Insects
The bird cherry-oat aphid, Rhopalosiphum padi L., one of the key pests of maize, and the black bean aphid, Aphis fabae Scop., one of the most common pests on bean plants, were selected as model insect herbivores. Rhopalosiphum padi was reared on barley, Hordeum vulgare L. (cv Golf) in multi-clonal cultures in a greenhouse under the same conditions as for plants. Aphis fabae was reared on broad bean, Vicia faba L. (cv Button dwarf). Both aphid species used in the experiments were wingless, mixed-instar individuals. They were collected from the cultures immediately prior to bioassay.
Adults of seven-spotted ladybirds C. septempunctata were collected from their natural habitat near Uppsala, Sweden (59°47′N, 17°39′E) and reared through several generations before being used in experiments. They were kept in cages (40 × 40 × 80 cm) with potted barley plants (cv Golf) infested with aphids, R. padi and Sitobion avenae (F.). Rapeseed, Brassica napus L. and white mustard, Sinapsis alba L. plants were used as a source of pollen. Insects and plants were kept in a room with a controlled environment: L16:D8 light cycle with one lamp (Hortilux Schréder, HPS 400 Watt, Holland) per square metre, 18–22 °C temperature and 80 % relative humidity.
Plants
Seeds of dwarf bean Phaseolus vulgaris L. (cv Saxa) (Bröderna Nelson, Tingsryd, Sweden) and maize seeds Zea mays L. (cv Delprim) (Delley Seeds and Plants Ltd Delley, Switzerland) were used in the experiments. Before sowing, the bean seeds were germinated in Petri dishes on filter paper for 24 h. Prior to sowing, maize seeds were sterilised in 70 % ethanol for 3 min and rinsed twice in deionized water, then the seeds were placed in a solution of chlorine and water in a ratio 1:1 for 15 min and rinsed again four times in deionized water.
Bean and maize plants were grown in plastic pots (9 × 9 × 7 cm) in garden potting soil (Hasselfors, Sweden) with one seed per pot in a greenhouse at 18–22 °C, with a L16:D8 light cycle. Natural light was supplemented by light from HQIE lamps (Hortilux Schréder, HPS 400 Watt, Holland)—one lamp per square metre. Each plant was watered via an automated water drop system daily at 8 a.m. (2 h into the photoperiod). Six days after sowing, maize plants at the two leaf stage and bean plants with two open leaves were selected for uniformity in size and moved into clear Perspex cages.
Touching treatment
Plants were placed inside modified Perspex cages (each 10 × 10 × 40 cm), with an opening (7 cm diameter) in the front side (Ninkovic et al. 2002). Pots with test plants were placed in Petri dish lids to prevent any contact with root exudates from neighbouring plants. Air entered the cage through an opening in the cage wall and was extracted through a Teflon tube attached to a vacuum tank. The extracted air was then vented outside the room by an electric fan to prevent volatile interaction between plants. Thus, plants in this system were not expected to interact with each other in any way. Airflow through the cages was 1.3 l min−1. Each of the treatments was repeated 18 times. Each block consisted of touched and untouched plants of maize and bean, respectively.
Plant touch treatments started after the plants had spent 24 h in the Perspex cages. A soft squirrel hair face brush (Rouge) (Lindex, Sweden) was used. The second leaves of maize were carefully brushed from the leaf base to the top, while both bean leaves were brushed back and forth, using the modified method previously described (Montgomery et al. 2004; Liu et al. 2007; Anten et al. 2010). This treatment was chosen to simulate the plant response to mechanical contact with a neighbouring plant. Until the last day of the experiment, leaves of maize and bean plants did not have any contact with cage walls. Treated maize and bean plants were brushed in the morning for 1 min/day for a period of 6 days. This period was based on the time needed by maize plants to reach the top of the cage. All maize and bean plants treated by touching did not have any visible damage at the end of the treatment period.
Aphid settling test
The objective was to test whether touch influences aphid settling on their host plants. An aphid no-choice settling test (Ninkovic et al. 2002) was used to investigate aphid behavioural response to touched and untouched plants. Both maize and bean plants were tested 24 h after the last touching treatment ended. The second maize leaf was placed inside a transparent 100-ml polystyrene tube (diameter 2.5 cm, length 25 cm). For this test, the second leaf of each treatment plant placed inside the tube represented a replicate. Touched and untouched plants had 18 replicates, respectively. Ten wingless R. padi of second to fourth larval instars were placed inside the polystyrene tube. The upper end of the tube was sealed with nylon net, and the lower end was plugged with a plastic sponge through which the leaf entered via a slit. To minimise mechanical damage to the plants, the test tube was attached to a wooden stick to support the plant. The number of aphids settled on the leaf was recorded after 2 h, which is sufficient time for aphids to settle and reach the phloem (Prado and Tjallingii 1997). Two parameters were used to evaluate whether aphids were settled on the leaf or not. The first parameter refers to slight leaf shaking during a period of approximately 10 s, after removing the leaf from the tube. The second parameter suggested by Powell et al. (1993) was used for the aphids remaining on leaf. If the aphid body did not move and the antennae were in the held-back position without any movement, the aphid recorded as settled.
Due to the morphological differences of bean leaves, another no-choice settling test was done on a bean leaf that was placed in a Petri dish (15 × 2 cm) through a side opening around the leaf petiole. The petiole was protected with a sponge prior to being placed in the Petri dish. Ten wingless A. fabae of second to fourth larval instars were placed into small tubes (diameter 5 mm, length 4 cm) and then carefully placed inside the Petri dish containing one bean leaf. The cover had a hole (diameter 6 cm) protected with nylon net to prevent condensation. Bean leaves of touched and untouched plants were treated in the same way as described above. To avoid any plant disturbance, all Petri dishes were placed on a bench at the same height as the second leaves of the bean plants. As A. fabae spent more time walking before accepting the plant, the period for testing aphid settling was prolonged. Thus, after 3 h, the number of aphids settled on the bean leaf was recorded using the same procedure as for R. padi. Aphid acceptance of bean leaves was tested on touched and control plants in 18 replicates. Data were expressed as a proportion of aphids settled on the leaves per tube/Petri dish.
Test of aphid olfactory response
The aim was to assess aphid olfactory preference when offered a choice between volatiles released by touched and untouched plants. Here, we tested olfactory preferences of R. padi for volatiles from touched and untouched maize plants and preferences of A. fabae for volatiles from touched and untouched bean plants. Olfactometry experiments were done 24 h after the last touching treatment. A two-way olfactometer was used, consisting of two stimulus arms (length 4 cm) directly opposite each other, with a central zone (2.5 × 2.5 cm) separating them.
Air was extracted from the centre of the olfactometer using a vacuum pump, establishing discrete air currents in the side arms. Airflow in the olfactometer was set to 250 ml min−1, measured with a flow meter at the arm inlets. Touched and control plants were placed into separate, clean Perspex cages. One arm of each olfactometer was connected to a cage containing a touched plant and the other arm to a cage containing an untouched plant (Fig. 1). The position of the treatments in the two-arm olfactometer was switched between the left and right arms in each olfactometer to account for any positional bias.
Thirty minutes before each olfactometry experiment started, aphids were randomly chosen from cultures. One aphid of second to fourth instar was tested per olfactometer, and, after 10 min of acclimation, its position in the arena was registered 10 times at 3-min intervals (Ninkovic et al. 2009). Data were expressed as mean of individual aphid visits per olfactometer arm during the observation period of 30 min. Observed positions of aphids in the middle part of olfactometer were excluded from the analyses. To prevent aphid visual responses, plants were surrounded by white paper cones (diameter 11 cm and height 15 cm). The accumulated number of visits in the arm zones after ten recordings was regarded as one observation. If an aphid did not move between three consecutive observations (was motionless), the replicate was discarded and a new one started with a new insect. The experiments were replicated with 16 individuals of A. fabae, giving them the choice between the odour of touched and untouched bean plants, and with 26 individuals of R. padi, with choice between the odour of touched and untouched maize plants. Each individual aphid was used only once. To avoid contamination between replicates, all olfactometers were cleaned between each trial.
Test of ladybird olfactory response
The olfactory preference of C. septempunctata males and females to volatiles from touched and untouched maize and bean plants was tested using a two-arm olfactometer consisting of an arena (6 × 6 cm) with two conical, extended arms (arm length 7 cm) (Ninkovic et al. 2001; Glinwood et al. 2009) with an airflow of 250 ml min−1. Ladybirds were randomly collected from culture 24 h before each experiment and separated by sex according to Baungaard (1980). During the 24-h period, males and females were kept in separate clean jars covered with net, without access to food and provided with a L16:D8 light cycle. Water was provided in a glass tube plugged with cotton wool. Tested plants were placed into clear Perspex cages and connected to the side arms of the olfactometer. An adult ladybird was placed in the central zone of the olfactometer and, after a 10-min acclimation period, its position was registered 10 times at 2-min intervals. The 2-min intervals are long enough to permit an adult ladybird to move from one end of the arena to the other (Ninkovic et al. 2001). For this purpose, 22 and 27 male and female ladybirds, respectively, were tested. Observations were done in the same way as described for aphids. For each ladybird tested, a new clean olfactometer was used.
Plant response to touching
Maize and bean plants were cut at a ground level using scissors and separated into stem, leaves and roots. Roots from each plant were washed carefully with water. Stem and leaves were scanned for each plant separately using a dual lens scanner (Epson 4490Pro). Leaf surface and stem height were calculated using WinRHIZO (Regent Instruments), an image analysis system specifically designed for plant morphological measurements. Leaves, stem and roots from each plant were separately packed into labelled aluminium bags. After drying for 48 h at 70 °C to constant mass dry weights, plants spent 24 h at room temperature and were then weighed. These data were used for the calculation of integral morphological indices specific leaf area (SLA) and shoot root ratio (S/R). SLA is calculated as ratio of leaf area to dry weight while S/R as ratio of shoots dry mass (leaf plus stem) to root dry mass.
Collection of volatiles
Prior to volatile collection, polyethyleneterephthalate (PET) oven bags (35 cm × 43 cm, Toppits®, Klippan, Sweden) were baked in an oven at 140 °C for 2 h to remove contaminants. Glass tubes (5 mm diameter) containing Tenax TA (Supelco, Bellefonte PA, USA; 60/80 mesh, 50 mg) were heated at 220 °C under nitrogen for 2 h to remove contaminants. Plants were subjected to brushing treatment as described above, and control plants were untreated. Twenty-four hours after this treatment, pots containing either one maize plant or one bean plant were carefully enclosed in oven bags, taking care not to touch the leaves and shoots. Charcoal-filtered air was pumped in at 400 ml min−1, and a tube containing Tenax was inserted through a hole in the top of the bag and air drawn through via PTFE tubing connected to a pump (300 ml min−1). The difference in flow rates created a positive pressure to ensure no air from the laboratory entered the system. A small hole cut in the top of the bag prevented build-up of pressure. Air was pumped in for 30 min prior to volatile collection to flush contaminating volatiles from the system.
Volatile collection was carried out for 48 h under controlled environmental conditions (22 °C, 16 h:8 h light–dark cycle). Seven replicates were carried out for each treatment, and two control treatments consisting of pots and soil without plants were included.
Chemical analysis
Volatiles were analysed by gas chromatography-mass spectrometry (GC/MS) on an Agilent 7890N (Agilent Technologies) GC coupled to an Agilent 5975C mass selective detector (electron impact 70 eV). The GC was equipped with an HP-1 column (100 % dimethyl polysiloxane, 30 m, 0.25 mm i.d. and 0.25 μm film thickness, J&W Scientific, USA) and fitted with an Optic 3 thermal desorption system (Atas GL Intl., Veldhoven, Netherlands). The liner containing the Tenax with absorbed volatiles was placed directly into the injector, and volatiles were thermally desorbed starting at 30 °C/0.5 min and rising at 30 °C/s to 250 °C. The GC temperature programme was 30 °C/2 min, 5 °C/min to 150 °C/0.1 min, 10 °C/min to 250 °C/15 min, using Helium as carrier with a flow rate of 1.3 ml/min. Volatile compounds were identified by comparison against a commercially available library (NIST 08) and by comparison of mass spectra and retention indices with commercially available authentic standards where available. Only compounds appearing in the headspace of plants and not pots with soil were quantified. Most of the compounds identified have been previously reported from Z. mays (Degen et al. 2004) and P. vulgaris (Wei et al. 2006).
Compounds were quantified using three-point response curves constructed using authentic standards where available. No authentic standards were available for α-bergamotene or the unknown sesquiterpenes, and these substances were quantified using the sesquiterpene (E)-β-caryophyllene. Chemical standards were obtained commercially as follows: (Z)-3-hexen-1-ol (Sigma-Aldrich 98 %), 6-methyl-5-hepten-2-one (Sigma-Aldrich 99 %), β-myrcene (Fluka 90 %), (Z)-3-hexenyl acetate (Sigma-Aldrich 98 %), linalool oxide (Fluka > 97 %), linalool (Sigma-Aldrich 97 %), indole (Sigma-Aldrich > 99 %), (+)-cyclosativene (Sigma-Aldrich 99 %), β-caryophyllene (Fluka > 98.5 %), (E)-β-farnesene (Fluka > 90 %), (+)-valencene (Sigma-Aldrich > 70 %), β-bisabolene (Alfa Aesar) and (E)-nerolidol (Fluka > 85 %). Standards of (E)-ocimene, (E,E)-4,8,12-Trimethyl-1,3,7,11-tridecatetraene (TMTT) and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) were kindly provided by Dr Mike Birkett, Rothamsted Research, UK.
Statistical analysis
Aphid acceptance was obtained in the form of proportions, and data were modelled using a generalised linear mixed model (e.g., Littell et al. 2006). In such models, data can take distributions other than normal, and the form of the relationship can be modelled through a link function. The following specifications were included in the model: the proportion of aphids settling in each tube/Petri dish was modelled using a binomial distribution, the logit link was used, the fixed part of the model included touch treatment (touched and untouched plants of maize and bean) and block, whereas the block × treatment interaction was regarded as a random factor. The hypothesis of no difference between treatments was tested at the 5 % level. Pairwise comparisons between least-square means were calculated and compared using Tukey’s HSD test. Proc GLIMMIX of the SAS Institute (2011) package was used.
Data from olfactory bioassays were analysed with Wilcoxon’s matched pairs tests. Differences in morphological parameters between touched and untouched plants were tested by t test. These analyses were performed with the Statistica software (Statsoft Inc. 2011).
One-way ANOVA (see e.g., Olsson 2011) was used to analyse whether any of the volatiles was related to treatment. Stepwise discriminant analysis (see e.g., Johnson and Wichern 2007) was used to conclude test any combination of volatiles could detect whether the plant was treated or not.