Tomato growth and physiology as well as soil physicochemical and biological properties affected by ozonated water in a saline agroecosystem

1 Tomato growth and physiology as well as soil physicochemical and biological 1 properties affected by ozonated water in a saline agroecosystem 2 Marta Díaz-López1,2 *, Lucas Galera3 Felipe Bastida2, Emilio Nicolás1 3 *corresponding author ([email protected]) 4 5 1Department of Irrigation, CEBAS-CSIC, Campus Universitario de Espinardo, Murcia, 6 30100, Spain. 7 2Department of Soil and Water Conservation and Waste Management, CEBAS-CSIC, 8 Campus Universitario de Espinardo, Murcia, 30100, Spain. 9 3Novagric (Novedades Agrícolas, S.A.), Bulevar de Vicar 743, 04738 Vicar, Almería, 10 Spain 11 12 Manuscript (double-spaced and continuously LINE and PAGE numbered)-for final publication Click here to view linked References 2 Abstract 13 Current trends in agriculture are focused on implementing sustainable practices that 14 avoid the use of chemical compounds. In this context, irrigation with ozonated water 15 could be a potential strategy to reduce some chemical compounds in soils due to the 16 degradative power of ozone. However, the effects of irrigation with ozonated water on 17 the soil microbial community and plant agrophysiology at the field scale are largely 18 unknown. Here, we studied the impact of irrigation with ozonated water on the microbial 19 community of a Mediterranean soil, and on Solanum lycopersicum L. agro-physiology 20 and productivity in a greenhouse experiment. To this end, we evaluated: i) soil 21 physicochemical properties, soil enzyme activities, microbial biomass via fatty acid 22 analysis, microbial diversity (via amplicon sequencing), and ii) the nutrient content, 23 physiology, phytohormone content, yield, and fruit quality of tomato plants. Our results 24 indicate that soil physicochemical properties were significantly affected by the irrigation 25 with ozonated water (OZ). We observed an increase in the content of total organic carbon 26 (TOC), water-soluble nitrogen (WSN) and ammonium, and a decrease in soil pH due to 27 the OZ treatment. In addition, a significant increase in alkaline phosphatase and fungal 28 and bacterial biomass was also observed in the OZ treatment. It was observed that the 29 prokaryotic community structure was affected by the OZ treatment, while that of fungi 30 was undisturbed. The OZ treatment increased the photosynthetic rates of tomato plants 31 and maintained water conditions when compared to control plants. The increased trans32 Zeatin riboside (tZ-Rib) could provide rapid apical and root growth allowing adaptation 33 to the new growing conditions. However, a more in-depth study on the physiological 34 response of the plant to this treatment would be of interest, as it would help with the 35 3 implementation of this strategy in agricultural fields in a safe manner, and with obtaining 36 higher plant yields. 37 Keywords: Soil microbial community; Ozone; Enzyme activity; Microbial biomass; 38 Plant physiology; Phytohormone; Tomato. 39 40 4 1. Introduction 41 Current trends in agriculture focus on applying sustainable practices that avoid the 42 use of chemical compounds such as pesticides, fungicides, etc. Some of these strategies 43 include i) bio-based sustainable intensification, ii) agroecological transformation, and iii) 44 polycultures and crop rotation (FAO, 2019). In this context, ozonation of irrigation water 45 could also be a potential strategy to reduce the content of some of these chemical 46 compounds in soils. Some studies have focused on this issue, but have paid less attention 47 on the effects of ozonated water on the soil microbial community, which plays a critical 48 role in soil fertility, and plant agro-physiological parameters (Ding et al., 2018; Ghahrchi 49 & Rezaee, 2020). Thus, it is important to evaluate the effects of ozonated water on soil 50 fertility and plant agro-physiology before implementing this strategy on a larger 51 agricultural scale. Nevertheless, there is little information of the effects on crops of ozone 52 applied to the irrigation water. 53 Some authors have pointed out that irrigation with ozonated water can promote the 54 accumulation of secondary metabolites, the activation of the antioxidant apparatus, and 55 the overproduction of phytohormones (Risoli and Lauria, 2022). Consequently, a higher 56 resistance against pathogen infection can be promoted (Landa Fernández et al., 2019; 57 Prigigallo et al., 2019). Thus, irrigation with ozonated water allows oxygenation in the 58 plant root area, and can help to sanitize irrigation systems, especially to prevent the spread 59 of pathogens (Zheng et al., 2020). However, when present in aqueous solutions, ozone 60 undergoes rapid breakdown into molecular oxygen and different reactive species of 61 oxygen (ROS), as it is an unstable compound (von Sonntag & von Gunten, 2015). The 62 5 increase in the concentration of ROS in the irrigation water can cause oxidative stress on 63 plants and ultimately affecting their growth and development (Zheng et al., 2020). In 64 comparison to its effect on plants, little is known about the effects of ozonated water 65 application on soils. Monitoring the effects of agricultural practices in the soil microbial 66 community is fundamental, given the important role of soil microbes in the maintenance 67 of soil fertility and ecosystem services (Delgado-Baquerizo et al., 2016). Indeed, 68 alterations in the composition and functionality of the soil microbial community can lead 69 to loss of soil health, resulting in the degradation of the soil for agricultural use, and 70 contributing to soil degradation and erosion (Usero et al., 2021). Several approaches have 71 been widely used to track the responses of the soil microbial community to agricultural 72 management, from the measurement of soil enzyme activities related to nutrient cycles 73 (Lehmann et al., 2020) to the composition and diversity of the bacterial and fungal 74 communities by amplicon sequencing. 75 This work aims to evaluate the effect of irrigation with ozonated water on the 76 performance of Solanum lycopersicum (tomato) and on the microbial community, of a 77 semiarid Mediterranean soil under salinity conditions. In particular, we expect changes in 78 the diversity and composition of the microbial community, but a reduced impact in 79 extracellular soil enzyme activities, which are usually protected in soil minerals and 80 organic matter (Burns et al., 2013). Moreover, we expect that irrigation with ozonated 81 water would increase the availability of nutrients in the soil, and consequently modify 82 both plant physiology and yield (Ikeura et al., 2018). Irrigation with ozonated water is 83 expected to influence several physiological parameters such as net photosynthesis, 84 stomatal conductance, and phytohormone content (Tahamolkonan et al., 2022). In 85 6 particular, the increase in salicylic acid content has been reported (Prigigallo et al. 2019), 86 which could decrease the stomatal conductance. Moreover, similar levels of net 87 photosynthesis (Martínez-Sánchez & Aguayo, 2019) can lead to higher levels of intrinsic 88 water use efficiency. For this purpose, we evaluated i) the soil microbial biomass, 89 composition, and enzyme activities, together with soil chemical parameters and nutrient 90 contents, and ii) the crop nutrient content, physiology, phytohormone content, yield, and 91 fruit quality. We hypothesized that changes in soil physico-chemical parameters (e.g. 92 reduction in soil pH, increase in electrical conductivity, etc.) upon ozone irrigation, will 93 affect the availability of nutrients and therefore the soil microbial community and plants. 94 In addition, we expect slight effect of irrigation with ozonated water on the plant nutrition 95 but some significant effect on physiological parameters due to the increase in oxidative 96 molecules. 97 2. Materials and methods 98 2.1. Experimental design 99 The experiment was conducted in a greenhouse located in Murcia, southeastern Spain 100 (37º28’N 1º32’W) during August-May 2020-2021. The selected soil was a sandy-loam 101 soil (14.7% clay, 21.3% silt, 64% sand) with the following characteristics: pH (H2O) 7.57 102 ± 0.20; electrical conductivity (dS m−1) 8.44 ± 3.28; organic C content (%) 1.89 ± 0.25; 103 total N (%) 0.39 ± 0.09; alkalinity (% CaCO3) 2.68 ± 1.59. The soil presented a high 104 concentration of Na, reaching 21% of the exchangeable sodium percentage, and can 105 therefore be considered a saline-sodic soil. 106 7 The treatments evaluated in this study were: 1) drip irrigation without ozonated water 107 (C), and 2) drip irrigation with ozonated water (OZ). The irrigation water was collected 108 and filtered using several procedures: i) multi-layered sand bed filtration, ii) disc system 109 filtration, and iii) ultrafiltration membranes filtration. The water ozonation was performed 110 in situ using commercial equipment provided by NOVAGRIC, S.A (utility model 111 ES1256014). Ozone, with a redox potential of 800-850mV, was generated from 112 atmospheric air and finally injected into the filtered irrigation water (Table S1). The 113 treatments were randomly arranged and distributed in the greenhouse (900 m2), resulting 114 in four replicates per treatment. Each replicate consisted of 120 Solanum lycopersicum 115 plants distributed in eight rows, with a distance of 1 m between rows and 40 cm between 116 plants in the same row (2.5 plants m−2). The plants were irrigated with drippers, with one 117 pressure-compensated emitter per plant discharging 2 L h-1, which resulted in an irrigation 118 of 2000 m3 ha-1 of water during the total growing season. In order to ensure the correct 119 development of the plants, all of them received the same amount of the main 120 macronutrients (N-P2O5-K2O): 240-105-405, and secondary macronutrients: 44 CaO and 121 26 MgO (kg ha-1), through the drip irrigation system. 122 The soil samples were collected as follows: three soil samples were taken (0–15 cm 123 of depth) and mixed to obtain one composite sample per replicate. Each soil replicate was 124 sampled in the middle of one row of tomato plants. Soil samples were taken at three 125 different times: December-2020 (t1), February-2021 (t2), and May-2021 (t3), 126 corresponding to the beginning of harvest (t1), middle of harvest (t2), and end of harvest 127 (t3) periods. The samples were sieved (2 mm) and kept at 4 °C for chemical analyses and 128 at -20 °C for fatty acid methyl ester analysis and DNA extraction. All the soil and plant 129 8 analyses were performed from the middle row of each replicate in order to avoid the edge 130 effect. Five plants from each replicate (2.5 plants m−2) were chosen for physiological and 131 nutrient content analyses. The productivity and quality of the fruits were also evaluated 132 in these plants. Gas exchange parameters, chlorophyll content, and phytohormone 133 determination were performed at the same time as t2 soil sampling. At this time, the 134 tomato plants were in full fruit production. 135 2.2. Soil physicochemical parameters, enzyme activities, and fatty acid methyl ester 136 (FAME) analysis 137 The pH and electrical conductivity (EC) of the soil were measured using a Crison 138 GLP 21 pH-meter and a Crison CM 2200 conductivity-meter (Crison Hach Lange, Alella, 139 Spain), respectively, from a soil:water extract (1:5, w:v). The total N (TN), total C (TC), 140 and total organic C (TOC) of the soil were analyzed with an Elemental Analyzer (C/N 141 Flash EA 112 Series-Leco Truspec). The soil water-soluble C (WSC) and water-soluble 142 N (WSN) were determined by an analyzer for liquid samples (Multi N/C 3100, Analytic 143 Jena, Germany) from a soil:water extract (1:5, w:v). The determination of the soil 144 ammonium content and the urease activity were analyzed by the Kandeler and Gerber 145 (1988) method. The alkaline phosphomonoesterase and β-glucosidase activities were 146 analyzed with the method by Tabatabai and Bremner (1969), and the Eivazi and Tabatabai 147 (1988) method, respectively. 148 Fatty acid methyl esters (FAMEs), hereafter fatty acids, were extracted from 3 g of 149 soil according to Schutter and Dick (2000), and were used as indicators of the soil 150 microbial biomass. The fatty acids representative of the bacterial biomass were i15:0, 151 9 a15:0, i16:0, i17:0, 16:1ω7, cy17:0, cy19:0, 10Me16:0, and 10Me18:0 (Dungait et al., 152 2011; Frostegård et al., 1993), and the fatty acids indicators of the fungal biomass were 153 18:2ω6,9t and 18:2ω6,9c (Brant et al., 2006; Rinnan & Bååth, 2009). The relative 154 abundances of all the fatty acids identified and the fungal: bacterial fatty acids ratio were 155 used for the analysis of the changes in the structure of the microbial community. 156 2.3. DNA extraction, amplicon sequencing, and indicator genera of the prokaryotic 157 community 158 Soil samples from t2 and t3 were selected to further explore the diversity and 159 composition of their bacterial and fungal communities by amplicon sequencing. The 160 amplicon sequencing provided us with a deeper understanding of the status of the soil 161 prokaryotic and fungal communities at a particular time. These soil sampling times were 162 chosen because they corresponded to the middle of harvest (t2) and the end of harvest (t3) 163 periods of tomato plants. DNA extraction was performed from 250 mg of soil using the 164 DNeasy PowerSoil Pro Kit (Qiagen). The 515F and 806R primer pair from (Caporaso et 165 al., 2012) was used to amplify the V4 region of the prokaryotic 16S rRNA gene, and the 166 gITS7 and ITS4 pair (Ihrmark et al., 2012) for the amplification of the fungal ITS2 region. 167 The PCR conditions and sequencing procedure were as described in Díaz-López et al. 168 (2021). 169 The sequences were processed using the USEARCH pipeline and the UPARSE-OTU 170 algorithm (Edgar, 2013). First, raw MiSeq paired-end reads from the 16S rRNA gene and 171 the ITS2 region were assembled separately. Then, the sequences were quality-filtered, 172 allowing a maximum e-value of 0.5 for the 16S library and 1.0 for the ITS2 library, 173 16 Brevibacillus, Microbispora, Idiomarina, and Tumebacillus were significantly reduced 306 by this treatment (p < 0.05). 307 3.4. Gas exchange parameters, chlorophyll content, and phytohormone content 308 The gas exchange parameters, chlorophyll content, and phytohormone content of 309 tomato leaves (Table 2) were measured in order to determine the response of the tomato 310 plants to the ozonated water treatment. All these analyses were conducted at t2, at the 311 same time that soil was sampled, coinciding with the middle of the harvest period. The 312 results obtained showed a significant (p < 0.05) increase in the net photosynthesis (A) 313 and the stomatal conductance (gs) in the plants irrigated with ozonated water. However, 314 we did not observe significant differences in the intrinsic water use efficiency (iWUE). 315 The increase in A was not coupled with a higher chlorophyll content, either chlorophyll 316 a (Chl a), b (Chl b), or total (Chl T) content. Nonetheless, there was a trend for the 317 ozonated water treatment plants to have a greater chlorophyll content. 318 In order to further study the physiological response of tomato plants to the ozonated 319 water treatment, we evaluated the phytohormone content (Table 3) at the same time as 320 we evaluated the gas exchange parameters. The OZ treatment significantly increased (p 321 < 0.05) the trans-Zeatin riboside (tZ-Rib) content. However, in the same treatment, a 322 significant decrease (p < 0.05) was observed in gibberellic acid 7 (GA7), abscisic acid 323 (ABA), and salicylic acid (SA) content. 324 3.5. Leaf mineral content, crop yield, and fruit quality 325 The nutritional status of tomato plants was analyzed at three different times 326 throughout the harvest period. We did not find significant differences in the elements 327 17 analyzed (Table S2), indicating that the ozonated water treatment did not influence the 328 nutritional status of the tomato plants under these study conditions. We also evaluated the 329 total yield and the fruit quality through the whole harvest period. No significant 330 differences were found in yield or fruit quality parameters (Table S3). Lastly, we analyzed 331 the cumulative yield and did not observe any significant differences between treatments 332 either (Figure 6). 333 4. Discussion 334 4.1. Soil chemical, biochemical and microbial responses to ozonated water 335 The evaluation of soil physicochemical and biochemical characteristics is of great 336 importance, as some of them are directly related to soil quality and health. When applied 337 to irrigation water, ozone rapidly decomposes into molecular oxygen and HO radicals 338 (von Sonntag & von Gunten, 2015). As a result, HO radicals react with the labile 339 compounds present in the soil and release protons (H+), salts, and other compounds into 340 the soil (Ghahrchi & Rezaee, 2020), which could explain the significant reduction in soil 341 pH with the OZ treatment. However, electrical conductivity was not affected by the OZ 342 treatment, possibly due to the high values observed in the initial measurement of the soil. 343 Maintaining or increasing the C and N content of the soil is key in future agricultural 344 strategies (Lekberg et al., 2021). In our case, the OZ treatment showed a significant 345 increase in TOC and WSN content, especially in ammonium content. Studies have shown 346 that the radicals released by the decomposition of ozone in irrigation water could have 347 enhanced the decomposition of organic matter and the generation of labile compounds in 348 the soil (Díaz-López et al. 2021; Wang and Chen 2020; Yu et al. 2005). Moreover, the 349 18 increase in TOC and WSN compounds could be due to the combination of several factors 350 that separately may not be significant but that finally affect in a significant way. For 351 example, increased ROS content could have led to increased exudate production by plants 352 (Risoli & Lauria, 2022), and on the other hand, the plant may absorb fewer nutrients from 353 the soil and cause them to accumulate to a greater extent than in the non-ozonated water 354 plants. Further, there are evidences that oxygenating the root area, which may also occur 355 here through quick ozone decomposition, can increase root growth and organic C in soil 356 (Zhou et al., 2022). 357 Soil enzymes and their activities catalyze many nutrient cycles in soils, and are 358 commonly used as indicators of soil quality and fertility (Bowles et al., 2014; Burns et 359 al., 2013). The enzyme activities tested (β-glucosidase, urease, and alkaline 360 phosphomonoesterase) in this study were maintained or even increased with the OZ 361 treatment. The significant increase in soil phosphomonoesterase activity in the OZ 362 treatment may indicate a greater enzyme production by the soil microbial biomass, larger 363 substrate availability, and/or greater phosphorus demand by tomato plants. The activity 364 of this enzyme is key for the phosphorus cycle, with this macronutrient being commonly 365 limited in soils. The observed enzyme activities correlated with the microbial biomass, as 366 estimated by fatty acids, which reinforce the idea that these enzymes produce easily 367 available substrates for both plant and microbial growth. 368 Microbial fatty acids provide an estimation of soil microbial biomass (Fanin et al., 369 2019), and can be useful for monitoring the potential effects of ozone, which has 370 recognized disinfectant role (Mitsugi et al., 2014), in the soil microbial community. 371 19 Overall, our results indicated that ozone did not negatively impact soil microbial biomass, 372 and this might be likely due to: i) the greater availability of nutrients as a result of the 373 higher degradation reactions carried out by the ozone-released radicals (Wang et al., 374 2019), ii) the reduction of the antimicrobial effect of ozone due to its quick breakdown in 375 water, and iii) the increase of soil and rhizosphere aeration due to the release of O2 on 376 decomposition of O3 in water (Zhou et al., 2022). 377 A deeper insight through amplicon sequencing analysis would shed light on the 378 effects of ozone application in irrigation water and how it affects soil microbial 379 communities (Usero et al., 2021). Previous studies observed a negligible effect of 380 irrigation with ozonated water on the structure of the soil microbial community (Díaz381 López et al., 2021). However, in this assay, we have evaluated a longer period of time, 382 provided a more realistic evidence of the long-term effects of the application of the 383 treatment under study. In this regard, the results of the NMDS analysis indicated that the 384 prokaryote community was significantly affected by the treatments analyzed, regardless 385 of the sampling time. The fungal community, on the contrary, was not affected either by 386 time or by treatment. These results are contradictory to those observed in previous trials, 387 in which similar growing conditions were evaluated (Díaz-López et al., 2021). However, 388 it should be noted that the characteristics of the soils studied were very different, so the 389 communities that inhabit these soils also have different characteristics and potential 390 responses to ozonated water. The soil in the present study had a high salinity, which 391 further limits the ability of microorganisms to adapt to new field conditions imposed by 392 ozonated water (Wang and Bao 2022). When analyzing the relative abundance of 393 prokaryotic orders, we observed that there were small differences between treatments in 394 20 the community composition. However, by studying genus indicators, we can discover 395 which genera are better adapted to the specific conditions created by the irrigation with 396 ozonated water. The OZ treatment, regardless of the sampling time, presented 13 genus 397 indicators, with some of them being bacteria with salinity tolerance, as Gracillibacillus, 398 Nocardiopsis, Prauserella, and Salinibacillus. However, little is known about these 399 genera and the potential impact they may have on both soil fertility and crop productivity. 400 Further investigation is needed to better understand the long-term effects of irrigation 401 with ozonated water. 402 4.2. Plant physiology and productivity under irrigation with ozonated water 403 It is well known that soil-plant interaction occurs, so that changes or alterations in 404 soil conditions can affect plant’s physiology (Sasse et al., 2018; Zak et al., 2003). As a 405 result, plant development can be positively or negatively modulated by changes in the 406 soil environment. It is therefore essential to jointly evaluate the soil-plant relationship 407 when implementing new strategies focused on a more sustainable agriculture. Moreover, 408 in commercial crops, it is very important to evaluate the agronomic parameters as well as 409 the plant’s physiology, since the main purpose is to look for a greater productivity without 410 affecting fruit quality. 411 Previous studies have observed imbalances in the nutritional content of plants in 412 crops grown with ozonated nutrient solution (Bou Jaoudé et al., 2008). This is mainly due 413 to the precipitation of some elements in the nutrient solution (Ikeura et al., 2018), making 414 them inaccessible to the plant. However, in our case, we did not find any alterations in 415 the nutrient content of the plants. The decomposition of ozone, in fact, favors the release 416 21 of labile compounds that are linked to the organic matter in the soil (Ghahrchi & Rezaee, 417 2020; Rizzo et al., 2020). Moreover, the increase in soil phosphomonoesterase activity 418 favors the release of simple P forms for plants. In this way, the oxidative stress that can 419 be potentially generated by its free radicals could be attenuated by the positive effects of 420 irrigation with ozonated water at the nutritional level. 421 Ozone gas exposure has been shown to damage photosynthetic processes (Cailleret 422 et al., 2018). However, the OZ treatment induced an increase in net photosynthesis (A) 423 and stomatal conductance (gs), leading to an intrinsic water use efficiency (iWUE) similar 424 to the control. In fact, an increased plant chlorophyll content was observed in the OZ 425 treatment. This could indicate that higher photosynthetic rates and better water conditions 426 can be achieved with the OZ treatment. In addition, crop yield is closely related to the 427 physiological and nutritional status of the plant. However, there is no general rule for 428 tomato crops, as yield varies depending on many environmental and varietal factors and 429 the agricultural practices adopted. In our case, the tomato plants had a similar productivity 430 with or without irrigation with ozonated water. Also, irrigation with ozonated water did 431 not significantly alter the productivity of tomato plants and fruit quality. 432 Delving deeper into the plant’s responses to ozone, we investigated the hormone 433 pattern. (Prigigallo et al., 2019; Risoli & Lauria, 2022). Irrigation with ozonated water 434 slightly affected the phytohormone content in this assay. The increase in stomatal 435 conductance could be explained by the reduction in ABA and SA content in the leaves 436 (Wilkinson & Davies, 2010). In contrast, tZ-Rib content increased with the OZ treatment. 437 The physiological role of tZ-Rib is to control leaf size, as well as traits related to meristem 438 22 activity, whereas tZ can only control leaf size (Osugi et al., 2017). Therefore, apical and 439 root growth can be quickly modulated by tZ-Rib, allowing the rapid adaptation to new 440 growing conditions. However, the physiological effect of irrigation with ozonated water 441 on crops must be studied further, to adequately implement this strategy in agricultural 442 fields. 443 5. Conclusions 444 Irrigation with ozonated water has been shown to have an influence on soil properties 445 and tomato physiology. At the soil level, the increase in bacterial biomass and in some of 446 the enzyme activities analyzed (e.g., alkaline phosphomonoesterase) could indicate that 447 irrigation with ozonated water has positive effect in the soil microbial community, which 448 is importantly linked to soil health and fertility, and also could improve plant 449 development. This could be due to i) increased nutrient availability, ii) a reduction in the 450 antimicrobial effect of ozone due to its rapid decomposition in water, and iii) increased 451 soil and rhizosphere aeration. At the plant level, irrigation with ozonated water seems to 452 modulate the plant’s response at the physiological level, while at the agronomic and 453 nutritional levels, there were no significant changes. The OZ treatment increased the 454 photosynthetic rates and maintained water conditions as compared to the control. In 455 addition, increased tZ-Rib could provide rapid apical and root growth allowing for a rapid 456 adaptation to the new growing conditions. However, a more in-depth study on the 457 physiological response of the plant to this treatment would be of interest, as it would help 458 with the implementation of this strategy in agricultural fields in a safe manner, and with 459 23 obtaining higher plant yields. Our results suggest that soil fertility and tomato plant 460 productivity were not significantly compromised by irrigation with ozonated water. 461 462 Acknowledgments 463 The authors acknowledge Mario G. Fon for his corrections and suggestions for the 464 English grammar in this manuscript. 465 Funding 466 This work was supported by the European Commission through the LIFE+ Program 467 (LIFE17 ENV/ES/000203 - LIFE AGREMSO3IL). The authors are also grateful to the 468 Fundación Séneca (19903/GERM/15 and 19896/GERM/15), the project PID2020469 114942RB-I00 funded by MCIN/AEI/10.13039/501100011033, and the project 470 PID2019–106226RB-C21/AEI/10.13039/501100011033). This study formed part of the 471 AGROALNEXT programme and was supported by MCIN with funding from European 472 Union NextGenerationEU (PRTR-C17.I1) and by Fundación Séneca with funding from 473 Comunidad Autónoma Región de Murcia (CARM). 474 Declarations 475 Competing interests. The authors declare no competing interests. 476 Supplementary information 477 Supplementary data for this article can be found online at 478 References 479 24 Albacete, A., Ghanem, M. 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Impacts and mechanisms of nanobubbles level in drip irrigation system on 669 soil fertility, water use efficiency and crop production: The perspective of soil 670 microbial community. Journal of Cleaner Production, 333(November 2021), 671 130050. https://doi.org/10.1016/j.jclepro.2021.130050 672 33 673 674 34 Figure captions: 675 Figure 1. Physicochemical and chemical properties of the studied soils: C (control), OZ 676 (irrigation with ozonated water). Time: t1 (December), t2 (February), and t3 (May). EC 677 (electrical conductivity), TOC (total organic C) and WSN (water-soluble nitrogen). Bars 678 represent the mean and the error bars represent the SD. For each time point, data followed 679 by different letters are significantly different (p <0.05). 680 Figure 2. Enzyme activities in the studied soils C (control), OZ (irrigation with ozonated 681 water). Time: t1 (December), t2 (February), and t3 (May). Bars represent the mean and 682 the error bars represent the SD. For each time point, data followed by different letters are 683 significantly different (p <0.05). 684 Figure 3. Fatty acid contents representative of different microbial groups, and the ratios 685 between microbial groups, in the studied soils: C (control), OZ (irrigation with ozonated 686 water). Time: t1 (December), t2 (February), and t3 (May). Bars represent the mean and 687 the error bars represent the SD. For each time point, data followed by different letters are 688 significantly different (p <0.05). 689 Figure 4. Non-metric dimensional scaling (NMDS) biplot of the Bray-Curtis 690 dissimilarity matrix of the prokaryotic (A) and fungal (B) community compositions. The 691 PERMANOVA P values are 0.0038 and 0.4107 for prokaryotes and fungi, respectively. 692 C-T2: control at t2; OZ-T2: irrigation with ozonated water at t2; C-T3: control at t3; OZ693 T3: irrigation with ozonated water at t3. Time: t2 (February), and t3 (May). 694 Figure 5. Relative abundances of prokaryotic phyla (A) and fungal (B) orders in the 695 studied soils with abundance > 1% in at least one treatment. C-T2: control at t2; OZ-T2: 696 35 irrigation with ozonated water at t2; C-T3: control at t3; OZ-T3: irrigation with ozonated 697 water at t3. Time: t2 (February), and t3 (May). 698 Figure 6. Cumulative yield of tomato fruits during the harvest period (Dec to May). C 699 (control), OZ (irrigation with ozonated water). 700 701 Tables Table 1. Prokaryotic genus indicator for treatments and sampling times analyzed, and also treatments regardless of sampling time (p <0.05). Sampling time: t2 (February) and t3 (May). Treatments Genus indicator Control t2 Ozone t2 Cytobacillus Isoptericola Oceanobacillus Streptomonospora Control t3 Halomonas Idiomarina Salinimicrobium Ozone t3 Control (t2 + t3) Caldovatus Microbispora Pedomicrobium Pseudogracilibacillus Thermomonospora Tumebacillus Ozone (t2 + t3) Aliifodinibius Flindersiella Gracilibacillus Jiangella Mycobacterium Nocardiopsis Phytoactinopolyspora Prauserella Saccharomonospora Saccharopolyspora Salinibacillus Streptomonospora Truepera Table 2. Gas exchange parameters and chlorophyll content at t2 (February). Values represent the mean and the SD (parenthesis). Data followed by different letters are significantly different (p <0.05). Table (Editable version) Click here to access/download;Table (Editable version);Tables_Diaz-Lopez2022.docx Gas exchange Control Ozone A (µmol CO2 m−2 s−1) 11.77 (3.55) b 20.27 (1.67) a gs (mol H2O m−2 s−1) 0.09 (0.03) b 0.17 (0.02) a iWUE (µmol CO2 mol H2O-1) 135.83 (25.18) 119.04 (13.06) Chlorophyll content Chl a (mg g-1) 1.49 (0.29) 1.70 (0.17) Chl b (mg g-1) 0.47 (0.10) 0.55 (0.06) ChlT (mg g-1) 1.96 (0.38) 2.25 (0.23) A: net photosynthesis; gs: stomatal conductance; iWUE: intrinsic water use efficiency. Table 3. Phytohormone content (ng g-1) at t2 (February). Values represent the mean and the SD (parenthesis). Data followed by different letters are significantly different (p <0.05) Phytohormone content Control Ozone ACC 34.86 (6.46) 30.84 (5.82) tZ 72.21 (13.08) 62.29 (12.25) tZ-Rib 1.14 (0.45) b 2.35 (0.64) a GA4 2.19 (1.17) 2.18 (1.55) GA7 95.88 (16.51) a 38.23 (11.51) b ABA 39.35 (4.10) a 28.91 (3.02) b SA 46.60 (14.22) a 15.91 (3.08) b ACC: 1-aminocyclopropane-1-carboxylic acid; tZ: trans-Zeatin; tZ-Rib: trans-Zeatin riboside; GA4: giberellic acid; GA7: giberellic acid; ABA: abscisic acid; and SA: salicylic acid Figure Click here to access/download;Figure;Fig1.jpg Figure Click here to access/download;Figure;Fig2.jpg Figure Click here to access/download;Figure;Fig3.jpg Figure Click here to access/download;Figure;Fig4.jpg