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Oecologia
DOI 10.1007/s00442-014-3104-6
PLANT-MICROBE-ANIMAL INTERACTIONS - ORIGINAL RESEARCH
Aboveground endophyte affects root volatile emission and host
plant selection of a belowground insect
Michael Rostás · Michael G. Cripps · Patrick Silcock
Received: 12 May 2014 / Accepted: 22 September 2014
© Springer-Verlag Berlin Heidelberg 2014
larvae of C. zealandica were more strongly attracted to
roots of uninfected than endophyte-harbouring grasses.
Combined gas chromatography–mass spectrometry and
proton transfer reaction-mass spectrometry revealed that
endophyte-infected roots emitted less VOCs and more
CO2. Our results demonstrate that symbiotic fungi in plants
may influence soil insect distribution by changing their
behaviour towards root volatiles. The well-known defen-
sive mutualism between grasses and Neotyphodium endo-
phytes could thus go beyond bioactive alkaloids and also
confer protection by being chemically less apparent for soil
herbivores.
Keywords Aboveground-belowground interactions ·
Costelytra zealandica · Neotyphodium uncinatum ·
Optimal foraging · Lolines
Introduction
Plants are an essential resource for a wide range of taxo-
nomically distant organisms including nematodes,
microbes, and herbivorous arthropods and therefore play
key roles in a multitude of interactions (Schoonhoven et al.
2005). Many of these plant-mediated relationships are con-
fined to the above (AG) or belowground (BG) space. How-
ever, an increasing number of studies have now highlighted
the importance of interactions that occur between AG and
BG organisms (Erb et al. 2009; Johnson et al. 2012; Van
Dam and Heil 2011). Owing to their spatial separation,
such interactions are mostly indirect, i.e. mediated by plant
physiological changes in response to herbivores or micro-
organisms (Pineda et al. 2010).
Cool-season grasses of the subfamily Pooideae are
frequently colonized by fungi belonging to the genus
Abstract Plants emit specific blends of volatile organic
compounds (VOCs) that serve as multitrophic, multi-
functional signals. Fungi colonizing aboveground (AG)
or belowground (BG) plant structures can modify VOC
patterns, thereby altering the information content for
AG insects. Whether AG microbes affect the emission of
root volatiles and thus influence soil insect behaviour is
unknown. The endophytic fungus Neotyphodium uncina-
tum colonizes the aerial parts of the grass hybrid Festuca
pratensis × Lolium perenne and is responsible for the pres-
ence of insect-toxic loline alkaloids in shoots and roots. We
investigated whether endophyte symbiosis had an effect on
the volatile emission of grass roots and if the root herbivore
Costelytra zealandica was able to recognize endophyte-
infected plants by olfaction. In BG olfactometer assays,
Communicated by Corné Pieterse.
Electronic supplementary material The online version of this
article (doi:10.1007/s00442-014-3104-6) contains supplementary
material, which is available to authorized users.
M. Rostás (*)
Bio-Protection Research Centre, Lincoln University,
Lincoln 7647, New Zealand
e-mail: michael.rostas@lincoln.ac.nz
M. G. Cripps
Department of Agriculture and Life Science, Lincoln University,
Lincoln 7647, New Zealand
M. G. Cripps
AgResearch Limited, Private Bag 4749, Christchurch 8140,
New Zealand
P. Silcock
Department of Food Science, Otago University, Dunedin 9016,
New Zealand
Oecologia
1 3
Neotyphodium (Clavicipitaceae) (Leuchtmann 1993).
These symbionts grow asymptomatically within the aerial
parts of the host tissue, including the seeds through which
they are vertically transmitted to the next generation. This
relationship, which is obligate for the endophytic fungus
and facultative for the plant, is considered a true mutualism
because of many reciprocal benefits such as stress toler-
ance, increased competitive abilities and, most well known,
resistance against herbivores (Clay and Schardl 2002;
Faeth and Saari 2012; Popay and Hume 2011; but see Faeth
2002). The latter is attributed primarily to the range of alka-
loids (peramine, ergot alkaloids, lolitrems, lolines) that the
various endophyte species produce in the aerial parts of the
plant and which in some associations are translocated to the
roots, albeit in lower concentrations (Omacini et al. 2012;
Patchett et al. 2008; Saikkonen et al. 2013). Enhanced
resistance against insects seems to be more prevalent in
agronomic grasses due to artificial selection for endophytes
with desired traits than in natural grassland communities
where greater variation exists as a result of evolutionary
adaptations by specialist herbivores (Faeth and Saari 2012).
However, evidence for the protective effects of endophytes
in wild grasses is increasing (Raman et al. 2012). While the
majority of studies on plant–endophyte–insect relationships
have focused on the role of toxic alkaloids (Saikkonen et al.
2013), it is clear that the fungus has further-reaching effects
on the host’s physiology which can also influence interac-
tions with insects (Rasmussen et al. 2008b). Endophyte-
infected hosts (E+) were shown to differ from non-infected
plants (E−) in their primary and secondary metabolism,
including the emission of leaf volatile organic compounds
(VOCs) that serve multiple ecological functions (Baldwin
2010; Panka et al. 2013; Rasmussen et al. 2008a).
Herbivorous insects are able to locate host plants from
a distance and to assess their value as a resource through
changes in the plant’s VOC bouquet. Qualitative and quan-
titative differences in VOC emission can result from her-
bivore and microbe activity or from abiotic stress factors.
Changes in the VOC composition can therefore be indi-
cators of competition, induced defences or poor-quality
host tissue (Ballhorn et al. 2013a; De Moraes et al. 2001;
Kalberer et al. 2001; Mayer et al. 2011; Winter and Ros-
tás 2010). In contrast to AG herbivores, very little is known
about the mechanisms of host selection in soil insects and
the role of root volatiles other than unspecific CO2 (John-
son and Gregory 2006; Johnson and Nielsen 2012; Wenke
et al. 2010). However, recent studies exploring the chemi-
cal ecology of root-feeding beetles have demonstrated that
BG larvae may employ similar mechanisms as AG adults
in finding hosts (Gfeller et al. 2013). Larvae of Melolon-
tha hippocastani (Scarabeidae) and Diabrotica virgifera
(Chrysomelidae) have been shown to orient towards roots
damaged by conspecifics which emitted a specific blend of
herbivore-induced VOCs (Robert et al. 2012; Weissteiner et
al. 2012). Conclusively, this correlated with the enhanced
performance of D. virgifera on infested roots (Robert et
al. 2012). Investigations into the olfactory apparatus and
chemosensory capabilities of Melolontha hippocastani
and Melolontha melolontha larvae suggested that these are
as highly developed as in many adult insects (Eilers et al.
2012; Weissteiner et al. 2012).
Managed grassland ecosystems for pastoral farming
is the major land use in New Zealand. Novel associations
between agronomic grass cultivars and naturally occurring
endophyte strains are used to control a number of insect
pests (Easton et al. 2001; Popay and Hume 2011). Among
the most abundant and damaging herbivores in grasslands
are larvae of the endemic grass grub, Costelytra zea-
landica (White) (Scarabeidae), which feed on the roots of
various pasture plants (Scott 1984). Meadow fescue (Fes-
tuca pratensis) is naturally associated with Neotyphodium
uncinatum (Clavicipitaceae), an endophyte that produces
a number of loline alkaloids. Field and laboratory experi-
ments have shown that root loline concentrations are posi-
tively correlated with plant resistance against C. zealandica
and negatively with larval body mass (Patchett et al. 2011;
Popay et al. 2003). Furthermore, grass grub larval densities
were found to be considerably lower in paddocks where
endophyte-infected F. pratensis was grown (Popay et al.
2003). In early studies that have pioneered the field, C. zea-
landica was shown to locate host plant roots by olfaction
and, although a generalist, to exhibit preferences for certain
plant species, indicating that volatiles more specific than
CO2 must play an important role (Sutherland and Hillier
1974).
In this study we carried out olfactometer experiments to
test whether shoot infection of a meadow fescue × peren-
nial ryegrass hybrid influences the host-locating behaviour
of soil-dwelling C. zealandica larvae when colonized by
the endophyte N. uncinatum. Furthermore, we analysed
the volatile spectrum emitted by the roots of intact E+ and
E− plants. Our data show that the grubs were attracted by
plants that were not colonized by the fungus and that endo-
phyte infection correlated with changes in the emission of
low molecular volatiles. These observations suggest that
soil insects are able to exploit plant volatiles as long-range
signals of host plant quality and those AG endophytes may
affect BG interactions.
Materials and methods
Plants and insects
A hybrid cultivar of Festuca pratensis × Lolium perenne
cv. Grubout (Cropmark Seeds, Templeton, New Zealand)
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with (E+) and without (E−) the endophyte Neotyphodium
uncinatum (Clavicipitaceae) (strain U2) was used. The
endophyte-free treatment was obtained by heat-treating
the seeds in a convection oven at 45 °C and 40 % relative
humidity for 30 days. Seeds were sown into 1.5-l plastic
pots containing potting mix with slow-release fertilizer.
Plants were grown in a greenhouse without supplemen-
tal light at 19.5 ± 3.8 °C and propagated vegetatively for
6–8 months by separating tillers until used in the experi-
ments. Plants were watered daily and maintained by weekly
clipping the leaves approximately 1 cm above the ligule.
Approximately 1 month before an experiment started,
grass plants were taken from their pots, and the roots were
washed to remove all the potting mix material. The shoots
were trimmed to 1 cm above the ligule and roots were
trimmed to 5 cm to stimulate growth in plants and endo-
phytes. Plants with 25–30 tillers were then transplanted
into 500-ml plastic pots containing fine-grade vermiculite
and supplied weekly with 50 ml hydroponic nutrient solu-
tion (Nutrient Film Technique, New Zealand).
Third-instar larvae of C. zealandica were collected in
May 2011 and February 2012 from a permanent pasture
at Ashley Dene pasture research farm (43°39′11.76″S,
172°19′50.97″E) near Lincoln (New Zealand). Ten to fif-
teen larvae were placed in a plastic pot (200 ml) with humid
soil and fed with slices of carrot. The insects were kept at
15 °C in a dark incubator for a maximum of 2 months until
used in the experiments.
Endophyte status of grasses
The endophyte status of the grasses to be used in the exper-
iment was checked by microscopic examination of leaf
sheath tissue stained with lactophenol cotton blue. Addi-
tionally, loline alkaloid concentrations were measured as
a more specific indicator of N. uncinatum colonization.
Briefly, 250 mg of freeze-dried root material was weighed
into an 8-ml scintillation vial and 5 ml of extraction solvent
(95:5 dichloromethane:ethanol) was added. The solvent
contained phenylmorpholine (30 mg) as an internal stand-
ard. Then, 250 µl of saturated NaHCO3 solution (200 mg−1
ml) was added to the vial. The vials were shaken on an
orbital shaker at 200 r.p.m. for 1 h. The extract was left to
settle and the supernatant filtered using a plugged Pasteur
pipette into a clean vial. One millilitre of the extract was
then transferred to a gas chromatography (GC) vial for
analysis. Samples were analysed based on the protocol by
Blankenship et al. (2001) using a Shimadzu GC-2010 GC
equipped with a flame ionization detector (FID). Loline
alkaloids were identified by comparison with authenticated
standards and quantified by relating the peak area of the
internal standard to the peak areas of the alkaloids. Six rep-
licates were carried out per treatment.
Host plant-selection behaviour
A modified BG olfactometer (Rasmann et al. 2005) was
used to test the attraction of C. zealandica larvae to roots of
E+ and E− plants. The olfactometer consisted of a central
glass chamber [inner diameter (i.d.) 10 cm, depth 11 cm]
with four equally distributed side arms (90° apart, i.d.
6 cm, depth 3.5 cm) at 0.5-cm height. Each side arm of the
central chamber was connected to a detachable glass cup
(i.d. 2.5 cm, depth 6 cm) with a side-arm (i.d. 5 cm, depth
11 cm).
Two sets of experiments were carried out. The first assay
assessed the combined gustatory and olfactory responses of
C. zealandica larvae while in the second assay only olfac-
tory responses were tested. Both experiments were carried
out in the same way, except that in the second experiment
access to the roots was blocked by a metal grid (mesh
3 mm) in the side arm of each olfactometer cup. The olfac-
tometer was filled with moderately moist, fine-grade ver-
miculite (400 g water added to 300 g vermiculite) to about
3 cm from the rim. Two glass cups with vermiculite served
as controls, the two remaining cups contained E+ and E−
plants, respectively. Control and test cups were randomly
positioned after each experimental run to avoid bias in grub
responses. At least 1 day before the olfactometer experi-
ment started, plants were removed from their pots. Same-
sized clusters of E+ and E− grasses were then transplanted
into the glass cups of the olfactometer. Eight grubs were
released into the central chamber and observed for 10 min
until they had burrowed into the substrate. Grubs that
showed no activity after this time were replaced. Twenty-
four hours after grub release, the olfactometer was disas-
sembled and the position of each grub was noted. A grub
was considered to have made a choice if recovered from
one of the side arms. Grubs found in the central cylinder
were excluded from the analysis. Each experiment was
repeated with a new set of plants until 55 grubs that had
made a choice were recovered. Three olfactometers were
run in parallel.
Gas chromatography–mass spectrometry of root volatiles
Dynamic headspace collection of root volatiles was car-
ried out with a modified push–pull system as described in
Turlings et al. (2004) and Rostás and Eggert (2008). Plants
were placed into odour-source vessels taken from their
pots and roots were carefully washed with water to remove
the vermiculite. The AG parts of E+ and E− meadow
fescues were enclosed in inert PE foil (Glad Oven Bags,
Australia) and the plants were then placed in an aluminium
foil-covered odour-source vessel. The cup of each vessel
featured two glass tubes (i.d. 0.8 cm, length: 2 cm) at 2.5-
cm height with screw cap attachments (GL 14; Schott) at
Oecologia
1 3
opposite ends. Filtered air (activated charcoal filter, 400 cc;
Alltech, Deerfield, IL) originating from a compressed air
cylinder was pushed into the cup through one of the tubes
at a rate of 300 ml min−1. With a vacuum pump (ILMVAC,
Germany) 200 ml min−1 of air was pulled out through a
trapping filter containing 30 mg SuperQ (ARS, Gaines-
ville, FL). Empty odour-source vessels and vessels filled
with 10 mg vermiculite were also sampled to check for
background contaminations. Before each experiment, the
traps were cleaned by rinsing with 1 ml methylene chlo-
ride. Each collection lasted 5 h. The adsorbed compounds
were eluted from the filter with 150 µl methylene chloride
and 200 ng tetralin (Sigma-Aldrich, Australia) was added
as an internal standard. The sample mixtures were sepa-
rated using a Shimadzu QP2010 GC–mass spectrometer
(MS) (Shimadzu, Japan) fitted with a Restek Rxi-1 ms
fused silica capillary column (30.0 m × 0.25 mm i.d. ×
0.25 μm). Of each sample, 3 µl were injected in pulsed
splitless mode at a temperature of 220 °C and with a pulse
of 168 kPa for 40 s. Oven temperature was held at 50 °C
for 3 min and then raised to 320 °C at 8 °C min−1 and held
at this temperature for 8 min. He was used as carrier gas
at a constant flux of 1.5 ml min−1. Compounds were iden-
tified using GC–MS Solution version 2.72 software (Shi-
madzu) with NIST 11 and Wiley 10 mass spectrum librar-
ies and by using the software MassFinder4/Terpenoids
library (Hochmuth Scientific Software, Hamburg). Stand-
ards were used to confirm identities of compounds that
were commercially available (Sigma-Aldrich; Treatt, UK).
Quantification was obtained by comparing the area of the
compounds to the area of the internal standard. For each
treatment the volatile collection and analysis was repeated
five times.
Proton transfer reaction–MS of root volatiles
Plants were taken from their pots. Roots were carefully
washed with water to remove the vermiculite and then
dried with soft tissue paper. The BG parts of E+ and
E− grasses were placed in an odour-source vessel and
inert biaxially oriented polyethylene terephthalate film
(Mylar; Dupont, USA) wrapped tightly around the junc-
tion between the AG and BG portions and secured around
the plants and across the cup so that the VOC analysis
was restricted to the root portion (Fig. S1). The cup of
each vessel featured two glass tubes (i.d. 0.8 cm, length
2 cm) at 2.5-cm height with screw cap attachments (GL
14; Schott) sealed by Teflon-faced septa. Two pieces
of Teflon tubing were inserted through the septa. The
VOCs were detected by a high-sensitivity proton trans-
fer reaction (PTR)-MS (Ionicon Analytik, Innsbruck).
All measurements were carried out under drift tube
conditions with a drift pressure of 2.23 mbar, chamber
temperature of 80 °C and voltage of 600 V, with an elec-
tric field to buffer the gas density ratio (E/N) of 139 Td
(Td = Townsend; 1 Td = 10–17 Vcm2). Instrument-grade
dry air (BOC, Dunedin, New Zealand) was introduced
through the Teflon tubing to the glassware at 60 stand-
ard cm3 min−1 (s.c.c.m.) and was drawn into the PTR-MS
through the other piece of Teflon tubing connected to a
heated 0.04″-i.d., 1/16″-outer diameter Silcosteel capil-
lary transfer line (Restek, Bellefonte, PA) at 110 °C at
a flow rate of 55 s.c.c.m. The mass range of mass/charge
ratios (m/z) 20–208 was scanned using a dwell time of
0.1 s per mass. Each sample was measured for six cycles
and a mean of cycles 2–6 was used for data analysis. The
VOC contribution of the glassware and air was measured
before a sample was introduced into the glassware by
securing a piece of biaxially oriented polyethylene tere-
phthalate film over the top of the glassware to provide
background spectra for data correction (see below). The
backgrounds were measured for six cycles and a mean of
cycles 3–6 was used for data analysis. For data process-
ing, VOC raw counts per second (c.p.s.) were normal-
ized to the sum of the primary ions (m/z 21 + 39) and
2.2 mbar drift pressure, yielding normalized counts per
second (n.c.p.s.). Mean n.c.p.s. values of the background
signal per m/z were subtracted from the VOC profile data
to filter out potential interference signals from within
the system and then normalized to the total dry mat-
ter (grams). The n.c.p.s. values were normalized to zero
mean–unit variance to account for differences in back-
ground noise as PTR-MS measurements were carried out
in two separate sets. Ionized molecules that differed in
quantity between treatments were tentatively ascribed to
compounds identified by GC–MS or volatiles reported in
published PTR-MS studies.
CO2 emission of roots
Root CO2 emission was determined by removing the root
system from a single tiller and placing it into a 20-ml gas-
tight vial. The vial was incubated at room temperature for
18 h. A gas-tight syringe was used to transfer the gas sam-
ple to an evacuated GC vial. Measurements were made
on an SRI 8610C GC using a Gilson 222 XL autosampler
fitted with a double concentric needle system (SRI Instru-
ments, Torrance, CA). The GC was fitted with a 1-m × 1/8″
stainless steel pre-column packed with Porapak Q 80/100
and a 6-m × 1/8″ analytical column also packed with Pora-
pak Q 80/100. A FID was used by activating the methan-
iser. The methaniser reduced the CO2 to CH4, which could
then be detected by the FID. The sample was flushed from
the vial using N2 carrier gas at a flow rate of 40 ml min−1.
The columns were kept at a constant 40 °C. The concentra-
tion was determined by comparing peak areas against those
Oecologia
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of known standard concentrations. Fifteen replicates were
carried out per treatment.
Statistical analysis
The functional relationship between the grubs’ behavioural
responses and the different odour sources offered in the
4-arm olfactometer was examined with a log-linear model
(a generalised linear model; GLM). As the data may not
conform to simple variance assumptions implied in using
the multinomial distribution, quasi-likelihood functions
to compensate for the overdispersion of grubs within the
olfactometer were used (Ricard and Davison 2007; Turl-
ings et al. 2004). The model was fitted by maximum quasi-
likelihood estimation in the software package R (version
2.15.2) and its adequacy was assessed through likelihood
ratio statistics and examination of residuals. Factors were
day, olfactometer and treatment. The entity computing
a repetition in the statistical analysis corresponded to the
response of a group of eight released grubs.
Principal component analysis (PCA) was used to charac-
terize the blend of emitted volatiles measured by PTR-MS
in an unsupervised manner (MVSP 3.22; Kovach Comput-
ing Services). The PTR-MS data were further evaluated by
comparing the sum of measured ions (m/z) from E− and
E+ roots using Student’s t-test (Statistica 12; StatSoft).
Subsequently, single m/z were analysed by using Student’s
t-tests with and without false discovery rate correction
(FDR) (Benjamini and Hochberg 1995). CO2 emission was
also analysed by Student’s test.
Results
Endophyte colonization
All E+ plants were infected by N. uncinatum hyphae
while no indication of endophytic colonization was found
in E− grasses. Confirming our microscopic observa-
tions, no loline alkaloids were measured in the roots of
E− grasses. In contrast, roots of E+ plants contained on
average 340 ± 125 µg g−1 dry mass N-formyl loline and
63 ± 40 µg g−1 dry mass N-methyl loline. Other loline
alkaloids produced by N. uncinatum and known to occur
in low concentrations, such as N-acetyl loline and N-acetyl
norloline (Patchett 2007), were not detected.
Orientation behaviour of grass grubs
In the first olfactometer assay, larvae of C. zealandica were
allowed access to plant roots. Grubs significantly preferred
E− (93 ± 6 %) compared to E+ (7 ± 6 %) plants or cups
without plants (0 ± 0 %, average of both controls) (GLM,
F2,37 = 77.884, P < 0.001) (Fig. 1). Overall, ten releases
(n = 80 grubs) were made until 55 grubs were recovered
that had made a choice, corresponding to a response rate
of ca 70 %. In the second experiment, where access to
plant roots was denied in order to test olfactory behaviour
only, the response rate dropped to ca. 30 %. To obtain 54
responding grubs, 23 releases (n = 184 grubs) were made.
Grubs making a choice in the second experiment also pre-
ferred E− plants (69 ± 8 %) over E+ (25 ± 8 %) plants or
Fig. 1 Response of Costelytra
zealandica grubs to grass root
volatiles in a four-arm olfac-
tometer. a Insects had access
to roots (n = 55), b access to
roots was blocked by a metal
grid (n = 54). Bars represent
percentage (mean ± SE) of
grass grubs found in each
arm. Pie charts show percent-
age of responding larvae (R).
E− Plants without endophyte,
E+ plants with endophyte, C
no plant (average of two arms).
Different letters above bars
indicate significant differ-
ences; generalised linear model,
P < 0.05
E- E+ C
Attracted grubs [%]
0
20
40
60
80
100
E- E+ C
Attracted grubs [%]
0
20
40
60
80
100
R
a
b
c
a
b
c
R
b
a
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1 3
controls (3 ± 3 %, average of both) (GLM, F2,89 = 23.172,
P < 0.001).
Root volatile emission
Analysis of root volatiles using dynamic headspace sam-
pling and GC–MS analysis resulted in the detection of
few compounds in low but not significantly different
amounts, such as the C6 compound hexanal (t = −0.899,
df = 8, P = 0.395), the benzenoid phenylacetaldehyde
(t = −1.233, df = 8, P = 0.252 and the two terpenoids,
camphor (t = −1.515, df = 8, P = 0.168) and α-fenchene
(t = 1.667, df = 8, P = 0.134). Targeted ion trace searches
for mono- and sesquiterpenes (m/z 136 and m/z 204, respec-
tively) revealed several monoterpenes (tricyclene, sabinene,
β-pinene, myrcene) present in trace amounts (Table 1);
quantification, which was also hampered by partial co-elu-
tion of terpenes with other compounds, was therefore not
attempted. No sesquiterpenes were detected.
PTR-MS analyses showed that the endophyte status
of the plant had a significant effect on root volatile emis-
sion. Overall, roots of E+ grasses emitted lower amounts
of VOCs compared to E− plants, as shown by PCA
and a t-test over the sum of all ions (t = −4.249, df = 8,
P = 0.003; Figs. 2, 3). PCA also indicated that numerous
single ions were negatively correlated with endophyte pres-
ence (Fig. 2b). Significantly (P < 0.05) attenuated emis-
sion rates were m/z 61 (acetic acid, t = −3.469, df = 8,
P = 0.026), m/z 65 (protonated ethanol–water cluster,
t = −3.032, df = 8, P = 0.016), m/z 83 (hexanal, hexe-
nols, t = −2.3703, df = 8, P = 0.046) and m/z 137 (sum
of monoterpenes, t = −4.488, df = 8, P = 0.002). Higher
abundances of m/z 43 and m/z 81 that are known to be frag-
ments of acetic acid and monoterpenes, respectively, were
not significant but correlated with E− plants in the PCA.
Other compounds showing a tendency for reduced emission
(P < 0.1) were m/z 45 (acetaldehyde, t = −2.209, df = 8,
P = 0.069) and m/z 63 (dimethylsulphide, t = −1.907,
df = 8, P = 0.092). No difference was found in m/z 169
(t = 0.654, df = 8, P = 0.532), which would correspond to
the protonated molecular ion [M+H]+ of N-methyl loline.
Likewise, no signal was measured at m/z 183 indicative of
N-formyl loline. All P-values were higher than their corre-
sponding critical FDR values.
Roots of E+ grasses were found to emit 25 % more
CO2 than roots from E− grasses (t = −2.919, df = 28,
P = 0.007; Fig. 4).
Discussion
Our study confirms earlier work that soil-dwelling C. zea-
landica larvae locate plants from a distance and that root
VOCs can play a crucial role as chemical signals in host-
choice behaviour (Sutherland 1972; Sutherland and Hillier
Table 1 Root volatile organic compounds measured by gas chroma-
tography–mass spectrometry
Average (±SE) emission rates are reported as ng g−1 fresh weight
5 h−1; n = 5
MW Molecular weight, RI(e) experimentally determined retention
index, RI(d) retention index in data bank, E−plants without endo-
phyte, E+ plants with endophyte, Tr. trace amounts
a Compounds were identified by comparison with authenticated
standard
Compound MW RI(e) RI(d) Treatment
E−E+
Green leaf volatiles
Hexanala100 808 806 15.5 ± 10.1 29.1 ± 11.1
Monoterpenes
Tricyclene 136 928 927 Tr. Tr.
α-Fenchene 136 940 941 26.2 ± 4.6 16.1 ± 3.8
Sabinenea136 964 973 tr. tr.
β-Pinenea136 966 978 tr. tr.
Myrcene 136 983 987 tr. tr.
Aromatic hydrocarbons
Phenylacetaldehydea120 1004 1012 3.0 ± 1.8 13.5 ± 8.2
Monoterpene ketone
Camphor 152 1116 1123 7.9 ± 0.8 11.8 ± 0.7 -1.0 -0.5 0.0 0.5 1.0 1.5
-1.5 -1.0 -0.5 0.0 0.5 1.0
PC2
PC1
20
22
26
28
30
35
36
37
38
3940
41
42
43
44
45
46
47
48
49
50
51
52
53
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Fig. 2 Bi-plot of principal component analysis (PCA) of volatile
organic compound (VOCs) emitted by roots of Festuca pratensis
× Lolium perenne. Numbers represent loadings of m/z values. Blue
arrows represent scores of E+ plants, red arrows scores of E− plants.
PC1 = 17 %, PC2 = 15 % (colour figure online)
Oecologia
1 3
1974; Osborne and Boyd 1974). Grubs of C. zealandica
clearly preferred F. pratensis × L. perenne plants that were
not colonized by N. uncinatum and therefore devoid of det-
rimental loline alkaloids. In the first experiment we recov-
ered most grubs from the rhizosphere where they were
able to assess the host roots using gustation and olfaction.
The second assay in which access to the root system was
blocked suggests that C. zealandica larvae can base their
foraging decision exclusively on olfactory signals. While
the insects displayed a strong preference for E− plants in
both bioassays, it was remarkable that fewer grubs were
found inside the olfactometer arms when root access was
denied. Low response rates can be due to many reasons and
are difficult to control by the experimenter. For example,
grass grubs could have been infected by parasites such as
m/z 61 [acetic acid]
E+ E-
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
nncps
m/z 65 [ethanol-water cluster]
E+ E-
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
nncps
m/z 83 [hexanols/hexanal]
E+ E-
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
nncps
m/z 137 [monoterpenes]
E+ E-
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
nncps
*
*
*
**
Total m/z
E+ E-
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
nncps
**
Fig. 3 VOCs (measured by proton transfer reaction-mass spectrome-
try) emitted in significantly different amounts by roots of E+ and E−
plants (n = 5). Boxes depict medians and quartiles, whiskers show
extreme values. nncps Normalized counts per second with values
normalized to zero mean unit variance. Student’s t-test, *P = 0.05,
**P = 0.01. For abbreviations, see Fig. 2
E- E+
2
CO [µl
-1
l
-1
g DW]
0
2000
4000
6000
8000
**
Fig. 4 Mean (±SE) CO2 emission of F. pratensis × L. perenne roots
(n = 15). Student’s t-test, **P = 0.01. For abbreviations, see Fig. 2
Oecologia
1 3
Serratia entomophila (the causal agent of bacterial amber
disease) or internal physiological factors may prevent feed-
ing. However, we did not observe any unhealthy grubs
before or after the experiments and insects were kept under
standardized conditions. Probably the most plausible expla-
nation is therefore the high mobility of the grubs, which
may have left the attractive arm at some stage as the food
source could not be reached. Additionally, roots damaged
by feeding grubs may emit high quantities of a qualitatively
different VOC bouquet. Such herbivore-induced volatiles
can be considerably more attractive to conspecific beetle
larvae (Robert et al. 2012). Both factors may explain the
stronger behavioural response observed in the first experi-
ment. To unequivocally prove this notion, further studies
will need to use a redesigned olfactometer with some kind
of trapping system that prevents the insects from leaving
the chosen arm.
In order to reveal the underlying mechanism for the
grubs’ behaviour, we compared the emission of root VOCs
from E+ and E− plants. A standard push–pull technique
for sampling plant volatiles followed by GC–MS analy-
sis was employed at first (Tholl et al. 2006; Winter et al.
2012). Compounds from different classes of plant volatiles
were detected, comprising derivatives of polyunsaturated
fatty acids, benzenoids and terpenoids. However, quanti-
ties for all compounds were generally low and gave no
indication as to which volatiles could have contributed to
the grubs’ host choice behaviour. These were emitted either
in similar amounts by E+ and E− plants or were too low
to be quantified. This is not surprising, given that undam-
aged plants often show lower emission rates compared to
mechanically or herbivore-damaged plants (Van Den Boom
et al. 2004). Also, from the little that is known about root
VOCs measured for the headspace of intact plants, diver-
sity and quantity of compounds seems to be greater in AG
plant parts (Danner et al. 2012). The headspace volatiles of
F. pratensis × L. perenne were therefore further analysed
using PTR-MS, a relatively new technique, in particular
for assessing root VOCs (Crespo et al. 2012; Danner et al.
2012; Steeghs et al. 2004). The use of PTR-MS in our study
had two main advantages: low molecular compounds could
be quantified with higher sensitivity while some previously
reported root volatiles were measured that was not detect-
able under the specific GC–MS conditions employed here.
However, PTR-MS does not allow for unequivocal iden-
tification of volatiles. Unlike electron impact ionization,
proton-transfer results in few or no fragments of the target
molecule and therefore only provides information about the
molecular weight of a compound. The identities of mole-
cules must be derived from combined GC–MS analyses or
by detailed studies of the fragmentation patterns of refer-
ence compounds. Hence, compounds in brackets following
m/z must be seen as founded suggestions. Our PTR-MS
results showed that colonization by N. uncinatum reduced
the total amount of F. pratensis × L. perenne root VOCs
by 22 %. PCA suggests that many ions apparently contrib-
ute to this general trend for lower emission but significant
differences of uncorrected t-tests were restricted to m/z 61
[acetic acid], m/z 65 [ethanol–water cluster], m/z 83 [hexa-
nal/hexanols] and m/z 137 [monoterpenes]. Differences
in the ion m/z 45 were marginally non-significant but it is
conceivable that in this case lower acetaldehyde emission
was masked by higher CO2 production in E+ roots as both
compounds have the same molecular mass. CO2 is typically
considered to be not measured by PTR-MS but under high
humidity it has been detected using PTR-TOF–MS (Beau-
champ et al. 2013; Herbig et al. 2009). Among the VOCs
that were identified at least β-pinene is known to be an
attractant for C. zealandica (Osborne and Boyd 1974). In
any case, FDR correction resulted in non-significant results
and therefore data on single m/z, in contrast to total m/z,
do not provide unequivocal evidence for differential emis-
sion in response to the endophyte. Rather, the results allow
for generating hypotheses about likely candidate molecules
which can be tested in further studies.
Changes in VOC emission due to endophyte coloniza-
tion have also been reported elsewhere, albeit these were
restricted to AG plant parts. The direction of change seems
to depend very much on the specific grass-fungus associa-
tion. Shoots of tall fescue, Schedonorus phoenix (syn. F.
arundinacea), infected with Neotyphodium coenophialum,
for instance, emitted less nonanal and (Z)-3-hexen-1-ol
acetate but higher amounts of two monoterpenes, while
most compounds remained unchanged (Yue et al. 2001).
A general increase in VOCs was found in L. perenne asso-
ciated with Neotyphodium lolii (Panka et al. 2013), while
a decrease in green leaf volatiles and monoterpenes was
reported for F. pratensis colonized by N. uncinatum, thus
corroborating our results for root VOC emission (Li et al.
2014). The only study the authors are aware of that has
linked endophyte-mediated changes in VOC emission and
herbivore behaviour is on a non-grass system. The pres-
ence of Acremonium strictum in tomato roots resulted in
significant attenuation of most leaf terpenes and correlated
with Helicoverpa armigera moths preferring to oviposit on
the leaves of E+ plants (Jallow et al. 2008). Furthermore,
microbial association with roots may also influence inter-
actions with higher trophic levels. Leaves of Phaseolus
vulgaris, for instance, emitted larger amounts of herbivore-
induced volatiles when colonized by a mycorrhizal fungus
which resulted in the attraction of more predatory mites
(Schausberger et al. 2012). Similar effects were reported
for N-fixing rhizobia in the related bean species P. lunatus
while the opposite was found in Arabidopsis thaliana asso-
ciated with non-pathogenic rhizobacteria (Ballhorn et al.
2013b; Pineda et al. 2013).
Oecologia
1 3
Notably, in our study roots of E+ plants released more
CO2, which is in contrast to the lower emission of other
VOCs. This ubiquitous compound is known to attract soil
herbivores, including C. zealandica, when offered in iso-
lation from a point source (Galbreath 1988; Johnson and
Gregory 2006). However, the effectiveness of CO2 as an
orientation cue in the soil has been questioned lately as it
is emitted by many organisms and not just the host plant
(Barnett and Johnson 2013). Also, the presence of other
more specific root metabolites may override any behav-
ioural attraction towards pure CO2 as demonstrated in the
scarab M. melolontha (Reinecke et al. 2008). In a another
study, larvae of D. virgifera displayed behaviour similar to
C. zealandica by preferring maize roots that emitted higher
amounts of VOCs and less CO2 (Robert et al. 2012).
We suggest that the proximate explanation why
C. zealandica was preferentially attracted to the roots of
E− grasses was their higher detectability due to larger
VOC emission in comparison to E+ plants. This may pro-
vide the plant-endophyte association with a further advan-
tage in addition to the protection already conferred by the
presence of toxic loline alkaloids. Given that these fungal
compounds are translocated to the roots, the question arises
whether they could be secreted into the rhizosphere and
perceived by the grubs. Although this scenario cannot be
categorically ruled out, it seems unlikely as loline alkaloids
have a very low volatility and were not detected in either
GC–MS or PTR-MS headspace analyses.
From an evolutionary standpoint, lower VOC emission
in the grass-endophyte system is certainly not a trait that
has evolved due to selective pressure from C. zealandica as
their common history in New Zealand is very recent. How-
ever, one can speculate whether emitting less volatiles that
act as kairomones for insects, and thus being less conspicu-
ous, is the result of coevolution with other soil herbivores
in the plant’s native range and would thus be part of the
defensive mutualism that characterizes fescue and Neoty-
phodium (Panaccione et al. 2014). On the other hand, soil
herbivores may have evolved the ability to ‘sniff out’ plants
that harbour specific endophytes. This would increase their
host-searching efficiency by reducing the cost of moving
through substrate. Further studies are necessary to establish
the physiological mechanisms of fungus-mediated changes
in VOCs and to elucidate whether this is an adapted
response or merely a side-effect with positive implications
for the grass-endophyte symbiosis.
Our results offer a new perspective on the factors that
determine the distribution of herbivorous insects in soil
by showing that root feeders respond to shifts in the con-
centrations of volatile plant secondary metabolites. To our
knowledge, this is the first evidence of an AG endophytic
microorganism affecting BG processes by changing plant
volatile emission. The agronomic implications of these
findings, taking into account the known effects of loline
alkaloids, are that protection of the pasture grass F. praten-
sis × L. perenne is a two-step process whereby endophyte-
colonized plants attract fewer herbivores through olfaction
in the first place and secondly deter feeding if roots are
attacked (Patchett et al. 2011).
Acknowledgments We thank Brian Patchett (CropMark Seeds) for
donating seed material and Jason Breitmeyer for assistance with GC–
MS analyses. Ana Nath Baral and Manu Somerville provided tech-
nical support. We also thank Franco Biasioli (Fondazione Edmund
Mach) for advice on PTR-MS and Dave Saville, Matthias Held and
Maki Ikegami for advice on statistics. This work was supported by a
grant from the Lincoln University Research Fund (INTD009).
Conflict of interest The authors declare that there are no conflicts
of interest and that the experiments comply with current laws in New
Zealand.
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