Duration of Emission of Volatile Organic Compounds From

Journal of Plant Physiology 188 (2015) 19–28

Contents lists available at ScienceDirect

Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Physiology

Duration of emission of volatile organic compounds from

mechanically damaged plant leaves

∗,1

Lincoln Smith , John J. Beck

USDA Agricultural Research Service, Western Regional Research Center, 800 Buchanan St. Albany, CA 94710, USA

a r t i c l e i n f o a b s t r a c t

Article history: Classical biological control of invasive alien weeds depends on the use of arthropod herbivores that

Received 7 July 2015

are sufficiently host specific to avoid risk of injuring nontarget plants. Host plant specificity is usually

Received in revised form 11 August 2015

evaluated by using a combination of behavioral and developmental experiments under choice, no-choice

Accepted 15 August 2015

and ﬁeld conditions. Secondary plant compounds are likely to have an important inﬂuence on host plant

Available online 21 August 2015

speciﬁcity. However, relatively little is known about the volatile organic compounds (VOCs) that are

emitted by target and nontarget plants, and how environmental conditions may affect their emission.

Keywords:

Previous studies have shown that mechanical damage of leaves increases the composition and content

Biological control

of VOCs emitted. In this study we measured the VOC emissions of ﬁve species of plants in the subtribe

Volatile organic compounds

Centaureinae (Asteraceae) – Carthamus tinctorius, Centaurea cineraria, Centaurea melitensis, Centaurea

Mechanical damage

Host plant speciﬁcity rothrockii, and Centaurea solstitialis – that have previously been used in host speciﬁcity experiments

Sesquiterpene for a prospective biological control agent of yellow starthistle (C. solstitialis). Leaves of each plant were

punctured with a needle and the VOCs were collected by solid-phase microextraction (SPME) periodically

over 48 h and analyzed by GC–MS. A total of 49 compounds were detected. Damage caused an immediate

increase of 200–600% in the composition of VOCs emitted from each plant species, and the amounts

generally remained high for at least 48 h. The results indicate that a very unspeciﬁc mechanical damage

can cause a prolonged change in the VOC proﬁle of plants.

Published by Elsevier GmbH.

1. Introduction growing evidence that secondary plant compounds play an impor-

tant role in determining which plants are acceptable to oligophagus

In order to use phytophagous arthropods as classical biolog- arthropods (Bernays and Chapman, 1994; Schoonhoven et al., 2006;

ical control agents to control invasive alien weeds, it is critical Wheeler and Schaffner, 2013). Volatile organic compounds (VOCs)

to determine that they are sufﬁciently host speciﬁc in order are known to attract both phytophagous arthropods and some of

to avoid the risk of attacking nontarget plants (Horner, 2004; their natural enemies, and there have been many reports on the lat-

Sheppard et al., 2005). Host plant speciﬁcity is usually evalu- ter phenomenon (e.g., Tumlinson, 1991; Turlings et al., 1990, 1993;

ated by a series of complementary no-choice, choice and ﬁeld Kugimiya et al., 2010; Mumm and Dicke, 2010; van Dam et al.,

experiments to assess the risk that the prospective agent will dam- 2010; Hare, 2011). However, relatively little is known about the

age nontarget plants (Withers et al., 1999; van Klinken, 2000; role of plant secondary metabolites and the speciﬁcity of biologi-

Schaffner, 2001; Spafford Jacob and Briese, 2003; Briese, 2005). cal control agents of weeds (Wheeler, 2005; Andreas et al., 2008;

Such experiments provide empirical evidence that they are useful Park et al., 2012; Rapo et al., 2012; Wheeler, 2012; Wheeler and

for obtaining regulatory approval to use the agent. Neverthe- Schaffner, 2013; Wheeler et al., 2014).

less, host speciﬁcity experiments do not provide an explanation VOCs emitted by plants can act as attractants or repellants to

why an agent prefers one plant species over another. There is phytophagous insects (e.g., Visser, 1986; Mitchell, 1994; Zhang and

McEvoy, 1995; Moyes and Raybould, 2001; van Tol et al., 2002;

Heil, 2004; Junker and Blüthgen, 2008; Otalora-Luna et al., 2009;

Piesik et al., 2011a,b, 2012). Although undamaged plant leaves typ-

∗

Corresponding author. Fax: +33 499623049. ically emit VOCs, physical damage usually causes them to emit

E-mail address: [email protected] (L. Smith).

additional compounds or greater amounts. Most of the studies

Present address: USDA Agricultural Research Service, European Biological Con-

of this phenomenon have focused on the multitrophic interac-

trol Laboratory, 810 Avenue du Campus Agropolis, 34980 Montferrier-sur-Lez,

France. tions between host plant, herbivore and natural enemies of the

http://dx.doi.org/10.1016/j.jplph.2015.08.003

0176-1617/Published by Elsevier GmbH.

20 L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28

herbivore (parasitoids and predators). However, experiments on 22-gauge needle then starting the collection of VOCs at 0, 4, 8, 24

the host speciﬁcity of biological control agents of weeds typically and 48 h afterward (Beck et al., 2008). Plants were damaged in the

use intact plants or plant cuttings without much concern that early morning so that samples for 0, 24 and 48 h were collected

this would substantially affect the relative attractiveness of the in the morning, 4 h was in the afternoon, and 8 h was after sun-

plants (Palmer, 1999). A previous study showed that mechanical set. Volatiles were collected from four replicates of C. tinctorius and

damage of leaves greatly changed the proﬁle (composition and from two replicates of each of the other plant species.

content) of VOCs emitted from three species of test plants (Beck

et al., 2008). Multivariate statistical analysis indicated that the plant 2.2. Collection of volatiles

species could be more easily distinguished when they had been

damaged, and that different VOCs were involved for discrimina- Volatiles were adsorbed onto solid-phase microextraction

tion of damaged versus undamaged plants (Smith and Beck, 2013). (SPME) (Supelco, Bellefonte, PA; 100 ␮m, polydimethylsiloxane)

Furthermore, the type of mechanical damage (cut, punctured or ﬁbers using methods identical to previously published methods

scraped) had relatively little effect on the VOC profiles, albeit cut (Beck et al., 2008; Smith and Beck, 2013). Briefly, a Teflon bag (SKC

leaves generally emitted fewer VOCs than punctured or scraped West, Fullerton, CA) was placed over a leaf and gently sealed over

leaves. the stem using a twist tie. Five minutes after leaf enclosure a SPME

There are few studies regarding the duration of the change in ﬁber was inserted and the control (undamaged) headspace volatiles

VOC emissions of damaged plants (Turlings et al., 1998; Schaub collected for 55 min. Immediately after the control headspace col-

et al., 2010). VOC emission is thought to occur during one of lection the bagged leaf was damaged as described above and 5 min

three time scales, depending on the synthetic pathway utilized and after damage the headspace volatiles collected onto the SPME ﬁber

whether the compounds are stored in plant structures (Baldwin, for 55 min.

1994). “Preformed-induced responses” occur immediately after

damage and are restricted to the damaged tissue. “Rapidly-induced 2.3. Analysis of collected volatiles

responses” occur within hours or days of the injury and can be

either systemic or restricted to the damaged leaf. “Delayed-induced For all experiments the adsorbed volatiles were immediately

responses” do not occur until the next growing season of a perennial analyzed (less than 1 min after collection) via gas chromatography-

plant. Herbivore-induced damage is expected to produce responses mass spectrometry (GC–MS) using identical methods previously

for long enough to improve plant ﬁtness by helping to protect published (Beck et al., 2008; Smith and Beck, 2013). NIST and

against herbivore damage (Karban and Baldwin, 1997; Agrawal, Wiley databases were initially used for fragmentation pattern

2000). However, simple mechanical damage is expected to release identiﬁcation. The retention indices (RIs) were calculated using

primarily “green leaf volatiles” that evaporate from the contents of a homologous series of n-alkanes on DB-Wax and DB-1 columns.

damaged cells, resulting in immediate emission that stops within Volatile identiﬁcations were veriﬁed by injection of authentic sam-

a few hours (Turlings et al., 1998; Dudareva et al., 2006). ples for comparison of retention times, retention indices, and

There have been few studies on the duration of changes in VOC fragmentation patterns. Compounds that we could not authenticate

proﬁles of plants that have been mechanically damaged. Given that with commercial or isolated standards were labeled as either tenta-

host speciﬁcity experiments are usually conducted for periods last- tive or unknown in Table 1. The labeling of unknown sesquiterpenes

ing several days, we wanted to determine if mechanical damage coincides with an earlier report (Smith and Beck, 2013) and lists the

would result in changes that persisted during most of this period. new unknown sesquiterpenes in alphabetical order and ascending

The purpose of this study was to measure changes in VOC emis- order of RI value.

sions during 48 h after mechanically puncturing leaves of ﬁve plant Data from GC–MS analyses were transferred to Microsoft Excel

species that represent a variety of phylogenetic clades within the for comparison of retention times and compound identiﬁcation for

tribe Centaureinae. These plant species have previously been used same-column analysis. Calculated RIs were used to assist in com-

to evaluate the host plant speciﬁcity of a weevil (Ceratapion basi- pound identiﬁcation and to perform comparison of DB-1 to DB-Wax

corne (Illiger), Coleoptera: Apionidae) as a prospective biological column results. Compounds consistent through all replicates are

control agent of yellow starthistle (Centaurea solstitialis L., Aster- included in Table 1. The GC–MS data were error-checked by plot-

aceae) (Smith, 2007; 2011; Cristofaro et al., 2013). ting the area of each identiﬁed peak from DB-Wax versus that from

DB-1. Outliers from the regression line were reviewed for errors

of interpretation or transcription and were corrected when appro-

2. Materials and methods

priate. The area of the peak on DB-Wax was used as the response

variable because it was generally larger than that on DB-1 (on aver-

Plants were grown from seed in ﬂower pots under outdoor 6

age by a factor of 2.1). Peaks with areas less than about 1 × 10 were

conditions and were tested indoors in the rossette stage of

not reliably detected, as was our previous experience (Smith and

development. The species that were evaluated – Carthamus tinc-

Beck, 2013).

torius L. (safﬂower varieties Seedtec 518 [oleic], CalWest 4440

[linoleic]), Centaurea cineraria L. (dusty miller), Centaurea melitensis

2.4. Statistical analysis

L. (tocalote; Napa thistle), Centaurea rothrockii Greenm. (Rothrock’s

basket ﬂower), and C. solstitialis – represent a variety of close rel-

The area of GC–MS peaks was transformed by log (area + 1) to

atives that were previously evaluated for host plant preference for

improve normality by reducing skewness and kurtosis, which also

a prospective insect biological control agent, the weevil Ceratapion

reduced the coefﬁcient of variation (CV = sd/mean). The signiﬁcance

basicorne, which feeds on and oviposits in leaves of C. solstitialis

of plant species, treatment and their interaction were analyzed by

(Smith, 2007).

GLM using plant species and treatment as classiﬁed effects fol-

lowed by 1-way GLM analysis of the effect of treatment on each

2.1. Leaf damage VOC for each species (SAS Institute, 2003). Contrasts were used to

test speciﬁc hypotheses: control (undamaged leaves) vs. 0 h to test

The effect of mechanical damage on production of VOCs by an the immediate effect of puncture damage; 0 h vs. 4 h, 0 h vs. 24 h

intact leaf was evaluated by use of six treatments: no damage (con- and 0 h vs. 48 h to test for delayed changes in VOCs after puncturing;

trol, Ctrl), and puncturing an intact leaf ten times with a sterile (4 h and 24 h) vs. 8 h to test the effect of morning vs. evening; and

L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28 21

Table 1

Identiﬁcation of VOCs absorbed on SPME that were detected by GC–MS (RT – retention time on DB-wax column (min), RI – retention index; sorted alphabetically by class,

structure, compound identity). The labeling of unknown sesquiterpenes coincides with an earlier report (Smith and Beck, 2013) and lists the new unknown sesquiterpenes

in alphabetical order and ascending order of RI value.

RT RI Compound identity Class Structure

20.24 1545 1-Pentadecene Alkene Acyclic

17.18 1446 1-Tetradecene Alkene Acyclic

16.06 1404 (E)-3-Hexen-1-ol GLV Alcohol

15.37 1381 (Z)-3-Hexen-1-ol GLV Alcohol

13.20 1312 (Z)-3-Hexenyl acetate GLV Ester

17.68 1458 (Z)-3-Hexenyl butyrate GLV Ester

18.53 1486 (Z)-3-Hexenyl isovalerate GLV Ester

18.06 1470 (Z)-3-Hexenyl 2-methylbutyrate GLV Ester

20.37 1550 Linalool Mono Acyclic

11.19 1245 (E)-␤-Ocimene Mono Acyclic

26.02 1744 (E,E)-␣-Farnesene Sesq Acyclic ␤

23.74 1662 (E)- -Farnesene Sesq Acyclic

17.93 1467 ␦-Elemene Sesq Monocyclic

24.85 1707 Germacrene-d Sesq Monocyclic

23.74 1666 ␣-Humulene Sesq Monocyclic

␣

21.39 1582 (E)- -Bergamotene Sesq Bicyclic

20.90 1565 (Z)-␣-Bergamotene Sesq Bicyclic

25.55 1732 Bicyclogermacrene Sesq Bicyclic

18.26 1478 Bicycloelemene Sesq Bicyclic

26.24 1752 ␦-Cadinene Sesq Bicyclic

26.56 1767 ␥-Cadinene Sesq Bicyclic

21.69 1594 ␤-Caryophyllene Sesq Bicyclic

␣

25.33 1722 -Muurolene (tentative) Sesq Bicyclic

24.33 1685 ␥-Muurolene Sesq Bicyclic

21.98 1605 Aromadendrene Sesq Tricyclic

21.54 1589 Calarene Sesq Tricyclic

32.05 1986 Caryophyllene oxide (tentative) Sesq Tricyclic

␣

18.57 1490 -Copaene Sesq Tricyclic

17.55 1456 ␣-Cubebene Sesq Tricyclic

19.98 1536 ␤-Cubebene Sesq Tricyclic

19.60 1525 Cyperene Sesq Tricyclic

19.72 1528 ␣-Gurjunene Sesq Tricyclic

19.60 1524 Sativene Sesq Tricyclic

21.02 1570 ␤-Ylangene Sesq Tricyclic

18.25 1480 Cyclosativene Sesq Tetracyclic

18.41 1485 Unknown sesquiterpene A Sesq –

19.62 1526 Unknown sesquiterpene A Sesq –

22.51 1623 Unknown sesquiterpene B Sesq –

23.59 1661 Unknown sesquiterpene B Sesq –

23.96 1674 Unknown sesquiterpene C Sesq –

24.21 1682 Unknown sesquiterpene D Sesq –

24.28 1684 Unknown sesquiterpene D Sesq –

24.54 1694 Unknown sesquiterpene D Sesq –

24.60 1696 Unknown sesquiterpene E Sesq –

25.14 1717 Unknown sesquiterpene E Sesq –

25.22 1718 Unknown sesquiterpene E Sesq –

25.41 1725 Unknown sesquiterpene F Sesq –

27.68 1805 Unknown hydrocarbon G Alkane –

27.63 1810 Unknown hydrocarbon H Alkane –

control vs. (0 h, 4 h, 8 h, 24 h and 48 h) to test the sustained effect general structures: 2 acyclic; 3 monocyclic, 9 bicyclic, 10 tri-

of puncturing. GLM was also used to test the effect of time since cyclic, 1 tetracyclic, and 12 unknown sesquiterpenes. The unknown

puncturing (using 0 h, 4 h, 8 h, 24 h and 48 h) as a quantitative vari- sesquiterpenes were classiﬁed as such due to their distinctive mass

able on peak size for each VOC for each plant species. Differences fragments (see Table 2) and are labeled in accordance with other

among plant species in the means of all punctured treatments (0 h unknown sesquiterpenes previously detected and reported from

to 48 h) were analyzed by multiple comparisons using the Ryan- several Centaurea species (Smith and Beck, 2013).

Einot-Gabriel-Welsch multiple range test (REGWQ) for each VOC The VOC peaks detected for each treatment of each plant

that occurred in at least two plant species. species are shown in Fig. 1. Overall, both plant species and

treatment signiﬁcantly affected VOC peaks (two-way GLM,

F = 33.49, P < 0.0001, plant: F = 36.54, P < 0.0001; treat-

3. Results (9,4358) (4,4358)

ment: F(5,4358) = 31.04, P < 0.0001). In general, VOC peaks for

undamaged leaves were signiﬁcantly lower than for punctured

A total of 49 volatiles were reliably detected among the ﬁve

leaves at 0 h or at 0 to 48 h (P < 0.001). All other contrasts

plant species and over the 48 h period of volatile analyses (Table 1).

were nonsigniﬁcant, indicating that, in general, there was no dif-

The 49 identiﬁed volatiles were delineated into four broad chemical

ference between morning and evening, nor among punctured

classes (number of compounds in that class): alkenes (2); unknown

leaves at different times (0–48 h after puncturing). Fig. 2 shows

hydrocarbons (2); green leaf volatiles and various associated ester

results for the eight most common VOCs detected from the ﬁve

analogs (6); monoterpenes (2), both acyclic; and sesquiterpenes

plant species ((Z)-3-hexen-1-ol, ␣-copaene, ␤-caryophyllene, ␣-

(37). The sesquiterpenes, which comprised the vast majority of

humulene, germacrene-d, bicylcogermacrene, (E,E)-␣-farnesene,

the volatiles emitted, were further delineated into the following

22 L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28

(A) 1.E+10

Carthamus tinctorius

ctr 0 4 8 24 48 1.E+09 area 1.E+08 peak 1.E+07 VOC 1.E+06

1.E+05

1381 1404 1446 1458 1470 1486 1545 1594 1666 1752 1805 1986

VOC DB-wax RI value

1.E+10

Centaurea solstitialis ctr 0 4 8 24 48 1.E+09 area

1.E+08 peak 1.E+07 VOC 1.E+06

1.E+05

1312 1381 1446 1458 1467 1490 1528 1545 1565 1594 1662 1682 1685 1707 1717 1725 1732 1744 1752 1767

VOC DB-wax RI value

(B) 1010

Centaurea cineraria

109 ctr 0 4 8 24 48 area 108

peak 7 10

6 VOC 10

105

1312 1381 1458 1470 1480 1485 1490 1526 1528 1536 1570 1582 1589 1594 1623 1666 1682 1684 1685 1696 1707 1722 1725 1732 1744 1752

VOC DB-w ax RI value

1.E+10

Centaurea melitensis

1.E+09 ctr 0 4 8 24 48 area 1.E+08 peak 1.E+07

VOC 1.E+06

1.E+05

1312 1381 1456 1480 1485 1490 1524 1528 1536 1545 1570 1589 1594 1605 1666 1674 1685 1694 1707 1717 1722 1725 1732 1744 1752

VOC DB-w ax RI value

1.E+10

Cen taurea rothrockii

1.E+09 ctr 0 4 8 24 48 area 1.E+08 peak 1.E+07

VOC 1.E+06

1.E+05

1245138114561458147014781490152515281536154515501570158915941605166116661685169417071717171817251732174417521810

VOC DB-w ax RI value

Fig. 1. Effect of puncturing and time elapsed after puncturing on VOCs emitted from intact leaves of ﬁve plant species. The 8 h reading was taken after sunset; the 0, 24 and

48 h readings were taken in the morning; and the 4 h reading was in the afternoon. “Ctrl” = undamaged leaf. Compound names corresponding to RI values on the x-axis are

listed in Table 1. (mean ± SE).

L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28 23

1E+10 1381 1E+9 1382: (Z)-3-hexen-1-ol

1E+8

1E+7

1E+6

1E+5

ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48

1E+10 1490:1490 α-copaene 1E+9

1E+8

1E+7

1E+6

1E+5

ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48

1E+10 1594 1E+9 1594: β-caryophyllene

1E+8

1E+7

1E+6

1E+5

ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 1E+10 1E+9 1666:1666 α-humulene 1E+8

1E+7

1E+6

1E+5

ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48

C. cineraria C . melitensis C. rothrockii C. solstitialis Ca. tinctorius 1E+10 1E+9 1707:1707 germacrene-D 1E+8 1E+7 1E+6

1E+5

ctr 048 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 048 24 48

1E+10 1732 1E+9 1732: bicylcogermacrene 1E+8 1E+7 1E+6

1E+5

ctr 0 4 8 24 48 ctr 0 4 8 24 48 ctr 0 4 8 24 48 ctr 0 4 8 24 48 ctr 0 4 8 24 48

1E+10

1E+9 1744:1744 (E,E)- α-farnesene 1E+8 1E+7 1E+6

1E+5

ctr 0 4 8 24 48 ctr 0 4 8 24 48 ctr 048 24 48 ctr 0 4 8 24 48 ctr 0 4 8 24 48

1E+10 1E+9 1752:1752 δ-cadinene 1E+8 1E+7 1E+6

1E+5

ctr 048 24 48 ctr 048 24 48 ctr 048 24 48 ctr 048 24 48 ctr 0 4 8 24 48

C. cineraria C . melitensis C. rothrockii C. solstitialis Ca. tinctorius

Fig. 2. Eight most commonly detected VOCs from intact leaves of ﬁve plant species (same data as in Fig. 1; mean ± SE).

24 L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28

Table 2

Mass fragmentation (m/z) patterns of the unknown sesquiterpenes. Detected by electron impact mass spectrometry. The fragment abundances relative to the base peak are shown in parentheses.

IDa RIb Fragments

A 1485 105 (100) 161 (82) 204 (55) 93 (53) 106 (42) 119 (36) 133 (36) 189 (27)

A 1526 137 (100) 121 (29) 95 (21) 136 (18) 138 (1) 180 (13) 161 (5) 204 (5)

B 1623 133 (100) 204 (39) 107 (30) 79 (30) 135 (28) 161 (20) 105 (22) 189 (10)

B 1661 161 (100) 79 (47) 105 (46) 91 (46) 119 (27) 137 (26) 204 (26) 189 (15)

C 1674 189 (100) 133 (56) 204 (44) 91 (44) 99 (44) 105 (44) 147 (29) 161 (25)

D 1682 69 (100) 93 (84) 133 (58) 161 (55) 120 (45) 41 (45) 79 (40) 204 (12)

D 1684 136 (100) 121 (64) 161 (46) 91 (33) 105 (24) 137 (24) 204 (13) 189 (10)

D 1694 105 (100) 107 (98) 161 (79) 93 (76) 119 (75) 135 (64) 189 (64) 204 (50)

E 1696 123 (100) 94 (45) 121 (32) 93 (30) 107 (20) 161 (11) 189 (11) 204 (8)

E 1717 107 (100) 93 (95) 105 (93) 79 (91) 204 (79) 189 (77) 121 (71) 133 (20)

E 1718 91 (100) 161 (90) 93 (86) 119 (85) 105 (85) 120 (76) 133 (72) 204 (64)

F 1725 93 (100) 119 (88) 69 (48) 107 (40) 91 (40) 79 (36) 105 (33) 204 (9)

G 1805 67 (100) 81 (93) 95 (62) 110 (40) 121 (23) 135 (22) 149 (13) 163 (10)

H 1810 68 (100) 81 (49) 41 (27) 79 (13) 95 (13) 107 (9) 137 (9)

Label corresponding to compound identities in Table 1.

Retention index values from a DB-Wax column.

and ␦-cadinene). In general, there were no differences among the lower amounts from undamaged plants than from damaged plants.

plant species in the effect of treatments on VOC peaks for a given Piesik et al. (2010) observed that a 24-h exposure to leaf feed-

compound, if the compound was present (plant × treatment inter- ing beetles stimulated higher production of VOCs than mechanical

action). In other words, all plant species responded similarly in that damage of several grasses, and that both types of damage resulted

the concentration of each VOC did not signiﬁcantly change dur- in much more emission than undamaged plants. Similarly, a 24-h

ing 0–48 h post damage within each species. The plant × treatment exposure of yellow starthistle plant leaves to slugs resulted in the

interaction was signiﬁcant only for ␦-cadinene (1752), which was detection of four sesquiterpenes from the damaged plant, whereas

emitted by undamaged control plants of only C. tinctorius. no volatiles were detected in the nondamaged plant (Oster et al.,

Results of statistical analyses of each VOC for each plant species 2014).

are summarized in Table 3 . There were few cases in which the

amount of a VOC detected from an undamaged leaf was similar to 4.2. Time response of damaged leaves

that of punctured leaves and greater than 1 × 10 (which was the

lower limit of reliable detectability) (contrast: ctrl vs. all others): Turlings et al. (1998) recorded VOC emissions of maize culti-

␤

(E)- -ocimene (RI = 1245), (Z)-3-hexenyl butyrate (1458), linalool vars every 30 min for 12 h after damaging a leaf and applying oral

(1550) and unknown hydrocarbon H (1810) in C. rothrockii, and regurgitant from Spodoptera littoralis (Boisd.) caterpillars. Green

␦

-cadinene (1752) in C. tinctorius. For the other species, the levels leaf volatiles, such as (Z)-3-hexen-1-ol were emitted immediately,

detected in undamaged leaves were below 1 10 , and were not but amounts rapidly decreased to undetectable levels within 2 to

signiﬁcantly different from zero. In these cases, the contrast ‘ctrl 3 h. On the other hand, terpenoids were not immediately emit-

v all others’ was not signiﬁcant because of the large error for ctrl. ted, but gradually increased, peaking at 3 to 8 h after leaf damage.

Only 14 to 33% of the VOCs that were detected in punctured leaves Our results are dramatically different. Green leaf volatiles were

were detected from undamaged leaves in these ﬁve species. emitted at similar levels from 0 to 48 h after damage. However,

No VOCs from C. cineraria or C. tinctorius differed in the evening those that were near or below 1 × 10 were not always detected.

versus morning (contrast: [4 h and 24 h] vs. 8 h). (Z)-3-Hexenyl All the sesquiterpenes that were observed in a given plant species

acetate (1312) was signiﬁcantly lower, and bicyclogermacrene were about as abundant in the 0 h sample as in the other post-

(1732) was signiﬁcantly higher, in the evening in C. solstitialis. For damage samples (see also Table 3: contrast 0 h vs. 4 h). We did not

␣ ␤

C. melitensis, -gurjunene (1528), -cubebene (1536), unknown observe a signiﬁcant increase or peaking of the VOCs, except for

␥

sesquiterpene C (1674), -muurolene (1685), unknown sesquiter- the 8 h (evening) samples for bicyclogermacrene in C. solstitialis and

␣ ␦

penes D (1694), -muurolene (1722), and -cadinene (1752) were seven VOCs (reported above) in C. melitensis. Thus, these ﬁve plants,

signiﬁcantly higher in the evening; however, none of these VOCs which are all in the subtribe Centaureinae of Asteraceae (Garcia-

showed a similar pattern in the other plants tested. Jacas et al., 2001), appear to behave quite differently from maize

The amount of VOCs emitted usually differed among species that (Poaceae). Although Turlings et al. (1998) were studying induction

emitted the same VOC (Table 3, Plant main effect; Fig. 2). of herbivore-speciﬁc VOC emissions and we are interested in gen-

eral emissions, the general difference in temporal pattern between

4. Discussion these two studies is noteworthy.

Pareja et al. (2007) reported that cutting the tip of a

4.1. Damaged versus undamaged leaves leaf of Centaurea nigra caused an increase in some VOCs (1-

pentadecene, 1-hexadecene, germacrene-d, (E,E)-␣-farnesene, and

Our ﬁndings were consistent with previous results regarding ␤-caryophyllene) during the ﬁrst 24 h, which then decreased to the

the low number of VOCs detected from undamaged leaves com- levels of undamaged plants on the second and third day. Centaurea

pared to those from mechanically damaged leaves (Beck et al., nigra is in the “Jacea pollen type”, a subgroup of the Centaureinae

2008; Piesik et al., 2010; Smith and Beck, 2013). In our study, that includes C. cineraria, C. melitensis and C. solstitialis (Villodre and

green leaf volatiles ((Z)-3-hexenyl acetate (1312), (Z)-3-hexen-1- Garcia-Jacas, 2000), so we would expect this plant to behave sim-

ol (1381), (E)-3-hexen-1-ol (1404), (Z)-3-hexenyl butyrate (1458), ilarly to the others that we tested. Thus, it is surprising that these

(Z)-3-hexenyl 2-methylbutyrate (1470), and (Z)-3-hexenyl iso- authors observed a decrease in VOCs at 48 h, whereas we did not.

valerate (1486)) were often not emitted by undamaged plants of However, Pareja et al. stated that their results were not quanti-

species that emitted them when damaged. Most sesquiterpenes ﬁed, nor were any values from the mechanically damaged plants

were either not emitted from undamaged plants, or were emitted in reported in their paper. The reporting of quantitative evidence by

L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28 25

Table 3

Statistical results from GLM analysis of the effect of plant species, treatment and their interaction on each VOC peak that occurred in at least two plant species, followed by

treatment contrasts within each plant species. “Trt” = P-value of main effect; contrasts: control (“Ctrl”) vs. 0 h to test the immediate effect of puncturing; control vs. (0 h, 4 h,

8 h, 24 h and 48 h) to test the sustained effect of puncturing; 0 h vs. 4 h to test for a delayed change in VOCs after puncturing; and (4 h and 24 h) vs. 8 h to test the effect of

morning vs. evening. P-values less than 0.05 are in bold text.

GLM model Plant Treatment Plant × Treatment

VOC df1 df2 MS F P df1 F P df1 F P df1 F P

1245

1312 17 18 10.2 1.7 0.015 2 8.6 0.002 5 1.4 0.288 10 0.4 0.919

1381 23 36 15.1 3.7 0.000 3 3.3 0.030 5 12.1 0.000 15 1.0 0.495

1404

1446 11 24 19.4 4.1 0.002 1 14.8 0.001 5 5.4 0.002 5 0.6 0.713

1456

1458 11 23 10.0 1.0 0.507 1 4.4 0.048 5 0.5 0.790 5 0.8 0.589 1467 1470

1478

1480 11 12 11.2 6.3 0.002 1 2.9 0.115 5 12.6 0.000 5 0.7 0.611

1485 11 12 14.2 198.8 0.000 1 16.4 0.002 5 432.9 0.000 5 1.2 0.350

1486

1490 23 24 12.6 16.2 0.000 3 3.5 0.032 5 70.0 0.000 15 0.9 0.581 1524 1525

1526

1528 17 18 15.0 3.3 0.008 2 6.1 0.010 5 7.3 0.001 10 0.8 0.632

1536 11 12 14.7 231.2 0.000 1 5.5 0.037 5 506.2 0.000 5 1.3 0.313

1545 17 30 21.1 5.3 0.000 2 16.7 0.000 5 10.2 0.000 10 0.6 0.767 1550 1565 1570

1582

1589 11 12 12.5 3.6 0.019 1 0.0 0.864 5 6.7 0.003 5 1.2 0.381

1594 29 42 10.7 5.6 0.000 4 9.1 0.000 5 19.9 0.000 20 1.3 0.235 1605 1623 1661

1662

1666 23 36 15.6 27.5 0.000 3 8.1 0.000 5 118.4 0.000 15 1.0 0.484 1674 1682

1684

1685 17 18 15.8 6.5 0.000 2 4.9 0.020 5 15.7 0.000 10 2.2 0.067 1694

1696

1707 23 24 7.1 2.8 0.007 3 1.2 0.329 5 11.0 0.000 15 0.4 0.963

1717 11 12 13.6 7.7 0.001 1 0.6 0.441 5 15.5 0.000 5 1.2 0.351

1718

1722 11 12 14.7 316.8 0.000 1 33.5 0.000 5 688.2 0.000 5 2.0 0.144

1725 17 18 15.5 6.4 0.000 2 5.9 0.010 5 17.7 0.000 10 0.8 0.595

1732 23 24 14.1 16.4 0.000 3 5.1 0.007 5 69.5 0.000 15 0.9 0.544

1744 23 24 15.5 4.3 0.000 3 6.1 0.003 5 14.7 0.000 15 0.5 0.935

1752 29 42 12.0 7.1 0.000 4 3.9 0.008 5 23.1 0.000 20 3.7 0.000 1767 1805 1810

1986

C. cineraria C. melitensis C. rothrockii

VOC Ctrl vs. Ctrl vs. all 0 h vs. 4 & Trt Ctrl vs. Ctrl vs. all 0 h vs. 4 & 24 h vs. Trt Ctrl vs. Ctrl vs. 0 h vs. 4 & 24 h vs. Trt

0 h others 4 h 24 h vs. 0 h others 4 h 8 h 0 h all 4 h 8 h

8 h others

1245 0.544 0.582 0.198 0.598 1.132

1312 0.097 0.046 0.962 0.774 0.7610.367 0.234 0.920 0.988 1.449

1381 0.278 0.044 0.270 0.452 0.4110.325 0.122 0.942 0.285 0.874 0.267 0.975 0.043 0.527 0.291 1404

1446

1456 0.340 0.239 0.963 0.121 0.825 0.020 0.009 0.902 0.122 0.146

1458 0.406 0.406 0.946 0.360 1.665 0.302 0.603 0.281 0.494 1.261

1467

1470 0.267 0.120 0.323 0.088 0.392 0.425 0.406 0.989 0.962 1.666

1478 1.000 0.025 0.018 0.133 0.072

1480 0.000 0.000 0.323 0.693 0.0000.052 0.022 0.938 0.790 0.460

1485 0.000 0.000 0.418 0.569 0.0000.000 0.000 0.563 0.078 0.000

1486

1490 0.000 0.000 0.495 0.764 0.0000.041 0.017 0.933 0.809 0.370 0.000 0.000 0.478 0.537 0.000

1524 0.260 0.251 0.970 0.075 0.415

26 L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28

Table 3 (Continued)

C. cineraria C. melitensis C. rothrockii

VOC Ctrl vs. Ctrl vs. all 0 h vs. 4 & Trt Ctrl vs. Ctrl vs. all 0 h vs. 4 & 24 h vs. Trt Ctrl vs. Ctrl vs. 0 h vs. 4 & 24 h vs. Trt

0 h others 4 h 24 h vs. 0 h others 4 h 8 h 0 h all 4 h 8 h

8 h others

1525 0.000 0.000 0.663 0.615 0.000

1526 0.271 0.111 0.271 0.087 0.341

1528 0.363 0.468 0.363 0.585 1.4080.000 0.000 0.624 0.030 0.000 1.000 0.246 0.365 0.610 0.887

1536 1.000 0.468 1.000 0.597 1.4080.000 0.000 0.477 0.027 0.000 0.000 0.000 0.432 0.644 0.000

1545 0.358 0.129 0.401 0.598 0.960 1.000 0.551 1.000 0.567 1.174

1550 0.260 0.255 0.925 0.948 1.139

1565

1570 1.000 0.550 1.000 0.503 1.1750.260 0.106 0.970 0.066 0.311 0.019 0.009 0.941 0.127 0.147

1582 0.000 0.000 0.611 0.559 0.000

1589 1.000 0.469 0.327 0.654 1.4060.014 0.007 0.135 0.268 0.111 0.020 0.009 0.902 0.122 0.144

1594 0.013 0.006 0.128 0.341 0.1010.046 0.018 0.938 0.766 0.397 0.000 0.000 0.314 0.475 0.001

1605 0.281 0.551 0.281 0.195 1.175 1.000 0.025 0.018 0.130 0.070

1623 0.270 0.105 0.270 0.074 0.309

1661 1.000 0.251 0.363 0.631 0.925

1662

1666 0.000 0.000 0.600 1.000 0.0000.045 0.017 0.978 0.755 0.379 0.000 0.000 0.481 0.917 0.000

1674 0.000 0.000 0.766 0.018 0.000

1682 0.053 0.049 0.941 0.546 0.485

1684 0.352 0.121 0.964 0.281 0.864

1685 0.013 0.021 0.013 0.988 0.0550.000 0.000 0.116 0.001 0.000 0.020 0.009 0.885 0.121 0.143

1694 0.134 0.219 0.134 0.006 0.097 1.000 0.110 0.052 0.226 0.338

1696 0.262 0.045 0.971 0.441 0.424

1707 0.026 0.009 0.936 0.988 0.2310.001 0.000 0.772 0.187 0.007 0.022 0.006 0.896 0.964 0.167

1717 0.012 0.006 0.917 0.218 0.090 0.000 0.000 0.272 0.658 0.000

1718 1.000 0.109 0.053 0.539 0.331

1722 0.000 0.000 0.463 0.746 0.0000.000 0.000 0.569 0.025 0.000

1725 0.057 0.051 0.957 0.548 0.5100.000 0.000 0.830 0.205 0.000 0.000 0.000 0.398 0.536 0.000

1732 0.000 0.000 0.651 0.441 0.0000.042 0.016 0.957 0.747 0.352 0.000 0.000 0.337 0.773 0.000

1744 0.000 0.000 0.649 0.860 0.0000.000 0.000 0.858 0.211 0.000 0.000 0.000 0.361 0.424 0.000

1752 0.000 0.000 0.620 0.773 0.0000.000 0.000 0.374 0.002 0.000 0.000 0.000 0.386 0.918 0.000 1767

1805

1810 0.121 0.538 0.028 0.195 0.415

1986

C. solstitialis C. tinctorius

VOC Ctrl vs. Ctrl vs. all others 0 h vs. 4 & 24 h vs. 8 h Trt Ctrl vs.0 h Ctrl vs. 0 h vs.4 h 4 & 24 h vs. 8 h Trt

0 h 4 h all others

1245

1312 0.035 0.210 0.347 0.005 0.056

1381 0.218 0.176 0.246 0.434 0.912 0.000 0.000 0.153 0.315 0.000

1404 1.000 0.217 0.136 0.802 0.898

1446 0.445 0.231 0.959 0.970 1.432 0.000 0.000 0.894 0.988 0.000

1456

1458 0.937 0.726 0.979 0.623 1.665 0.203 0.086 0.919 0.879 1.291

1467 1.000 0.406 0.429 0.952 1.665

1470 0.177 0.153 0.955 0.368 0.854 1478 1480

1485

1486 0.200 0.081 0.967 0.782 1.222

1490 0.000 0.000 0.172 0.406 0.000 1524 1525

1526

1528 0.136 0.008 0.141 0.893 0.120

1536

1545 0.000 0.000 0.820 0.519 0.000 0.001 0.000 0.903 0.972 0.006

1550

1565 0.364 0.120 0.356 0.561 0.860 1570 1582

1589

1594 0.000 0.000 0.261 0.201 0.000 0.006 0.001 0.960 0.829 0.060 1605 1623

1661

1662 0.000 0.000 0.356 0.134 0.000

1666 0.000 0.000 0.875 0.114 0.000

1674

1682 0.429 0.222 0.978 0.323 1.341

L. Smith, J.J. Beck / Journal of Plant Physiology 188 (2015) 19–28 27

Table 3 (Continued)

C. solstitialis C. tinctorius

VOC Ctrl vs. Ctrl vs. all others 0 h vs. 4 & 24 h vs. 8 h Trt Ctrl vs.0 h Ctrl vs. 0 h vs.4 h 4 & 24 h vs. 8 h Trt

0 h 4 h all others

1684

1685 0.361 0.114 0.987 0.525 0.790 1694

1696

1707 0.040 0.012 0.877 0.767 0.288

1717 1.000 0.236 1.000 0.058 0.346 1718

1722

1725 1.000 0.406 0.445 0.927 1.664

1732 0.000 0.000 0.317 0.020 0.000

1744 0.443 0.230 0.990 0.936 1.424

1752 0.267 0.043 0.267 0.410 0.392 0.687 0.755 0.521 0.247 0.722

1767 0.062 0.047 0.883 0.480 0.447

1805 0.461 0.212 0.981 0.495 1.279

1810

1986 0.000 0.000 1.000 0.975 0.000

Paraja et al. may have provided more support to their statement. orous insects are exposed to in laboratory host plant speciﬁcity

The differences in the type of damage applied in these two studies, experiments. Our results suggest not only that slightly damaged

cutting versus puncturing, should not make a very big difference leaves provide dramatically different VOC proﬁles, but that such

in the responses of the plants (Smith and Beck, 2013). We did not differences persist for at least 48 h after damage. Thus, attractancy

measure VOC emission at 72 h, so we do not know if VOC levels of a plant might be underestimated if it has not been damaged,

would have changed after 48 h, but the data in Fig. 1 show no sign because it is releasing fewer VOCs. On the other hand, repellancy of

of decrease by 48 h. a plant that has been damaged might deter insects from attacking

Monoterpenes and sesquiterpenes are synthesized by the iso- nearby plants that otherwise might be acceptable (Marohasy, 1998;

prenoid pathway (Gershenzon and Croteau, 1989, 1991). However, Briese, 2005). However, experiments need to be done to determine

some constitutive defensive secondary metabolites (e.g., ␣- and ␤- whether such injury can change the outcome of multichoice host

pinene, ␣-caryophyllene, and ␣-humulene) are known to be stored plant speciﬁcity experiments. Regarding responses of parasitoids

in specialized glands of some plants, such as cotton (Elzen et al., or predators to plant odors, it would be interesting to determine if

1985; Loughrin et al., 1994). Unlike the induced terpenoids, these mechanical damage (either of plants with host damage or nearby

compounds are not synthesised de novo in response to herbivory plants without host damage) affects attractancy, and whether it

(Paré and Tumlinson, 1997). Rather, they are emitted as soon as the may interfer with the search for herbivorous hosts.

plant is damaged and cease soon after damage stops (Loughrin et al., Any study to quantitatively measure VOCs emitted by plants

1994). The rapid emission of sesquiterpenes observed in the ﬁve should take into consideration the large difference between those

Centaureinae species in our study suggests that the compounds may with damaged and undamaged leaves (Smith and Beck, 2013; Beck

be stored in the plant leaves in structures that permit their release et al., 2014). However, our results suggest that once plants have

when mechanically damaged. However, the fact that VOC levels did been damaged, the VOC proﬁles are stable for at least 2 days. All

not decrease within 48 h of the end of mechanical damage suggests the plants that we have evaluated are in the subtribe Centaureinae

that de novo synthesis may also have been occurring. Although the (Asteraceae), so it would be interesting to determine if other taxa

method of mechanical damage was not intended to mimic insect behave similarly.

feeding damage, it is superﬁcially similar to that caused by adults

of the weevil, Ceratapion basicorne, which is oligophagous on C. Acknowledgements

soltitialis and C. cyanus (Smith, 2007, 2011). Such chewing damage

evidently exposes such herbivorous insects to a wider variety and

The authors thank Glory Merrill, Wai Gee and M. Irene Wibawa

higher concentrations of secondary compounds that are likely to

(USDA-ARS) for their technical assistance. USDA is an equal oppor-

affect acceptance of the plant (Piesik et al., 2011b). Further research

tunity provider and employer.

should be done to determine if such a specialist herbivore causes

different responses from their host plant.

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