Plant Soil https://doi.org/10.1007/s11104-023-06421-9 RESEARCH ARTICLE Combined effects of drought and simulated pathogen attack on root exudation rates of tomatoes Catherine Preece · Kaijun Yang · Joan Llusià · Jana Barbro Winkler · Jörg‑Peter Schnitzler · Josep Peñuelas Received: 18 August 2023 / Accepted: 24 November 2023 © The Author(s) 2023 Abstract Methods We conducted a controlled environment Background and aims Food production is threatened experiment to investigate the effects of moderate by direct climate change effects including drought. drought and simulated pathogen attack (using pipec- Indirect effects, including changes in plant-pathogen olic acid, an inducer of systemic acquired resistance) dynamics and increased susceptibility to pathogens, on the rates of root exudation of total organic carbon further exacerbate the risks. Root exudation, which (TOC) and total nitrogen (TN) of four tomato culti- plays a crucial role in plant defence against drought vars grown in potting soil. and pathogens, is influenced by both water stress and Results Drought increased the exudation of TOC pathogens. However, the interactive effects of these and TN per unit of root area, while pipecolic acid did abiotic and biotic factors are rarely studied. not have any significant effect. Furthermore, there was no interaction observed between the abiotic and biotic factors. However, due to the reduction in plant and root biomass caused by drought, the total exuda- Responsible Editor: Eva Oburger. tion per plant remained similar between control and Supplementary Information The online version water-limited plants. Additionally, pipecolic acid contains supplementary material available at https://​doi.​ reduced the carbon-to-nitrogen ratio of exudates and org/​10.​1007/​s11104-​023-​06421-9. increased the total exudation of TN. Conclusion The increased exudation observed C. Preece (*) Institute of Agrifood Research (IRTA), Programme in drought-stressed plants may serve as a strategy of Sustainability in Biosystems, Torre Marimon, to maintain root and rhizosphere activity despite Caldes de Montbui 08140, Spain reduced root growth. Notably, the impact of drought e-mail: catherine.preece09@gmail.com differed among the tested cultivars, highlighting their K. Yang · J. Llusià · J. Peñuelas diverse levels of drought tolerance. This emphasises CSIC, Global Ecology Unit CREAF-CSIC-UAB, the importance of preserving a wide range of crop Bellaterra, Catalonia 08193, Spain cultivars to ensure food security under increasing drought. K. Yang · J. Llusià · J. Peñuelas CREAF - Ecological and Forestry Applications Research Centre, Cerdanyola del Vallès, Catalonia 08193, Spain Keywords Rhizodeposition · Pipecolic acid · Total organic carbon · Total nitrogen · Solanum J. B. Winkler · J.-P. Schnitzler lycopersicum Research Unit Environmental Simulation, Helmholtz Zentrum München, 85764 Neuherberg, Germany Vol.: (0123456789) 13 Plant Soil Introduction has been shown to increase in response to mild and moderate drought (Preece and Peñuelas 2016), and Maintaining secure food supplies involves under- may represent a plant strategy to change the soil envi- standing how the plants we eat are affected by abi- ronment to reduce drought stress (Williams and De otic and biotic stress and, ultimately, selecting vari- Vries 2020). The mechanisms for this may include eties that are more tolerant to such stress. Drought exudation of the structural carbohydrate mucilage is an increasing problem for agriculture, as lower which keeps the soil near the roots moist and main- rainfall across large regions of the world means that tains hydraulic conductivity (Ahmed et al. 2014), there is less water available for irrigation. Around the formation of a rhizosheath (soil aggregation on 75% of the global harvested area has experienced the surface of the root) (Ndour et al. 2020; Chai and yield losses related to drought (Kim et al. 2019), Schachtman 2022), and the promotion of interactions leading to food shortages, which increase prices and with rhizosphere microbes that can improve water threaten the nutrition of millions of people. At the relations (Naylor and Coleman-Derr 2018). Other same time, pathogens that attack crops reduce yields studies have indicated specific compounds in exu- by up to 30%, depending on the crop and the loca- dates that change under drought. For example, experi- tion (Savary et al. 2019). While plants have in-built mental drought stress increased exudation of proline defence systems to fight pathogen attacks, the alloca- and abscisic acid in barley (Calvo et al. 2017). tion of resources to this process can mean a decrease The role of root exudates in pathogen defence is in growth due to growth–defence trade-offs (He also key (Haichar et al. 2014; Vives-Peris et al. 2019). et al. 2022). Moreover, plant pathogen infection risk Defence compounds in exudates may be released is predicted to increase in some regions due to cli- both constitutively (phytoanticipins) and in response mate warming altering the geographical and seasonal to specific threats (phytoalexins) (Baetz and Marti- niches of pathogens (Chaloner et al. 2021), leading to noia 2014). There is a large diversity of antimicro- a higher requirement for pesticide use, with negative bial compounds released by roots, with phenolics impacts on the environment and human health (Tang and terpenoids being particularly notable (Badri et al. et al. 2021; Wuepper et al. 2023). New strategies to 2013; Baetz and Martinoia 2014), including in tomato adapt our agricultural systems to be able to resist (Steinkellner et al. 2005). For example, various cin- water stress and pathogen attack are needed. namic acid derivatives (e.g., t-cinnamic acid) have Root exudation is a main link between plants and anti-fungal activity, and have been shown to increase soils, with exuded compounds playing key roles in both inside barley root tissues and in root exudates, interactions with other plants, with microbes and during infection by the soil fungal pathogen Fusarium fauna, and with soil properties (Bais et al. 2006; (Lanoue et al. 2010). In a separate study with barley, Sasse et al. 2018). Root exudation is the release of roots infected with Fusarium graminearum recruited organic compounds into the soil (including sugars, bacteria with antifungal properties, which was pro- organic acids, amino acids and structural carbohy- posed to be linked to the root exudates (Dudenhöffer drates). This represents a significant proportion of the et al. 2016). Pathogen attack, even above ground, can carbon budget of a plant, making up approximately also stimulate the release of root exudates that attract 11% of net fixed C (Jones et al. 2009), although this beneficial root microbes. For example, in Arabidop- varies a lot between plant functional types and tend- sis, infection by the foliar pathogen Pseudomonas ing to be higher in younger plants (Gransee and Wit- syringae increases exudation of l-malic acid, which tenmayer 2000; Pausch and Kuzyakov 2018). Root causes increased root colonisation by Bacillus subtilis exudation patterns have been shown to be very vari- which is a plant-beneficial rhizobacterium (Rudrappa able, between species and individuals, and also to et al. 2008). Similarly, tomato plants that were change (in quantity and composition) in response to exposed to foliar pathogen attack released root exu- biotic and abiotic factors (Walker et al. 2003; Bais dates that attracted a beneficial fungus (Trichoderma et al. 2006; Gargallo-Garriga et al. 2018; Tiziani et al. harzianum) (Lombardi et al. 2018). 2022). While the impact of individual abiotic and biotic In relation to water availability, overall root exuda- stressors on exudation is well-documented, the inter- tion, usually measured as total organic carbon (TOC), active effects of multiple stressors remain largely Vol:. (1234567890) 13 Plant Soil unexplored, creating a significant knowledge gap. providers (Bella Rosa: Agroleg Varo SRL, Bogata The main objective of our study was to investigate the de Mures, Nr. 486D, Rumania; Estrella del Balcón: combined influence of moderate drought and simu- http://​www.​seeds​galle​ry.​shop; Flor de Baladre: Semi- lated pathogen attack on root exudation rates in four lleros Corbalán CB, La Carrasquilla, Murcia, Spain, tomato cultivars of varying drought tolerance. Tomato via suhuertoencasa.com; Valenciano: Fitoagrícola, (Lycopersicon esculentum) was selected as the focal Castellón de la Plana, Castellón, Spain). Tomato crop due to its global significance, ranking as the sec- plants were grown from seed in the greenhouse of the ond most important vegetable crop worldwide, with a Research Unit Environmental Simulation at Helm- current production of approximately 189 million tons holtz Munich (Germany) in January 2020. Sowing of fresh fruit from 5 million hectares (FAOstat 2021). of seed was staggered by cultivar, with a week in Future climate projections indicate that increasing between, due to constraints on the space available air temperatures and decreased water availability in in the ‘VOC-SCREEN’ platform (Jud et al. 2018) major tomato-growing regions will limit production, where the later stages of the experiment took place. potentially resulting in a 6% decrease by 2040 com- Thus, for all parts of the experiment, cultivars were pared to the 1980–2009 period (Cammarano et al. separated in time. Plants were grown in potting soil 2022). Additionally, tomato is highly susceptible to (Einheitserde CL-Topf, a peat-based mixture), which various plant diseases caused by bacterial, fungal, and consisted of 70% organic material, and contained 340 viral pathogens (Panno et al. 2021), and that can be mg ­L−1 nitrogen, 260 mg L ­ −1 phosphorus, 330 mg −1 increased due to water stress. ­L potassium oxide, and 100 mg L ­ −1 magnesium. Our hypotheses were as follows: (1) Moderate Plants grew in 1 L pots, with optimum watering, drought would increase specific exudation rates (exu- until they were 14 days old. At that stage, 30 plants dation per root area), consistent with previous stud- per cultivar were transferred to the ‘ExpoSCREEN’ ies in other plant species that have demonstrated (Vanzo et al. 2015) controlled climate chamber (27 increased exudation under drought conditions. (2) ºC/20 ºC day/night temperature with 13-hour photo- Simulated pathogen attack would also stimulate spe- period and 600µmol PPFD ­m−2 ­s−1). Plants were kept cific root exudation as a response to produce higher in that chamber for 14 additional days, to acclimatise levels of antimicrobial compounds, thus (3) com- to being out of the greenhouse environment. bined drought and simulated pathogen attack would therefore produce the greatest changes in exudation. Experimental design Furthermore, we hypothesised (4) that there would be cultivar-specific variations in exudation rates due to Two treatments of moderate drought and simulated different resource allocation patterns and varying lev- pathogen attack were applied in a factorial design. els of resistance to water stress and pathogen attack. Drought was applied by limiting watering and patho- gen attack was simulated by the addition of pipecolic acid to the irrigation water. Pipecolic acid is a crucial Methods regulator of plant systemic acquired resistance, that confers long-lasting protection against a broad spec- Plant material and initial growth conditions trum of microorganisms (Ding et al. 2016). There- fore, pipecolic acid was used as a proxy for pathogen Four cultivars of tomato (Solanum lycopersicum attack, being much more controllable than the addi- (L.) H.Karst.) were selected to represent commer- tion of a soil pathogen. There were four treatment cially available varieties with different stated levels combinations: control, drought, pipecolic acid, and of drought tolerance, namely cv. Bella Rosa, Estrella drought + pipecolic acid. For each cultivar, on day 28 del Balcón, Flor de Baladre and Valenciano. The at 16:00, 16 individuals were selected, being those cultivars that were stated (by the seed providers) as that were healthy and most similar in size and appear- being more drought resistant were Estrella del Bal- ance. The 16 selected plants were randomly assigned cón and Valenciano, and the two cultivars that were to one of the four treatments, with four replicates more drought sensitive were Bella Rosa and Flor de per treatment (see the schematic diagram in Supple- Baladre. Seed was acquired from different comercial mentary Information Fig. S1, for a summary of the Vol.: (0123456789) 13 Plant Soil experimental steps). Plants receiving pipecolic acid Plants were kept in the VOC-SCREEN cuvettes were separated from the other plants by placing them for seven days, and during that time, there were in a separate sub-chamber, to prevent the compound constant measurements of ­CO2 exchange, air tem- from affecting the other plants through the emission perature and humidity (using the sensor DKRF400, of stress-related volatile organic compounds (VOCs) Driesen + Kern GmbH, Bad Bramstedt, Germany), induced by pipecolic acid. For each of those plants, and soil temperature and humidity (using 5TM soil 40 mL of 2.06 mmol L ­ −1 pipecolic acid was applied moisture and temperature sensors, Decagon Devices to different points on the soil surrounding the stem. Inc., Pullman, WA, USA). Temperature did not vary This made 82.4 µmol of pipecolic acid per pot, with between treatments with 27 ºC/19 ºC (day/night) this amount reflecting other experiments that have temperatures and a 13-hour photoperiod with 600 used pipecolic acid to study systemic acquired resist- µmol PPFD ­m−2 ­s−1. Plants in the control water- ance (Lenk et al. 2019). The remaining plants were ing treatment received 30 mL twice per day (morn- given 40 mL of deionised water instead. ing and evening) totalling 420 mL of water during On day 29, those 16 plants were photographed for the seven days. The drought treatment was imple- determination of leaf area, by placing them on a rotat- mented by reducing the daily watering, but exact ing plate inside a cubicle with a blue background. amounts of water addition varied slightly between Two fixed cameras were used to capture images: one cultivars, to ensure an effective drought treatment was positioned above the plant and the other had a for all. This was evaluated by checking the visual horizontal view. This setup allowed for standardized status of the plants as well as gas exchange meas- lighting conditions during image acquisition. Nine urements. Therefore, Estrella del Balcón received images were taken of each plant to capture the plant 85 mL under drought (no watering during the first from all angles (360º rotation), and this was used later three days, then 25 mL on the fourth day and 20 to calculate the total leaf area using the Leaf Area mL on the remaining three days) whereas the other Calculator as described by Jud et al. (2018). Plants three cultivars received 120 mL under drought (no were then transferred to the VOC-SCREEN platform watering during the first three days, then 30 ml (Jud et al. 2018), which is a customised cuvettes sys- on the remaining four days). All plants from the tem set-up in a climate chamber of the ExpoSCREEN drought treatment, across all four varieties, showed facility. This platform consists of 24 cuvettes, visual signs of drought stress (i.e., wilting) by the equipped with two C ­ O2/H2O gas analysers (LI-640, end of the experiment. On day 35, the plants were LICOR, Lincoln, Nebraska, USA) that measure the removed from the chamber and photographed for a absolute ­CO2 and ­H2O concentration of the air at the second determination of leaf area (example images inlet and outlet of the cuvettes, allowing calculation for each cultivar can be seen in the Supplementary of carbon balance (assimilation and evapotranspira- Information Fig. S3). tion rates). Air humidity within the cuvettes was on aver- In this platform, each plant was placed inside a age 51% (SE = ± 4%) in well-watered cuvettes and glass cuvette (Supplementary Information Fig. S2), 42% in drought cuvettes (SE = ± 5%), when group- each with a volume of ~ 40 L (height of 60 cm and ing together all cultivars. Mean soil volumetric water an inner diameter of 29 cm). The cuvettes were con- content over the seven days, across all cultivars, was tinuously purged with particle- and carbon-filtered air 27.0% (SE = ± 6.2%) in well-watered cuvettes and from the chamber, which had the ambient C ­ O2 con- 16.5% (SE = ± 6.6%) under drought. Therefore, there centration. The inlet flow was set to 10 L ­min−1 for was a significant effect of drought on soil volumetric Estrella del Balcón and Valenciano measurements but water content (linear model, F = 72.8, p < 0.001). Cul- had to be adjusted to 12 L m ­ in−1 for Bella Rosa and tivar also had a significant effect on soil water con- Flor de Baladre to avoid condensation of water vapor tent (linear model, F = 6.9, p < 0.001), but there was in the cuvettes. Because the soil compartment in the no interaction between cultivar and the drought treat- cuvette is not separated from the above-ground space, ment, with the drought consistently reducing water four additional cuvettes contained pots that had only content. There was no significant effect of the pipec- soil but no plant to correct for high ­CO2 and ­H2O gas olic acid addition on air humidity or soil volumetric exchange rates that might occur from the soil. water content. Mean soil temperature was 22.5 ºC Vol:. (1234567890) 13 Plant Soil (SE = ± 2.5 ºC) in well-watered cuvettes and 23.3 ºC They were then placed in a 50 mL falcon tube con- (SE = ± 2.8 ºC) in drought cuvettes. taining 45 mL of milli-Q water and secured in posi- tion so that roots were fully submerged. No microbial Gas exchange measurements activity inhibitor was used, as this has been shown to modify exudation patterns, although this means that During the last seven days of the experiment for each exudation rates may be slightly higher than measured tomato cultivar, gas exchange measurements were due to potential microbial degradation of exudates. performed in the VOC-SCREEN platform. Meas- Aluminium foil was used to cover the top of the urements were taken for one cuvette at a time, for tube to prevent anything else from entering the exu- 5 min and 20 s each time, before sampling the next date solution. Plants were transferred to a growth cuvette. Net C­ O2 assimilation and evapotranspiration chamber with constant light (600 µmol PPFD rates were calculated according to von Caemmerer ­m−2 ­s−1) and temperature (27 ºC) and remained there and Farquhar (1981) and related to leaf area, that was for two hours. After this time, roots were carefully interpolated from the images taken at the beginning removed from the solution, and the sample was fil- and the end of each experiment. The soil C ­ O2 gas tered (0.22 μm), then stored in a freezer (-80 ºC) until exchange in the background (soil-only) cuvettes was further analysis. Total (non-purgeable) organic car- very low and hence considered negligible. Daily sums bon (TOC) and total nitrogen (TN) were measured for net photosynthetic C­ O2 assimilation (mmol C ­ O2 (Analytik Jena-Analyser multi N/C 3100, Analytik ­m−2 ­d−1, and later referred to as net-C assimilation) Jena, Jena, Germany). Root area was measured for the and evapotranspiration (mol ­H2O ­m−2 ­d−1) were esti- subset that was used for the exudate measure using mated per cuvette during the daylight period by inte- a flatbed scanner with images later analysed using grating each measurement for the time between two ImageJ software. All plant samples were dried at 60 measurements. These daily sums were calculated for ºC for two days and dry mass was measured. This was the five days where the full day was measured, which separated into root, stem and leaf fractions, with the excluded the first and last day in the VOC-SCREEN, root fraction further separated into the part that was therefore included days 30–34. used for the exudate measurement and the rest. The specific rate of C and N released from the root Plant traits and root exudation measurements to be calculated per root area, as µg C or N ­cm−2 root ­h−1, or alternatively per dry root mass (mg C or N ­g−1 To measure root exudation, we followed a modi- root ­h−1). In addition to specific exudation rate, total fied version of the soil-hydroponic-hybrid method exudation rate per pot (mg of TOC or TN per hour) (Oburger and Jones 2018) detailed by Williams was calculated by multiplying the exudation rate per et al. (2021). For each intact plant, the roots were root mass by the total mass of roots per plant. By cleaned by submerging them in distilled water, but measuring the root exudation rates immediately after not removed from the rest of the plant. A subset of the experiment we aimed to capture the effects of the the roots was more carefully cleaned by further sub- drought and simulated pathogen treatments before the merging them in distilled water and carefully remov- plant adapted to the post-experiment conditions. A ing any remaining pieces of substrate with tweezers. drawback of this short acclimation time is that exuda- The subset of the roots for exudate analysis made up tion may have been overestimated if roots had been 30% of the total fresh root mass on average. Exudates subjected to minor wounding during the root washing were sampled from this subset, rather than the full process. However, any root damage was minimal, fol- root system to ensure a careful yet effective clean- lowing visual assessment, and we therefore deemed ing within a few hours at the end of each round of the this a worthwhile trade-off. experiment. It should be noted that a recovery period of several days has been shown to be beneficial in Statistical analyses avoiding the measurement of carbon released from damaged root tissue (Williams et al. 2021). The roots All statistical analyses were carried out using R for exudate analysis were briefly rinsed with water to v3.6.1 (R Core Team 2019). Plant biomass and root remove metabolites released by the washing process. exudation data were log-transformed to correct for Vol.: (0123456789) 13 Plant Soil positive skewness and to achieve normality of the linear mixed effects models (cultivar as a random residuals in subsequent models. The effect of drought effect). and pipecolic acid on plant biomass was analysed using linear mixed-effects models with tomato culti- var as a random effect. The effect of the cultivar and Results the drought and pipecolic acid treatments on net-C assimilation and evapotranspiration was analysed in Drought and simulated pathogen attack on plant two different ways. Firstly, linear models were used biomass and gas exchange with data from the final full sampling day (day 34), when differences between treatments were expected Drought had a negative effect on total dry mass (lin- to be greatest, with cultivar and treatment as fixed ear mixed effects model, t = -18.2, p < 0.001). Plant effects. Secondly, mixed effects models were used to biomass in the two treatments with adequate water analyse the data from days 30–34, with cultivar and was 1.8 times higher than in treatments includ- treatment as fixed effects and sampling day as a ran- ing drought, with mean biomass (± SE) being 2.98 dom effect. This was to account for the time series (± 0.11) g under drought compared with 5.2 (± 0.18) and the fact that different cultivars may have started g with adequate water. This pattern of a negative the experiment with different sizes or rates of net-C effect of drought on total biomass was the same for assimilation and evapotranspiration. the root dry mass (t = -11.7, p < 0.001), stem dry Specific root exudation was calculated per root mass (t = -11.0, p < 0.001), and leaf dry mass (t area and root mass, with results showing the same = -20.2, p < 0.001) (Supplementary Information patterns. Data are presented for exudation per root Fig. S4). We observed no effect of pipecolic acid (and area to allow us to explore the relationship between no interaction between pipecolic acid and drought) specific exudation and root biomass without the two for plant biomass. Plant dry biomass varied strongly variables being confounding variables. One data point between the tomato cultivars (linear model, F = 6.6, was removed for exudation of TN as it was unusually p < 0.001), with mean (± SE) total dry biomass being low and deemed to be an outlier. The effect of cultivar 3.37 (± 0.27) g for Estrella del Balcón, 3.47 (± 0.28) on exudation rate of C and N per root area was tested g for Valenciano, 4.59 (± 0.36) g for Bella Rosa and with linear models. The overall effect of drought and 5.03 (± 0.36) g for Flor de Baladre. However, there pipecolic acid treatments on C and N exudation rates was no interaction with the drought treatment, such was analysed using linear mixed-effects models with that plants under drought were always smaller than tomato cultivar as a random effect. Subsequently, well-watered plants, within each cultivar. cultivars were also analysed separately, using linear Net-C assimilation on the final full day of meas- models, to see the effect of drought and pipecolic urements (after six days of the drought and pipecolic acid on exudation, and a Tukey test was used to see if acid treatments) was strongly affected by both culti- there were significant differences between treatments. var (linear model, F = 17.7, p < 0.001) and treatment To assess the relationship between two continuous (linear model, F = 51.4, p < 0.001) (Table 1). There variables (e.g., TOC and TN exudation, and TOC was no interaction between cultivar and treatment. exudation and biomass) the function r.squaredGLMM Net-C assimilation was lower in drought-treated (from the package MuMIn) was used and the condi- cuvettes, providing evidence for the effectiveness tional R2 was reported. of the drought treatment. Mean values (± SE) of The overall effects of drought and pipecolic acid net-C assimilation on that final day, for all cultivars on total exudation rate per pot were then tested using together, were 271 (± 15) mmol ­CO2 ­m−2 ­d−1 in well- a linear mixed model as before, with cultivar as a ran- watered cuvettes and 88 (± 12) mmol ­CO2 ­m−2 ­d−1 dom effect, and linear models and Tukey tests were in drought cuvettes. The pipecolic acid treatment did used for testing effects within cultivars. Additionally, not appear to affect net-C assimilation. The cultivar the C:N ratio of root exudates was calculated (using that had the highest C assimilation rates on day 34 values for specific exudation). The effect of cultivar was Estrella del Balcón, followed by Valenciano, and on C:N was tested using linear models, and the effects then with Flor de Baladre and Bella Rosa having the of drought and pipecolic acid were analysed with lowest rates (Table 1). Analysis of the five full days Vol:. (1234567890) 13 Plant Soil Table 1  Net-C assimilation Mean net-C assimilation mmol C ­ O2 Mean evapotranspiration and evapotranspiration ­m−2 ­d−1 (± SE) mol ­H2O ­m−2 ­d−1 (± SE) values on day 34 of the experiment for each of the Bella Rosa four tomato cultivars under Control 247 (± 29) 49 (± 5) the different treatments Drought 39 (± 20) 17 (± 3) PA 237 (± 22) 53 (± 5) Drought + PA 11 (± 11) 10 (± 3) Estrella del Balcón Control 334 (± 78) 81 (± 14) Drought 208 (± 38) 62 (± 8) PA 384 (± 23) 77 (± 2) Drought + PA 142 (± 18) 43 (± 3) Flor de Baladre Control 222 (± 12) 42 (± 2) Drought 55 (± 15) 20 (± 1) PA 193 (± 18) 37 (± 3) Drought + PA 79 (± 19) 24 (± 3) Valenciano Control 262 (± 31) 54 (± 4) Drought 81 (± 21) 28 (± 3) PA 292 (± 31) 69 (± 9) Drought + PA 93 (± 20) 34 (± 3) PA pipecolic acid of sampling identified an interaction between cultivar higher evapotranspiration under the drought only and treatment (mixed effects model, ChiSq = 28.0, treatment (Supplementary Information Fig. S5b). p < 0.001), with the cultivar Estrella del Balcón maintaining slightly higher C assimilation under the The effect of tomato cultivar on root exudation drought only treatment (Supplementary Information Fig. S5a). There was a significant effect of cultivar on root exu- Similar to net-C assimilation, evapotranspiration dation of TOC per root area (linear model, F = 4.4, on day 34 was strongly affected by both cultivar (lin- p < 0.01), with mean (± SE) exudation rates high- ear model, F = 30.1, p < 0.001) and treatment (lin- est for Estrella del Balcón (15.9 ± 2.4 ­ cm−2 root ear model, F = 30.0, p < 0.001), with no interaction. ­h ) and Valenciano (12.9 ± 1.1 ­cm root ­h−1) fol- −1 −2 Evapotranspiration was lower in cuvettes treated with lowed by Flor de Baladre (11.5 ± 1.7 ­ cm−2 root drought, with means (± SE) for all cultivars together ­h−1), and with lowest TOC exudation for Bella Rosa being 58 (± 3) mol H ­ 2O ­m−2 ­d−1 in well-watered (10.4 ± 3.2 ­cm−2 root h­ −1). A significant difference in cuvettes and 30 (± 3) mol H ­ 2O ­m−2 ­d−1 in drought- the specific TOC exudation rate was found between treated cuvettes. There was no noticeable effect of Bella Rosa and both Estrella del Balcón and Valen- pipecolic acid. Cultivar differences in evapotranspi- ciano (Fig. 1). ration rates were very similar to those for C assimi- Mean (± SE) exudation rate of TN was high- lation, with Estrella del Balcón having the highest est for Estrella del Balcón (1.4 ± 0.9 ­cm−2 root h­ −1), values, followed by Valenciano, then Bella Rosa and followed by for Flor de Baladre (1.1 ± 0.2 ­cm−2 root Flor de Baladre having lower rates (Table 1). Analysis ­h−1), then Valenciano (1.0 ± ­ cm−2 root h­−1), and of evapotranspiration across the five sampling days lastly Bella Rosa (0.8 ± 0.2 ­cm−2 root h­ −1) (Fig. 1). found an interaction between cultivar and treatment Although there was no significant effect of cultivar (mixed effects model, ChiSq = 50.1, p < 0.001), again in TN exudation per root area, there was a significant with the cultivar Estrella del Balcón maintaining difference in exudation rates between Estrella del Vol.: (0123456789) 13 Plant Soil Fig. 1  Root exudation of TOC and TN per root area per hour ences (p < 0.05) from a Tukey Test. Cultivar names are abbre- (on a natural log scale) for the four tomato cultivars. Each viated to: BR (Bella Rosa), EB (Estrella del Balcón), FB (Flor point represents a plant. Letters a and b show significant differ- de Baladre) and V (Valenciano) Balcón and Bella Rosa (Tukey Test, p < 0.05). There de Baladre showed a positive drought effect (linear was a positive correlation between TOC and TN exu- model, t = 2.5, p < 0.05), whereas the other two cul- dation rates per root area (linear mixed effect model, tivars did not show significant effects (Fig. 3). t = 8.0, p < 0.001, conditional R2 = 0.51) (Supplemen- Results were very similar for the exudation rate of tary Information Fig. S6). N per root area, with an overall drought effect (lin- ear mixed effects model, t = 5.0, p < 0.001) but no Drought and simulated pathogen attack on exudation interaction with pipecolic acid, or single pipecolic per root area acid effect (Fig. 2b). Mean TN exudation rate per root area was 1.9 times higher in the treatments with Drought had a positive effect on the specific exu- drought compared to those without (1.42 ± 0.2 ­cm−2 dation rate of TOC per root area (mixed effects root ­h−1 compared with 0.73 ± 0.1 ­cm−2 root ­h−1). model, t = 3.8, p < 0.001, Fig. 2a). Mean TOC exu- The results for the separate cultivars showed that dation rate per root area was 1.7 times higher in drought increased specific exudation for Bella Rosa the treatments with drought compared to those (linear model, t = 3.9, p < 0.01), and also for Flor de without (15.6 ± 1.9 ­cm−2 root ­ h−1 compared with Baladre (linear model, t = 2.8, p < 0.05) (Fig. 4). −2 −1 9.4 ± 0.9 ­cm root h­ , respectively). However, there There was a strong negative correlation between was no effect of the pipecolic acid treatment and no exudation rate per root area and dry root mass, for interaction between the drought treatment and the both TOC (linear mixed effects model, t = -5.6, pipecolic acid application. p < 0.001, conditional R2 = 0.41) and TN (linear When considering the four cultivars separately, mixed effects model, t = -5.7, p < 0.001, conditional Bella Rosa showed a drought * pipecolic acid inter- R2 = 0.39) (Supplementary Information Fig. S7). action (linear model, t = -2.3, p < 0.05), and Flor Vol:. (1234567890) 13 Plant Soil Fig. 2  Exudation rates by treatment, shown as specific exuda- exudation (p < 0.05) and pipecolic acid had a positive effect on tion rate per root area of TOC (a) and TN (b), and total exu- total N exudation (p < 0.05). For all panels, each point repre- dation rate per plant of TOC (c) and TN (d). Drought had a sents a plant, and exudation is shown on a natural log scale, positive effect on specific exudation rates of TOC (p < 0.001) with all tomato cultivars grouped together and TN (p < 0.05). Drought had a negative effect on total TOC This negative relationship was not affected by either increased exudation rate per root area but decreased the drought or the pipecolic acid treatment. root mass, and total exudation per plant also showed a small negative effect of drought (linear mixed Total exudation per plant effects model, t = -2.2, p < 0.05) (Fig. 2c). How- ever, there was a smaller effect size, with plants The total exudation rate of TOC per plant was also under drought releasing 1.3 times less TOC than calculated, representing the product of the exu- plants receiving adequate water (total biomass was dation rate per root mass and the total root mass 1.8 times less). This indicated that the decrease of that plant. As already shown above, drought in root (and overall plant) biomass was partially Vol.: (0123456789) 13 Plant Soil Fig. 3  Exudation rate of TOC per root area on a natural log scale for the four different treatments, with cultivars shown separately. Letters a and b show significant differences (p < 0.05) from a Tukey Test. Each point represents a plant compensated for by the increase in the rate of spe- F = 8.6, p < 0.05). For Bella Rosa there was an inter- cific exudation (summarised in Fig. 5). action between drought and pipecolic acid, such that There was also an effect of cultivar (linear model, drought alone increased exudation, however, drought F = 3.9, p < 0.05), with total exudation rate of TOC and pipecolic acid decreased exudation (linear model, per plant being 1.7 times higher in Valenciano, the F = 4.9, p < 0.05) (Fig. 6). cultivar with highest exudation (2.8 mg ­h−1) com- A different result was shown for total exudation of pared with Bella Rosa, the cultivar with lowest exu- nitrogen per plant, with a positive effect of pipecolic dation (1.6 mg ­h−1). Moreover, treatment effects var- acid (linear mixed effects model, t = 2.2, p < 0.05), ied by cultivar. Estrella del Balcón demonstrated a when all cultivars were combined, and no effect of positive effect of pipecolic acid on total exudation of drought, nor any interaction (Fig. 2d). There was no TOC (linear model, F = 6.6, p < 0.05), and this same significant effect of cultivar on total exudation of N, pattern was shown for Flor de Baladre, but the effect but cultivars nonetheless did behave differently from was not significant (linear model, F = 3.9, p = 0.07) each other. In particular, Estrella del Balcón showed (Fig. 6). However, a significant negative effect of an interaction between drought and pipecolic acid drought was found for Valenciano (linear model, (linear model, F = 4.8, p < 0.05), such that while Vol:. (1234567890) 13 Plant Soil Fig. 4  Exudation rate of TN per root area on a natural log scale for the four different treatments, with cultivars shown separately. Letters a and b show significant differences (p < 0.05) from a Tukey Test. Each point represents a plant drought alone decreased exudation, when drought and signifying a relative decrease in TOC and a relative pipecolic acid were both added, exudation increased increase in TN in exudates (Supplementary Informa- (Fig. 7). This was the same pattern shown for total tion Fig. S8). TOC exudation for the same cultivar and in Flor de Baladre (Fig. 6). Discussion C:N ratio of root exudates Biomass and net‑C assimilation was decreased by The C:N ratio of root exudates was very variable drought between samples, ranging from 2.5 to 30.5. There was no cultivar effect, but there was a marginally sig- Changes in biomass are often found in experiments nificant interaction effect of the drought and pipecolic with abiotic and biotic stresses, and in this experi- acid (t = -1.9, p = 0.06). This resulted from the com- ment, the drought treatment dramatically reduced bined drought and pipecolic acid treatment having a the overall plant biomass across all tissue types (root, lower C:N composition than the other treatments, stem and leaf), as well as net-C assimilation. It is Vol.: (0123456789) 13 Plant Soil also expected that the induction of systemic acquired resistance (SAR) should result in lower allocation to biomass (Heil et al. 2000; Walters and Heil 2007), although SAR can be induced without having an impact on biomass. In this study, the pipecolic acid addition did not affect plant biomass or net-C assimi- lation which may indicate that the treatment was not fully effective in simulating a pathogen infection, or that the duration of the treatment was not long enough to see such an effect. However, this would have to be confirmed with a pathogen assay and molecular anal- ysis of pathogenesis-related (PR) genes, which was not carried out in this experiment. Also, there were other effects of the pipecolic acid addition, particu- larly on the exudation of N, suggesting that it was effective. Drought increased exudation of C and N per root area The drought treatment was the dominant factor affect- ing the specific exudation rates of both TOC and TN, when considering all tomato cultivars together. Drought increased exudation rates, in line with our first hypothesis. Increases in exudation rates under water stress have been shown before in many stud- ies with a variety of plant species and measurement techniques (Henry et al. 2007; Preece and Peñuelas 2016; Karlowsky et al. 2018; Preece et al. 2018). It is important to note that while there was this overall drought effect, when tomato cultivars were consid- ered individually, results were more nuanced, and cultivar-specific effects were evident, in agreement with our fourth hypothesis. For specific exudation of both TOC and TN, two cultivars (Estrella del Balcón and Valenciano) did not show significant effects of the drought treatment (or pipecolic acid), indicating that drought responses vary between tomato culti- vars and that these two cultivars are potentially more drought resistant. This agrees with the net-C assimi- lation and evapotranspiration measurements which Fig. 5  Summary of the effects of drought and pipecolic acid also indicated that these two cultivars had higher gas on exudation of TOC and TN from tomato roots. Root biomass exchange rates over all treatments. Further evidence under drought was lower than under adequate watering (control that Estrella del Balcón was potentially the most conditions), but specific exudation of TOC and TN per root area was increased. Total exudation of TOC per plant was lower under resistant to drought, was that net-C assimilation and drought but exudation of TN per plant was maintained at control evapotranspiration did not decrease as much as in levels. Addition of pipecolic acid did not affect root biomass or other cultivars. The other two cultivars, Bella Rosa specific exudation but did increase total exudation of nitrogen per plant due to a decrease in the C:N ratio of exudates, particularly and Flor de Baladre, did show increased exudation when drought and pipecolic acid were combined. There was no rates due to drought, although for TOC exudation in significant interaction between drought and pipecolic acid when all Bella Rosa this was only evident in the drought-only cultivars were grouped together Vol:. (1234567890) 13 Plant Soil Fig. 6  Total exudation rate of TOC per plant on a logged scale for the four different treatments, with cultivars shown separately. Let- ters a and b show significant differences (p < 0.05) from a Tukey Test. Each point represents a plant treatment, and when drought was combined with the in allocation either to making roots or to increasing pipecolic acid addition there was no change from the exudation. This implies that under drought plants are control. unable to allocate the same resources (in terms of car- Previous studies have searched for general pat- bon) to constructing roots, but by up-regulating root terns between root exudation and root morphology, exudation in the roots that are present, the physiologi- but results are variable with both positive and nega- cal activity of the roots can be maintained to some tive relationships found between exudation rates and extent. root length or root area (Wen et al. 2022). A recent Our hypothesis that the addition of pipecolic acid study with 17 common perennial European grassland would stimulate specific exudation rates was not con- species showed positive relationships between exuda- firmed, as there was no overall effect of the simula- tion and root diameter which is linked to microbial tion of pathogen attack. This could indicate that collaboration and outsourcing of nutrient acquisition the drought treatment had a stronger effect on plant (Williams et al. 2022). In our experiment, the strong physiology, at least at this scale. Clear evidence sup- negative correlation between root biomass and root porting the hypothesis that the combined treatment exudation per root area clearly indicates a trade-off (drought and simulated pathogen attack) had the Vol.: (0123456789) 13 Plant Soil Fig. 7  Total exudation rate of TN per plant on a logged scale for the four different treatments, with cultivars shown separately. Let- ters a and b show significant differences (p < 0.05) from a Tukey Test. Each point represents a plant strongest effect on root exudation (hypothesis 3) was in Williams et al. 2021), might have led to an overes- not found. When interacting factors are present, such timation of exudation. This overestimation could be as in this study, the overall effects on plant physi- attributed to the additional measurement of carbon ological processes, such as exudation, are likely to and nitrogen released from damaged roots during the be complex and potentially antagonistic at the level root washing process. However, great care was taken of overall exudation rates. Future studies that also to limit the manipulation of the roots and any minor consider changes in root exudate composition might damage would have been equal between the treat- provide a clearer understanding of these complexities. ments and should not have affected the drought effect As we discuss below, results at the whole-plant level that we observed. More studies are warranted to fur- did reveal a response to pipecolic acid, in terms of ther support these results. higher total TN exudation. It should be noted that all methods of measure- Drought effects on total exudation of carbon and ment of root exudates are subject to drawbacks. In nitrogen are minimal this case, the collection of exudates so soon after the experiment concluded (without allowing for a By calculating the total exudation per plant, we lengthy hydroponic recovery period as recommended showed that, when all cultivars were pooled, increased Vol:. (1234567890) 13 Plant Soil specific exudation rate of TOC under drought offsets higher proportion of N-containing compounds. This the decrease in root biomass, resulting in total exu- lowering of the C:N ratio in exudates was particularly dation rates per plant that were only slightly lower driven by the combined treatment of both drought and than under well-watered conditions. For nitrogen, pipecolic acid addition. A higher proportion of N in this compensation effect was even greater, with no root exudates may indicate a shift towards exudation of significant difference in total exudation found due to amino acids, though this was not specifically measured the drought treatment. This reflects results in trees, in this study. Other studies have found exudation of where the maintenance of total exudation has been amino acids to increase during drought (Bobille et al. shown following experimental drought in a Fagus 2019), during recovery following drought (Gargallo- sylvatica and Picea abies forest (Brunn et al. 2022). Garriga et al. 2018), and following infection with the A review of drought effects on rhizodeposition also bacterial pathogen Pseudomonas syringae pv tomato found that a drought-related reduction in root biomass (Yuan et al. 2018), and to decrease when plants are was often the main driver of plant-level reductions in under N deficiency (Carvalhais et al. 2011). rhizodeposition (Preece and Peñuelas 2016). Decreases in exudate C:N ratio increase the As with other measured parameters, there were decomposability of root exudates, which constitute differences between cultivars, and while drought a labile fraction of soil organic matter. Lower C:N decreased total exudation at the species level, at the ratios in the labile soil fraction, including exudates, cultivar level, drought was only the main driver of were found to be positively correlated with higher total exudation of TOC for Valenciano. In contrast, N mineralization in a N-limited grassland (Fornara for Estrella del Balcón and Flor de Baladre, pipecolic et al. 2011). Labile exudates induce soil priming, acid increased exudation (although only significant leading to additional effects on soil microbial turno- for Estrella del Balcón), and the two treatments inter- ver and community structure (Liu et al. 2017; Zhou acted with each other in Bella Rosa. This underlines et al. 2021). Moreover, this change in the C:N ratio the important phenotype effect. The importance of suggests that alterations in exudate composition are differences between accessions has previously been likely to be occurring even when there are no changes shown for root exudate composition in genetically in exudation rates, as found in a recent study that diverse accessions of Arabidopsis, further emphasis- observed microbial responses to exudates produced ing the importance of considering multiple popula- by drought-stressed plants, despite overall C content tions (Mönchgesang et al. 2016). Given that only four being accounted for (De Vries et al. 2019). tomato cultivars were tested here, all of which are usu- Significant effects of the treatments on total N exuda- ally grown under conditions with irrigation, it is likely tion were more difficult to see at the cultivar level, likely that we would have found an even greater variability due to the large variability in measurements and the in responses if more cultivars had been included, such relatively low replication rate. Only Estrella del Balcón as those normally grown outside in rainfed systems. showed a significant effect, with the combined drought This also highlights the need to preserve diversity in and pipecolic acid treatment inducing more exudation of crop cultivars, in order to increase the chances of iden- N per plant than the drought only treatment. tifying cultivars with advantageous root traits, such as for higher drought or pathogen tolerance. Conclusion Total exudation of nitrogen is increased under simulated pathogen attack Under moderate drought conditions, the specific rates of TOC and TN exudation per root area increased when Pipecolic acid generally had fewer effects on plant considering all four tomato cultivars. However, the simu- growth and root exudation compared with drought, lated pathogen attack did not have a discernible effect on but, when considering all cultivars together, simulated specific exudation. The total exudation of TOC per plant pathogen attack did increase the total amount of nitro- slightly decreased due to reduced root biomass caused by gen exuded per plant. This was apparently achieved, drought, but because of the higher specific exudation this not by an increase in the specific exudation rate, but was to a much lesser extent than the biomass differences. by a change in the composition of exudates, to have a In contrast, the total exudation of nitrogen increased Vol.: (0123456789) 13 Plant Soil following the addition of pipecolic acid, resulting in a the permitted use, you will need to obtain permission directly shift towards a higher N content and lower C content in from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. the exudates. Notably, there were noticeable variations among the different tomato cultivars in response to the treatments, both in terms of specific and total exudation References rates. These differences indicate varying levels of resist- ance to water stress and pathogen attack among the culti- Ahmed MA, Kroener E, Holz M et al (2014) Mucilage exuda- vars. Overall, drought had a more pronounced impact on tion facilitates root water uptake in dry soils. Funct Plant total C exudation, while the simulated pathogen attack Biol 41:1129–1137 exerted a greater influence on total N exudation. Badri DV, Chaparro JM, Zhang R et al (2013) Application of natural blends of phytochemicals derived from the root exudates of Arabidopsis to the soil reveal that phenolic- Acknowledgements We acknowledge the support of the related compounds predominantly modulate the soil staff of the Research Unit Environmental Simulation (Helm- microbiome. J Biol Chem 288:4502–4512 holtz Munich) for assisting with the implementation of the Baetz U, Martinoia E (2014) Root exudates: the hidden part experiment. of plant defense. Trends Plant Sci 19:90–98 Bais HP, Weir TL, Perry LG et al (2006) The role of root Author contributions All authors contributed to the study exudates in rhizosphere interations with plants and other conception and design. Material preparation, data collec- organisms. Annu Rev Plant Biol 57:233–266 tion and analysis were performed by Kaijun Yang, Catherine Bobille H, Fustec J, Robins RJ et al (2019) Effect of water Preece, Joan Llusià and Jana Barbro Winkler. The first draft of availability on changes in root amino acids and associ- the manuscript was written by Catherine Preece and all authors ated rhizosphere on root exudation of amino acids in commented on previous versions of the manuscript. All authors Pisum sativum L. Phytochemistry 161:75–85 read and approved the final manuscript. Brunn M, Hafner BD, Zwetsloot MJ et al (2022) Carbon allo- cation to root exudates is maintained in mature temper- Funding Open Access funding provided thanks to the ate tree species under drought. New Phytol 235:965–977 CRUE-CSIC agreement with Springer Nature. We acknowl- Calvo OC, Franzaring J, Schmid I et al (2017) Atmospheric edge the support of the EPPN2020 (European Plant Phenotyp- CO2 enrichment and drought stress modify root exuda- ing Network) for the PhenoTOMVOC project that enabled us tion of barley. Glob Chang Biol 23:1292–1304 to use the facilities at the Research Unit Environmental Simu- Cammarano D, Jamshidi S, Hoogenboom G et al (2022) Process- lation (Helmholtz Munich). This research was also financially ing tomato production is expected to decrease by 2050 due supported by the grant TED2021-132627B-I00 funded by the to the projected increase in temperature. Nat Food 3:437–444 Spanish MCIN/AEI/https://​doi.​org/​10.​13039/​50110​00110​33 Carvalhais LC, Dennis PG, Fedoseyenko D et al (2011) Root and European Union NextGenerationEU/PRTR, the Fundación exudation of sugars, amino acids, and organic acids by Ramón Areces grant CIVP20A6621, and the Catalan Govern- maize as affected by nitrogen, phosphorus, potassium, ment grant SGR 2021 − 1333. and iron Deficiency. J Plant Nutr Soil Sci 174:3–11 Chai YN, Schachtman DP (2022) Root exudates impact plant Data availability The datasets generated during the current performance under abiotic stress. Trends Plant Sci 27:80–91 study are available from the corresponding author on reason- Chaloner TM, Gurr SJ, Bebber DP (2021) Plant pathogen able request. Infection risk tracks global crop yields under climate change. Nat Clim Chang 11(8):710–715 Declarations De Vries FT, Williams A, Stringer F et al (2019) Changes in root-exudate-induced respiration reveal a novel mecha- nism through which drought affects ecosystem carbon Competing interests The authors have no relevant financial cycling. New Phytol 224:132–145 or non-financial interests to disclose. Ding P, Rekhter D, Ding Y et al (2016) Characterization of a pipecolic acid biosynthesis pathway required for sys- temic acquired resistance. Plant Cell 28:2603–2615 Open Access This article is licensed under a Creative Com- Dudenhöffer JH, Scheu S, Jousset A (2016) Systemic enrich- mons Attribution 4.0 International License, which permits ment of antifungal traits in the rhizosphere microbiome use, sharing, adaptation, distribution and reproduction in any after pathogen Attack. J Ecol 104:1566–1575 medium or format, as long as you give appropriate credit to the FAOstat (2021) In: https://​www.​fao.​org/​faost​at/​en/#​home. original author(s) and the source, provide a link to the Crea- Accessed 19 April 2023 tive Commons licence, and indicate if changes were made. The Fornara DA, Bardgett R, Steinbeiss S et al (2011) Plant effects images or other third party material in this article are included on soil N mineralization are mediated by the composition in the article’s Creative Commons licence, unless indicated of multiple soil organic fractions. Ecol Res 26:201–208 otherwise in a credit line to the material. If material is not Gargallo-Garriga A, Preece C, Sardans J et al (2018) Root exu- included in the article’s Creative Commons licence and your date metabolomes change under drought and show limited intended use is not permitted by statutory regulation or exceeds capacity for recovery. Sci Rep 8:12696 Vol:. 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