Download Effect of leaf herbivory by spider mite Tetranychus urticae on flower

Effect of leaf herbivory by spider mite
Tetranychus urticae on flower
characteristics of different cucurbits
Marit A.M. Rutten (910125718060)
Dr. ir. Iris F. Kappers, Laboratory of Plant Physiology, Wageningen
March – December 2014
Master Biology Thesis (PPH-80430)
Effect of leaf herbivory by spider mite Tetranychus
urticae on flower characteristics of different cucurbits
Marit A.M. Rutten
Abstract
Cucumber, melon, watermelon, courgette, pumpkin and bitter gourd are some economically
very important (cultivated) fruits that belong to the Cucurbitaceae family, which is troubled
by the spider mite Tetranychus urticae. This herbivore causes chlorotic lesions in the leaves,
which can have a tremendous effect on the plant’s performance, for example by changes in
flower morphology and metabolism. Most cucurbits depend on pollination for fruit and seed
production. In this study the variation among different cucurbit species with respect to flower
characteristics that may be influenced by herbivory on leaves by spider mites is explored.
With LC-MS and GC-MS the production of endogenous metabolites and volatiles are
investigated, respectively. For the non-bitter cucumber and the bitter Chinese cucumber the
RNA of flowers and leaves will be isolated to test which terpene synthase family (TPS) genes
(TPS1, TPS9 and TPS19) are expressed in control and infested samples, and therefore may be
responsible for the volatile emission identified with GC-MS. This study demonstrates a large
range of qualitative and quantitative variation in volatile emission induced by leaf herbivory
of spider mites (Tetranychus urticae) in leaves of different cucurbit. But there was no
significant effect on flower volatile emission. The TPS genes investigated in this study were
presumably not (always) the ones that were responsible for the emission of certain volatiles.
Some flowers were most probably not able to produce plant-defence related volatiles, because
of the tremendous drop in JA-Ile/JA ratio in flowers when plants were infested with spider
mites.
1
Introduction
Some economically very important cultivated fruits, which are produced worldwide, belong
to the Cucurbitaceae family. Examples of such fruits are cucumber, melon, watermelon,
courgette, pumpkin and bitter gourd. In 2012 the world produced about 65,134,078 tonnes of
cucumbers and gherkins of which 86.5% was produced in Asia, The Netherlands accounts for
0.6% of total cucumber production (FAO, 2014).
The Cucurbitaceae family, like other plant families, is troubled by various herbivores
eating plant parts, especially the leaves (Barber et al., 2011; Park and Lee, 2005). Fortunately,
the plant evolved some ways to protect itself from such harm. However, as Herms and
Mattson (1992) already stated, a plant must find a balance between investing in growth and
development on the one hand and defence on the other hand, because both are essential for
life. Searching for this balance often results in a dilemma for the plant: to compete with
neighbouring plants it has to grow fast enough, but on the other hand it has to maintain its
defence mechanisms to survive in an environment with herbivores and pathogens (Herms and
Mattson, 1992).
Spider mite (Tetranychus urticae)
Plants are attacked by herbivores and pathogens that belong to various taxons (e.g.
arthropods, insects and mammals). Arthropods and insects represent the largest group of
phytophagous organisms, approximately 1-3 million species (Schoonhoven et al., 2005). In
this research a member of this large herbivore-group is taken into account, the two-spotted
spider mite, Tetranychus urticae Koch (Fig. 1). An important characteristic of this mite is a
dark spot at both sides of the body. This spider mite is a generalist herbivore and a serious
pest in many crops, for instance cucumber (Park and Lee, 2005), but also in other cucurbits.
Spider mites belong to the group of sucking feeders. They feed on the mesophyll in leaves by
piercing cells with their stylet. The cells are sucked out which results in chlorotic lesions
(reductions in chlorophyll concentration) on the leaves of the plant. Because of this, plant
development will be less successful and when the damage caused by the spider mites is severe
enough, it can be devastating for the plant. Spider mites prefer to live in colonies on the
downside of leaves where they spin silk webs, which protect both mites and eggs against
predators. A female spider mite can lay hundreds of eggs during her life. The small, spherical
eggs hatch after 2-3 days. The time needed to complete the whole life cycle (Fig. 1) depends
on the temperature of the climate the spider mites live in. A natural enemy of this spider mite
species is the predatory mite Phytoseiulus persimilis (Fig. 1), which can eliminate whole
spider mite populations (Fasulo and Denmark, 2009).
Figure 1 Left up: Spider mite Tetranychus urticae
Right: The life cycle of spider mites
Left down: Predatory mite Phytoseiulus persimilis
2
Plant defence mechanisms
Plants have evolved a multitude of defence mechanisms to prevent serious damage caused by
herbivores, which can be divided in the main groups direct and indirect defence. Plant traits
that affect the performance (survival and growth) and behaviour of attacking herbivores
themselves are direct defences, those that attract the natural enemies of herbivores are indirect
defences. Both strategies can be either constitutive (always expressed/present) or inducible
(activated by herbivory) (Arimura et al., 2005; Kessler and Baldwin, 2002).
Constitutive direct defences can be physical and/or chemical. An example of a
physical one are trichomes (plant hairs), which are grown from the epidermal cells on
numerous plant species. Trichomes are a good defence mechanism against piercing-sucking
arthropods, like spider mites. When hairs are dense, they form a physical barrier that makes it
very difficult for this kind of herbivores to reach the plant’s epidermis. To make it even more
difficult, some plant hairs (glandular trichomes) can produce chemicals which are sticky
(Wagner, 1991; Werker, 2000). As a response to herbivore attack (induced response), plants
can change their phenotype (phenotypic plasticity) by making changes in their physical
characteristics, like increased trichome density, which is an induced direct defence
mechanism (Traw and Dawson, 2002). However, this also has an influence on the natural
enemies (e.g. predatory mites) of piercing-sucking arthropods (e.g. spider mites). Those
predators often walk large distances over the leaf surface to find their prey/host and trichomes
are tough obstacles (Krips et al., 1999).
Plants can also change their phenotype by making changes in their chemical
characteristics, for example the production of secondary metabolites (Kessler and Baldwin,
2002; Mumm and Dicke, 2010).
Secondary metabolites
Secondary metabolites are organic compounds with a low molecular mass which have no
function in plant development, growth or reproduction but in plant defence (Kessler and
Baldwin, 2002; Mumm and Dicke, 2010). Some secondary metabolites will directly decrease
the number of herbivores on the plant. Therefore, they are called direct defences. Examples of
such secondary metabolites are toxins and alkaloids which are toxic to herbivores, and
phenolic acids, tannins and lignin which reduce the digestibility and/or palatability of the
plant material. These compounds are called endogenous metabolites, because they stay in the
plant tissue (Dicke, 2009; Kessler and Baldwin, 2002; Lambers et al., 2008). Not all
secondary metabolites produced in direct defence mechanisms are endogenous, some are
volatile (De Moraes et al., 2001).
Other secondary metabolites will not have a direct negative effect on the number of
herbivores, but attract the natural enemies of those herbivores. Therefore, they are called
indirect defences where specific volatile organic compounds (VOCs) are involved (Kessler
and Baldwin, 2002; Dicke and Baldwin, 2010).
VOCs, signal transduction pathways and HIPVs
During their life cycle, plants communicate with organisms in their surroundings through
volatile organic compounds (VOCs). These compounds are produced in every part of the
plant, from roots underneath the soil till flowers at its top. VOCs determine the smell of the
plant and can diffuse easily through the air or the soil. Therefore, they can be detected by
organisms from a distance. Which kinds of organisms are attracted by a volatile depends on
the plant part that emits the volatile. The volatile-profile of a leaf blend differs from one of a
flower blend. This is because of the different functions of the plant parts. The flowers of most
plants are meant to attract pollinators with their visual, tactile and olfactory signals. The scent
of a flower can be a complex blend containing many compounds from different chemical
3
classes. The seven main classes are: aliphatics, benzenoids/phenyl propanoids, C5-branched
compounds, terpenoids, nitrogen-containing compounds, sulphur-containing compounds and
a class of various cyclic compounds. However, little is known about which individual
compound attracts a specific pollinator (Knudsen et al., 1993; Knudsen et al., 2006; LucasBarbosa et al., 2011; Pichersky and Gershenzon, 2002).
Herbivores can detect specific volatiles emitted by leaves and because of that locate
their (host) plant. When herbivorous arthropods eat plants’ tissue, their oral secretions
(regurgitant) contacts the plant. This regurgitant consists of elicitors that are species specific
components that can be recognised by the plant’s membrane receptors (e.g. Hilker and
Meiners, 2010). As a result three endogenous interconnecting signal transduction pathways in
the plant are activated: the octadecanoid pathway with jasmonic acid (JA) as a key compound,
the shikimic acid pathway with salicylic acid (SA) as a key compound, and the ethylene
pathway. Jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are phytochemicals which
have protective characteristics: they activate plant defence genes (e.g. Pieterse and Dicke,
2007). These plant defence genes are activated by transcription factors. In the JA signalling
pathway, members of the jasmonate-ZIM domain (JAZ) protein family repress these
transcription factors. It is known that jasmonic acid conjugated with the amino acid isoleucine
(jasmonoyl-isoleucine or JA-Ile) is able to degrade the JAZ proteins, which results in free
transcription factors and eventually the expression of plant defence genes (Thines et al.,
2007). Some of those genes will induce the production of VOCs. As a consequence, the VOC
composition of herbivore-damaged plants differs from the VOC composition of intact plants.
This change in volatile mixture can be either quantitative (different emission level of
components also present in non-infested plants) and/or qualitative (release of other
components).
Herbivore-damaged plants emit VOCs that can attract the natural enemies (arthropod
predators and parasitoids) of the herbivorous arthropods. Those VOCs are also called HIPVs
(herbivore-induced plant volatiles) and they are used by natural enemies to locate their prey
(Dicke and Baldwin, 2010; Mumm and Dicke, 2010; Dicke et al., 2003). HIPVs are emitted
locally at the site of damage (often a leaf), but can also be emitted systemically from
undamaged parts of attacked plants, so there is a connection between plant parts. The aim of
this connection is to prevent the attacker from spreading through the whole plant (Heil and
Ton, 2008). Carnivores foraging for prey or hosts perceive HIPVs with their peripheral
olfactory system that can be located for instance on antennae or palpi. The peripheral
olfactory system consists of olfactory sensilla containing receptor neurons that are sensitive to
specific HIPVs within a HIPV blend (Schoonhoven et al., 2005). Those specific HIPVs
provide carnivores, for example, with information about the plant species or cultivar that
emits the odour (Takabayashi and Dicke, 1996) and which herbivore species is damaging the
plant (e.g. De Moraes et al., 1998). HIPV blends are often complex mixtures with multiple
different compounds that are also from the same seven chemical classes as compounds in the
scent of a flower. However, most compounds belong to one of the four main groups: fatty
acid derivatives, phenylpropanoids, benzenoids and terpenoids (Dudareva et al., 2004; Mumm
and Dicke, 2010).
Terpenoids
Terpenoids, also called isoprenoids or terpenes, are a large group of various compounds that
are produced by many plant species. A relatively small number of terpenoids are involved in
primary metabolism. Most terpenoids are secondary metabolites, which have a function in
communication and defence (Aharoni et al., 2005; Trapp and Croteau, 2001). The terpene
synthase family (TPS) is a set of related genes, approximately 20 to 150 (Chen et al., 2011),
that encodes for enzymes which synthesize different terpenes (Bohlmann et al., 1998). All
4
terpenoids, primary metabolites as well as secondary metabolites, are derived from
isopentenyl diphosphate. Via different pathways (mevalonate (MVA) pathway or
methylerythritol 4-phosphate (MEP) pathway) consisting of terpene synthases monoterpenes,
sesquiterpenes, diterpenes, triterpenes and tetraterpenes are produced. Monoterpenes and
sesquiterpenes are volatile under ambient temperature and atmospheric pressure (Aharoni et
al., 2005; Trapp and Croteau, 2001).
Many plant species infested with the spider mite Tetranychus urticae emit volatile
blends containing terpenoids (see Van den Boom et al., 2002). It is known that some terpenes
are attractive to predatory mites. For example (E)-β-ocimene, linalool, (E)-4,8-dimethyl1,3,7-nonatriene [(E)-DMNT] and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene [(E,E)TMTT] (Dicke et al., 1990; De Boer et al., 2004). Some examples of plants emitting
terpenoids as a response to spider mite attack are lima bean (Phaseolus lunatus L.) and the
cultivated cucumber (Cucumis sativus L.) (Bouwmeester et al., 1999; Dicke et al., 1990;
Kappers et al., 2010; Mercke et al., 2004; Takabayashi et al., 1994). It is likely that other
cucurbit-species will also produce terpenoids as a response to herbivory by the spider mite
Tetranychus urticae.
Cucurbits
For the cultivated cucumber already a lot is known about its interaction with spider mites.
However, the non-bitter cucumber (C. sativus var corona) does not need to be pollinated to
produce fruits (seedless fruits) while other cucurbits depend on pollination (often by insects)
for fruit and seed production. Previous research has shown that leaf herbivory does lead to
changes in flower morphology and metabolism (e.g. Kessler and Halitschke, 2009; Theis et
al., 2009; Thomson et al., 2004). As already mentioned, HIPVs are produced in leaves as a
response to herbivory and they can be emitted systemically because plant parts are connected.
As a consequence, HIPVs may also be expressed in the flowers, which will result in a
different flower volatile blend. This may have an influence on the behaviour of flower visitors
like pollinators (e.g. Adler et al., 2001), which may result in less or no pollinated flowers and
thus less or no fruit production. For growers this is devastating. Studying volatile emission of
vegetative and flower tissues of a plant may give a more complete picture of what the effect
of plant defence against herbivores is and eventually could be on pollination (Lucas-Barbosa
et al., 2011).
The cucurbits cucumber (Cucumis sativus var corona, Cucumis sativus var Chinese
long, 9930 and Cucumis sativus var hardwickii), melon (Cucumis melo), watermelon
(Citrullus lanatus), courgette (Cucurbita pepo), pumpkin (Cucurbita maxima) and bitter
gourd (Momordica charantia) will be studied to look at variation in interaction with spider
mites within the Cucurbitaceae family (Fig. 2). Van Poecke and colleagues (2001) had shown
that the model plant Arabidopsis thaliana is capable of emitting volatiles, as a response to
herbivory, to attract carnivorous arthropods. Examples of emitted volatiles by A. thaliana are
methyl salicylate and terpenoids (van Poecke et al., 2001). Both are known to be induced by
herbivory in many plant species (Dicke et al., 1990). The entire genome of Arabidopsis
thaliana is known. It also contains known terpenoid synthase genes (Aubourg et al., 2002;
Chen et al., 2004; Tholl et al., 2005). Therefore Arabidopsis will also been grown and infested
with spider mites.
So there is strong evidence that flower morphology and metabolism change when there are
herbivores attacking the plant. This leads to the following research question: Does herbivory
by spider mites (Tetranychus urticae) on the leaves of different cucurbits have influence
on flower/leaf characteristics?
5
First of all it must be confirmed that the spider mites really cause damage to the plant
they are feeding on. Damage can consist of the chlorotic lesions the spider mites create on the
leaves. Therefore, changes in the amount of chlorophyll will be measured on leaves of all
cucurbit species.
The objective of this study is to explore the variation among members of the plant
family Cucurbitaceae with respect to flower characteristics that may be influenced by
herbivory on leaves by spider mites (Tetranychus urticae). The characteristics that will be
investigated are the production of endogenous metabolites and volatiles (especially terpenes)
in flowers as well as leaves of the different cucurbits with and without leaf herbivory. With
the methods liquid chromatography–mass spectrometry (LC-MS), and headspace collection in
combination with gas chromatography–mass spectrometry (GC-MS) it is determined which
kind of endogenous metabolites and volatiles are produced, respectively. For cucumber
(Cucumis sativus var corona and Cucumis sativus var Chinese long, 9930) the RNA of
flowers and leaves will be isolated to test which terpene synthase family (TPS) genes (TPS1,
TPS9 and TPS19) are expressed in control and infested samples, and therefore may be
responsible for the volatile emission identified with GC-MS.
The genome of cucumber (Cucumis sativus var. sativus L.) is recently discovered (Huang et
al., 2009) as well as a substantial part of the genome of melon (Cucumis melo L.) (GarciaMas et al., 2012). Some terpene synthase genes are already characterised in cucumber
(Mercke et al., 2004), for other terpenoids it is currently investigated by which terpene
synthase genes they are produced. Until now 26 TPS are known for cucumber (personal
comment dr. ir. Iris F. Kappers). The known genomes and terpene synthase genes in
Arabidopsis and cucumber can be helpful in studying the signal transduction pathways that
induce direct and indirect defence mechanisms in the other plant species. It can be expected
that there will be an overlap between cucumber and the other Cucurbitaceae members,
because they are genetically closely related.
Members of the family Cucurbitaceae are able to produce cucurbitacins. Cucurbitacins
are bitter triterpenoid compounds that are responsible for the bitter taste of the foliage, the
fruits are normally not bitter. These triterpenoid compounds are toxic to most organisms (can
also be toxic to mammals, including man) and therefore are part of the defence of cucurbits
against herbivores (Miró, 1995). Cucurbitacins are present constitutively in the plant, but can
also be induced by herbivores (Agrawal et al., 1999). Plants that taste bitter contain the gene
Bi, non-bitter plants contain the recessive allele bi (bitterfree) (Pierce and Wehner, 1990). The
fruits of cucumber plants can become bitter as a result of stress. The bi gene in non-bitter
plants prevents this from happening (Balkema-Boomstra et al., 2003). Cucurbitacins occur
widely in wild and cultivated Cucurbitaceae (Miró, 1995). It is speculated that cucurbitacins
are involved in the resistance towards the cucurbit-pest: the two-spotted spider mite,
Tetranychus urticae Koch. Much research about this resistance is done in cucumber (Cucumis
sativus). The research of Da Costa and Jones (1971) demonstrated that non-bitter cucumber
plants suffered much more from feeding by the two-spotted spider mite than did bitter plants.
The spider mite-mortality on bitter cucumber varieties was higher because mites did not
evolve mechanisms to detoxify the bitter cucurbitacins in the leaves (Da Costa and Jones,
1971; Pierce and Wehner, 1990). Gould (1978) also found results supporting the hypothesis
that cucurbitacins in the family Cucurbitaceae are involved in resistance against the twospotted spider mite. Even did Balkema-Boomstra and colleagues (2003) with cucurbitacin C.
In the cultivated non-bitter cucumber Cucumis sativus (cultivar Eversweet) so far, only one
cucurbitacin-form is identified. This cucurbitacin-form is similar to cucurbitacin C (Rice et
al., 1981). However, spider mite resistance in cultivated non-bitter cucumbers (and other
cucurbit species) is not (yet) reality (Balkema-Boomstra et al., 2003).
6
Figure 2 Phylogenetic tree of the family Cucurbitaceae
In this research the tribes Momordiceae, Cucurbiteae and Benincaseae are important. To the Momordiceae-tribe belongs the plant Momordica
charantia L. (bitter gourd). Cucurbita pepo L. (courgette/zucchini) and Cucurbita maxima Duchene (pumpkin) are in the Cucurbiteae-tribe. To the
Benincaceae-tribe belongs Citrullus lanatus Thunb. (watermelon) and the genus Cucumis with the species Cucumis melo L. (melon) and Cucumis
sativus L. with the varieties hardwickii, corona and Chinese long, 9930.
7
Material and methods
Plant and mite material
Seeds of six different cucurbit species were sown and grown in 1 L pots with potting compost
at 22°C during 12 hours of light, 18°C at night and 70% relative humidity in a climate
chamber at Wageningen University, The Netherlands (Table 1). TL lamps were used which
relatively burned with 55% at an intensity of 175 µmoll. The seeds of Momordica charantia
L. were sown and grown in 1 L pots with potting compost in a box made of glass in a
greenhouse to create a humid, warm environment which they prefer (Njoroge and Van Luijk,
2004). Here they grew at 18-24°C, 20-85% relative humidity under a natural daylight
photoperiod (March-April) for 2.5 weeks before being transferred to the same climate
chamber as the other cucurbits. The dormancy in the seeds of Cucumis sativus L. var.
hardwickii had to be broken by putting them in an Eppendorf tube with sterile water and
warm them till 55°C in a warm water bath for 2 hours. They were germinated on water agar
(2%) with 30 mg/L MS (Murashige & Skoog medium) in an oven of 28°C. After germination
the hardwickii seedlings were grown further in a climate chamber with a temperature of 24°C
till they had grown about 4 leaves. Then they were transferred to the same climate chamber as
the other cucurbits. Before the Arabidopsis thaliana L. seeds were able to germinate on
Rockwool, they needed a pre-treatment. The seeds were sterilized by washing them with 70%
EtOH, 10% bleach and three times with sterile water to wash off the remaining EtOH and
bleach. After sterilization, the seeds were germinated on Rockwool blocks in the same climate
chamber as the other plant species.
The phytophagous two-spotted spider mite Tetranychus urticae Koch was reared on
lima bean plants (Phaseolus vulgaris L.) in a greenhouse at Wageningen University, The
Netherlands.
Table 1 Cucurbitaceae members (cucurbits) used in this research
Bitter/non-bitter
leaves
Flowers
♀/♂/
Scientific name
Trivial name
Cultivated/wild
Cucumis sativus L.
var. corona
cucumber
cultivated
(hybrid)
non-bitter
♀
Cucumis sativus L.
var. Chinese long,
9930
cucumber
cultivated
bitter (cucurbitacin
C)
♀+♂
Cucumis sativus L.
var. hardwickii
cucumber
wild
bitter (cucurbitacin
C)
♀+♂
Cucumis melo L.
melon
cultivated
Citrullus lanatus
Thunb.
watermelon
cultivated
♀+♂
Cucurbita pepo L.
courgette
cultivated
♀+♂
Cucurbita maxima
Duchesne
pumpkin
cultivated
♀+♂
Momordica
charantia L.
bitter gourd
bitter
♀+♂
(pentanorcucurbitacin
A & B, momordicine
II & IV)
8
Plant treatments
The cucurbit plants used for experiments were 3-5 weeks old
and had four or five fully expanded leaves (C. sativus var
hardwickii did not germinate). Arabidopsis thaliana L. plants
used for experiments had a rosette that was not fully grown and
did not form a peduncle yet. Of each species (about 10 plants
per species) the plants were split into two separate, but
comparable compartments (Table 2). One half stayed in the
climate chamber as control plants and the other half was put in
tents in a greenhouse at Wageningen University, The
Netherlands (Fig. 3). These tents were suitable for infestations
with spider mites, which was done by laying a piece of lima
bean leaf, infested with spider mites, on top of each plant. The Figure 3 Tent containing plants
mites occupied the leaves themselves. The spider mites were on infested with spider mites
the plants for approximately 10 days before further experiments
were carried out.
Table 2 Number of plants per treatment for all plant species
Plant species
C. sativus var corona
C. sativus var Chinese long, 9930
C. melo (melon)
C. lanatus (watermelon)
C. pepo (courgette)
C. maxima (pumpkin)
M. charantia (bitter gourd)
A. thaliana
Plant number per treatment (biological replicates)
Control
Infested
5
5
5
5
5
5
5
4
5
5
5
5
4
3
3
3
Harvest leaf and flower material
About 15 days after the start of the plant treatment, leaf number 4 or 5 (or some leaves of the
rosette of A. thaliana) of each plant was harvested and ground with a mortar and pestle in
liquid nitrogen. Material of each leaf was divided over Eppendorf tubes, around 100 mg leaf
material per tube, for different experiments: chlorophyll measurement, RNA isolation and
liquid chromatography–mass spectrometry (LC-MS). Another Eppendorf tube was filled with
leftover material as back-up.
Of each plant species flowers were harvested and ground with a mortar and pestle in
liquid nitrogen. If applicable, male and female flowers were harvested separately. The time
points at which flowers were harvested varied between plant species because of the difference
in plant development rate. Material of each flower was divided over Eppendorf tubes, around
100 mg flower material per tube, for different experiments: RNA isolation and liquid
chromatography–mass spectrometry (LC-MS). Another Eppendorf tube was filled with
leftover material as back-up.
9
Chlorophyll analysis
The amount of chlorophyll in leaves of control and infested plants was measured as an
indication of leaf damage caused by spider mites. Spider mites suck out the cells of a leaf,
which results in chlorotic lesions (Fasulo and Denmark, 2009). The amount of chlorophyll in
the samples was determined with a spectrophotometer, which measured the absorbance of
chlorophyll at various wavelengths. For each plant species there were about 5 control plants
and 5 infested plants, which function as biological replicates, from which leaves were
investigated (see Table 2).
Per sample around 100 mg ground leaf material was put in an Eppendorf tube. 1 mL
of 96% EtOH (around 4°C) was added as a solvent to each sample, which was homogenized
on a vortex till the leaf material thawed. All samples were stored in the dark in a fridge
(around 4°C) overnight. After homogenizing on a vortex again, the samples were centrifuged
at low speed. For each sample a part of the supernatant was diluted 10 times with 96% EtOH,
the other part stayed undiluted. Of both the diluted and undiluted extractions 200 µL was
added in wells of a 96-well plate. As a control, some wells were filled with 200 µL 96%
EtOH. This 96-well plate was read by a spectrophotometer at wavelengths 665 nm and 649
nm which gave information about the light absorbance by chlorophyll A and B and the
controls. With the formulas provided by Rowan (1989),
𝐶ℎ𝑙 𝐴 (𝜇𝑔 𝑚𝐿−1 ) ≈ 13.70 × 𝐴665 − 5.76 × 𝐴649
𝐶ℎ𝑙 𝐵 (𝜇𝑔 𝑚𝐿−1 ) ≈ −7.60 × 𝐴665 + 25.8 × 𝐴649
the concentration of chlorophyll A and B in each sample was calculated (Raymond, 2006).
Those concentrations were corrected for the solvent by subtracting the concentration of 96%
EtOH. To get to know the amount of chlorophyll per fresh leaf weight (µg/mg FW), the
concentrations for chlorophyll A and B were divided by the weight of the ground leaf
material. For each plant species the average amount of chlorophyll per fresh leaf weight from
the biological replicates was calculated. These average amounts of chlorophyll in the control
and infested leaves were compared with each other by setting the amount of the controls at
100%. Therefore, the chlorophyll percentage in the infested leaves was calculated which gave
an indication for the chlorophyll reduction caused by spider mites sucking out the contents of
the cells in leaves.
Dynamic headspace collection
Dynamic headspace (airspace) collection was applied in the climate chamber as well as in the
greenhouse to analyse the headspace profile and its quantity of herbivory-induced volatiles in
the different cucurbits. In this research, the headspace collection was done in different ways
depending on the size of the plant parts from which the scents were collected. Ideally, the
investigated leaves and flowers stayed attached to the plant, because mechanical damage by
cutting the leaf or flower from the plant could influence the volatile emission of the plant part.
This was done by developing a new headspace set-up using a PET (polyethylene
terephthalate) bag (Toppits® Melitta) instead of a glass jar (Fig. 5). This bag was placed
around the leaf or flower on a living cucurbit plant and was loosely attached to the stem of the
leaf or flower to close it a bit. There were two holes in the bag that functioned as an inlet and
an outlet, the same as in a glass jar. In both the inlet and outlet a stainless steel cartridge was
connected, which contained 200 mg of the adsorbent Tenax TA (20/35 mesh; GraceAlltech,
Deerfield, MI, USA). Using a pump air was blown into the bag with a constant flow of 150±5
mL min-1 through the inlet cartridge which filtered the air. On the outlet cartridge a portable
battery-operated air sampler was placed that drew the air through the cartridge for 1 hour with
a constant flow of 100±5 mL min-1 (Ametek/du Pont de Nemours & Co., type Alpha-2,
10
DEHA International, Huizen, The Netherlands). Here the volatiles emitted by the plant part
were trapped. The difference in air flow strength was necessary to prevent dirty air leaking
out from the bag from the hole around the stem of the leaf or flower where the bag did not
close well enough. However, not all plant parts of all investigated plant species were big
enough to cover with the PET bag. Therefore, the plant part needed to be harvested and the
new headspace collection set-up recently developed by The Laboratory of Plant Physiology at
Wageningen University & Research Centre was used (Fig. 5). This set-up does not make use
of the portable battery-operated air samplers anymore and it has 12 positions to collect the
headspace at the same time. In principle it works the same as with the PET bag. A plant or
plant part was covered with a glass jar (2.5 L). The inner space was connected with an inlet
and outlet with cartridges which contained 200 mg Tenax TA. A pump blew air into the jar
with a constant flow of 150±5 mL min-1 through the inlet cartridge which filtered the air.
Another pump was connected to the outlet cartridge that drew air from the inside of the jar
through the outlet cartridge with a constant flow of 100±5 mL min-1 for 1 hour. Here the
volatiles emitted by the plant part were trapped. Also in this set-up the difference in flow
strength was necessary to prevent dirty air leaking from the outside of the jar into the system.
A computer was connected to both pumps to control the air flow and other environmental
conditions.
Figure 5 Headspace collection set-up
Left: Headspace collection set-up with a PET bag. The bag was placed around the leaf or flower. With a
pump air was blown into the bag through the inlet Tenax cartridge which filtered the air. The portable batteryoperated air sampler drew the air through the outlet Tenax cartridge to trap the emitted volatiles. A higher
inlet airflow than outlet airflow was necessary to prevent dirty air from leaking into the PET bag.
Right: New headspace collection set-up developed by The Laboratory of Plant Physiology at Wageningen
University & Research Centre. The glass jar was placed on the flower. With a pump air was blown into the jar
through the inlet Tenax cartridge which filtered the air. Another pump drew the air through the outlet Tenax
cartridge to trap the emitted volatiles. A higher inlet airflow than outlet airflow was necessary to prevent dirty
air from leaking into the glass jar.
11
When the new headspace set-up was not available, the headspace was collected in the
old fashioned way by putting the plant part of interest in a glass jar (0.5 L). The jar was closed
with a lid equipped with an inlet and an outlet. In both the inlet and outlet a stainless steel
cartridge was put, which contained 200 mg Tenax TA. Air was drawn through the cartridges
for 1 hour using a portable battery-operated air sampler with a constant flow of 100±5 mL
min-1.
Gas chromatography–mass spectrometry
Headspace samples of cucurbit leaves and flowers were analysed with a gas chromatography–
mass spectrometry (GC-MS) machine to identify the volatiles that were trapped (analyte).
This machine consisted of Thermo Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA,
USA) connected to a Thermo Trace DSQ (Thermo Fisher Scientific, Waltham, MA, USA)
quadrupole mass spectrometer. Before being analysed, the Tenax cartridges were dry-purged
with nitrogen (pressure 20 PSI) for 10 minutes at room temperature to remove any water. The
cartridges were heated at 250°C by a thermal desorption system with a helium flow of 30 mL
min-1 to elute the volatiles from the adsorbent Tenax. To concentrate the analytes, they were
cooled down by an electronically-cooled sorbent trap at 3°C and brought splitless onto the
analytical column by rapidly increasing the temperature in the cold trap to 250°C. This
column started with a temperature of 40°C and increased with 10°C min-1 until 280°C
(Kappers et al., 2011). Gas chromatography is based on a mobile phase which carries the
analyte with it through a column containing stationary phase materials as a film at its wall.
The analyte consists of molecules with specific chemical and physical characteristics which
determine the rate at which they are transported through the column by the mobile phase, and
their interaction (adsorption) by the stationary phase materials in the column. So, the column
retains the molecules, elutes them eventually when the temperature of the column got high
enough and therefore the different molecules reach the end of the column at specific time
points (retention time). In this way the compounds are separated from each other and are
further analysed by the mass spectrometer (Tholl et al., 2006).
In the mass spectrometer the compounds that exit the column were ionized by electron
impact (EI) at 70 eV, which resulted in charged molecule fragments. They were separated by
the quadrupole according to their mass-to-charge ratio of 45-400 m/z. With its specific mass
spectrum on a certain retention time it was clear how abundant the molecule (fragment) was
present in the headspace collection sample. To align the peaks in the chromatograms of all
headspace collection samples, Metalign software (PRI-Rikilt, Wageningen, The Netherlands)
was used which resulted in two excel files: a noise and an amplitude file. The noise was
subtracted from the amplitude with the IF-formula: IF (amplitude value > 5000, amplitude
value, 10 (a random number)). Eventually the peaks with mass 93 (important terpene-mass)
were selected for, the average and standard deviation was calculated and a Students t-test was
used to see at which retention times the control and infested samples were significantly
different (p<0.05). For those significantly different samples the fold change was calculated. If
the amplitude of infested plant material was higher than that of control plant material, than
there was an increase in volatile emission level by the infested plant (shown in tables 4-7 as
upward arrows). When not, there was a decrease in volatile emission level by the infested
plant (shown in tables 4-7 as downward arrows). A fold change higher than 10 did indicate an
important peak in the volatile blend. Peaks were visualized in the chromatograms (shown in
figures 8-12 and in Appendix 3), which were also consulted for the determination of volatile
emission level by comparing the relative abundances of the control and infested peak with
each other. For peaks with a fold change higher than 10, the putative identity of the volatiles
was searched for by using Xcalibur and by comparing their retention times and mass spectra
with those in NIST and Wiley spectral libraries (Kappers et al., 2011). Some other peaks
12
without a fold change higher than 10 could also be interesting to know the volatile identity of.
For example when at a specific retention time the peaks of both treatments differ (a lot) in
their relative abundance.
Liquid chromatography–mass spectrometry
Leaf and flower material of C. sativus var Chinese long, 9930 and C. sativus var corona was
examined for endogenous stress-related phytohormones by a liquid chromatography–tandem
mass spectrometry (LC-MS/MS). The metabolites of interest were jasmonic acid (JA), its
bioactive amino acid conjugate (JA-Ile), salicylic acid (SA) and abscisic acid (ABA). The leaf
or flower sample was homogenized in a mortar using liquid nitrogen and divided over three
2ml microcentrifuge tubes (technical replicates). About 15 mg of fresh plant weight was
extracted with 1 mL 10% ice cold methanol/water. To control the extraction recovery and
validate the quantification of the selected metabolites, stable isotope-labelled compounds
were used as internal standards: [2H6]-JA and [2H2]-JA-Ile (Olchemim), [2H4]-SA and [2H6]ABA (Cambridge Isotope Laboratories). Tungsten carbide balls were added to each sample.
The samples were homogenized in a mixer mill MM400 at a frequency of 27 Hz for 3
minutes and further sonicated in an ultrasonic bath for 2 minutes. Samples were extracted 25
min/4°C using a rotator shaker. To separate the plant material from the extraction solvent, the
tubes were centrifuged for 4 minutes at 14,000xg at 4°C. The supernatant was transferred to a
new tube. The plant material was re-extracted by adding 1 mL 10% ice cold methanol/water,
quickly spin stirring by vortex and centrifuging it again. Both supernatants were combined
and further purified by solid phase extraction. The Oasis® HLB (1 cc/30 mg cartridge, Waters
Co., Milford, MA, USA) sorbent was conditioned with 1 mL of 100% methanol (organic
solvent) and equilibrated with 1 mL of MiliQ water. These liquids were used to activate
functional groups of the sorbent. The supernatant was loaded onto the column and the sorbent
was subsequently washed with 1 ml of extraction solvent (10% methanol/water). The
compounds were eluted off the cartridge by adding 3 mL 80% methanol. Samples were
evaporated under the stream of nitrogen till dryness and reconstructed with 40 µL of 15%
ACN: 15 mM HCOOH, v/v (initial conditions of LC gradient) (Flokova et al., 2014).
Samples were further analysed by the ultra-high performance liquid chromatography–
tandem mass spectrometry, consisting of the ACQUITY UPLC System (Waters) and Xevo
TQ S (Waters) triple quadrupole mass spectrometer. The solvent used as a mobile phase was
composed of 15mM HCOOH (A, aqueous part) and ACN (B, organic part). The compounds
were separated by ACQUITY UPLC CSH C18 column (100x2.1 mm, 1.7 μm; Waters) at a
flow rate 0.4 ml/min and temperature 40°C with binary gradient as follows: isocratic elution
at 15% B (from 0 to 1 min), linear increase to 60% B (1-7 min), linear increase to 80% B (7-9
min), logarithmic increase to 100% B (9-10 min). Finally the column was washed with 100%
B for 1 min and equilibrated for initial conditions for 2 min. The effluent was introduced into
the electrospray ion source of the mass spectrometer. The capillary voltage was set on 3kV.
The source block/desolvation temperature was 120°C/ 550°C with desolvation gas flow 650
l.h-1. Compounds were quantified by multiple ion monitoring mode (MRM) with settings
listed in Flokova et al., 2014.
Gene expression of TPS1, TPS9 and TPS19 genes
The RNA in leaf as well as flower material of the plant species C. sativus var corona and C.
sativus var Chinese long, 9930 was isolated to test which terpene synthase family (TPS) genes
(TPS1, TPS9 and TPS19) were expressed in control and infested samples. Per sample around
100 mg ground leaf or flower material was put in an Eppendorf tube to which 1 mL TriPure
was added. This mixture was homogenized on a vortex and was allowed to stand at room
temperature for 5 minutes before 200 µL of chloroform was added. This mixture was again
13
homogenized on a vortex and allowed to stand at room temperature for 5 minutes. TriPure
and chloroform form a phenol-chloroform extraction which is a liquid-liquid extraction
technique for purifying RNA and eliminating plant proteins. After centrifugation at 12,000xg
at 4°C for 15 minutes the supernatant containing the RNA was pipetted into a new Eppendorf
tube, while the pellet containing proteins and other plant cell debris was thrown away. To the
new Eppendorf tube 0.5 times the supernatant volume of 96% ethanol (EtOH) was added and
mixed by inverting. This mixture was applied to an RNeasy spin column placed in a 2 mL
collection tube and centrifuged at 8000xg for 15 seconds. The flow-through was discarded.
From this point onwards the protocol “Purification of Total RNA from Plant Cells and
Tissues and Filamentous Fungi” was used starting with step 7 on page 53 (RNeasy Mini
Handbook 06/2012, QIAGEN). During this protocol the RNA on the RNeasy spin column
was washed with RW1 and RPE buffer. Every time a specific buffer was applied to the
column, a waiting step of 5 minutes was introduced to give the buffer time to work, instead of
centrifuging immediately. At step 11 of the protocol 100-200 µL RNase-free water was used
to elute the RNA from the RNeasy spin column, it depends on if a back-up was preferred.
With NanoDrop (Nanodrop 1000 3.7.1) the RNA concentration and the A260/A280 and
A260/A230 values were measured. The A260/A280 and A260/A230 values should be around
2.0. When these values were sufficient the RNA was used for the DNase treatment. 89 µL
RNA was applied to an Eppendorf tube together with 10 µL DNase buffer and 1 µL DNase.
This was allowed to stand at room temperature for 15 minutes. From this point onwards the
protocol “RNA Cleanup” was used starting with step 1, second line on page 54 (RNeasy Mini
Handbook 06/2012, QIAGEN). During this protocol the RNA was washed with RLT and
RPE buffer and with 96% ethanol (EtOH). Also within these washing steps, a waiting step of
5 minutes was introduced to give the buffer or the ethanol time to work, instead of
centrifuging immediately. At step 7 of the protocol 30-100 µL RNase-free water was used to
elute the RNA from the RNeasy spin column. Again with NanoDrop the RNA concentration
and the A260/A280 and A260/A230 values were measured. The A260/A280 and A260/A230
values should again be around 2.0 (see Appendix 1).
With reverse transcription polymerase chain reaction (RT-PCR) complementary DNA
(cDNA) transcripts from RNA were created, which were eventually used to detect gene
expression. This cDNA was made by using 1 µg of RNA. A total volume of 20 µL, of which
4 µL 5x iScript reaction mix and 1 µL iScript Reverse Transcriptase, was added to Eppendorf
PCR tubes, supplemented with Nuclease free water. The PCR machine (GeneAmp PCR
System 9700, PE Applied Biosystems) followed the reverse transcription programme: 5
minutes at 25°C, 30 minutes at 42°C, 5 minutes at 82°C and hold at 10°C (see Appendix 1).
With NanoDrop the concentration of created cDNA was measured, which should be around
1000 ng/µL and the difference in concentration among the cucurbit samples should be less
than 100 ng/µL (see Appendix 2, step 7). The cDNA was diluted to a volume of 200 µL. The
samples were run on the qPCR machine (Bio-RAD MyiQTM Single colour Real-Time PCR
Detection System) in duplicate (see Appendix 2). Forward and backward primers of TPS1,
TPS19 and TPS9 and actin were used in combination with the cDNA of the flowers of C.
sativus var Chinese long, 9930 and C. sativus var corona. TPS1, TPS19 and TPS9 encode for
enzymes which synthesize the terpenes linalool, α-farnesene and β-ocimene, respectively
(personal comment dr. ir. Iris F. Kappers). Actin was used as a reference gene, because it is a
housekeeping gene that is expressed in each plant cell under normal conditions, and its
expression is quite stable under different conditions. The threshold cycles of the genes of
interest (TPS1, TPS19 and TPS9) were compared to the threshold cycle of the housekeeping
gene actin to see how relative transcripts of each of the TPS genes were.
14
Results
Chlorophyll analysis
Figure 6 Left: control plants 15 days after germination. Right: plants infested with spider mites for 20 days. On both
pictures C. pepo (courgette) plants are shown.
Figure 6 showed what the treated plants of C. pepo (courgette) looked like after a certain
period. The control plants of this species were greener than the plants infested with spider
mites.
In Table 12 (see Appendix 4) the amount of chlorophyll per fresh leaf weight (µg/mg FW) is
shown. To check if the amount of chlorophyll A and B per fresh leaf weight (µg/mg FW) for
the diluted and undiluted samples was sufficient, the values for the diluted samples (diluted
10 times), when multiplied with 10, should be the same as for the undiluted samples.
However, for chlorophyll A this was not the case, the values were often much higher.
Therefore, the values of the diluted samples were used for further calculations.
The amount of chlorophyll per fresh leaf weight (µg/mg FW) for the diluted samples
of all control plants were seen as the maximum amounts of chlorophyll that could be present
in leaves without damage caused by herbivores. Therefore, these amounts were set at 100%.
For the infested plants it was calculated what percentage their amount of chlorophyll per fresh
leaf weight was, compared to the control plants. So for example for chlorophyll A in the nonbitter cucumber C. sativus var corona, 0.045 is set at 100%, and 0.011 is then
(0.011 ∗ 100)
= 25%
0.045
So the amount of chlorophyll A per fresh leaf weight in infested non-bitter cucumber plants
was 25%. This calculation was done for all plant species and showed that for all species,
plants infested with spider mites had a lower chlorophyll A and chlorophyll B percentage than
control plants (Table 3).
The percentages of chlorophyll A and B in infested leaves of A. thaliana and different
cucurbits from table 3 were shown in figure 7. The chlorophyll percentage of the control
plants was always 100% and therefore not shown. For all plant species a decrease in
chlorophyll percentage can be seen when the plant was infested with spider mites. Most of the
time there was less chlorophyll A left in the leaves than chlorophyll B. How large the effect of
herbivory was on the amount of chlorophyll left in the plant varied between the plant species.
15
Table 3 Amount of chlorophyll per fresh leaf weight (µg/mg FW) in percentages for control and infested leaves
in A. thaliana and different cucurbits in diluted samples.
Plant
species
C. sativus
var corona
C. sativus
var Chinese
long, 9930
C. melo
(melon)
C. lanatus
(watermelon)
C. pepo
(courgette)
C. maxima
(pumpkin)
M. charantia
(bitter gourd)
A. thaliana
Treatment Diluted
Amount of chlorophyll per fresh leaf weight
(µg/mg FW) in percentages
Chlorophyll A
Chlorophyll B
Average
± SD Average
± SD
(%)
(%)
(%)
(%)
control
infested
control
infested
diluted
diluted
diluted
diluted
100
25
100
71
10
5
18
6
100
25
100
80
10
5
21
4
control
infested
control
infested
control
infested
control
infested
control
infested
control
infested
diluted
diluted
diluted
diluted
diluted
diluted
diluted
diluted
diluted
diluted
diluted
diluted
100
55
100
68
100
28
100
66
100
62
100
52
35
5
4
4
4
7
23
13
23
22
6
18
100
57
100
73
100
28
100
76
100
71
100
63
28
8
3
5
5
6
25
16
26
25
6
13
compared to
Figure 7 Chlorophyll A and B percentage in leaves of A. thaliana and the different cucurbits that were
infested with spider mites for about 15 days. The chlorophyll percentage of the control plants was always
100% and therefore not shown.
16
Gas chromatography–mass spectrometry
The headspace of leaves and flowers of different cucurbits, induced by spider mites feeding
on the leaves, consisted of 25 volatile compounds that did represent most of the volatile blend
emitted by leaves and flowers (Table 4, 5, 6 and 7). The composition of the volatile blend
varies between the plant species. Most of these compounds can be found in the infested as
well as the control plants, especially in the flowers. In leaves, some volatiles were only
emitted by infested plants: (-)-β-pinene, ((E)-β-)ocimene, linalool, (E)-4,8-dimethyl-1,3,7nonatriene [(E)-DMNT], methyl salicylate (MeSA), α-farnesene and (E,E)-4,8,12-trimethyl1,3,7,11-tridecatetraene [(E,E)-TMTT] (Table 4). The majority of the volatile compounds in
both leaves and flowers were terpenes: about 75% of the whole blend, consisting of eight
monoterpenes (α-pinene, (-)-β-pinene, (E)-β-ocimene, ocimene, linalool, linalool tetrahydride,
nerol and (E)-2-decanal), nine sesquiterpenes (α-bourbonene, cedrene, α-cubebene,
caryophyllene, α-farnesene, β-farnesene, β-ylangene, germacrene and nerolidol) and two
homoterpenes (DMNT and TMTT). The remaining compounds were a phenylpropanoid
(MeSA) and two aldehydes (nonanal and decanal). In flowers an aromatic alcohol (benzyl
alcohol) and another aldehyde (benzaldehyde) were found, however, these compounds and the
two aldehydes (nonanal and decanal) did not contain mass 93 like the other volatile
compounds, which are terpenoids.
In figures 8 and 9 the chromatograms of the volatile blends of the infested and control
leaves of the cucumber species C. sativus var Chinese long, 9930 and C. sativus var corona
are shown. For the Chinese bitter cucumber the volatile profile of the control and the infested
leaves was quite different (Fig. 8). Mite feeding on leaves induced the emission of five
volatiles not seen in control plants: (E)-β-ocimene, (E)-DMNT, methyl salicylate (MeSA), αfarnesene and (E,E)-TMTT. For compounds which are not in the biochemical class of
terpenes like the aldehydes nonanal and decanal, the relative abundance was decreased when
comparing the blend of infested to control leaves. Geranylacetone, on the other hand, had an
increased relative abundance.
17
Table 4 Important volatiles in headspace of cucurbit leaves according to Metalign results and chromatograms. Within each plant species the headspace of infested leaves is
compared with the headspace of control leaves. The production of a specific volatile in infested plants can be increased, decreased or is about the same level as in control
plants. This is determined with the values given by Metalign and with comparing the relative abundance of the control and infested peaks in the chromatogram.
Leaves
Plant species
C. sativus var
corona
Code
Compound
Biochemical class
f
g
h
o
u
v
F
G
M
N
S
y
U
A
α-pinene
(-)-β-pinene
β-ocimene
monoterpene
monoterpene
monoterpene
monoterpene
monoterpene
monoterpene
sesquiterpene
sesquiterpene
sesquiterpene
sesquiterpene
sesquiterpene
homoterpene
homoterpene
phenylpropanoid
(E)-β-ocimene
x
B
I
P
linalool tetrahydride
linalool
α-bourbonene
α-cubebene
cedrene
caryophyllene
α-farnesene
(E)-DMNT
(E,E)-TMTT
methyl salicylate
(MeSA)
nonanal
decanal
methyl perillate
geranylacetone
↑
↓
−
?
*
increase
decrease
about the same level
this or other compound
only in infested plant
aldehyde
aldehyde
−
C. sativus var
Chinese long,
9930
−
C. melo
(melon)
−
↑*
−
↑*
−
↑*
↑*
↑*
↑*
↑*
↑*
↑*
↑*
↑*
↑*
? ↑*
C. lanatus
(watermelon)
↑
↑ and ↑*
↑*
↑*
↑*
C. pepo
(courgette)
C. maxima
(pumpkin)
M. charantia
↑*
−
? ↑*
? ↑*
? ↑*
? ↑*
↑*
↑*
↑
?↑
?↑
−
↑*
↑*
↑*
↑*
? ↑*
↑*
↑*
↓
↓
↓
↓
↓
↓
−
↓
↑
↑
↑
↑
−
↑
−
↑
↓
↑
↓
−
↑*
−
■ Significant difference infested and control plant, fold change > 10
■ Significant difference infested and control plant, fold change > 10, not always clear peak
■ Significant difference infested and control plant, fold change < 10
■ No significant difference infested and control plant
compared to
18
Table 5 Important volatiles in headspace of cucurbit male flowers according to Metalign results and chromatograms. Within each plant species the headspace of male flowers
grown on infested plants is compared with the headspace of male flowers grown on control plants. The production of a specific terpene can be increased or decreased in
flowers of infested plants or is about the same level as in flowers of control plants. This is determined with the values given by Metalign and with comparing the relative
abundance of the control and infested peaks in the chromatogram.
♂ flowers
Biochemical
class
Plant species
C. sativus var Chinese
long, 9930
Code
Compound
C. pepo (courgette)
C. maxima (pumpkin)
g
m
n
v
O
Q
R
S
T
x
B
P
β-pinene-(1S)-(-)
nerol
(E)-2-decanal
linalool
β-ylangene
β-farnesene
germacrene
α-farnesene
nerolidol
nonanal
decanal
geranylacetone
monoterpene
monoterpene
monoterpene
monoterpene
sesquiterpene
sesquiterpene
sesquiterpene
sesquiterpene
sesquiterpene
aldehyde
aldehyde
↑
↓
increase
decrease
■ Significant difference infested and control plant, fold change > 10
■ Significant difference infested and control plant, fold change > 10, not always clear peak
−
about the same level
■ Significant difference infested and control plant, fold change < 10
?
*
this or other compound
only in infested plant
■ No significant difference infested and control plant
M. charantia
?↓
?↓
↓
−
↑
↑
−
−
↑
?↓
↑
↓
↑
↑
−
↑
↑
↑
↑
↓
−
compared to
19
Table 6 Important volatiles in headspace of cucurbit female flowers according to Metalign results and chromatograms. Within each plant species the headspace of female
flowers grown on infested plants is compared with the headspace of female flowers grown on control plants. The production of a specific terpene can be increased or
decreased in flowers of infested plants or is about the same level as in flowers of control plants. This is determined with the values given by Metalign and with comparing the
relative abundance of the control and infested peaks in the chromatogram.
Code
♀ flowers
Compound
f
g
h
n
v
Q
S
T
A
α-pinene
β-pinene-(1S)-(-)
β-ocimene
(E)-2-decanal
linalool
β-farnesene
α-farnesene
nerolidol
methyl salicylate (MeSA)
x
B
P
nonanal
decanal
geranylacetone
↑
increase
■ Significant difference infested and control plant, fold change > 10
↓
decrease
■ Significant difference infested and control plant, fold change > 10, not always clear peak
−
about the same level
■ Significant difference infested and control plant, fold change < 10
Biochemical class
monoterpene
monoterpene
monoterpene
monoterpene
monoterpene
sesquiterpene
sesquiterpene
sesquiterpene
phenylpropanoid
aldehyde
aldehyde
Plant species
C. sativus var corona
C. sativus var Chinese long, 9930
M. charantia
?−
?−
↑
−
?−
↑
↑
↑
↑
↑*
↓
↓
−
↑
↑
↑
this or other compound
■ No significant difference infested and control plant
?
only
in
infested
plant
*
No results of female flowers of the plant species C. pepo and C. maxima because no adult female flowers
were grown on infested plants.
↓
↓
↓
compared to
20
Table 7 Important volatiles in headspace of cucurbit bisexual flowers according to Metalign results and
chromatograms. Within each plant species the headspace of bisexual flowers grown on infested plants is
compared with the headspace of bisexual flowers grown on control plants. The production of a specific terpene
can be increased or decreased in flowers of infested plants or is about the same level as in flowers of control
plants. This is determined with the values given by Metalign and with comparing the relative abundance of the
control and infested peaks in the chromatogram.
Code
flowers
Compound
g
u
x
B
P
β-pinene-(1S)-(-)
linalool tetrahydride
nonanal
decanal
geranylacetone
Biochemical class
Plant species
C. melo (melon)
monoterpene
monoterpene
aldehyde
aldehyde
−
−
↑
↑
↓
■ Significant difference infested and control plant, fold change > 10
■ Significant difference infested and control plant, fold change > 10, not always clear peak
■ Significant difference infested and control plant, fold change < 10
■ No significant difference infested and control plant
↑ increase
↓ decrease
− about the same level
compared to
? this or other compound
* only in infested plant
In the headspace of the non-bitter cucumber C. sativus var corona there was not much
difference between the blend of control and infested leaves (Fig. 9). Mite feeding on leaves
induced the emission of the volatile (E)-DMNT, which was not seen in control plants. For
compounds which are not in the biochemical class of terpenes like the aldehydes nonanal and
decanal, the relative abundance was decreased when comparing the blend of infested to
control leaves. For geranylacetone the relative abundance stayed at about the same level.
In figures 10, 11 and 12 the chromatograms of the volatile blends of the infested and
control male and female flowers of the cucumber species C. sativus var Chinese long, 9930
and C. sativus var corona are shown. Two important volatiles which not belong to the
terpenoid class were only seen in chromatograms of flowers: benzaldehyde and benzyl
alcohol. For the Chinese bitter cucumber the volatile profile of the male flowers grown on
control and infested plants was the same (Fig. 10). The relative abundance of the volatiles
linalool, nonanal1 and geranylacetone was decreased when comparing the blend of male
flowers on infested plants to male flowers on control plants. The compounds decanal, βfarnesene and nerolidol, on the other hand, had an increased abundance and for α-farnesene it
stayed at about the same level.
1
Not sure nonanal is the right identity name for this volatile compound
21
Figure 8 Chromatogram of control and infested leaves of C. sativus var Chinese long, 9930 with the
important volatile compounds (Table 4).
Figure 9 Chromatogram of control and infested leaves of C. sativus var corona with the important volatile
compounds (Table 4).
22
Figure 10 Chromatogram with the important volatile compounds emitted by male flowers grown on infested and
control plants of the plant species C. sativus var Chinese long, 9930 (Table 5). And two important volatiles only
emitted by the flowers: i = benzaldehyde and q = benzyl alcohol. (?x? not sure about identity).
Figure 11 Chromatogram with the important terpene volatiles emitted by male flowers grown on infested and
control plants of the plant species C. sativus var Chinese long, 9930 (Table 6). And two important volatiles only
emitted by the flowers: i = benzaldehyde and q = benzyl alcohol.
23
Figure 12 Chromatogram with the important terpene volatiles emitted by female flowers grown on infested and
control plants of the plant species C. sativus var corona (Table 6). And two important volatiles only emitted by
the flowers: i = benzaldehyde and q = benzyl alcohol. (?S? not sure about identity).
The female flowers of C. sativus var Chinese long, 9930 had the same volatile profile as the
male flowers except for the compound linalool (Fig. 11). Linalool was not emitted and the
infested female flowers produced methyl salicylate (MeSA) instead. The relative abundance
of the volatiles β-farnesene, α-farnesene, nerolidol, nonanal, decanal and geranylacetone was
increased when comparing the blend of female flowers on infested plants to female flowers on
control plants.
The volatile profile of female flowers of the non-bitter cucumber C. sativus var corona
was the same for flowers grown on control and on infested plants (Fig. 12). It contained some
of the compounds also found in female flowers of the Chinese bitter cucumber: nonanal,
decanal, α-farnesene2 and geranylacetone. And it contained volatiles not seen in the volatile
profile of the bitter cucumber: (E)-2-decenal and α-pinene or ocimene. For compounds which
are not in the biochemical class of terpenes like the aldehydes nonanal and decanal, the
relative abundance was decreased when comparing the blend of female flowers on infested
plants to female flowers on control plants. The volatile (E)-2-decenal, on the other hand, had
an increased abundance and for α-farnesene, geranylacetone and α-pinene or ocimene it
stayed at about the same level.
The chromatograms of leaves and flowers of the other cucurbits can be found in
Appendix 3. The volatile profiles of leaves and flowers of A. thaliana are not shown, because
something went wrong with the GC-MS machine while analysing the Tenax tubes containing
the captured headspaces of this plant species.
2
Not sure α-farnesene is the right identity name for this volatile compound
24
Liquid chromatography–mass spectrometry
Table 8 Amount of the endogenous secondary metabolites jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), salicylic acid (SA) and abscisic acid (ABA) in pmol/g fresh
sample weight in control and infested leaves and flowers of the cucumbers C. sativus var corona and C. sativus var Chinese long, 9930.
JA
JA-Ile
SA
Amount of endogenous secondary metabolite (pmol/g fresh weight)
Plant species
C. sativus var
corona
Treatment
control
infested
control
infested
Plant
material
leaf
leaf
♀ flower
♀ flower
C. sativus var
Chinese long, 9930
control
infested
control
infested
control
infested
leaf
leaf
♂ flower
♂ flower
♀ flower
♀ flower
ABA
Average
± SD
Average
± SD
Average
± SD
Average
± SD
0.939
6.127
0.461
0.603
0.072
0.578
0.067
0.043
ND*
1.326
2.844
0.483
ND*
0.103
0.314
0.060
13994
4077
10809
13904
5240
1576
1014
1037
5751
19531
58396
70677
589
1476
4283
4750
1.192
3.100
0.793
0.516
0.677
0.496
0.110
0.302
0.087
0.041
0.055
0.068
0.052
0.597
6.050
0.390
0.475
0.657
0.004
0.063
0.983
0.056
0.050
0.039
ND*
7920
23165
12874
13891
7155
ND*
5935
1448
1705
3039
752
52784
12451
76385
84540
53342
79562
3662
797
4765
5331
5170
11743
* ND = not detected
25
Figure 13 The amount of the endogenous secondary metabolites jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), salicylic acid (SA) and abscisic acid (ABA) in control and
infested leaves and female flowers of C. sativus var corona.
26
Figure 14 The amount of the endogenous secondary metabolites jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), salicylic acid (SA) and abscisic acid (ABA) in control
and infested leaves, male and female flowers of C. sativus var Chinese long, 9930.
27
The amount (pmol/g fresh weight) of the endogenous stress-related phytohormones jasmonic
acid (JA), its bioactive amino acid conjugate (JA-Ile), salicylic acid (SA) and abscisic acid
(ABA) in leaf and flower material of C. sativus var Chinese long, 9930 and C. sativus var
corona, was shown in table 8. For each plant species plant material of three control and three
infested plants was harvested and every sample was divided over three tubes, which served as
technical replicates. For those technical replicates the average amount of each metabolite was
taken, which did result in an amount of each metabolite within every plant. Eventually the
average amount of each metabolite was calculated within the three control and the three
infested plants of each plant species, which was shown in table 8. Figures 13 and 14 showed
the content of table 8. The amount of the endogenous secondary metabolites JA and ABA in
leaves and female flowers of C. sativus var corona increased when plants were infested with
spider mites. The amount of salicylic acid in female flowers grown on infested non-bitter
cucumber plants increased; however, in the leaves it decreased (Fig. 13). The amount of the
metabolites JA, and SA in both male and female flowers of C. sativus var Chinese long, 9930
decreased when plants were infested with spider mites. In the leaves of those plants the
amount of JA increased. Furthermore, the amount of ABA in male and female flowers grown
on bitter cucumber plants increased; however, in the leaves it decreased (Fig. 14).
The ratio of jasmonoyl-isoleucine (JA-Ile) and jasmonic acid (JA) in all plants parts of
the plant species C. sativus var corona and C. sativus var Chinese long, 9930 is shown in
table 9. The female flowers grown on infested plants of the non-bitter cucumber had a lower
JA-Ile/JA ratio than the flowers on the control plants. The same applied to male flowers of the
bitter cucumber. Female flowers grown on infested plants of the bitter cucumber and the
leaves of these infested plants had a higher JA-Ile/JA ratio than the controls.
Table 9 Ratio JA-Ile/JA in leaves, male and female flowers of C. sativus var corona and C.
sativus var Chinese long, 9930.
Plant species
Treatment
Plant material
JA-Ile/JA ratio
C. sativus var corona
control
infested
control
infested
leaf
leaf
♀ flower
♀ flower
NP*
0.216
6.163
0.801
C. sativus var Chinese long, 9930
control
leaf
0.043
infested
leaf
0.192
control
♂ flower
7.625
infested
♂ flower
0.755
control
♀ flower
0.701
infested
♀ flower
1.326
* NP = not possible, because JA-Ile not detected for control leaves of C. sativus var corona.
28
Gene expression of TPS1, TPS9 and TPS19 genes
The RNA concentration and the A260/A280 and A260/A230 values in control and infested
leaf and flower material of the plant species C. sativus var corona and C. sativus var Chinese
long, 9930 was checked with NanoDrop. For the leaf material of both species the RNA
concentration and the A260/A280 and A260/A230 values were not sufficient. Therefore,
leaves were not used for further experiments. The values for flowers on the other hand, were
sufficient.
For the flowers of each plant species and treatment, the threshold cycles of the genes
of interest (TPS1, TPS19 and TPS9) were compared to the threshold cycle of the
housekeeping gene actin to see how relative transcripts of each of the TPS genes were. The
difference in number of threshold cycles was used to calculate the relative gene expression
(RGE) and eventually the log2 ratio (Table 10). When the gene of interest needed a lower
number of cycles to cross the threshold than actin, there was a higher amount of cDNA in the
sample coding for that particular gene. Therefore, the RGE value was high. A gene of interest
had a low RGE value when it needed more cycles to cross the threshold than actin, because of
its low amount of cDNA present in the sample. The log2 ratio of a specific gene compared the
RGEs of that gene between an infested sample and a control sample. TPS1 and TPS9 in the
female flowers of the non-bitter cucumber had a positive log2 ratio while TPS19 had a
negative log2 ratio (Fig. 15). The TPS1 and TPS19 genes had a negative log2 ratio in both
male and female flowers of the Chinese cucumber (Fig. 16).
Table 10 Average relative gene expression (RGE) of TPS genes TPS1, TPS19 and TPS9 in male and female
flowers grown on control and infested plants of the plant species C. sativus var corona and C. sativus var
Chinese long, 9930. And the log2 ratio calculated with the formula: log (RGE infested/RGE control;2).
TPS
gene
Average RGE
(relative gene
expression)
Plant species
Treatment
Plant
material
C. sativus var corona
control
infested
control
infested
control
infested
♀ flower
♀ flower
♀ flower
♀ flower
♀ flower
♀ flower
1
1
19
19
9
9
0.482
0.911
0.117
0.035
0.002
0.006
C. sativus var Chinese control
long, 9930
infested
control
infested
control
infested
control
infested
♂ flower
♂ flower
♂ flower
♂ flower
♀ flower
♀ flower
♀ flower
♀ flower
1
1
19
19
1
1
19
19
23.772
2.428
0.087
0.041
27.617
0.816
0.367
0.024
log2 ratio
0.917
-1.756
1.834
-3.292
-1.106
-5.081
-3.933
29
compared to
Figure 15 The log2 ratio of TPS genes TPS1, TPS19 and TPS9 in female flowers of the plant species C. sativus
var corona.
Figure 16 The log2 ratio of TPS genes TPS1and TPS19 in female flowers of the plant species C. sativus var
Chinese long, 9930. No TPS 9 measurement for this cucumber species, because primers were not ready.
30
Discussion
The objective of this study was to explore the variation among members of the plant family
Cucurbitaceae with respect to flower characteristics that may be influenced by herbivory on
leaves by spider mites (Tetranychus urticae). First of all it had to be confirmed that spider
mites really caused damage to the plant they were feeding on. Damage did consist of chlorotic
lesions the spider mites created on the leaves, which resulted in loss of chlorophyll content.
Park and Lee (2002) show a reduction in chlorophyll content of leaves of C. sativus infested
with spider mites. This loss of chlorophyll was also the case for all cucurbit species and A.
thaliana included in this study. In most species, the reduction in chlorophyll A was larger than
that of chlorophyll B. This is because in most green plants the total chlorophyll content has a
chlorophyll A/B ratio of 3:1 (Raven et al., 2005). Therefore, in proportion to this ratio more
chlorophyll A will be gone in infested leaves than chlorophyll B. How large the effect of
herbivory was on the total amount of chlorophyll left in the plant varied between the plant
species. Most probably, this is because of differences in the plant defence mechanisms that try
to avoid that plants are eaten by herbivores. For C. sativus var Chinese long, 9930 it is known
it contains cucurbitacins that are responsible for the bitter taste of the leaves. This will repel
most individual herbivores (Miró, 1995). C. sativus var corona, on the other hand, does not
contain these cucurbitacins and is therefore not bitter. This difference in bitterness most
probably leads to the higher and lower amount of chlorophyll left in infested leaves of the
bitter Chinese cucumber and the non-bitter cucumber, respectively. Leaves of the cultivated
plant species C. melo (melon), C. lanatus (watermelon), C. pepo (courgette) and C. maxima
(pumpkin) also showed a reduction in chlorophyll content. According to Miró (1995) melon,
courgette and pumpkin contain cucurbitacins in different compositions (watermelon was not
encountered). Courgette only contains one type of cucurbitacin (cucurbitacin E), while
pumpkin and melon contain 3 (cucurbitacin B, D and E) and 4 types (cucurbitacin B, D, E and
I), respectively. The amount of chlorophyll left in infested courgette leaves was lower than in
infested leaves of pumpkin and melon, most probably because the latter have a better defence
mechanism against herbivory. Cucurbitacins may play a role in plant defence, and the number
of cucurbitacin types may have an effect on the effectiveness of plant defence against
herbivores. There are also other plant components that can help protect the plant against
attackers: M. charantia contains for example momordicines which make the leaves of the
plant very bitter (Raman and Lau, 1996). Nevertheless, also this plant species showed a
reduction in chlorophyll content when infested with spider mites. It can be concluded that
spider mites really caused damage to all the plant species they were feeding on in this study.
In a previous study about Cucumis sativus by Thomson and colleagues (2004) it is
found that leaf herbivory by Helix aspersa during the flowering phase leads to a reduction in
male flowers. In the study of Sigrid Dassen (2012) on the effect of leaf herbivory by spider
mites on flower number on Cucumis sativus, it seemed there were a slight reduction and a
slight increase in female flower and male flower number, respectively. In Cucurbita pepo
(courgette), leaf herbivory resulted in a more male-biased sex ratio (Krupnick et al., 2000).
So, it can be suggested that leaf herbivory could have an effect on flower number in different
cucurbits. Therefore, flower number could be seen as another way to determine plant damage
caused by herbivores. It would be interesting to investigate in a future study what the effect of
leaf herbivory by spider mites is on flower number on the different cucurbits studied in this
study. The non-bitter cucumber C. sativus var corona does not need to be pollinated to
produce fruits (seedless fruits), however, other cucurbits depend on pollination (often by
insects) to produce fruits (and seeds). If the flower sex ratio of those latter cucurbits is
changed because of leaf herbivory, it could eventually have an effect on fruit production on
the plant.
31
One of the characteristics that were investigated was the volatile emission by flowers and
leaves grown on plants with and without leaf herbivory for the different cucurbits. The
headspace of the flowers and leaves was analysed using TD-GCMS and a total of 25 volatile
compounds were identified that did represent most of the volatile blend emitted by flowers
and leaves. The majority of the volatile compounds in both leaves and flowers consisted of
terpenes. The question is if spider-mite infestation changes the volatile blend emitted by
flowers and leaves. And if so, is this change qualitative (release of new components) and/or
quantitative (different emission level of components also present in non-infested plants)?
With respect to the leaves there was quite some difference in the volatile profile of
control and infested plants. Some volatiles were only emitted by infested plants, so induced
by mite feeding: (-)-β-pinene, ((E)-β-)ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene
[(E)-DMNT], methyl salicylate (MeSA), α-farnesene and (E,E)-4,8,12-trimethyl-1,3,7,11tridecatetraene [(E,E)-TMTT]. This indicates that the change in leaf volatile profile when a
plant is attacked by herbivores is qualitative (release of new components). There is also a
quantitative change in leaf volatile profile: some volatiles were emitted by control as well as
infested leaves and for some of those volatiles the emission level did decrease, while for most
others it did increase. All cucurbits except M. charantia did emit some of those spider miteinduced volatile compounds. Most of these volatiles are known to attract the predatory mite
Phytoseiulus persimilis, among them (E)-β-ocimene, linalool (E)-DMNT and methyl
salicylate (Dicke et al., 1990) and it is reported that the presence of (E,E)-TMTT in a volatile
blend does improve the attraction (De Boer et al., 2004). The results of this study did indicate
that herbivore-damaged cucurbits emit herbivore-induced plant volatiles (HIPVs) in their
leaves that can be used by natural enemies to locate their prey. Behavioural bioassays with
predatory mites can provide significant evidence that the changed volatile profile of infested
leaves of the different cucurbits really help attracting natural enemies. It can also be used to
compare infested plants between the cucurbit species to see what kind of volatile blend, with
their specific volatile profile, is the most effective to attract predatory mites. In this way there
will maybe become more understanding about which compounds are used by natural enemies
to locate their prey.
In flowers on the other hand, there was almost no difference in the volatile profile of
flowers grown on control compared to those grown on infested plants. Female flowers of
mite-infested Chinese bitter cucumber plants did emit methyl salicylate while this was not
emitted from these flowers when leaves were not infested. However, there was no significant
difference between the amplitudes of this volatile in flowers grown on control and infested
plants (Students t-test, P=0.49). This suggests that in flowers the change in volatile profile is
not qualitative when a plant is attacked by herbivores (release of new components).
Furthermore, there was not a convincing quantitative (different emission level of components
also present in non-infested plants) change in volatile emission levels in flowers.
Although spider mite-damaged vegetative tissue emitted herbivore-induced plant
volatiles (HIPVs), there was no effect of herbivore damage on flower volatile emission in
these cucurbits. Lucas-Barbosa and colleagues (2011) summarise that in several plantherbivore-combination studies plants respond to herbivory by making changes in flower
volatile emission. In one of these studies it was found that leaf damage induces a significant
increase of volatiles in male flowers, but not in female flowers of C. pepo (Theis et al., 2009).
However, leaf damage was not caused by herbivores, but by making holes in the leaves that
mimicked beetle damage. The flower volatile blend of tomato plants whose leaves where
damaged by the caterpillar species (Manduca sexta) differed significantly form the blend of
non-damaged plants (Solanum peruvianum) (Kessler and Halitschke, 2009). However,
Effmert and colleagues (2008) found no effect of chewing damage by caterpillars (Manduca
sexta) on flower volatiles of Nicotiana suaveolens. They did find an increase in leaf volatiles
32
like in this study. However, the study is about chewing insects and the timing of damage is
much later (just before anthesis) than in this study.
Mite-infestation already started when plants had developed about four or five
expanded leaves, flower buds where just starting developing in the axils of the leaves.
Headspace collection of male and female flowers of the plant species was about 20-30 days
after first spider mite infestation. In the study of Sigrid Dassen (2012) on the effect of leaf
herbivory by spider mites on volatile emission in flowers on Cucumis sativus, the infestation
duration was about the same. This most probably indicates that infestation duration may not
be the reason why there is almost no difference in the volatile profile of flowers grown on
control compared to those grown on infested plants. However, in that study it was found that
flowers of plants suffering from spider-mite herbivory released different volatile compounds
and also at a different intensity (Sigrid Dassen, 2012).
In a future study about the effect of leaf herbivory by spider mites on flower volatile
emission in different cucurbits, it would be interesting to follow the volatile emission of
leaves and flowers in time, because the total amount of volatile compounds emitted by a plant
increases during spider-mite infestation and when herbivory progresses, other volatile
compounds are released (Kappers et al., 2010). This indicates that during spider-mite
infestation the emission of volatiles changes quantitatively and qualitatively.
There could also be the possibility that the flowers grown on plants infested with
spider mites emit the right volatile profile, but the control flowers do not. The control plants
were grown in a clean climate chamber. However, there is always a chance herbivores
manage to enter the room with accidental help by people, or when new plant material enters
the room that might not be totally free of herbivores. There was no herbivore-damage visible
on the leaves of the cucurbits, but thrips, for instance, are herbivores that feed on (developing)
flowers, not on leaves. This florivory may induce an herbivore-induced volatile profile.
Another characteristic that was investigated was the production of endogenous stress-related
phytohormones examined with liquid chromatography–mass spectrometry. Leaf and flower
material of the plant species C. sativus var Chinese long, 9930 and C. sativus var corona was
examined for: jasmonic acid (JA), its bioactive amino acid conjugate (JA-Ile), salicylic acid
(SA) and abscisic acid (ABA). These are phytohormones related to stress and their signalling
pathways are responsible for plant resistance against: water and salt stress (ABA), pathogen
attack (SA) and herbivore attack (JA), nevertheless the pathways can interact (Thaler and
Bostock, 2004). Spider mites are herbivores and therefore induce specifically JA. However,
these cell piercing herbivores may also induce SA-mediated defence genes (Zhang et al.,
2009).
The amount of the endogenous secondary metabolites JA and ABA in leaves and
female flowers of C. sativus var corona was increased when plants were infested with spider
mites. This increase in JA is to be expected when plants are attacked by herbivores. The
amount of SA in infested leaves was decreased compared to control leaves. These results
correspond to the interaction of the signalling pathways shown in figure 17: JA and SA act
antagonistically (increase in JA, than decrease in SA); ABA has a positive relationship with
JA (increase in ABA, than increase in JA); ABA has a negative relationship with SA (increase
in ABA, than decrease in SA) (Thaler and Bostock, 2004). In contrast, the amount of SA in
female flowers grown on infested non-bitter cucumber plants was increased compared to
flowers on control plants. So, spider mites induced the production of JA in leaves of the nonbitter cucumber, while in the flowers both JA and SA are induced. There was a tremendous
drop in JA-Ile/JA ratio in flowers when plants were infested. JA-Ile is the active form of JA
and can make sure that plant defence genes are expressed (Thines et al., 2007). When there is
less JA-Ile produced in infested flowers, less plant defence genes are expressed. This may
33
support the not-changed volatile profile of flowers grown on infested plants: infested flowers
on non-bitter cucumber were most probably not able to produce plant-defence related
volatiles.
The volatile profile of infested leaves changed compared to the profile of control
leaves: infested leaves on the non-bitter cucumber were able to produce herbivore-induced
plant volatiles (HIPVs) that are able to attract natural enemies of the herbivore. This
production of plant defence volatiles might suggests that in infested plants, JA turns into its
active form JA-Ile, to make sure that defence genes come to expression (Thines et al., 2007).
This supports an increase in JA-Ile/JA ratio when plants are infested by spider mites, and
therefore a lower JA-Ile/JA ratio in control leaves (despite it was not possible to measure JAIle in leaves).
Figure 17 Diagram of proposed relationship between abscisic acid, salicylic acid and jasmonic acid signalling
pathways. Solid arrows indicate a positive regulating relationship between stresses and hormones. Dashed
arrows indicate a positive (+) or negative (-) relationship between the hormonal signalling pathways (Thaler and
Bostock, 2004).
The amount of the endogenous secondary metabolite JA in infested leaves of C.
sativus var Chinese long, 9930 was increased when compared to control leaves. This is also
seen in the results of Sigrid Dassen, 2012. This increase in JA is to be expected when plants
are attacked by herbivores. However, in both male and female flowers grown on infested
plants the amount of JA was decreased. The ABA level in the infested plant parts was the
opposite of the JA level in those plant parts: ABA was decreased in leaves and increased in
flowers when infested with spider mites. These results do not correspond to the interaction of
the signalling pathways shown in figure 17: ABA should have a positive relationship with JA
(increase in ABA, than increase in JA) (Thaler and Bostock, 2004). This decrease of ABA in
infested leaves is also in contradiction with the increased ABA level in infested leaves found
by Sigrid Dassen, 2012. The amount of the metabolite SA in both the male and female
flowers was decreased when plants were infested with spider mites. This increase in ABA and
the decrease in SA in flowers correspond to the interaction of the signalling pathways shown
in figure 17: ABA has a negative relationship with SA (increase in ABA, than decrease in SA)
(Thaler and Bostock, 2004). The JA-Ile/JA ratio in infested leaves was increased compared to
control leaves, which gives the indication that more JA turns into its active form to make sure
that defence genes come to expression (Thines et al., 2007). This may support the change in
volatile profile of infested leaves: infested leaves on the Chinese bitter cucumber were able to
produce herbivore-induced plant volatiles (HIPVs) that are able to attract natural enemies of
the herbivore. The JA-Ile/JA ratio in female flowers grown on infested plants was increased
compared to those on control plants. In male flowers, however, there was a tremendous drop
in JA-Ile/JA ratio when plants were infested. When there is less JA-Ile produced in infested
male flowers, less plant defence genes are expressed. This may support the not-changed
34
volatile profile of male flowers grown on infested plants: infested male flowers on the
Chinese bitter cucumber were most probably not able to produce plant-defence related
volatiles.
The last characteristic that was investigated was the gene expression of the terpene synthase
family (TPS) genes TPS1, TPS9 and TPS19 in control and infested flower material of the
plant species C. sativus var corona and C. sativus var Chinese long, 9930. TPS1, TPS9 and
TPS19 encode for enzymes which synthesize the terpenes linalool, β-ocimene and αfarnesene, respectively (personal comment dr. ir. Iris F. Kappers).
The log2 ratios of TPS1 and TPS9 in female flowers of C. sativus var corona were
positive, indicating higher relative gene expressions of TPS1 and TPS9 in infested flowers. In
theory, this would result in an induced emission of linalool and β-ocimene. TPS19 on the
other hand, had a negative log2 ratio, which indicates a relative decrease of the transcription
of the gene so in theory the emission of α-farnesene by infested flowers would be reduced.
The log2 ratios of TPS1 and TPS19 in both male and female flowers of C. sativus var
Chinese long, 9930 were negative, indicating lower relative gene expressions of TPS1 and
TPS19 in infested flowers. In theory, this would result in a reduced emission of linalool and
α-farnesene. In the study of Sigrid Dassen (2012) on the effects of spider-mite infestation of
leaves on flower characteristics it was found that the expression of TPS19 was increased in
female flowers, but reduced in male flowers.
When comparing gene expression with volatile emission, it is shown that some results
confirm each other while others are contradictory (Table 11). The volatile emission results
showed that the male flowers of C. sativus var Chinese long, 9930 did emit less linalool when
spider mites attacked the plant. On gene level, this observation is confirmed by a relative
decrease in TPS1 transcripts in infested male flowers. This could indicate that this particular
TPS gene could be responsible for the emission of linalool in male flowers of the bitter
Chinese cucumber. However, the difference between infested and control male flowers for
linalool in the volatile emission results was not significant (Students t-test, P=0.076).
The female flowers of C. sativus var Chinese long, 9930 did emit more linalool and αfarnesene when spider mites attacked the plant. However, the difference between infested and
control female flowers for linalool and α-farnesene in the volatile emission results was not
significant (Students t-test, linalool P=0.438, α-farnesene P=0.526). On gene level, this
observation of more linalool and α-farnesene emission is not confirmed, because of the
relative decreases in TPS1 and TPS19 transcripts in infested female flowers. So the gene
expression and volatile emission for female flowers of the bitter Chinese cucumber are
contradictory.
The terpene synthase family (TPS) is a set of related genes that encodes for enzymes
which synthesize different terpenes (Bohlmann et al., 1998). Until now 26 TPS genes are
known for cucumber. However, it is not yet known for all genes which terpene(s) they can
synthesize. TPS1, TPS9 and TPS19 encode for enzymes which synthesize the terpenes
linalool, β-ocimene and α-farnesene, respectively. Nevertheless, it can be that one TPS gene
encodes for various terpenes and that the production of a specific terpene can be activated by
more than one individual TPS gene. The latter is true for linalool (personal comment dr. ir.
Iris F. Kappers). Therefore, the increase in linalool and α-farnesene emission in infested
female flowers of the bitter Chinese cucumber could maybe be confirmed on gene level when
the gene expression of other TPS genes is investigated.
35
Table 11 Comparison of the results of the gene expression and the volatile emission experiments. Flowers grown
on infested plants are compared with flowers grown on control plants.
Plant species
Plant
material
TPS
gene
Compound
Experiment
Gene
Volatile
expression emission
C. sativus var corona
♀ flower
1
19
9
1
linalool
α-farnesene
β-ocimene
linalool
↑
↓
↑
↓
−
?−
?−
↓
19
1
19
α-farnesene
linalool
α-farnesene
↓
↓
↓
−
↑
↑
C. sativus var Chinese long, ♂ flower
9930
♀ flower
↑
↓
−
?
increase
decrease
about the same level
this or other compound
■ results of experiments are the same
■ results of experiments are opposite
In conclusion, this study demonstrates a large range of qualitative and quantitative variation in
volatile emission induced by leaf herbivory of spider mites (Tetranychus urticae) in leaves of
different cucurbit species. Furthermore, this study found no significant effects of herbivore
damage on flower volatile emission in these cucurbits. The production of endogenous stressrelated phytohormones varied between the plant species C. sativus var corona and C. sativus
var Chinese long, 9930 and between the plant parts. The female flowers of C. sativus var
corona and the male flowers of C. sativus var Chinese long, 9930 grown on infested plants
were most probably not able to produce plant-defence related volatiles, because of the
tremendous drop in JA-Ile/JA ratio in flowers when plants were infested. When comparing
the gene expression of TPS1, TPS9 and TPS19 with the volatile emission of linalool, βocimene and α-farnesene in flowers of C. sativus var corona and C. sativus var Chinese long,
9930, it was shown that spider mites reduced the emission of linalool as well as the gene
expression responsible for the production of linalool in male flowers of the Chinese bitter
cucumber. However, for the other compound-flower combinations the comparison ended in
contradictions.
36
Acknowledgements
I would like to thank Harro for allowing me to do this research at the Plant Physiology group.
It is a big group with people from all kinds of places having knowledge on various topics.
Still, it is one group and I met nice people and had very nice experiences during lunch time
and coffee breaks, and especially during the barbeque and lab trip.
Iris, thank you very much for introducing me to this interesting topic with cucumber and its
relatives in the spotlights. I learned many new things about cucurbits, which I could not have
learned during my part-time job at the greengrocery De Komkommerin on Saturdays. I did
appreciate it that you just let me figure out things by myself, because that is what research is
all about: make mistakes, learn from them and hopefully do things right the next time. I did
also appreciate it when you pushed me in the right direction in case I was stuck or too focused
on one thing and almost forgetting the rest.
I would also like to thank Kristyna, Yuanyuan and Jun for helping me with experiments and
advising or teaching me when I had questions.
This study stimulates me to get to know more about plants and the interaction with their
environment (especially insects). The next challenge will be a consultancy project about plant
metabolites in sweet pepper plants.
37
Appendix 1
RNA isolation from Cucumber and cDNA synthesis for qPCR
Grind ± 100 mg leaf material in liquid nitrogen to powder
In the fumehood:
Add 1 mL TriPure & homogenize on the vortex,
Let stand for 5 min at RT
(Tripure contains phenol!)
Add 200 µl Chloroform, vortex, let stand for 5 min at RT
Centrifuge for 15 min, 12.000xg, 4 °C
Pipet the supernatant in a fresh tube
Add 0.5 Volume of 96 % EtOH, Mix by inverting
Apply to the column of the RNA box (red box)
Wash with the wash buffer (Follow the protocol in the Kit!)
Step 1: RW1 (1x)
Step 2: RP (2x)
Elute RNA with 200 µl water from the kit
DNAse treatment:
89 µl RNA
10 µl DNAse buffer
1 µl DNAse
15 min at 25 °C
Apply onto a RNA column to purify (Follow the protocol in the Kit!)
Wash and elute as described above
Measure concentration on the nanodrop
Use 1 µg of RNA to synthesize iScript DNA:
4 µl 5X iScript reaction Mix
1 µl iScript Reverse Transcriptase
X µl Nuclease free water
X µl RNA-template (100 fg-1µg total RNA)
5 min
25 °C
30 min
42 °C
5 min 85 °C
38
Appendix 2
Protocol for Quantitative Real-Time PCR
1. Isolate RNA following the protocol from Ralph “RNA isolation Tripure-small
sample”.
For Research Use Only. Ralph Litjens © All rights reserved _ 1 _ R--11
RNA isolations ((TriPure) – small sample)
1) Harvest 2 leave disks, (diameter 1.5 cm – weight about 80 mg)
2) and freeze in liquid nitrogen (store -80ºC)
3) Grind leave disks in 1.5 ml tube, using the “blue potter”
4) Add 500 ul TriPure (Roche)
5) Homogenize using the homogenizer with the “steel white tip potter”.
6) Add 500 ul TriPure and vortex
7) Centrifuge 1 min microcentrifuge max speed at room temperature
8) Transfer supernatant into a fresh tube (2 ml) (Then, prepare chloroform,
isopropanol and 75% ethanol in fresh 50 ml blue-cap tubes.)
9) Add 0.2 ml chloroform and vortex for 15 seconds
10) Incubate at room temperature for 2-15 minutes
11) Centrifuge 15 min microcentrifuge 12,000 g at 4ºC
12) Transfer the supernatant to a fresh tube (1.5 ml)
13) Add 500 ul isopropanol and vortex
14) Incubate at room temperature for 10 minutes
15) Centrifuge 10 min microcentrifuge max speed at 4ºC
16) Remove supernatant and wash the pellet with 800 ul 75% EtOH
17) Centrifuge 5 min microcentrifuge max speed at 4ºC
18) Remove supernatant and dry the pellet briefly （less than 3 min）
19) (not too dry because it will be hard to dissolve the RNA pellet)
20) Dissolve pellet in 95 ul MQ (keep on ice from this moment) (Normally,
dissolve pellet in 30-50 µl MQ. However, 90 µl perfectly fit the following
steps.)
21) Incubate 5 minutes at 60ºC to dissolve the RNA completely
22) Centrifuge 10 min microcentrifuge max speed at 4ºC
23) Transfer the supernatant to a fresh tube (1.5 ml).
24) Store the RNA at -70ºC
For tobacco leaves………….. up to 100 µg total RNA could be isolated
2. Check the RNA quality by running 5 µl of RNA samples on gel to see whether
there are sharp bands. (The RNA loading buffer is in the general stock in -20°C.
The gel and running buffer are preferably to be freshly prepared.)
3. Treated the RNA samples with DNAse.
- RNA
90 µl
- DNAse buffer
10 µl
- DNAse I
1 µl
 Then incubate 15 min at 25 °C
 Then add EDTA (25mM) 1 µl, incubate 10 min at 50 °C to stop the
reaction. （if immediately clean RNA, EDTA treatment is not necessary!
）
39
4. Purify the RNA by “RNA easy Mini Handbox” following the protocol in the box.
5. Measure the concentration of RNA after purification by Nanodrop. If the values
of 260/280 and 260/230 are above 1.8, the purity and quality of RNA is good for
real time PCR. (If the quality is not good enough, such as the value of 260/230 is
quite low, purify that RNA sample with the kit again. )
6. Reverse transcription on PCR machine.
a. Calculate the amount of RNA solution needed for 1 µg RNA. The system
was optimized for 1 µg RNA. (The maximum amount of RNA solution is
15 µl in the following reaction system. If the RNA concentration is too low
and even 15 µl RNA solution can not reach 1 µg, this RNA sample should
be concentrated by “Speed Vacuum”.)
- iScript cDNA synthese kit from Biorad (20µl)
- 5×iScript reaction Mix
4 µl
- iScript reverse transcriptase
1 µl
- RNA sample+ MQ water
15µl
b. Reverse transcription programme:
 25 °C 5min (annealing of primer)
 42 °C 30 min (extension)
 85 °C 5 min (kill the RTase)
 Hold at 10 °C
7. Measure the concentration of cDNA by Nanodrop. The concentrations are
around 1000 ng/µl. The differences of concentration among all samples should be
less than 100 ng/µl.
8. Dilute cDNA to the volume of 200ul, or 400 (Depending) with fresh MQ
water.
9. Prepare RT-PCR mixture.
a. Get fresh MQ water in fresh 50 ml blue-cap tube. (First turn on the tap of
MQ machine, and let the MQ water run for a few seconds. Then load the
MQ water in the tube.)
b. Prepare RT-PCR mixture in a flow cabinet, but do not turn on the flow
cabinet.
triplicates
duplicates
- Fresh MQ water
13 µl
11.5µl
- RT-PCR Buffer (2×)
32.5 µl
22.5µl
- Forward primer (3 µM)
6.5 µl
4.5µl
- Reverse primer (3 µM)
6.5 µl
4.5µl
- cDNA template
6.5 µl
2µl
- ------------------------------------------------------------------------+) 65 µl
+) 45 µl
Prepare this mixture in a 0.5 ml tube.
Note:
i. “RT-PCR Buffer” mentioned above was “BIO-RAD iQTM
SYBR@ Green Supermix 2× Mix for Real-Time PCR 100 × 50 µl
reactions, Catalog 170-8880”. This buffer should be stored at -20
40
°C. But once it’s opened, it should be stored in 4 °C. It will be
stable at 4 °C for at least 6 months.
ii. The primers were dissolved in MQ water. The primers were
designed by the software “Beacon Designer” which is especially for
designing primers for RT-PCR. For more details, please see the
“Protocol for designing primers for RT-PCR”.
iii. The tips used in this and following steps are special tips which have
filters inside.
iv. The pipettes used in this and following steps are preferably those
pipettes only used for RT-PCR work. (One set of these pipettes can
be found in the drawer near qPCR machine.)
v. Always prepare a mixture of “no template control (NTC)” by
adding MQ water instead of cDNA template and a mixture of
“RNA template control” by adding RNA instead of cDNA
template.
10. Then divide the 65 µl mixture into 3 wells (20 µl/ well) in the RT-PCR plate as
triplicates. When dividing the mixture, cover the other wells you are not working
with!
11. Carefully seal the RT-PCR plate with special film (Microseal, adhesive
sealer). When taking out the film, only hold its edge which is not transparent. Do
not touch the transparent part of the film! After the film sticking to the RP-PCR
plate, use a small wooden board (can be found in the top drawer near RT-PCR
machine) to scrape the film several times.
12. Centrifuge the RT-PCR plate in Taqman centrifuge gently in the shaker room
for several minutes to spin down the droplets attaching to the wall of the wells.
13. Run RT-PCR machine.
a. Turn on the RT-PCR machine at least 10 min in advance to preheat it. Then
turn on the computer connected to the machine.
b. Put your RT-PCR plate in the machine.
c. The short cut of the software “Bio-red iQ5” can be found on the desktop of
the computer. Double click it.
d. Click “Protocol”. Then choose the file ”C:\ Program Files\ Bio-Rad\ iQ5\
users\ Jos (or Liping)\ 2stepAmp+MelJos.tmo”
e. Click “Plate”.
i. Then choose the file “C:\ Program Files\ Bio-Rad\ iQ5\ users\ Jos
(or Liping)\ JosEVSol5.pls”
ii. Click “Create New”.
a) “Sample volume” is 20 µl.
b) “Seal Type” is film.
c) “Vessel Type” is plates.
d) Click button
. Then choose the well positions of NTC. (Left
click mouse, then drag the mouse through those 3 wells. Do not
41
click each well one by one. )
e) Click button
. Then choose the well positions of samples.
(Left click mouse, then drag the mouse through 3 wells which are
triplicates of one sample. Then drag the mouse through another 3
wells which belong to another sample.)
f) If there are some wrongly chosen wells, just click
to erase
them.
g) Then click “Save & Exit Plate Editing”. Save it in your own file
folder.
f. Click “Run”. Choose “Collect well…”
g. Click “Begin Run”.
14. View the RT-PCR results about one and a half hour later.
a. Click “Data File”. Then choose the data file you saved.
b. Click “Data analysis”. Now you are in the “PCR Quant” window.
c. Click “Results”. Then copy the result table and paste it in an Excel file for
further data analysis.
Note: If you would like to know whether the primers are good, click “Melt
Curve/Peak”. In “Melt Peak Chart”, if there is only one peak for each sample,
that means there is an unique product and there is not unspecific amplification.
15. Analyze data in Excel file.
a. Copy the raw data to another Excel sheet, and name it “RGE” (relative
gene expression). Do not work on the raw data directly.
b. Fill in the “Identifier” column with sample name and primer name (such as
“WT- actin”). Then it’s easy to see which well is which.
c. Keep “Identified”, “Threshold”, “Replicate #” and “Ct Std. Dev” columns,
and remove the other columns which are not necessary for data analysis.
d. Calculate the “δ Ct”. Ct value (threshold cycle) of gene of interest
subtracts the Ct value of house-keeping gene for the same sample.
e. Calculate the “RGE” by using the function of “f(x)=POWER(2,- δ Ct)”.
42
f. Calculate the “mean RGE”. Since there are 3 replicates for each sample, so
there are 3 “RGE” value for each sample. The “mean RGE” is the average
of those 3 “RGE” value.
g. Calculate the “RGE Sta. Dev.” by using the function of “f(x)=
=STDEV(RGE1, RGE2, RGE3)”
h. Make a chart with type of “Clustered column”. The χ-axis is the sample
name and the у-axis is the “mean RGE” value. The error bar is the “RGE
Sta. Dev.” value. Do log scale for у-axis. (Right click on у-axis→ click
“Format Axis” → click “Scale” → choose “Logarithmic scale” .) For
example (fig 1)
i. The data of REG value can also be *100 (or 1000 times, or more), then do
the Log(X, 2) to make the columns better to show, the last data show the
fold number of different genes.
j. The REG of the transgenic can also be divided to WT to show the fold
difference. For example: (fig 2)
100.00
10.00
1.00
w t-OC
1-OC
2-OC
3-OC
4-OC
5-OC
6-OC
7 OC1
8-OC
9-OC
10
OC1
11
OC1
12-OC 13-OC 14-OC 15-OC
0.10
0.01
0.00
chry FDS, CDS
16
REG fold
14
12
10
1581JA-leaf
1581JA+leaf
8
1581JA-ovary
6
1581JA+ovary
4
2
0
chrCDS
chrFDS1
chrFDS2
For
more details about analyzing data, please refer to “Kenneth J. Livak and Thomas D.
Schmittgen . Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR
and the 2- δCt CT Method. METHODS 25, 402–408 (2001) ”
Written by: Ting
Modified by Jing
43
Appendix 3
Chromatograms of leaves and flowers of other cucurbit plant species.
Figure 18 Chromatogram of control and infested leaves of C. melo (melon) with the important volatile
compounds (Table 4).
Figure 19 Chromatogram of control and infested leaves of C. lanatus (watermelon) with the important
volatile compounds (Table 4).
44
Figure 20 Chromatogram of control and infested leaves of C. pepo (courgette) with the important volatile
compounds (Table 4).
Figure 21 Chromatogram of control and infested leaves of C. maxima (pumpkin) with the
important volatile compounds (Table 4).
45
Figure 22 Chromatogram of control and infested leaves of M. charantia (bitter gourd) with the
important volatile compounds (Table 4).
Figure 23 Chromatogram with the important volatile compounds emitted by male flowers grown
on control and infested plants of the plant species C. pepo (courgette) (Table 5).
46
Figure 24 Chromatogram with the important volatile compounds emitted by male flowers grown
on control and infested plants of the plant species C. maxima (pumpkin) (Table 5).
Figure 25 Chromatogram with the important volatile compounds emitted by male flowers grown
on control and infested plants of the plant species M. charantia (Table 5).
47
Figure 26 Chromatogram with the important volatile compounds emitted by female flowers grown
on control and infested plants of the plant species M. charantia (Table 6).
Figure 27 Chromatogram with the important volatile compounds emitted by bisexual flowers
grown on control and infested plants of the plant species C. melo (melon) (Table 7).
48
Appendix 4
Table 12 Amount of chlorophyll per fresh leaf weight (µg/mg FW) for control and infested leaves in A. thaliana
and different cucurbits in undiluted and diluted samples
Amount of chlorophyll per fresh leaf
(µg/mg FW)
Plant
Chlorophyll A
Chlorophyll B
± SD
Average
species
Treatment (Un)diluted Average
C. sativus
control
undiluted
0.188
0.018 0.318
*
var corona
diluted
0.045
0.005 0.020
infested
undiluted
0.105
0.020 0.055
*
diluted
0.011
0.002 0.005
C. sativus
control
undiluted
0.192
0.035 0.231
*
var Chinese
diluted
0.035
0.006 0.014
long, 9930
infested
undiluted
0.183
0.017 0.163
*
diluted
0.025
0.002 0.011
C. melo
control
undiluted
0.185
0.028 0.263
(melon)
diluted
0.038*
0.013 0.018
infested
undiluted
0.188
0.017 0.120
diluted
0.021*
0.002 0.010
C. lanatus
control
undiluted
0.174
0.021 0.408
(watermelon)
diluted
0.054*
0.002 0.025
infested
undiluted
0.217
0.018 0.262
*
diluted
0.037
0.002 0.019
C. pepo
control
undiluted
0.149
0.011 0.240
*
(courgette)
diluted
0.034
0.002 0.014
infested
undiluted
0.095
0.022 0.047
*
diluted
0.010
0.002 0.004
C. maxima
control
undiluted
0.213
0.032 0.157
*
(pumpkin)
diluted
0.029
0.007 0.012
infested
undiluted
0.168
0.025 0.105
diluted
0.019*
0.004 0.009
M. charantia control
undiluted
0.199
0.012 0.464
(bitter
diluted
0.063*
0.015 0.029
gourd)
infested
undiluted
0.211
0.011 0.304
diluted
0.039*
0.014 0.020
A. thaliana
control
undiluted
0.212
0.035 0.294
*
diluted
0.040
0.002 0.019
infested
undiluted
0.169
0.040 0.155
*
diluted
0.021
0.007 0.012
weight
± SD
0.029
0.002
0.011
0.001
0.063
0.003
0.014
0.001
0.101
0.005
0.016
0.001
0.020
0.001
0.029
0.001
0.017
0.001
0.013
0.001
0.046
0.003
0.027
0.002
0.097
0.007
0.140
0.007
0.033
0.001
0.048
0.003
*
Values of diluted samples multiplied with 10 should give the same values as for the undiluted samples,
however, they did not.
49
References
Adler LS, Karban R, Strauss SY (2001) Direct and indirect effects of alkaloids on plant
fitness via herbivory and pollination. Ecology 82: 2032-2044
Agrawal AA, Gorski PM, Tallamy DW (1999) Polymorphism in plant defense against
herbivory: constitutive and induced resistance in Cucumis sativus. Journal of Chemical
Ecology 25: 2285-2304
Aharoni A, Jongsma MA, Bouwmeester HJ (2005) Volatile science? Metabolic engineering
of terpenoids in plants. Trends in Plant Science 10: 594-602
Arimura G, Kost C, Boland W (2005) Herbivore-induced, indirect plant defences. Biochimica
et Biophysica Acta 1734: 91-111
Aubourg S, Lecharny A, Bohlmann J (2002) Genomic analysis of the terpenoid synthase
(AtTPS) gene family of Arabidopsis thaliana. Molecular Genetics and Genomics 267: 730745
Balkema-Boomstra AG, Zijlstra S, Verstappen FWA, Inggamer H, Mercke PE et al. (2003)
Role of cucurbitacin in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis
sativus L.). Journal of Chemical Ecology 29: 225–235
Barber NA, Adler LS, Bernardo HL (2011) Effects of above- and belowground herbivory on
growth, pollination, and reproduction in cucumber. Oecologia 165: 377-386
Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: Molecular biology
and phylogenetic analysis. Proceedings of the National Academy of Sciences of the United
State of America 95: 4126-4133
Bouwmeester HJ, Verstappen FWA, Posthumus MA, Dicke M (1999) Spider mite-induced
(3S)-(E)-nerolidol synthase activity in cucumber and lima bean – the first dedicated step in
acyclic C11-homoterpene biosynthesis. Plant Physiology 121: 173-180
Chen F, Ro D-K, Petri J, Gershenzon J, Bohlmann J et al. (2004) Charaterization of rootspecific Arabidopsis terpene synthase responsible for the formation of the volatile
monoterpene 1,8-cineole. Plant Physiology 135: 1956-1966
Chen F, Tholl D, Bohlmann J, Pickersky E (2011) The family of terpene synthases in plants: a
mid-size family of genes for specialized metabolism that is highly diversified throughout the
kingdom. The Plant Journal 66: 212-229
Da Costa CP, Jones CM (1971) Cucumber beetle resistance and mite susceptibility controlled
by the bitter gene in Cucumis sativus L. Science 172: 1145-1146
De Boer JG, Posthumus MA, Dicke M (2004) Identification of volatiles that are used in
discrimination between plants infested with prey or nonprey herbivores by a predatory mite.
Journal of Chemical Ecology 30: 2215-2230
De Moraes CM, Lewis WJ, Pare PW, Alborn HT, Tumlinson JH (1998) Herbivore-infested
plants selectively attract parasitoids. Nature 393: 570-573
50
De Moraes CM, Mescher MC, Tumlinson JH (2001) Caterpillar-induced nocturnal plant
volatiles repel nonspecific females. Nature 410: 577-580
De Vos RC, Schipper B, Hall RD (2012) High-performance liquid chromatography-mass
spectrometry analysis of plant metabolites in Brassicaceae. Methods in Molecular Biology
860:111-28.
Dicke M (2009) Behavioural and community ecology of plants that cry for help. Plant, Cell
and Environment 32: 654-665
Dicke M, Baldwin IT (2010) The evolutionary context for herbivore-induced plant volatiles:
beyond the ‘cry for help’. Trends in Plant Science 15: 167–175
Dicke M, van Beek TA, Posthumus MA, Dom NB, van Bokhoven H et al. (1990) Isolation
and identification of volatile kairomone that affects acarine predator-prey interactions involvement of host plant in its production. Journal of Chemical Ecology 16: 381-396
Dicke M, van Poecke RMP, de Boer JG (2003) Inducible indirect defence of plants: from
mechanisms to ecological functions. Basic and Applied Ecology 4: 27-42
Dudareva N, Pickersky E, Gershenzon J (2004) Biochemistry of plant volatiles. Plant
Physiology 135: 1893-1902
Effmert U, Dinse C, Piechulla B (2008) Influence of green leaf herbivory by Manduca sexta
on floral volatile emission by Nicotiana suaveolens. Plant Physiology 146: 1996-2007
Fasulo TR, Denmark HA (2009). Featured creatures, twospotted spider mite. Available:
http://entomology.ifas.ufl.edu/creatures/orn/twospotted_mite.htm. Consulted in March 2014.
Flokova K, Tarkowska D, Miersch O, Strnad M, Wasternack C et al. (2014) UHPLC-MS/MS
based target profiling of stress-induced phytohormones. Phytochemistry 105: 147-157
Garcia-Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G et al. (2012) The genome of
melon (Cucumis melo L.). Proceedings of the National Academy of Sciences of the United
State of America 109: 11872-11877
Gould F (1978) Resistance of cucumber varieties to Tetranychus urticae: Genetic and
environmental determinants. Journal of Economic Entomology 71: 680-683
Heil M, Ton J (2008) Long-distance signalling in plant defence. Trends in Plant Science 13:
264-272
Herms DA, Mattson WJ (1992) The dilemma of plants: to grow or to defend. The Quarterly
Review of Biology 67: 283–335
Hilker M, Meiners T (2010) How do plants “noticce” attack by herbivorous arthropods?
Biological Reviews 85: 267-280
Huang S, Li R, Zhang Z, Li L, Gu X et al. (2009) The genome of the cucumber, Cucumis
sativus L. Nature Genetics 41: 1275-U29
51
Kappers IF, Hoogerbrugge H, Bouwmeester HJ, Dicke M (2011) Variation in herbivoryinduced volatiles among cucumber (Cucumis sativus L.) varieties has consequences for the
attraction of carnivorous natural enemies. Journal of Chemical Ecology 37: 150-160
Kappers IF, Verstappen FWA, Luckerhoff LLP, Bouwmeester HJ, Dicke M (2010) Genetic
variation in jasmonic acid- and spider mite-induced plant volatile emission of cucumber
accessions and attraction of the predator Phytoseiulus persimilis. Journal of Chemical
Ecology 36: 500-512
Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular
analysis. Annual Review of Plant Biology 53: 299-328
Kessler A, Halitschke R (2009) Testing the potential for conflicting selection on floral
chemical traits by pollinators and herbivores: predictions and case study. Functional Ecology
23: 901-912
Knudsen JT, Eriksson R, Gershenzon J, Ståhl B (2006) Diversity and distribution of floral
scent. Botanical Review 72: 1-120
Knudsen JT, Tollsten L, Bergström G (1993) Floral scents – a checklist of volatile
compounds isolated by head-space techniques. Phytochemistry 33: 253-280
Krips OE, Kleijn PW, Willems PEL, Gols GJZ, Dicke M (1999) Leaf hairs influence
searching efficiency and predation rate of the predatory mite Phytoseiulus persimilis (Acari:
Phytoseiidae). Experimental and Applied Acarology 23: 119-131
Krupnick GA, Avila G, Brown KM, Stephenson AG (2000) Effects of herbivory on internal
ethylene production and sex expression in Cucurbita texana. Functional Ecology 14: 215-225
Lambers H, Chapin III FS, Pons TL (2008) Plant physiological ecology, 2nd edn. Springer
Science+Business Media, New York, USA. pp. 445-477.
Lucas-Barbosa D, van Loon JJA, Dicke M (2011) The effects of herbivore-induced plant
volatiles on interactions between plants and flower-visiting insects. Phytochemistry 72: 16471654
Mercke P, Kappers IF, Verstappen FWA, Vorst O, Dicke M et al. (2004) Combined transcript
and metabolite analysis reveals genes involved in spider mite induced volatile formation in
cucumber plants. Plant Physiology 135: 2012-2024
Miró M (1995) Cucurbitacins and their pharmacological effects. Phytotherapy Research 9:
159-168
Mothershead K, Marquis RJ (2000) Fitness impacts of herbivory through indirect effects on
plant-pollinator interactions in Oenothera macrocarpa. Ecology 81: 30-40
Mumm R, Dicke M (2010) Variation in natural plant products and the attraction of
bodyguards involved in indirect plant defense. Canadian Journal of Zoology 88: 628–667
52
Njoroge GN, Van Luijk MN (2004) Momordica charantia L. Available:
http://www.prota4u.org/protav8.asp?h=M4&t=Momordica,charantia&p=Momordica+charanti
a#Synonyms. Consulted in March 2014.
Park YL, Lee JH (2002) Leaf cell and tissue damage of cucumber caused by twospotted
spider mite (Acari: Tetranychidae). Journal of Economic Entomology 95: 952-957
Park YL, Lee JH (2005) Impact of twospotted spider mite (Acari: Tetranychidae) on growth
and productivity of glasshouse cucumbers. Journal of Economic Entomology 98: 457–463
Pichersky E, Gershenzon J (2002) The formation and function of plant volatiles: perfumes for
pollinator attraction and defence. Current Opinion in Plant Biology 5: 237-243
Pierce LK, Wehner TC (1990) Review of genes and linkage groups in cucumber. Hortscience
25: 605-615
Pieterse CMJ, Dicke M (2007) Plant interactions with microbes and insects: from molecular
mechanisms to ecology. Trends in Plant Science 12: 564-569
Raman A, Lau C (1996) Anti-diabetic properties and phytochemistry of Momordica charantia
L. (Cucurbitaceae). Phytomedicine 2: 349-362
Raven PH, Evert RF, Eichhorn SE (2005) Biology of plants, 7th edn. W.H. Freeman and
Company, New York, USA, pp 119-121.
Rice CA, Rymal KS, Chambliss OL, Johnson FA (1981) Chromatographic and mass-spectral
analysis of cucurbitacins of 3 Cucumis sativus cultivars. Journal of Agricultural and Food
Chemistry 29: 194-196
Schoonhoven LM, van Loon JJA, Dicke M (2005) Insect-plant biology. Oxford University
Press, Oxford
Sigrid Dassen (2012) Volatiles of male and female flowers of Cucumis sativus are
differentially affected by Tetranychus urticae leaf herbivory. Thesis.
Takabayashi J, Dicke M (1996) Plant-carnivore mutualism through herbivore-induced
carnivore attractants. Trends in Plant Science 1: 109–113
Takabayashi J, Dicke M, Takahashi S, Posthumus MA, van Beek TA (1994) Leaf age affects
composition of herbivore-induced synomones and attraction of predatory mites. Journal of
Chemical Ecology 20: 373-386
Thaler JS, Bostock RM (2004) Interactions between abscisic-acid-mediated responses and
plant resistance to pathogens and insects. Ecology 85: 48-58
Theis N, Kesler K, Adler LS (2009) Leaf herbivory increase floral fragrance in male but not
female Cucurbita pepo subsp. texana (Cucurbitaceae) flowers. American Journal of Botany
96: 897-903
53
Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A et al. (2007) JAZ repressor proteins are
targets of the SCFCOI1 complex during jasmonate signalling. Nature 448: 661–666
Tholl D, Boland W, Hansel A, Loreto F, Röse USR et al. (2006) Practical approaches to plant
volatile analysis. The Plant Journal 45: 540-560
Tholl D, Chen F, Petri J, Gershenzon J, Pickersky E (2005) Two sesquiterpene synthases are
responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. The
Plant Journal 42: 757-771
Thomson VP, Nicotra AB and Cunningham SA (2004) Herbivory differentially affects male
and female reproductive traits of Cucumis sativus. Plant Biology 6: 621-628
Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular
evolutionary implications. Genetics 158: 811-832
Traw MB, Dawson TE (2002) Differential induction of trichomes by three herbivores of black
mustard. Oecologia 131: 526-532
Van den Boom CEM, van Beek TA, Dicke M (2002) Attraction of Phytoseiulus persimilis
(Acari: Phytoseiidae) towards volatiles from various Tetranychus urticae-infested plant
species. Bulletin of Entomoligical Research 92: 539-546
Van Poecke RMP, Posthumus MA, Dicke M (2001) Herbivore-induced volatile production by
Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: chemical,
behavioral, and gene-expression analysis. Journal of Chemical Ecology 27: 1911-1928
Wagner GJ (1991) Secreting glandular trichomes: More than just hairs. Plant Physiology 96:
675-679
Werker E (2000) Trichome diversity and development. Advances in Botanical Research 31:
1-35
Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A et al. (2009) Whiteflies interfere
with indirect plant defense against spider mites in Lima bean. Proceedings of the National
Academy of Sciences USA 106: 21202-21207
http://faostat.fao.org/
54