DOI: 10.5061/dryad.kkwh70s78
title: Effect of water availability on volatile-mediated communication between potato plants in response to insect herbivory

description: Airborne plant communication is a widespread phenomenon in which volatile
 organic compounds (VOCs) from damaged plants boost herbivore resistance in
 neighbouring, undamaged plants. Although this form of plant signalling has
 been reported in more than 30 plant species, there is still a considerable
 knowledge gap on how abiotic factors (e.g., water availability) alter its
 outcomes. We performed a greenhouse experiment to test for communication
 between potato plants (Solanum tuberosum) in response to herbivory by the
 generalist insect Spodoptera exigua and whether communication was affected
 by water availability. We paired emitter and receiver potato plants, with
 half of the emitters damaged by S. exigua larvae and half serving as
 undamaged controls. Both emitter and receiver plants were subjected to one
 of two water availability treatments: high (i.e., well-watered) vs. low
 (i.e., reduced watering) availability, thus effectively teasing apart
 water availability effects on the emission and reception components of
 signalling. After four days of herbivore feeding, we collected emitter
 VOCs and receivers were subjected to feeding by S. exigua to test for
 effects of signalling on induced resistance. Herbivory by S. exigua led to
 increased VOCs emissions as well as changes in VOCs composition in emitter
 plants. Furthermore, emitters subjected to low water availability
 exhibited a weaker induction of VOCs in response to herbivory relative to
 well-watered emitters. Results from the feeding bioassay indicated that
 receivers exposed to VOCs from herbivore-induced emitters showed lower S.
 exigua damage (i.e. higher induced resistance) compared to receivers
 exposed to undamaged emitters. However, we did not observe a significant
 effect of water availability in either emitters or receivers on plant
 communication. Overall, our study contributes to the understanding of how
 the abiotic context affects plant communication by providing evidence of
 water availability effects on the induction of VOCs that may act as
 airborne signals between plants. The observed changes in induced VOCs had
 no visible consequences for plant communication. These findings thus
 suggest that the induction of key compounds mediating communication was
 not compromised by our experimental conditions.
description: In April 2021, we sowed 168 tubers from three different
 <em>Solanum tuberosum</em> varieties (cv. Baraka, cv. Desiree,
 and cv. Monalisa) in 4-L pots containing potting soil and peat (Gramoflor
 GmbH & Co. KG Produktion, Vechta, Germany). We grew plants in a
 glasshouse under controlled light (minimum 10 h per day,
 Photosynthetically Active Radiation = 725 ± 19 μmol m-2 s-1) and
 temperature (10°C night, 25°C day), and watered them twice a week up to
 field capacity. Five weeks after germination, we assigned half of the
 plants to one of two water availability treatments: high (i.e.,
 well-watered) vs. low (i.e., reduced watering) water availability (Fig.
 1). We watered plants in the high water availability treatment every three
 days to replenish the 100% of their water demand, whereas for plants in
 the low water availability treatment watering was reduced to meet the 25%
 of the total water demand. We estimated water demand gravimetrically.To
 corroborate that plants in the low water availability treatment were under
 higher physiological stress in comparison to well-watered plants, two
 weeks after the start of the treatments (right before applying the
 herbivory treatment, see below) we used a subset of 24 plants (half high
 and half low water availability; four of each potato variety) to measure
 stomatal conductance and photosynthesis. We measured stomatal conductance
 and net photosynthetic rate on a leaflet of a young, fully-expanded leaf
 from 11:30 to 12:30 am at an irradiance of 1500 μmol m<sup>-2</sup>
 s<sup>-1</sup> and CO<sub>2</sub> concentration of
 400 μmol mol-1 with a
 portable photosynthesis system Li-6400XT (Li-Cor Inc., Lincoln, NE, USA).
 Plants in the low water availability treatment exhibited significantly
 lower stomatal conductance (F<sub>1,22</sub> = 22.1, P
 &lt; 0.001) and photosynthetic rates (F<sub>1,22</sub> =
 31.5. P &lt; 0.001) compared to well-watered plants. Specifically,
 reduced watering resulted in a 90 % and an 80% decrease in stomatal
 conductance (high water availability: 0.073 ± 0.01 mol
 H<sub>2</sub>O m<sup>-2</sup>
 s<sup>-1</sup>; low water availability: 0.008 ± 0.01 mol
 H<sub>2</sub>O m<sup>-2</sup>
 s<sup>-1</sup>) and photosynthesis rates (high water
 availability: 9.31 ± 0.94 µmol CO<sub>2</sub>
 m<sup>-2</sup> s<sup>-1</sup>; low water
 availability: 1.88 ± 0.94 µmol CO<sub>2</sub>
 m<sup>-2</sup> s<sup>-1</sup>), respectively (Fig.
 S1a, b). Seven weeks after germination (two weeks after
 establishing the water availability treatments), we paired 144 potato
 plants in 37.5 × 37.5 × 96.5 cm plastic cages to prevent VOCs
 cross-contamination between replicates. One plant of each pair (i.e.,
 replicate) acted as the emitter (average height ± SE = 51.17 ± 0.64 cm)
 and the other served as the receiver (48.52 ± 0.70 cm). Within each cage,
 emitter and receiver plants were placed 20 cm apart so that they did not
 touch each other. Plants in each water stress treatment were randomly
 selected as either receiver or emitter plants resulting in a factorial
 design consisting on four combinations of water availability treatment in
 the emitter (two levels; high vs. low) and water availability treatment in
 the receiver (two levels; high vs. low) (Fig. 1).  In addition, we
 randomly assigned emitter plants within each cage to one of the following
 herbivory treatments: (1) subjected to S. exigua feeding
 (“herbivore-induced plants” hereafter) or (2) control (intact; no
 herbivory) plants (Fig. 1). Overall, the experiment consisted in 72
 replicate cages, namely 36 for the herbivore-induced treatment (nine per
 emitter vs. receiver water availability combination) vs. 36 for the
 control (nine per emitter vs. receiver water availability combination).
 Emitter and receiver plants were always of the same variety and varieties
 were similarly distributed across treatment combinations. For
 herbivore-induced emitters, we placed two third-instar larvae of S. exigua
 on each of three fully expanded leaves per plant using a fine paintbrush
 and covered these leaves with a nylon bag to prevent herbivore dispersal.
 For control plants, we covered three fully expanded leaves with a nylon
 bag but without adding the larvae to control for a possible bagging
 effect. After four days of herbivore feeding, we removed all emitter
 plants from cages and collected VOCs from each emitter (see below). After
 VOCs sampling, we collected leaves subjected to larvae feeding and
 photographed them with a Samsung Galaxy A30s (25 effective megapixels, 4×
 digital zoom). We estimated the percentage of leaf area consumed using the
 mobile application BioLeaf - Foliar Analysis™ (Brandoli Machado et al.,
 2016). Average percentage leaf area consumed by S. exigua for
 herbivore-induced emitters was 77.58% (± 3.72) and was homogeneously
 distributed among plants in the high (80.46% ± 3.50) vs. low (73.63% ±
 4.25) water availability treatments (F1,33 = 0.7; P = 0.399).
 We collected aboveground VOCs produced by emitter plants
 following Rasmann et al. (2011). Briefly, we bagged plants with a 2-L
 Nalophan bag and trapped VOCs on a charcoal filter (SKC sorbent tube
 filled with anascorb CSC coconut-shell charcoal) for two hours at a rate
 of 0.25 L min-1. We eluted traps with 150 μL dichloromethane (CAS#75-09-2,
 Merck, Dietikon, Switzerland) to which we had previously added one
 internal standard (tetralin [CAS#119-64-2], 200 ng in 10 μL
 dichloromethane). We then injected 1.5 μL of the extract for each sample
 into an Agilent 7890B gas chromatograph (GC) coupled with a 5977B mass
 selective detector (MSD) fitted with a 30 m × 0.25 mm × 0.25 μm film
 thickness HP-5MS fused silica column (Agilent, Santa Clara, CA, USA). We
 operated the GC in pulsed splitless mode (250 ºC, injection pressure 15
 psi) with helium as the carrier gas (constant flow rate 0.9 mL min-1). The
 GC oven temperature programme was: 3.5 min hold at 40ºC, 5ºC min-1 ramp to
 230ºC, then a 3 min hold at 250ºC post run. Transfer line was set at 280
 ºC. In the MS detector (EI mode), a 33-350 (m/z) mass scan range was used
 with MS source and quadrupole set at 230ºC and 150ºC, respectively. We
 identified volatile terpenes using the NIST MS Search Program v.2.3 and by
 comparison with the terpenes reference database developed at the
 University of Neuchâtel and based on pure standards. We quantified total
 emission of individual VOCs using normalized peak areas and expressed it
 as nanograms per hour (ng h-1). We obtained the normalized peak area of
 each individual compound by dividing their integrated peak area by the
 integrated peak area of the internal standard (Abdala-Roberts et al.,
 2022). The total emission of VOCs was then calculated as the sum of
 individual VOCs. The same day after collecting emitter
 VOCs, we set up an herbivore bioassay for receiver plants to test whether
 prior exposure to VOCs from herbivore-induced emitters increased herbivore
 resistance. For this, we placed one third-instar <em>S.
 exigua</em> larvae on each of two fully expanded leaves per plant
 following the same procedure described above for emitter induction. We
 kept larvae on receivers for three days and then estimated the percentage
 of leaf area consumed by S. exigua (‘leaf damage’ hereafter) using the
 same procedure described above for emitter plants.