information more than females. In the field, H. obsoletus males show to develop plant volatile-based systems that attract males greater activity than females in their search for mates and and females of this planthopper vector. However, further host plants, while females tend to remain sedentary on their studies need to be carried out to investigate the roles of host plants (Bressan et al. 2007; Mazzoni et al. 2010; Minuz these active synthetic mixtures in the field and their et al. 2013). The attraction of females to the nettle-derived potential applications to pest monitoring and management. mixture in the present study was expected as in Italy nettle is Furthermore, improved understanding of the interactions a primary host plant of H. obsoletus (Bressan et al. 2007; among the BN phytoplasma, its vector H. obsoletus, and Riolo et al. 2007). Previous studies have shown that H. ob- their host plants is needed to determine whether pathogen- soletus females show preference for plant volatiles emitted induced plant responses facilitate the spread of the stolbur by their primary hosts (Sharon et al. 2005; Riolo et al. 2012) phytoplasma. and that this affiliation is established during its last nymphal instar (Kessler et al. 2011). Furthermore, limited offspring Acknowledgements This study was funded by the Italian Ministry of mobility and egg laying in batches have been highlighted as Education, Universities and Research (MIUR), Research Project of National Interest (PRIN) ‘Mechanisms of host-plant location and potentially important factors that promote female host-plant host-plant preferences in two leafhoppers (Hemiptera: Auchenor- choice (Gripenberg et al. 2010). On the other hand, the rhyncha), vectors of grapevine yellow diseases’. Comments and attraction of H. obsoletus males and females to chaste tree- suggestions made by three anonymous reviewers have helped us to derived mixtures that was observed during the present study improve the manuscript. 123 Behavioral responses of Hyalesthes obsoletus to host-plant volatiles cues 77

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