Effect of water availability on volatile-mediated communication between potato plants in response to insect herbivory

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.

Methods
In April 2021, we sowed 168 tubers from three different Solanum tuberosum 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-2 s-1 and CO2 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 (F1,22 = 22.1, P < 0.001) and photosynthetic rates (F1,22 = 31.5. P < 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 H2O m-2 s-1; low water availability: 0.008 ± 0.01 mol H2O m-2 s-1) and photosynthesis rates (high water availability: 9.31 ± 0.94 µmol CO2 m-2 s-1; low water availability: 1.88 ± 0.94 µmol CO2 m-2 s-1), 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 S. exigua 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.

植物空中通讯是一种广泛存在的自然现象：受损植株释放的挥发性有机化合物（Volatile Organic Compounds，缩写为VOCs）可提升邻近未受损植株的植食性昆虫抗性。尽管这类植物信号传导已在30余种植物中被报道，但关于非生物因子（如水分可利用性）如何调控其传导效果的认知仍存在显著空白。

本研究开展温室试验，以探究马铃薯（*Solanum tuberosum*）在面对广食性昆虫甜菜夜蛾（*Spodoptera exigua*）取食时的植株间通讯现象，以及水分可利用性是否会对该通讯过程产生影响。我们将马铃薯植株分为信号释放株与信号接收株，其中半数释放株经甜菜夜蛾幼虫取食受损，另一半作为未受损对照。所有释放株与接收株均接受两种水分可利用性处理之一：高水分（即充分灌溉）与低水分（即减量灌溉），借此可有效解析水分可利用性对信号传导的释放与接收两个环节的调控作用。在植食性昆虫取食4天后，我们收集释放株释放的VOCs，并将接收株暴露于甜菜夜蛾取食环境中，以验证信号传导对诱导抗性的影响。

甜菜夜蛾取食可使释放株的VOCs释放量显著提升，同时改变其VOCs组成。此外，与充分灌溉的释放株相比，低水分处理的释放株在应对植食性昆虫取食时，其VOCs的诱导水平显著更低。取食生物测定结果显示，相较于暴露于未受损释放株VOCs的接收株，暴露于经植食性昆虫诱导的释放株VOCs的接收株，其受甜菜夜蛾取食造成的损伤显著更低（即诱导抗性更强）。然而，本研究未观察到释放株或接收株的水分可利用性对植物空中通讯产生显著影响。

总体而言，本研究证实了水分可利用性对介导植物间空中通讯的VOCs诱导过程的调控作用，从而加深了学界对非生物环境如何影响植物通讯的认知。本研究观测到的诱导VOCs变化并未对植物通讯产生可观测的影响，因此该结果表明，本实验条件并未损害介导植株间通讯的关键化合物的诱导过程。

## 材料与方法

2021年4月，我们将来自3个不同马铃薯品种（品种名Baraka、Desiree与Monalisa）的168个块茎播种于装有营养土与泥炭的4升花盆中（基质购自德国费希塔的Gramoflor GmbH & Co. KG Produktion公司）。植株种植于可控环境温室中，光照条件为每日最少10小时，光合有效辐射为725±19 μmol·m⁻²·s⁻¹，温度设置为夜间10℃、日间25℃，每周浇水两次至田间持水量水平。发芽5周后，我们将半数植株随机分配至两种水分可利用性处理之一：高水分（即充分灌溉）与低水分（即减量灌溉）（图1）。高水分处理组植株每3天浇水一次，以补充其100%的需水量；而低水分处理组的浇水量被削减至仅满足其总需水量的25%。本研究通过重量法估算植株需水量。

为验证低水分处理组植株相较于充分灌溉植株处于更高的生理胁迫水平，在水分处理开始两周后（即开展植食性昆虫处理前，详见下文），我们选取24株植株作为样本（高、低水分处理组各12株，每个马铃薯品种各4株），测定其气孔导度与光合速率。我们于当日11:30至12:30期间，在辐照度1500 μmol·m⁻²·s⁻¹、CO₂浓度400 μmol·mol⁻¹的条件下，使用便携式光合仪Li-6400XT（美国Li-Cor公司，林肯市，内布拉斯加州）测定植株幼嫩完全展开叶片的气孔导度与净光合速率。结果显示，低水分处理组植株的气孔导度（F₁,₂₂=22.1，P<0.001）与光合速率（F₁,₂₂=31.5，P<0.001）均显著低于充分灌溉组。具体而言，减量灌溉使气孔导度与光合速率分别下降90%与80%：高水分处理组气孔导度为0.073±0.01 mol·H₂O·m⁻²·s⁻¹，低水分处理组为0.008±0.01 mol·H₂O·m⁻²·s⁻¹；高水分处理组光合速率为9.31±0.94 μmol·CO₂·m⁻²·s⁻¹，低水分处理组为1.88±0.94 μmol·CO₂·m⁻²·s⁻¹（图S1a、b）。

发芽7周后（即水分处理开始两周后），我们将144株马铃薯植株放置于37.5×37.5×96.5 cm的塑料笼中，以避免不同重复组间的VOCs交叉污染。每对植株（即一个重复单元）中，一株作为信号释放株（平均株高±标准误：51.17±0.64 cm），另一株作为信号接收株（48.52±0.70 cm）。每个塑料笼内，释放株与接收株间距为20 cm，确保二者无接触。我们将各水分胁迫处理组的植株随机分配为释放株或接收株，由此形成析因设计，包含释放株水分处理（两个水平：高、低）与接收株水分处理（两个水平：高、低）的4种组合（图1）。此外，我们将每个塑料笼内的释放株随机分配至以下两种植食性昆虫处理之一：(1) 经甜菜夜蛾取食（以下简称“植食诱导株”），或(2) 对照（完整无损伤，无植食）植株（图1）。本实验共设置72个重复塑料笼，其中36个为植食诱导处理组（每个释放株-接收株水分组合各9个），36个为对照组（每个释放株-接收株水分组合各9个）。释放株与接收株始终为同一品种，且各品种在各处理组合中分布均匀。对于植食诱导的释放株，我们使用细毛刷将2只甜菜夜蛾三龄幼虫放置于植株的3片完全展开叶片上，并用尼龙袋套住这些叶片以防止幼虫逃逸。对于对照植株，我们同样用尼龙袋套住3片完全展开叶片，但不添加幼虫，以排除套袋本身可能产生的影响。植食性昆虫取食4天后，我们将所有释放株移出塑料笼，并收集其释放的VOCs（详见下文）。VOCs采样完成后，我们收集被幼虫取食的叶片，使用三星Galaxy A30s手机（有效像素2500万，4倍数字变焦）进行拍照，并通过移动应用BioLeaf - Foliar Analysis™（Brandoli Machado等，2016）估算叶片被取食的面积百分比。植食诱导释放株的甜菜夜蛾取食叶面积百分比均值为77.58%±3.72，且在高水分处理组（80.46%±3.50）与低水分处理组（73.63%±4.25）间分布均匀（F₁,₃₃=0.7，P=0.399）。

我们参考Rasmann等（2011）的方法收集释放株地上部释放的VOCs。简要步骤如下：使用2升Nalophan采样袋包裹植株，以0.25 L·min⁻¹的流速将VOCs捕获于装有活性炭吸附剂的SKC吸附管（填充anascorb CSC椰子壳活性炭）中，捕获时长为2小时。随后，我们用150 μL二氯甲烷（CAS#75-09-2，瑞士Dietikon的Merck公司）洗脱吸附管，洗脱液中预先加入内标物四氢萘（CAS#119-64-2，10 μL二氯甲烷中含200 ng四氢萘）。之后，我们将每份样品的1.5 μL萃取液注入安捷伦7890B气相色谱仪（GC），该色谱仪联用5977B质谱选择检测器（MSD），并配备30 m×0.25 mm×0.25 μm膜厚的HP-5MS熔融石英毛细管柱（安捷伦公司，圣克拉拉，加利福尼亚州，美国）。气相色谱采用脉冲不分流进样模式（进样口温度250℃，进样压力15 psi），以氦气作为载气，恒定流速为0.9 mL·min⁻¹。柱温箱升温程序为：40℃保持3.5分钟，以5℃·min⁻¹升温至230℃，随后在250℃保持3分钟完成后运行。传输线温度设置为280℃。质谱检测器采用电子轰击电离（EI）模式，质量扫描范围为33~350（m/z），离子源与四级杆温度分别设置为230℃与150℃。我们使用NIST MS Search Program v.2.3软件，并结合纳沙泰尔大学基于纯标准品构建的萜类参考数据库，对挥发性萜类化合物进行定性分析。我们通过归一化峰面积对各VOCs组分的释放量进行定量，单位以纳克每小时（ng·h⁻¹）表示。各化合物的归一化峰面积通过其积分峰面积除以内标物的积分峰面积计算得到（Abdala-Roberts等，2022）。VOCs总释放量为各单体VOCs释放量的总和。

在收集释放株VOCs的当日，我们针对接收株开展植食性昆虫生物测定，以验证预先暴露于植食诱导释放株VOCs是否可提升接收株的植食性昆虫抗性。具体操作如下：参照上述释放株诱导的方法，将1只甜菜夜蛾三龄幼虫放置于接收株的2片完全展开叶片上。让幼虫在接收株上取食3天后，采用与上述释放株相同的方法估算甜菜夜蛾造成的叶面积取食百分比（以下简称“叶片损伤率”）。