Journal of Chemical Ecology

, Volume 39, Issue 5, pp 620–629 | Cite as

Synthetic Cis-Jasmone Exposure Induces Wheat and Barley Volatiles that Repel the Pest Cereal Leaf Beetle, Oulema melanopus L.

  • Kevin J. Delaney
  • Maria Wawrzyniak
  • Grzegorz Lemańczyk
  • Danuta Wrzesińska
  • Dariusz Piesik
Article

Abstract

The plant semiochemical cis-jasmone primes/induces plant resistance that deters herbivores and attracts natural enemies. We studied the induction of volatile organic compounds (VOCs) in winter wheat and spring barley after exposure of plants to three synthetic cis-jasmone doses (50 μl of 1, 100, and 1 × 104 ng μl−1) and durations of exposure (1, 3, and 6 h). Cereal leaf beetle, Oulema melanopus, adult behavioral responses were examined in a Y-tube olfactometer to cis-jasmone induced plant VOC bouquets and to two synthetic blends of VOCs (3 green leaf volatiles (GLVs); 4 terpenes + indole). In both cereals, eight VOCs [(Z)-3-hexanal, (Z)-3-hexanol, (Z)-3-hexanyl acetate, (Z)-β-ocimene, linalool, β-caryophyllene, (E)-ß–farnesene, and indole] were induced 100- to 1000-fold after cis-jasmone exposure. The degree of induction in both cereals was usually positively and linearly associated with increasing exposure dose and duration. However, VOC emission rate was only ~2-fold greater from plants exposed to the highest vs. lowest cis-jasmone exposure doses (1 × 104 difference) or durations (6-fold difference). Male and female O. melanopus were deterred by both cereal VOC bouquets after plant exposure to the high cis-jasmone dose (1 × 104 ng μl−1), while females were also deterred after plant exposure to the low dose (1 ng μl−1) but attracted to unexposed plant VOC bouquets. Both O. melanopus sexes were repelled by terpene/indole and GLV blends at two concentrations (25 ng · min−1; 125 ng · min−1), but attracted to the lowest dose (1 ng · min−1) of a GLV blend. It is possible that the biologically relevant low cis-jasmone dose has ecological activity and potential for inducing field crop VOCs to deter O. melanopus.

Keywords

Hordeum vulgare Triticum aestivum cis-jasmone Volatile Cereal leaf beetle Induction 

Introduction

Plants are attacked by many herbivorous insects, and almost half of described insect species are herbivorous. However, plants are not merely passive victims of herbivore attack and have evolved a variety of mechanisms that withstand the damage and stresses caused by biotic attack. One defensive mechanism involves volatile organic compound (VOC) induction in response to biotic attack (Birkett et al., 2000; Thaler et al., 2002; Schoonhoven et al., 2005; Dicke et al., 2009; Dicke and Baldwin, 2010; Hare, 2011). Herbivore induced plant volatiles (HIPVs) can repel future herbivores and/or act as an indirect plant defense mechanism by attracting other insects that prey on or parasitize the herbivores (Turlings et al., 1990; Birkett et al., 2000; Kessler and Baldwin, 2001). However, it is difficult to demonstrate that such indirect defenses improve plant fitness (Dicke and Baldwin, 2010). Plants defend themselves against multiple attackers (Bruce and Pickett, 2007; Dicke et al., 2009), so responses to a current attacker may influence effectiveness of future responses to attack by the same or different species. Knowledge of plant interactions with attacking organisms has advanced considerably in the last decade, and it has been realized that plant defenses are quite sophisticated and intricate (Hare, 2011). An example of this sophistication is priming of antiherbivore defense, in which plants respond to VOCs emitted from their own injured tissues, or from neighboring plants, so that they respond more strongly and rapidly to future attack (Engelberth et al., 2004; Conrath et al., 2006; Kessler et al., 2006; Frost et al., 2007).

Since plants potentially can be exposed to a variety of VOCs from their own tissues and neighboring plants, it would be adaptive for plants to detect and respond to induced VOCs that reliably indicate attack from nearby biotic enemies. Common plant phytohormones produced after biotic attack include non-volatile jasmonic acid (JA) and salicylic acid (SA), as well as volatile phytohormones, i.e., ethylene and methyl jasmonate (Me-JA) and methyl salicylate (Me-SA) (Farmer and Ryan, 1990; Thaler et al., 1996). Thus, Me-JA and Me-SA are possible reliable VOC indicators of biotic attack to nearby plants, herbivores, and natural enemies.

Another possible reliable VOC indicator of biotic attack is cis-jasmone (CJ), a compound that results from one biosynthetic step downstream from JA (Koch et al., 1997). Initially, CJ was suggested to serve as a biological sink for JA (Koch et al., 1997) because JA was the known liquid phytohormone for plant wound responses at the time, and Me-JA was an active volatile form. However, it has been realized since that CJ might also result from other processes besides simple JA catabolism (see Matthes et al., 2010). Studies have measured CJ in plant VOC blends induced by insect herbivory (Loughrin et al., 1995; Birkett et al., 2000; Bruce et al., 2003; Röse and Tumlinson, 2005). Even if CJ is a ‘waste’ product after JA induction, it might still reliably indicate biotic attack, alerting recipient plants when their neighbors are being damaged by phytophagous insects (Chamberlain et al., 2000; Pickett and Poppy, 2001). However, CJ also has been found to be constitutively released by flowers or leaves of several plant species, where this VOC acts as an attractant for pollinators and as a chemical cue for host localization (or avoidance) by insect herbivores (Schlotzhauer et al., 1996; El-Sayed et al., 2009; Tanaka et al., 2009). Plant exposure to CJ also has been demonstrated to induce plant defense metabolism (Pickett et al., 2007a, b; Bruce et al., 2008; Moraes et al., 2008). Moreover, CJ induces a discrete and distinctive suite of genes in Arabidopsis thaliana that are involved in primary and defensive metabolic responses, with only partial overlap in genes induced by CJ and Me-JA (Matthes et al., 2010). Thus, CJ may be a reliable semiochemical to prime or induce defenses of neighboring plants, and attract or repel herbivorous and predatory insects.

Our experiments aimed to elucidate how the emission of VOCs that are induced by exposure of wheat (Triticum aestivum L.) and spring barley (Hordeum vulgare L.) depends on the CJ dose and duration of exposure. We characterized and compared the CJ-induced blends of VOCs in both cereals. We applied three CJ exposure doses (cover 10,000-fold range in quantity) that were crossed with three durations (cover 6-fold range). Studies have reported 0 to 602 ng in CJ emission levels h−1 (Loughrin et al., 1994, 1995; Schlotzhauer et al., 1996; Röse and Tumlinson, 2004, 2005), so one of our test doses fell within this range while two doses were larger and well outside this range. This experimental procedure allowed us to determine whether plant sensitivity of each cereal species is high (dramatic increases in VOC induction) or low (small or no increases in VOC induction) to increasing CJ exposure doses and duration, and whether exposure concentration and time interact to influence degree of plant VOC induction.

The second aim of our experiments was to determine how the cereal leaf beetle (Oulema melanopus L.), an important small grain pest in Europe (Dimitrijević et al., 1999) and North America (Phillips et al., 2011), responds to CJ-induced plant VOCs. So far, few studies have addressed the question whether O. melanopus prefers injured or uninjured host grasses that it encounters in the field (Phillips et al., 2011). Prior studies have examined O. melanopus (Piesik et al., 2011a) or O. cyanella (Piesik et al., 2013) responses to a single VOC in the lab, but not to synthetic VOC blends or plant VOC bouquets. Thus, we conducted Y-tube experiments to test O. melanopus behavioral responses to VOC bouquets from barley and wheat exposed or not exposed to CJ. A subsequent experiment examined O. melanopus dose responses to one synthetic blend of three green leaf volatiles (GLV), and a second synthetic blend of four terpenes and indole (IND); these two blends were chosen based on the 8 VOCs induced in barley and wheat after CJ exposure.

Methods and Materials

Plant Culture

Experiments were performed in 2011 at the Plant Growth Center (PGC; University of Technology and Life Sciences, Bydgoszcz, Poland). Winter wheat cv. ‘Nutka’ and spring barley cv. ‘Antek’ were planted and grown in a greenhouse with supplemental light and ambient humidity (75–85 %). The photoperiod was16L:8D, day temperature was 22 ± 2 °C, and the night temperature was 18 ± 2 °C. Plants were grown with two individuals per pot in sterilized soil, watered four times weekly, and fertilized twice weekly with Peters® General Purpose Fertilizer (J.R. Peters Inc., Allentown, PA, U.S.A.) at 100 ppm in aqueous solution as part of the watering. Fertilizing commenced when the plants reached the 3rd leaf stage. All plants were at the Zadoks (ZGS) 32 stage, with the emergence of an elongating stem section separating the first two nodes. Thus, seedlings were ~6-week-old for spring barley seedlings and 12-week-old (6 week for vernalization) for winter wheat seedlings. At ZGS 32, there are 5 large leaves for wheat and 6 large leaves for barley, projecting upwards from the area of the elongating stem in addition to 4–6 older leaves lower on the main stem and some leaves of younger tillers.

CJ Application

One of three quantities (5 × 105, 5 × 103, or 50 ng) of synthetic CJ (95 % purity; Sigma-Aldrich, Inc., St. Louis, MO, USA) was added to 50 μl hexane (1, 100, and 1 × 104 ng μl−1), the mix poured onto filter paper, and the paper placed into a microcentrifuge tube. There were three durations of CJ exposure tested at each concentration: 1, 3, and 6 h. Dose and duration of CJ exposure were fully crossed factors, and there were 8 plants measured for each treatment combination with spring barley (72 experimental plants and 4 unexposed controls) and winter wheat (72 experimental plants and 4 unexposed controls). GC-MS analysis showed that only 70 to 20 % (median 34.5 %) of an initial CJ dose were measured after 1 h (D. Piesik, unpublished data). Therefore, for the treatments where plant exposure to CJ lasted 3 or 6 h, an old filter paper was replaced every h with a new one. When plants were to be exposed to CJ, they were transferred from the greenhouse to the laboratory, and thus separated from all other raised plants during CJ exposure. A 27.5-gauge syringe needle (Allison Medical Inc, Englewood, CO, USA) was pushed through each microcentrifuge tube cap immediately prior to adding the first filter, and the tube was placed on the soil adjacent to an experimental plant in a pot. Four control plants for each cereal were exposed to 50 μl hexane that did not contain CJ for 6 h. Control (hexane-exposed) plants have comparable VOC emission levels to uninjured plants unexposed to hexane (Piesik et al., 2011b).

Volatile Collection System

Volatiles were collected from the main stem of each experimental plant immediately after the end of the CJ exposure period, where the main stem was enclosed within a Nalophan bag (~50 cm high × 30 cm diam.), while the tillers and soil were kept outside of each volatile collection chamber. These polyethylene terephtalate bags are odor- and taste-free cooking bags made of a plastic film resistant to decomposition in the temperature range from −60 °C to +220 °C (Charles Frères, Saint Etienne, France). In order to collect odors from four plants simultaneously, a volatile collector trap (6.35-mm outside diameter, 76-mm-long glass tube; Analytical Research Systems, Inc., Gainesville, FL, USA) containing 30 mg of Super-Q adsorbent (Alltech Associates, Inc., Deerfield, IL, USA) was inserted into each of four Tygon tubes, each of which served to connect the collector trap to a common airflow meter. Purified, humidified air was delivered at a rate of 1.0 L · min−1 over the plants, and a vacuum pump sucked 20 % less (0.8 L · min−1) to avoid collecting odors from any gap in the closed system. The volatile collection sequence lasted for 3 h, from 10:00 to 13:00. This time period corresponded to the period of peak barley and wheat diurnal VOC emission (D. Piesik, unpublished data). Thus, plant exposure durations to CJ of 1, 3, and 6 h began at 9:00, 7:00, and 4:00, respectively.

Analytical Methods

The methods used follow those of previous studies (Piesik et al., 2010a, b, 2011a, b; 2013). Briefly, volatiles were eluted from Super-Q in each trap with 225 μl hexane, followed by adding 7 ng decane as an internal standard. Previous research showed that this quantity of hexane was sufficient to extract all trapped volatiles (D. Piesik, unpublished data). Individual samples (1 μl) were injected and analyzed by coupled gas chromatography–mass spectrometry (GC/MS). A GC/MS Auto System XL/Turbomass (Perkin Elmer Shelton, CT, USA) fitted with a 30-m Rtx-5MS capillary column (0.25 mm ID, 0.25 μm film thickness; Restek, USA) was used. The temperature program increased from 40 °C to 200 °C at 5 °C/min. Identification of volatiles was verified by comparison of GC retention times and mass spectra with authentic standards purchased from commercial sources (Sigma-Aldrich). Concentrations were calculated by comparing each VOC peak area in a chromatogram with the peak area for the internal standard and expressed as a rate (ng · h−1).

Ouelema melanopus Behavior

In the first insect behavior experiment, plants that provided experimental VOC bouquets during trials had been exposed to the highest (10 μg μl−1) or lowest (1 ng μl−1) test CJ concentrations in 50 μl hexane for 0 (unexposed plants), 1, or 6 h Preliminary work indicated that the best airflow delivery rate was 150 ml · min−1 (D. Piesik, unpublished data), so this rate was used. Thus, there were 6 exposure treatment combinations (2 concentrations * 3 durations). A plant began to be used as a VOC bouquet source for behavioral trials immediately after the CJ exposure period ended. For each cereal species, one plant was used to provide a VOC bouquet for each treatment combination to test responses of 20 female and 20 male O. melanopus adults to that combination (it took 2–3 h to complete the 40 trials using the odor from a test plant). A total of 6 winter wheat plants and 6 barley plants were used to conduct all trials of beetles responding to plant odors. Volatiles were provided directly from plants enclosed in Nalophan bags to one arm of the Y-tube, while only humidified and purified air was delivered to the second arm (control).

There were 8 VOCs measured from barley and wheat exposed to CJ that were chosen for Y-tube experiments involving O. melanopus adult behavioral responses. Beetles were tested with one blend of three synthetic GLVs [(Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexen-1-yl acetate], and a second blend consisting of two monoterpenes [(Z)-β-ocimene and linalool], two sesquiterpenes [β-caryophyllene, and (E)-β-farnesene] and a shikimic acid pathway derivative (indole). Each VOC was purchased from Sigma-Aldrich and had 95 % purity. The Y-tube system used was similar to that described by Piesik et al. (2008). Each blend was tested at 5 concentrations (0, 1, 5, 25, 125 ng · min-1). Each individual VOC was present in a blend at the specified concentration. Thus, for the GLV blend 1 ng · min−1 means that 1 ng (Z)-3-hexenal + 1 ng (Z)-3-hexenol + 1 ng (Z)-3-hexenyl acetate was added to 50 μl hexane. A dose of a blend was placed in one arm of the Y-tube and tested against 50 μl hexane without the blend (0 ng · min−1). The odor source was replaced, and the Y-tube was cleaned with hexane, after each trial. Of each sex, 20 newly-emerged (≤1-day-old) adults were tested at each concentration of each blend, with each insect tested once. All tested adults chose an arm of the Y-tube within 3 min.

Data Analysis

A mixed model MANOVA was conducted with the data from all 8 VOCs for spring barley, and a separate analysis for wheat VOC data. These two MANOVA were conducted using PROC GLM in SAS version 9.2. The barley and wheat CJ exposure dose and duration were fixed main effects, and their interaction was a fixed effect too, while groups of four measured plants served as random, incomplete blocks. If overall MANOVA results were significant for a cereal species, then univariate mixed model ANOVA were conducted for each VOC separately. Since the CJ exposure dose x duration was significant for all VOCs from both cereals (except one case), LSD post-hoc tests were used to indicate which dose x duration treatment combinations were significantly different from each other for each VOC.

A 3 × 2 goodness of fit test (G-test) was conducted to examine whether total counts of O. melanopus adults choosing a Y-tube arm was influenced by three tested plant exposure to CJ durations (0, 1, or 6 h) for both barley and wheat, with the odor source coming from a plant compared to hexane solvent only. Then, separate G-tests were conducted for adult counts only from low vs. high CJ exposure dose trials, or from only female or male trials, to indicate how both exposure doses and beetle sexes contributed to the overall analysis results for each species. Finally, G-tests were conducted for counts from trials from each dose x sex combination to examine whether certain combinations contributed most strongly to the results of the overall analysis for each species.

A 5 × 2 goodness of fit test (G-test) was conducted to examine whether total counts of O. melanopus adults choosing a Y-tube arm was influenced by 5 tested concentrations (0 ng min−1 serving as a control) of a synthetic blend (3 GLVs or 4 terpenes + IND), with the odor source coming from a blend compared to hexane solvent only. Separate G-tests were conducted for adult counts only from female or male trials, to indicate how both beetle sexes contributed to the overall analysis results for each blend.

Chi-square goodness of fit tests (Χ2-test), with the Yates correction for small samples (1 × 2), were conducted to indicate whether choice of Y-tube arms was influenced by a preference for odor source (plant bouquet vs. hexane solvent; or synthetic blend vs. hexane solvent) at each exposure concentration x sex x exposure duration combination. Non-significant tests indicated that the observed beetle counts did not significantly deviate from an expected ratio of 10:10 (hexane solvent only arm : plant bouquet or synthetic blend). Significant tests indicated attraction (more individuals chose Y-tube arm with a plant bouquet or synthetic blend) or repellency (more individuals chose the Y-tube arm with only hexane solvent). All statistical tests were conducted using SAS version 9.2 (significance threshold α = 0.05).

Results

Barley or Wheat VOC Emission after CJ Exposure

Wheat and barley plants exposed to CJ usually showed 100- to 1000-fold higher VOC emissions than control plants that were not exposed to CJ, but to hexane for 6 h. The major VOCs detected from CJ-exposed wheat and barley plants were (Z)-3-hexenal (Z3HAL), (Z)-3-hexenol (Z3HOL), (Z)-3-hexenyl acetate (Z3HAC), (Z)-β-ocimene (ZβOCI), linalool (LIN), β-caryophyllene (βCAR), (E)-β-farnesene (EβFAR), and indole (IND) (Figs. 1, 2). These VOCs were hardly detected in the headspace of control plants. When these VOCs were detected in measurable quantities from control plants unexposed to CJ, emission rates were ≤0.9 ng · h−1 from 4 control barley plant, and <0.4 ng · h−1 from 4 control wheat plants (data not shown). Thus, control plants were not included in analyses of plant VOCs from either cereal species after CJ exposure.
Fig. 1

Mean (± 1 SE) concentration of spring barley emission rate of the following VOC: a βFAR, b Z3HAC, c LIN, d Z3HAL, e βCAR, f βOCI, g IND, and h Z3HOL, at 1, 3, and 6 h following aerial plant exposure to concentrations of 1 ng, 100 ng, or 10 μg, (Z)-jasmone μl−1 in 50 μl hexane. Treatments with the same letter were not significantly different based on LSD post-hoc tests

Fig. 2

Mean (± 1 SE) concentration of winter wheat emission rate of the following VOC: a βFAR, b Z3HAC, c LIN, d Z3HAL, e βCAR, f βOCI, g IND, and h Z3HOL, at 1, 3, and 6 h following aerial plant exposure to concentrations of 1 ng, 100 ng, or 10 μg, (Z)-jasmone μl−1 in 50 μl hexane. Treatments with the same letter were not significantly different based on LSD post-hoc tests

Overall duration and dose of CJ exposure, and their interaction, had significant effects on VOC emissions from winter wheat (Duration- Wilks’ λ = 0.095, F16,106 = 15, P < 0.001; Concentration- Wilks’ λ = 0.003, F16,106 = 110, P < 0.001; Concentration x Duration- Wilks’ λ = 0.023, F32,197 = 11, P < 0.001) and from spring barley (Duration- Wilks’ λ = 0.093, F16,106 = 15, P < 0.001; Concentration- Wilks’ λ = 0.003, F16,106 = 120, P < 0.001; Concentration x Duration- Wilks’ λ = 0.058, F32,197 = 7.2, P < 0.001).

In general, plants exposed to higher CJ doses had higher emissions of the 8 reported VOCs from spring barley and winter wheat (F2,60 ≥ 103, P < 0.001). With the same pattern from both cereal species, the top four emission level VOCs included one sesquiterpene (EβFAR), two GLVs (Z3HAC and Z3HAL), and one monoterpene (LIN), while the bottom four emission level VOCs included one sesquiterpene (βCAR), one monoterpene (ZβOCI), one aromatic compound (IND), and one GLV (Z3HOL) (Figs. 1, 2). Test cereal emission levels usually were lowest after exposure to the 1 ng μl−1 dose, intermediate from the 100 ng μl−1 dose, and often doubled from the 10 μg μl−1 dose compared to the 1 ng μl−1 dose for spring barley (Fig. 1) and winter wheat (Fig. 2). Also, longer durations of exposure to CJ resulted in higher emissions of all 8 reported VOCs from spring barley and winter wheat (F2,60 ≥ 11, P < 0.001). Levels were lowest after 1 h, intermediate after 3 h, and often doubled after 6 h of exposure received by spring barley (Fig. 1) and winter wheat (Fig. 2) compared to 1 h. A general pattern was that VOC emission concentrations were higher from spring barley than winter wheat (Figs. 1, 2).

Moreover, interactions between CJ exposure dose and duration were significant for all VOCs for winter wheat (F4,60 ≥ 5.8, P < 0.001), and spring barley (F4,60 ≥ 2.6, P < 0.05) except for Z3HAL (F4,60 = 1.4, P = 0.23). The most striking interactions related to non-linear dose responses where the highest emission levels of Z3HOL from barley (Fig. 1h) and IND from wheat (Fig. 2g) came after the intermediate, rather than highest, CJ exposure dose. In other cases, interactions were due to either nonsignificant or relatively small differences between 1 vs. 3, or 3 vs. 6, h exposure durations at one or more exposure doses, or between 1 ng μl−1vs. 100 ng μl−1, or 1 ng μl−1vs. 10 μg μl−1, CJ doses at one or more exposure durations received by barley and wheat (Figs. 1, 2).

Ouelema melanopus Responses to Cereal VOCs after CJ Exposure

Adult beetles displayed significant responses (G2df = 25, P < 0.001) to spring barley VOC bouquets (Table 1). Choices by females, were influenced by the VOC bouquets depending on whether barley had been exposed to CJ (G2df = 41, P < 0.001), but not the choices by males (G2df = 3.6, P = 0.16). Also, pooled choices by males and females depended on whether barley had CJ exposure with low (1 ng μl−1) and high (10 μg μl−1) exposure doses (left column in Table 1). A significant proportion of females was attracted to VOC bouquets from barley unexposed to CJ, but avoided barley VOC bouquets when plants had 1 or 6 h exposure duration with both low and high CJ exposure doses (Table 1). There was no significant male attraction to VOC bouquets of barley unexposed to CJ. A significant proportion of males avoided barley VOC bouquets when plants had 1 or 6 h exposure duration to the high CJ dose, but the proportions of males that avoided plants exposed or not to CJ were not significantly different at either low or high CJ doses (Table 1).
Table 1

Effect of spring barley plant volatiles after (Z)-jasmone exposure (or not) on the number of Oulema melanopus adult females and males choosing to enter a Y-tube arm containing the odor of a test plant (Plant odor) or the Y-tube arm containing purified, humidified air and hexane solvent (No odor)

Plant exposure

No. of females

χ2 (1)

No. of males

χ2 (1)

Plant odor

No odor

Plant odor

No odor

1 ng/μl

Control

16

4

6.1* (a) 3

8

12

0.5 ns

After 1 h

5

15

4.1* (r) 2

13

7

1.3 ns

After 6 h

4

16

6.1* (r) 2

7

13

1.3 ns

G2df = 8.8**

G2df = 19***

G2df = 4.2ns

10 μg/μl

Control

15

5

4.1* (a) 3

7

13

1.3 ns

After 1 h

4

16

6.1* (r) 2

5

15

4.1* (r) 2

After 6 h

2

18

11.3*** (r) 2

3

17

8.5** (r) 2

G2df = 19***

G2df = 22***

G2df = 2.2ns

1ns not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

2r repellent

3a attractant

Adult beetles also displayed significant responses to winter wheat VOC bouquets (G2df = 19, P < 0.001) (Table 2). Again, choices by females were influenced by emitted VOC bouquets depending on whether wheat had CJ exposure (G2df = 21, P < 0.001), but not the choices by males (G2df = 4.0, P = 0.14). A significant proportion of females avoided wheat VOC bouquet that had 6 h of low dose CJ exposure, but the proportion of females that avoided plants exposed to the low CJ dose was not significantly different (Table 2). In contrast, a significant proportion of females avoided wheat plants exposed for 1 or 6 h of high CJ dose, and a significantly greater proportion of females was attracted to wheat plants unexposed to CJ (Table 2). There was no significant male attraction to VOC bouquets of wheat unexposed to CJ. Also, even though a significant proportion of males avoided wheat VOC bouquets when plants had 1 or 6 h exposure duration to the high CJ dose, the proportions of males that avoided plants exposed or unexposed to CJ were not significantly different at either exposure dose (Table 2).
Table 2

Effect of winter wheat plant volatiles after (Z)-jasmone exposure (or not) on the number of Oulema melanopus adult females and males choosing to enter a Y-tube arm containing the odor of a test plant (Plant odor) or the Y-tube arm containing purified, humidified air and hexane solvent (No odor)

Plant exposure

No. of females

χ2 (1)

No. of males

χ2 (1)

plant odor

no odor

plant odor

no odor

1 ng/μl

control

11

9

0.1 ns

10

10

0.1 ns

after 1 h

8

12

0.5 ns

14

6

2.5 ns

after 6 h

5

15

4.1* (r) 2

8

12

0.5 ns

G2df = 5.0ns

G2df = 3.8ns

G2df = 3.8ns

10 μg/μl

control

16

4

6.1* (a) 3

10

10

0.1 ns

after 1 h

5

15

4.1* (r) 2

5

15

4.1* (r) 2

after 6 h

3

17

8.5** (r) 2

4

16

6.1* (r) 2

G2df = 23***

G2df = 21***

G2df = 4.7ns

1ns not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

2r repellent

3a attractant

Ouelema melanopus Y-tube Responses to One GLV Blend and One Terpene Blend (+ IND)

Both sexes were significantly attracted to the lowest test concentration (1 ng · min−1) and were significantly repelled by the highest tested GLV blend concentration (125 ng · min−1; also 25 ng · min−1 for females; Table 3). Female and male beetles both had dose-dependent responses to the blend of the three GLVs (significant G-tests in Table 3). However, while the proportion of females attracted to the lowest GLV blend test dose was different from the proportion of females attracted to the 0 dose (G1df = 8.6, P = 0.003), the same comparison was not significant for males (G1df = 1.0, P = 0.31). Males were significantly repelled by the terpene/IND blend at the two highest test concentrations (25 and 125 ng · min−1), and these responses were different compared to individuals responding to the lowest test concentration (1 ng · min−1) and the 0 dose (significant G-test in Table 3). In contrast, even though females were repelled by the three highest test concentrations of the terpene/IND blend (5, 25, and 125 ng · min−1), these responses were not different when compared to individuals responding to the lowest test concentration (1 ng · min−1) or the 0 dose (nonsignificant G-test in Table 3).
Table 3

Effect of one synthetic blend of three GLV ((Z)-3-hexenal- Z3HAL; (Z)-3-hexenol- Z3HOL; and (Z)-3-hexenyl acetate- Z3HAC), and a second synthetic blend of four terpenes ((Z)-β-ocimene- ZOCI; linalool- LIN; β-caryophyllene- β-CAR; and (E)-β-farnesene- β-FAR) with indole (IND), on the number of Oulema melanopus adult females and males choosing to enter a Y-tube arm containing the odor of a test plant (plant odor) or the Y-tube arm containing purified, humidified air and hexane solvent (no odor)

Blend compounds

Dose

ng.min-1

No. of females

No. of males

VOC blend

No blend

χ2 (1)

VOC blend

No blend

χ2 (1)

 

Control

0

7

13

1.3 ns

12

8

0.5 ns

Z3HAL

1

1

16

4

6.1* (a) 3

15

5

4.1* (a) 3

Z3HOL

2

5

8

12

0.5 ns

9

11

0.1 ns

Z3HAC

3

25

3

17

8.5** (r) 2

10

10

0.1 ns

4

125

2

18

11.3*** (r) 2

4

16

6.1* (r) 2

G4df = 37***

G4df = 28***

G4df = 14**

ZOCI

Control

0

8

12

0.5 ns

11

9

0.1 ns

LIN

1

1

7

13

1.3 ns

11

9

0.1 ns

IND

2

5

4

16

6.1* (r) 2

6

14

2.5 ns

βCAR

3

25

3

17

8.5** (r) 2

5

15

4.1* (r) 2

βFAR

4

125

3

17

8.5** (r) 2

3

17

8.5** (r) 2

G4df = 17**

G4df = 5.8ns

G4df = 12*

1ns not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

2r repellent

3a attractant

Discussion

Cis Jasmone Exposure: Wheat and Barley VOC Induction

In the first part of our experiment, we confirmed that CJ application dramatically induced the same eight VOCs from barley and wheat, all of which are common injury-induced GLVs, terpenes, and an aromatic compound (Kessler and Baldwin, 2001; Engelberth et al., 2004; Dicke et al., 2009; Hare, 2011). Mechanical injury often induces GLVs, while terpene and indole induction often requires actual herbivore feeding injury (see Dicke et al., 2009; Hare, 2011). However, pathogen infection also can induce GLVs and terpenes (Piesik et al., 2011a, b; 2013). Terpenes have been suggested to be involved in mediating herbivore deterrence (Kessler and Baldwin, 2001) or attraction (Carroll et al., 2006), or parasitoid attraction (Dicke and Baldwin, 2010). GLVs can deter herbivorous arthropods (Kessler and Baldwin, 2001), suppress pathogen infection (see Dicke and Baldwin, 2010), and they can prime/induce neighboring plant defenses (Farag and Paré, 2002; Engelberth et al., 2004; Farag et al., 2005).

The degree of induction of all eight cereal VOCs was positively dependent on the CJ exposure dose and duration. However, a doubling in the degree of induction was disproportionately small given the 10,000-fold range in tested CJ exposure doses and six-fold range in durations. The emission level ranking of these VOCs was generally consistent across all exposure dose x duration combinations for each test cereal. There were minor exposure dose x duration interactions that usually reflected plant emission not increasing with increasing CJ exposure duration (1 to 3, or 3 to 6, h). However, it was striking that the highest IND emission levels were detected in the headspace of winter wheat exposed to the medium, rather than the highest tested CJ dose.

Plant CJ emission has been reported at the ng h−1 scale (Loughrin et al., 1994, 1995; Schlotzhauer et al., 1996; Röse and Tumlinson, 2004, 2005). For ecological relevance, the 1 ng μl−1 and 100 ng μl−1 CJ exposure doses are probably more realistic to represent amounts from an injured plant than the 10 μg μl−1 dose. For potential agricultural relevance, the 1 ng μl−1 dose is the closest dose to levels in previous plant exposure to CJ in lab or field studies (Bruce et al., 2003, 2008; Moraes et al., 2008), while the 100 ng and 10 μg μl−1 matched or were closer to doses of 100 ng and 1 μg μl−1 tested in aphid olfactometer choice tests in the lab (Birkett et al., 2000). Overall, the degree of barley and wheat VOC emission was not highly sensitive to CJ exposure dose or duration, so greatly increasing field plant CJ exposure would not be expected to dramatically increase plant VOC production or potential herbivore resistance.

Chamberlain et al. (2000) reported that induced wheat plants are more resistant to attack by the grain aphid, Sitobion avenae, with CJ possibly acting as a semiochemical by alerting plants to nearby insect attack. Another study has demonstrated that CJ exposed bean plants induce VOCs, including the monoterpene (E)-β-ocimene, a common HIPV across plant species (Birkett et al., 2000). Birkett et al. (2000) also report that lettuce aphids, Nasonovia ribis-nirgi, are repelled by CJ, while aphid natural enemies like the seven-spot lady beetle, Coccinella septumpunctata, and the aphid parasitoid, Aphidius ervi, are attracted to CJ. Bruce et al. (2003) observed that when CJ exposure activates wheat defenses, there is a reduction in attack by S. avenae, and (E)-β-ocimene is an HIPV. More recent studies have shown that plant exposure to CJ leads to defense gene upregulation and metabolism (Pickett et al., 2007a, b; Bruce et al., 2008; Moraes et al., 2008; Matthes et al., 2010). This helps to explain why CJ exposed plants can have improved herbivory resistance (Chamberlain et al., 2000; Bruce et al., 2003).

Ouelema melanopus Behavioral Responses to VOC Bouquets from CJ Exposed Barley or Wheat

Our bioassays showed that both adult O. melanopus sexes could be repelled by test cereal VOC bouquets when plants had been exposed to CJ, but only females could be attracted to plants not exposed to CJ. The latter result suggests that O. melanopus might be attracted to constitutive (uninjured, uninfected, unexposed) plant VOC emission. Females were somewhat more prone to be repelled than males, as only females were repelled by plants exposed to the low (1 ng μl−1) CJ dose. The biology, life history, and host cereal species preferences of O. melanopus have been studied intensively (Phillips et al., 2011). However, only a few field studies have addressed the mechanisms that mediate attraction of O. melanopus adults to host plants, their choice to consume a host plant, and their behavioral preferences for injured or uninjured host plants. Barley and wheat VOC induction after CJ exposure might have biological relevance in terms of O. melanopus deterrence, with the caveat that our results come from greenhouse and lab studies.

In the second Y-tube behavioral experiment, we chose to use equal amounts of each VOC in the blend for a given test dose. Thus, the ratios of synthetic blend VOCs encountered by test herbivores likely differ from the ratio of these compounds in natural plant VOC bouquets. Adult O. melanopus were attracted to the lowest tested concentration (1 ng · min−1 of each GLV = 60 ng · h−1) of a synthetic blend of three GLVs. This amount is comparable to emission levels after CJ exposure in the plant VOC induction experiment, and to emission levels from barley or wheat infected by Fusarium spp. (Piesik et al., 2011b; 2013). When Z3HAC or Z3HAL (two of three GLVs in our test blend) was presented singly, adult O. melanopus were attracted to three test doses (Piesik et al., 2011a). However, adults were repelled by two much higher doses (25 ng · min−1 = 1500 ng · h−1 (only females); 125 ng · min−1 = 7500 ng · h−1) of our GLV blend. These levels are much higher than previously reported from barley and wheat plants after O. melanopus herbivory (Piesik et al., 2010a), or maize with Fusarium spp. infection (Piesik et al., 2011a).

Female O. melanopus were significantly repelled by the three highest test concentrations (two for males) of a second synthetic blend of 4 terpenes + IND. When LIN or EβFAR (two terpenes in this test blend) was presented singly, adult O. melanopus were attracted to three doses or one test dose, respectively (Piesik et al., 2011a). It may be that a blend of terpenes and IND provide a greater contribution than GLVs to plant repellency after CJ exposure or biotic attack. Nonetheless, GLV still may influence O. melanopus host plant attraction. A single VOC can be attractive to an herbivore, and when added to a VOC blend of an uninjured plant, may render an otherwise constitutive plant VOC bouquet attractive to the same herbivore (e.g., LIN for Spodoptera frugiperda; Carroll et al., 2006). There are often synergistic effects of VOC blends on insect attraction or repellence, so only certain VOCs of a plant bouquet may influence insect behavior (Tasin et al., 2007; Bruce and Pickett, 2011). Our work contributes to understanding how O. melanopus behaviorally responds to cereal plant VOC bouquets, and whether certain synthetic, subset VOC blends can generate the same responses in this beetle as complete plant bouquets.

Notes

Acknowledgments

We thank R.B. Srygley for performing the ARS paper review, as well as two anonymous reviewers for providing comments that improved previous drafts of this manuscript. This research was partly supported with funds provided by the Ministry of Science and Higher Education (contract number 1648/B/P01/2010/39 entitled, ‘Effect of volatile organic compounds released by green and floral parts of Brassica napus on behavior of Meligethes aeneus F.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kevin J. Delaney
    • 1
  • Maria Wawrzyniak
    • 2
  • Grzegorz Lemańczyk
    • 2
  • Danuta Wrzesińska
    • 2
  • Dariusz Piesik
    • 2
  1. 1.Pest Management Research Unit, Northern Plains Agricultural Research LabUSDA-ARSSidneyUSA
  2. 2.Department of Entomology and Molecular PhytopathologyUniversity of Technology and Life SciencesBydgoszczPoland

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