On testing the effectiveness of soil microbial inoculants in integrated pest management for commercial tomato production

On testing the effectiveness of soil microbial inoculants in integrated pest management for commercial tomato production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article On testing the effectiveness of soil microbial inoculants in integrated pest management for commercial tomato production Zhivko Minchev, Beatriz Ramírez-Serrano, Laura Dejana, Ana S. Lee Díaz, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3953202/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Research is showing that soil-borne beneficial microorganisms can enhance plant growth, productivity, and resistance against pests and pathogens, and could thus serve as a sustainable alternative to agrochemicals. To date, however, the effect of soil beneficial microbes under commercial crop production has not been fully assessed. We here investigated the effect of root inoculation with 11 well-characterized bacterial and fungal strains on tomato performance under intensive tomato crop management practices. We measured the impact of these strains on plant growth, fruit quality, yield, and pest and pathogen incidence. While most microbial strains showed weak effects, we found that the fungal strains Trichoderma afroharzianum T22 and Funneliformis mosseae significantly increased marketable tomato yield. Moreover, we found that inoculation with most of the fungal strains led to a significant reduction in the incidence of the devastating leaf mining pest Tuta absoluta , while this effect was not observed for bacterial inoculants. In addition, we found that microbial inoculations did not impact the incidence of introduced natural enemies, supporting their compatibility with well-established integrated pest management strategies in horticulture. In sum, the observed general positive effects of soil microbes on tomato yield and resistance reinforce the move toward a broader adoption of microbial inoculants in future crop production, ultimately improving agricultural sustainability. Beneficial soil-borne microorganisms bioinoculants crop protection field research microbe-induced resistance plant-microbe-insect interaction Tuta absoluta yield improvement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The urgent need to enhance agricultural sustainability has compelled scientists, agroindustry, growers, and consumers to seek for innovative approaches to mitigate the reliance on agrochemicals, all while ensuring optimal crop yields (Arora, 2018 ). One emerging environmentally friendly biotechnology is the use of soil-beneficial microbes, applied as bioinoculants, to improve plant growth and productivity across a variety of systems (Berg, 2009 ; Martínez-Medina et al., 2011 ; Barea, 2015 ; Trivedi et al., 2017 ; Ab Rahman et al., 2018 ; Compant et al., 2019 ; Singh et al., 2020 ). Furthermore, several of these microbes have been shown to antagonize soil pathogens (Minchev et al., 2021 ) and boost the resistance of crops to a broad spectrum of pests and diseases, a phenomenon known as induced resistance (IR) (Pieterse et al., 2014 ; De Kesel et al., 2021 ). Therefore, the multifaceted functionality of soil beneficial microbes could be harnessed to potentially increase crop productivity via direct boosting of resource acquisition, and indirectly, by reducing yield losses due to pest and pathogen attack (Li et al., 2022 ). Yet, soil beneficial microbes constitute a complex and diverse community of bacteria or fungi (Bakker et al., 2018 ), with their cumulative effect shown to be context and crop dependent (Lee Díaz et al., 2021 ), thereby currently limiting their competitive commercial exploitation against agrochemicals. Consequently, more studies in proper commercial settings are needed to test the efficacy of soil microbes for more sustainable agriculture practices. Beneficial soil microbes used as bioinoculants include several functional groups, such as the plant growth-promoting rhizobacteria (PGPR), the plant growth-promoting fungi (PGPF), and the arbuscular mycorrhizal fungi (AMF) (Woo et al., 2014 ; Aamir et al., 2020 ; Bitterlich et al., 2020; Bamisile et al., 2021 ). Besides their growth promoting properties, PGPR have been shown to act as efficient biological control agents, either by direct pathogen or disease suppression or through IR (Orozco-Mosqueda et al., 2021 ). Bacteria from the genera Bacillus and Pseudomonas are among the most studied and best characterized PGPR (Santoyo et al., 2012 ; Orozco-Mosqueda et al., 2021 ; Elnahal et al., 2022 ), as evidenced by the high number of commercial biofertilizer and biocontrol products in the market containing them (Aamir et al., 2020 ). Similarly, the ability of some PGPF species from the genus Trichoderma to promote plant growth, and resistance to pests and pathogens has been widely acknowledged during the last decades (Harman et al., 2004 ; Martínez-Medina et al., 2014 ; Guzmán-Guzmán et al., 2019 ; Poveda, 2021 ; Papantoniou et al., 2021 ; Woo et al., 2022 ; Modrzewska et al., 2022 ). However, their current success in the market as bioinoculants is primarily attributed to their mycoparasitic capacity, constituting 64.8% of available products claiming to be fungicidal (Woo et al., 2014 ). AMF are obligate biotrophs that establish symbiotic associations with the roots of most terrestrial plants, constituting one of the most studied plant-fungal interactions (Pozo et al., 2021 ). This symbiosis has been shown to improve plant nutrient uptake (van der Heijden et al., 2015 ; Sardans et al., 2023 ) and increases plant tolerance to biotic and abiotic stresses (Pozo & Azcón-Aguilar, 2007 ; Rivero et al., 2018 ; Rivero et al., 2021 ; Dejana et al. 2022 ; Ramírez-Serrano et al. 2022 ). Accordingly, the use of AMF has been amply proposed in sustainable agriculture (Smith & Smith, 2011 ; Jeffries & Barea, 2012 ; Barea, 2015 ; Salomon et al., 2022 ; Martin & van der Heijden 2024 ). AMF-based inoculants are commercially available in the market and the number of companies selling them has steadily increased (Bitterlich et al., 2020). The commercial products containing AMF are mostly used in agriculture as biofertilizers, mainly for nutrient and growth promotion benefits, but also for stress alleviation (Basiru et al., 2021 ). Finally, EPF constitute another important group of fungi in agroecosystems because of their well-known ability to infect and kill insect and mite pests (Quesada-Moraga, 2020). Besides this direct antagonism, EPF can interact and colonize plants endophytically, promoting plant growth and negatively affecting pathogens and phytophagous insects without direct contact with them (Gange et al., 2019 ; Quesada-Moraga, 2020; Rasool et al., 2021 ; Bamisile et al., 2021 ). EPF have been used in biological control of insects for more than 150 years, and several products are currently commercially available, with more than 170 species formulated as biopesticides (Bamisile et al., 2021 ). Besides single microbe applications, the design of synthetic microbial communities (SynComs) for improving plant growth and health is receiving increasing interest within the scientific community and on the market (Liu et al., 2020 ; Trivedi et al., 2020 ; Batista & Singh, 2021 ; Minchev et al., 2021 ). SynComs can offer extended functionality compared to single strain inoculations for the biocontrol of foliar and soil pathogens, as they can simultaneously combine different modes of action (Minchev et al., 2021 ). For instance, the combined application of EPF and AMF showed functional complementarity for plant protection and growth promotion (Zitlalpopoca-Hernandez et al., 2022 ). Nonetheless, despite the widespread optimism for using these microbial inoculants to improve plant growth and health, most of the research has been performed under highly controlled conditions, posing challenges to the successful transfer and adoption of this technology in agriculture (Mitter et al., 2019 ; Saad et al., 2020 ). Indeed, plant-microbe interactions and their effect on plant growth and health are often conditioned by environmental factors (Saad et al., 2020 ; Lee Díaz et al., 2021 ). For example, temperature (Di Lelio et al., 2021 ), nitrogen or phosphorous fertilization (Ramírez-Serrano et al., 2022 ; Dejana et al., 2022 ), soil water content (Orine et al., 2022 ) and light intensity (de La Hoz et al., 2021 ) and quality (Saha et al., 2022 ) have all been reported to impact plant-microbe interactions and microbial benefits for host plants. In other words, the intricate nature and context dependency of these interactions emphasizes the need to consider how variable environmental conditions and agricultural practices can potentially impede the success and reproducibility of microbial inoculation results under field conditions (Compant et al., 2019 ). Consequently, it is essential to perform rigorous assessments of previously characterized plant beneficial microorganisms within real production systems. Such evaluations serve not only to gauge microbial impacts on the target crop under commercial production conditions, but also to ascertain their compatibility with commonly used crop management practices. Our aim here was to address this gap by investigating how various microbial inoculations impact plant productivity and the interaction between plants and pests and pathogens under commercial production conditions (Fig. 1 ). We used tomato ( Solanum lycopersicum , Fig. 1 ), the second most produced vegetable crop worldwide, as model system. We selected well characterized strains of bacteria and fungi, and previously designed SynComs to test their impact on yield and pest resistance in a commercial greenhouse that uses standard tomato management practices, including integrated pest management (IPM) methods (Acebedo et al., 2022 ). We hypothesized that microbial inoculations would benefit plant performance by reducing susceptibility to biotic stressors without major costs in plant growth or production even under standard crop management practices. We show that microbial inoculation can increase plant resistance to pests without compromising yield, thus supporting the inclusion of microbe-induced resistance in IPM programs. MATERIAL AND METHODS Microbial treatments We performed a large-scale experiment in a commercial greenhouse with a total of 12 microbial treatments, including two bacteria: Bacillus amyloliquefaciens CECT8238 (BA) and Pseudomonas azotoformans F30A (PA), two strains of Trichoderma afroharzianum : T22 (T2) and T. harzianum T78 (T7), two EPF: Beauveria bassiana KVL 13–39 (BB) and Metarhizium robertsii KVL 12–35 (MR), and three AMF: Rhizophagus irregularis MUCL57021 (RI), Funneliformis mosseae BEG12 (FM) and Claroideoglomus etunicatum EEZ163 (CE). In addition, two SynComs (M1, M2), described below, and a control treatment without soil microbe addition (non-inoculated, NI) were included. The bacterium B. amyloliquefaciens was cultured on tryptone soy agar (TSA) and grown at 28°C for 24 hours. For spore production, liquid Difco sporulation medium (Nicholson & Setlow, 1990 ) was inoculated with a single bacterial colony and incubated at 28°C for 48 hours with a rotary shaking at 200 rpm. Spore concentration of the liquid culture was quantified using a Neubauer hemocytometer, then the culture was centrifuged for 15 min at 5000 rpm to separate the spores from the growing medium. Finally, the recovered spores were resuspended in sterile water to a concentration of 1 x 10⁷ spores/mL. For inoculation, 1 mL of spore solution was applied to each plant in the root system during transplanting (Minchev et al., 2021 ). The bacterium P. azotoformans was cultured on TSA and grown at 28°C for 24 hours. A pre-culture was prepared in tryptone soya broth (TSB) inoculated with a single colony and incubated overnight at 28°C with rotary shaking at 200 rpm. Next, 1 mL of pre-culture was added to 25 mL of TSB and incubated at 28°C for 2h 30 min with rotary shaking at 200 rpm to reach the exponential growth phase. Then, the cell concentration was quantified measuring the optical density (620 nm) of the bacterial culture using a spectrophotometer. The bacterial culture was centrifuged for 15 min at 5000 rpm to separate the bacterial cells from the growing medium. Finally, the obtained cells were resuspended in sterile water to a concentration of 1 x 10⁷ CFU/mL. For inoculation, 1 mL of bacterial solution per plant was applied to the root system during transplanting (Minchev et al., 2021 ). The fungus T. afroharzianum strain T22 was cultured on potato dextrose agar (PDA) and grown at room temperature for seven days. The sporulated plates were scraped using a sterile spatula and sterile water. The resulting spore suspension was filtered using a sterile miracloth filter to remove remaining mycelia and the spore concentration was quantified using a Neubauer hemocytometer and adjusted to 1 x 10⁷ spores/mL. For inoculation 1 mL of spore suspension was added to the root system of each plant during transplanting (Minchev et al., 2021 ). The fungus T. harzianum strain T78 was cultured on PDA. The fungal inoculum was prepared by adding aseptically a square piece of the fungal culture on a sterile mix of vermiculite and oat (Martínez-Medina et al., 2009 ) and incubated at 28°C in the dark for five days. The inoculum, containing 1x10 9 spores/g, was mixed with the substrate in a proportion of 1 g per Kg of substrate (Martínez-Medina et al., 2013 ). The EPF B. bassiana and M. robertsii were cultured in Sabouraud dextrose agar (SDA) and grown at 24° C in darkness for three weeks. The sporulated plates were scraped using a sterile spatula and the spores were recovered in a sterile solution of Triton X (0.05%). The spore concentration was quantified using a Neubauer hemocytometer and adjusted to 1 x 10 8 spores/mL. Inoculation was done by adding 1 mL of spore suspension per plant directly to the roots during transplanting (Zitlalpopoca-Hernandez et al., 2022 ). The AMF R. irregularis was grown in vitro on a minimal (M) medium with Agrobacterium rhizogenes -transformed carrot ( Daucus carota ) roots as host (St-Arnaud et al., 1996 ). Spore extraction was performed by adding citrate buffer (0.01 M, pH = 6) to the AMF culture in a proportion of 3:1 (v/v) and maintained for 1 hour on a rotary shaker to dissolve the agar. The spores were recollected using sieves with mesh size of 250 and 53 µm and resuspended in sterile water at 1000 spores/mL. For inoculation 1 mL of spore solution was applied to the root system of each plant (Minchev et al., 2021 ). The AMF F. mosseae and C. etunicatum were maintained as living inocula on mixed cultures of Trifolium repens and Sorghum vulgare in vermiculite-sepiolite substrate. The inoculants consisted of substrate containing colonized root fragments, mycelia and spores. For inoculation, 10% (v/v) of mycorrhizal inocula were mixed with the substrate at transplanting (Rivero et al., 2018 ). Further, two SynComs were used. The M1 inoculum included B. amyloliquefaciens , P. azotoformans and T. afroharzianum T22 at concentration of 1 x 10⁷ CFU/mL each, and R. irregularis at a concentration of 1000 spores/mL (Minchev et al., 2021 ). The M2 inoculum included M. robertsii and B. bassiana both inoculated at a concentration of 1 x 10 8 spores/mL, and R. irregularis at a concentration of 1000 spores/mL. For both SynComs 1mL/plant was applied to the root system during transplanting. Plant material and growing conditions Solanum lycopersicum cv Money maker seeds (Vreeken’s Zaden, The Netherlands) were surface sterilized by immersion in 5% Sodium hypochlorite solution for 10 min and rinse three times in sterile water for 10 min each. The surface sterilized seeds were sown in sterile vermiculite and incubated for seven days in a greenhouse at 24°C : 16°C day : night with a photoperiod 16 h: 8 h light : dark and 70% of relative humidity. Experimental set up One-week old tomato seedlings were transferred to starting trays, with cell dimensions 2.9 x 2.9 x 6.8 cm- containing blond seedling peat (Kekkilä LSM 0 R8406, Projar, Valencia, Spain): zeolite: perlite (1:1:1) mixture and inoculated with the microbial treatments described previously. Inoculated seedlings were grown in a commercial nursery (ACRENA SAT 251, El Ejido, Spain; 36°, 47', 52.9''N; 2°, 43', 36.3''W) for four weeks. On September 3rd, 2020 the plants were transplanted to a commercial production greenhouse (Estación experimental Cajamar, Paraje las Palmerillas, El Ejido, Almería; 36°, 47', 36.3"N; 2°, 43', 15.2"W) and maintained during the whole crop cycle from September 2020 to March 2021. The greenhouse consisted of a typical "raspa y amagado" type (Ávalos-Sánchez et al., 2022 ), 37.8 m long and 23.2 m wide with a total area of 877 m 2 and usable area of 720 m 2 , passive ventilation (25.0% window surface) with side windows (north and south sides) and zeniths, covered with anti-trip mesh. The microbial inoculation treatments were organized following a randomized complete block design, with four blocks. Each block contained all 12 treatments, and each treatment in all blocks was replicated with six plants ( Figure S1 ; N = 12 treatments x 4 blocks x 6 replicates = 288 plants). Biological control, pheromone and pollinator application Two weeks after transplanting, the predatory mirid bug Nesidiocoris tenuis (Hemiptera: Miridae) (NESIDIOcontrol, Agrobio, Spain) was released in the greenhouse with a density of 0.5–1.5 individuals/m2 following the product label recommendation, to reduce incidence of whiteflies (Hemiptera: Sternorrhyncha) and Tuta absoluta (Lepidoptera: Gelechiidae) on tomato plants. In addition, pheromones for the mating disruption of T. absoluta were released during the whole cropping season. For ensuring pollination of tomato flowers from the start of flowering, Bombus terrestris bumblebees (Hymenoptera: Apidae) were released three weeks post transplantation (wpt), placing one hive (Agrobio, Spain) in the middle of the greenhouse. Irrigation and fertilization The irrigation scheme during the whole cropping season and nutrient supply are shown in Table S1 . Nutrient content in soil and irrigation water (nutrient solution) was evaluated periodically to adjust to the crop needs for nutrient supply. Specifically, phosphorus was measured by visible spectrophotometry using the compound phosphorous vanadate molybdate (Tandon et al., 1968 ). Nitrates were measured spectrophotometrically at 220 and 275 nm (Norman & Stucki, 1981 ). Ammonia was measured by the Nessler reagent method (Yuen & Pollard, 1954 ). Sodium, calcium, potassium, magnesium, iron, copper, manganese and zinc, were determined by atomic absorption / emission (Isaac & Kerber, 2015 ). Carbonates and bicarbonates were measured by titration with 0.01 N sulfuric acid (Allison et al., 1954 ). Chlorides were also measured by volumetry with silver nitrate between 0.01 and 1 N using potassium chromate as an indicator (Mohr's titration). Boron was determined by spectrophotometry with the azomethine reaction (John et al., 2006 ). Sulfates were measured by precipitation of barium sulfate. Response variables and data collection In total we measured 17 response variables related to plant growth and yield, and plant resistance to pathogens and insect pests (See below, and Table S2 ). Plant growth, nutritional status and yield As a proxy of plant growth, plant height from soil surface to the top of the shoot of each plant (6 plants per treatment per block) was measured on December 3rd, 2020 (12 wpt). As a proxy for plant productivity, we quantified the number of inflorescences per plant (6 plants per treatment per block) on October 26th, 2020 (8 wpt). Total leaf carbon and nitrogen content was measured on the January 21st, 2021 (19 wpt), evaluating 3 plants per treatment per block. Leaves were sampled, immediately frozen in liquid nitrogen and lyophilized. Then lyophilized leaves were ground in a Tissue Lyser II (Qiagen, Germany) using metal beads at a maximum speed for 3 min. Two milligrams were weighed from each sample for analysis of carbon (C) and nitrogen (N) content in the Flash 1112 Elemental Analyzer (Thermo Scientific, MA, USA). Fruit productivity (average g/plant) and quality were evaluated every week between November 12th, 2020 (10 wpt) and February 4th, 2021 (21 wpt) on 6 plants per treatment per block. Tomato fruits were classified by size (size GG 82-102 mm; size G 67‐82 mm; size M 57‐67 mm; size MM 47‐57 mm) and by categories (first, second and non‐commercial). Fruits were considered non-commercial when their size was too small (< 45 mm of diameter), when they showed presence of pathogen damage, cracks, blossom-end rot or blotchy ripening, or when they were misshapen. Fruit quality and nutraceutical value Parameters such as fruit dry weight (determined after drying the fruits in a forced air stove at 70 ºC for 48 hours), acidity % (Acid-base volumetry using 1 N NaOH as base and phenolphthalein indicator), ºBrix or total soluble solids (manual refractometer), maturity index (the ratio between the content of total soluble solids and assessable acidity) were assessed on December 16th, 2020 (14 wpt) on one fruit per treatment per block (n = 4). Polyphenol and carotenoid content in fruits were evaluated on February 25th, 2021 (23 wpt) on one fruit per treatment per block (n = 4). Polyphenols were measured by the spectrophotometric method of Folin-Ciocalteau (Georgé et al., 2005 ) using a standard curve of gallic acid from 0 to 1000 ppm at 760 nm (double ultraviolet‐visible beam, Unicam Helios Alpha) and expressed as mg of gallic acid/100g dry fruit biomass. Lycopene and beta-carotene content of fruits was measured with an acetone‐hexane extraction and spectrophotometric determination at 487.5 nm (Sadler et al., 1990 ) with modifications (Rousseaux et al., 2005 ) and expressed as mg/100g fresh fruit. Pest and disease incidence. The incidence of thrips, T. absoluta , whiteflies and powdery mildew was evaluated on December 3rd, 2020 (12 wpt). For thrips, occurrence was assessed by counting the number of leaves per plant presenting lacerations caused by thrips. The incidence of T. absoluta was estimated as the percentage of plants per treatment displaying mines (the typical lesions caused by the larvae of this species). Whiteflies were evaluated using yellow sticky traps for a period of 4 weeks until December 3rd, 2020 (12 wpt), placing one sticky trap per treatment per block and assessing the number of whiteflies per trap. Finally, powdery mildew prevalence was measured as the percentage of infected plants per treatment. Abundance of natural enemies The abundance of the predatory mirid bug Nesidiocoris tenuis , released in the greenhouse at the beginning of the cropping season for the control of whiteflies and T. absoluta , was evaluated on December 3rd, 2020 (12 wpt), based on evaluation of the same yellow sticky traps as for whitefly incidence (see above) and counting the number of N. tenuis per trap. Statistical analysis Data were analysed using R statistical language, version 4.1.1 (R Development Core Team 2021) and figures were produced using the package ggplot2 (Wickham, 2009 ). The effects of the 12 microbial treatments (including the control) on the 17 different response variables were analyzed using linear (lm) or generalized linear models (glm) following the details as shown Table S2 . Treatment effects were always compared to the control (non-inoculated) treatment, as shown by the asterisks in Figures presented in the results section. Moreover, to measure the overall effect of soil microbial inoculation on plant resistance, we produced radar plots using scaled values for each of the four pests and pathogens studied (function ggradar in ggplot2), and calculated the area of each polygon generated for each treatment using R. RESULTS Effect of soil beneficial microbes on plant growth, nutritional status and flowering To assess the effect of soil beneficial microbes on tomato plants’ growth, nutritional status and reproduction potential, we measured plant height, leaf carbon and nitrogen content and their ratio (C/N) and counted the number of inflorescences. For plant height, we found weak negative effects of the soil microbial treatments, in which only the AMF C. etunicatum decreased tomato plant height on average by 12.96 cm compared to the control plants (Fig. 2 A, treatment effect; F 11,264 = 2.29, p = 0.01). We however found no effect of soil microbial treatments on C and N leaf content, resulting in an unaltered C/N (F 11,116 = 1.3, p = 0.23; Fig. 2 B), nor on the number of inflorescences (F 11,130 = 1, p = 0.45; Fig. 2 C). Effect of soil beneficial microbes on fruit yield To evaluate the impact of the microbial inoculants on fruit production, we assessed the fruit yield during the whole fruit production period, quantifying the total production as well as the commercial quality production. Soil microbial inoculations had a prominent effect on total tomato production (Chisq 11,464 = 23.1, p = 0.017; Fig. 3 A), as well as on commercial quality tomato production (Chisq 11,464 = 25.1, p = 0.009; Fig. 3 B). Specifically, F. mosseae and T. afroharzianum T22 inoculated plants showed a 13% and 15% higher total productivity than control plants, respectively (Fig. 3 C). Even more so, the same soil microbes increased commercial quality fruit production by 12% and 14% at the end of the experiment as compared to the non-inoculated control plants (Fig. 3 D). Effect of soil beneficial microbes on fruit quality and nutraceutical value Fruit quality was evaluated by assessing different parameters: ºBrix, %Acidity, Maturity index and %Dry weight. We did not observe any effect of the microbial inoculations on ºBrix (F 11,33 = 1.00, p = 0.47), Maturity index (F 11,33 = 1.19, p = 0.31) and %Dry weight (F 11,33 = 0.86, p = 0.59). The only fruit quality parameter significantly affected by the microbial treatments was the %Acidity (F 11,33 = 2.46, p = 0.02), in which fruits from C. etunicatum inoculated plants showed an increase of 0.04% in acidity compared to the control treatment (t = 2.25, p = 0.03; Table S3 ). Furthermore, regarding fruit nutraceutical value, we found that fruit polyphenols’ content was not significantly affected by the soil microbial treatment (F 11,36 = 0.61, p = 0.81, Figure S2A ), as well as carotenoids’ content of fruits (lycopene, beta-carotene and total carotenoids) were also not significantly altered by soil microbes (F 11,36 = 1.25, p = 0.29, Figure S2B; F 11,36 = 0.66, p = 0.77, Figure S2C and F 11,36 = 1.18, p = 0.34, Figure S2D , respectively). Effect of soil beneficial microbes on natural enemies To evaluate any potential impact of the microbial inoculants on applied beneficial insects, we evaluated the abundance of the predatory mirid bug N. tenuis , released in the greenhouse at the beginning of the cropping season for the control of whiteflies and T. absoluta . We did not find significant differences in N. tenuis abundance among the different microbial treatments, indicating no negative effect of the microbial inoculants on the predator (F 11,36 = 0.55, p = 0.86; Figure S3 ). Effect of soil beneficial microbes on pest and disease incidence We next assessed the natural incidence of different pests and diseases appearing during the cropping season. We found that the incidence of T. absoluta was significantly impacted by microbial inoculation (Chisq 11,273 = 24.37, p = 0.01). In particular R. irregularis (z = -2.698, p = 0.007), F. mosseae (z = -2.31, p = 0.02), C. etunicatum (z = -2.31, p = 0.02), T. afroharzianum T22 (z = -2.31, p = 0.02), T. harzianum T78 (z = -1.98, p = 0.05), M. robertsii (z = -2.69, p = 0.007) and the consortium M2 (z = -1.98, p = 0.05) treatments significantly decreased the percentage of plants damaged by the leaf miner as compared to the control treatment, with average reductions ranging from 60% for T. harzianum T78 up to 90% for R. irregularis and M. robertsii (Fig. 4 A). Thrips and whitefly incidences were not significantly affected by the microbial treatments (Chisq 11,131 = 15.82, p = 0.15; Fig. 4B and F 11,36 = 1, p = 0.46; Fig. 4 C). Regarding diseases, the powdery mildew was the only pathogen that naturally appeared on the crop. No significant effect of the microbial treatments was observed on the incidence of powdery mildew (Chisq 11,132 = 9.01, p = 0.62; Fig. 4 D). Compound effect of soil beneficial microbes on plant resistance We visualized the overall effect of each different soil microbial inoculation with radar plots (Fig. 5 ). For this analysis, smaller areas indicate higher resistance induced by the soil microbial inoculations. Notably, the polygon for the control (0.8; red polygons of Fig. 5 ) show the highest area compared to rest of treatments (expect for C. etunicatum ; see area values in Fig. 5 ). Of those, the smallest area is with R. irregularis , with a decrease of 95% from the control treatment. Those results suggest that besides C. etunicatum , soil microbial inoculations increase general resistance of tomato plants (smaller areas in our model) against the four pest and pathogens studied. DISCUSSION In this study, by testing diverse plant beneficial microorganisms under commercial settings, we demonstrated the viability of using microbial inoculants for crop protection and yield improvement for a commercial crop production system. We have identified microbial strains that can be used as biostimulants and bioprotectors under real tomato production conditions, thus confirming their potential as bioinoculants to improve agricultural sustainability. Considering all measured traits, our results point out a prominent effect of fungal inoculants (including different AMF, EPF and Trichoderma strains) in promoting plant resistance, and in some cases, improving crop yield, while the tested bacterial inoculants did not show any significant effects on the evaluated parameters. Soil beneficial microbe effect on tomato growth and yield Plant beneficial microbes such as PGPR, Trichoderma , AMF and EPF have been widely reported to improve plant growth and nutritional status (Quesada Moraga, 2020 ; Orozco-Mosqueda et al., 2021 ; Salomon et al., 2022 ; Woo et al., 2022 ). Contrary to previous observations, the microbial inoculation did not impact plant height or leaf C/N under commercial production conditions. Although plants inoculated with the AMF C. etunicatum were significantly smaller than control plants, this reduction did not negatively affect tomato yield, which is the most relevant parameter for tomato producers. The lack of microbial effects on plant nutritional levels can be related to the standard periodic application of fertilization, so that the plant nutritional needs were sufficiently covered, making the role of nutrient acquisition by the beneficial microbes redundant. Indeed, nutritional benefits by interaction with beneficial microorganisms are usually visible only under limiting conditions (Martínez-Medina et al., 2011 ). Specifically, under reduced fertilization dosage, some of the tested microbes such as T. harzianum T78, R. irregularis and F. mosseae , has shown to improve plant growth, nutrient acquisition and fruit production in melon plants under field conditions, while such effects were absent under conventional fertilization conditions (Martínez-Medina et al., 2011 ). Yet, when evaluating the potential of microbial inoculants as biostimulants, crop yield and fruit quality are the most relevant parameters, particularly for the economy of producers. A recent meta-analysis conducted on 97 peer-reviewed articles (69% conducted under greenhouse and 31% under field conditions) that examined the effect of different microbial inoculants -mostly PGPR- on crop productivity concluded that microbial inoculants can overall improve crop productivity, mainly by stress alleviation or by improving nutrient availability for plants (Li et al., 2022 ). Our findings demonstrate that while none of the treatments influenced flower production, two fungal treatments, the AMF F. mosseae and the fungus T. afroharzianum T22, increased the total and marketable tomato yield during the cropping season. Increased tomato yield after microbial inoculants application can derive from a better alleviation of stress (Li et al., 2022 ). In agreement with this hypothesis, we observed that the two fungal treatments that increased yield also caused significant pest reduction of the leaf miner T. absoluta (see below). Of particular interest is the fact that tomato plants with increased yield did not show a trade-off by reducing fruit quality, suggesting a net benefit for the farmers under these conditions. Soil microbial effect on tomato resistance against pests Soil-borne beneficial microbes are widely reported to improve plant resistance by triggering defenses against a broad range of attackers, including pathogens and herbivorous insects (Pieterse et al., 2014 ). Here, we evaluated the impact of microbial inoculation on the incidences of powdery mildew, the phloem and cell-content feeders whiteflies and thrips respectively, and the leaf miner T. absoluta . While we found no effect of soil microbes on powdery mildew, thrips or whiteflies, the percentage of plants damaged by the leafminer T. absoluta was significantly reduced by most of the fungal inocula. This benefit was not observed upon inoculation with PGPR, underscoring the positive outcomes associated with fungal inoculations. All three mycorrhizal strains, both Trichoderma strains, the EPF M. robertsii and the M2 SynCom (a fungal consortia including the EPF B. bassiana and M. robertsii , and the AMF R. irregularis ) reduced the natural incidence of T. absoluta , in some cases ( R. irregularis and M. robertsii ) up to 90%. These results agree with recent studies showing induced resistance against T. absoluta under controlled conditions by strains of AMF (Shafiei et al., 2022 ), T. afroharzianum (Aprile et al., 2022 ) and by the EPF B. bassiana and M. anisopliae (Giannoulakis et al., 2023 ). While AMF and Trichoderma are widely documented to induce plant resistance against very diverse pathogens and pests (Martínez-Medina et al., 2013 ; Coppola et al., 2019 ; Sanmartín et al., 2020 ; Di Lelio et al., 2021 ; Rivero et al., 2021 ; Dejana et al., 2022 ), only few recent studies have demonstrated their negative impact on the performance of the leafminer T. absoluta (Aprile et al., 2022 ; Shafiei et al., 2022 ). For EPF, most studies are focused on the direct biological control action of fungal conidia resulting in the infection or reduction of the performance and fitness of T. absoluta (Chouikhi et al., 2022 ). More recently, evidence on the induction of plant resistance by EPF against insects and pathogens is increasing (Raad et al., 2019 ; Rivas-Franco et al., 2020 ; Rasool et al., 2021 ; Zitlalpopoca-Hernandez et al., 2022 ). This effect is particularly important for concealed pest stages such as mining larvae, which are hidden for contact with sprayed biopesticides. Thus, the results illustrate the ability of AMF, Trichoderma and EPF to enhance plant resistance and reduce T. absoluta incidence in commercial production conditions, and thus, to improve or complement current IPM practices in tomato crop protection. Worldwide, T. absoluta is a major pest of tomato (Biondi et al., 2018 ) so the present results are encouraging for its management. The high reproductive capacity of T. absoluta allows for rapid population growth and widespread infestation, and its miner lifestyle difficult the use of surface applied (bio)pesticides (Abd El-Ghany et al., 2016 ). Moreover, T. absoluta is known for its ability to develop resistance to chemical pesticides, making control measures against this pest even more challenging (Guedes et al., 2019 ). Hence, applying effective soil microbes that can hinder the development of this pest, without reducing fruit quality nor yield -and even improving it-, can be an efficient and ecologically-sound solution for tomato crop protection. However, in real crop production several different pests and diseases could emerge at the same time or sequentially during the cropping season, challenging the crop performance and productivity. Thus, we explored the impact of each microbial inoculant on the global pest and disease incidence, considering all four aggressors, to get an insight on the impact of microbial inoculations on the overall plant resistance to pests and pathogens. We found that most of the beneficial microbes used in our study increased the global plant resistance, prominently reducing the overall pest and disease incidence in the crop. Remarkably, for some microbial strains as the AMF R. irregularis and the PGPF T. harzianum the reduction was around 95% as compared to the non-inoculated control plants. These finding are in agreement with the general idea that IR by beneficial microbes such as AMF and Trichoderma could be effective against a broad range of pest and pathogens (Martínez-Medina et al., 2013 ; Sanmartín et al., 2020 ; Di Lelio et al., 2021 ; Rivero et al., 2021 ). The successful implementation of microbe-induced resistance in agriculture does not only rely on its effectiveness to control pests and pathogens but also on its compatibility with other strategies regularly used in IPM (Stenberg, 2017 ). One point of caution would be if the microbes, by modifying plant defenses, may negatively impact auxiliary fauna, as for example biocontrol insects. As this study was performed under common pest management practices in Spanish tomato production based on IPM (Acebedo et al., 2022 ), we also evaluated the effect of the microbial inoculation on the pest predator N. tenuis . Our results did not show any negative effects of the inoculations on the abundance of the predator, suggesting that microbe-induced resistance is compatible with the release of this predator, a generalist biocontrol agent commonly used in IPM programs. Conclusions Overall, this study highlights the potential of soil-inoculated microorganisms, particularly fungi, to improve tomato crop productivity and resistance to important pests such as the devastating leaf miner T. absoluta . By testing microbial strains -previously characterized under controlled lab conditions- in agronomic settings we identified beneficial microbes that are competent and functional under commercial growing conditions. The identification of microbes that effectively improve plant health and productivity in real crop production system will contribute to a faster and wider adoption of microbial inoculants for environmentally safe crop protection and to improve future agricultural sustainability. Declarations Funding: This study was funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 765290 in the project “MiRA – Microbe-induced Resistance to Agricultural Pests”. Conflict of interest: The authors have no conflicts of interest to declare that are relevant to the content of this article. Ethics approval: Not applicable Consent to participate: Not applicable Consent for publication: Not applicable Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors contribution: " Conceptualization, M.J.P, S.R, A.B., T.H., N.V.M and Z.M; Investigation, Z.M., B.R.S., L.D, A.L.D, G.Z.H., D.O., H.S., D.P., J.M.G., A.G.; Formal analysis, S.R. and Z.M; Writing – Original Draft, Z.M. and M.J.P.; Writing –Review & Editing, M.J.P., S.R., A.B., A.M.M., N.V.M., D.O., B.R.S. and Z.M; Visualization, S.R. and Z.M; Funding Acquisition, T.H., A.B., M.J.P., S.R., A.M.M, N.V.D. , N.V.M, R.S., D.G., P.G; Project administration, M.J.P. and Z.M.; Supervision, M.J.P." 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Biol Control 175:1–8. 10.1016/j.biocontrol.2022.105034 Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 07 Mar, 2024 Reviewers invited by journal 05 Mar, 2024 Editor invited by journal 22 Feb, 2024 Editor assigned by journal 15 Feb, 2024 First submitted to journal 12 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3953202","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276466503,"identity":"70844977-ac01-4e04-aa19-da86e545f87d","order_by":0,"name":"Zhivko Minchev","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYDACCcYGxgYgzQZGDDZQYQPitaQRo4WBAawFoovhMGF3yc9ubpOcUcEQzSfd/OzBx7bzcvz8Bxg//iiwYwAysGphnHOwTXLDGYbcNplj5oYz224bSzYcYJbmMUhmkJyRgFULs0Ris+HDNqAWiRw2aZ4ztxM3HGxgY2YwOMBgcAO7w9hQtPw5c65+/2EGNsYfQC3257E7jEcisfHhRpgWhooDCQZswHDgAdnCgN1hEiAtM85IALWkmUn2VCQbzjjD2AzyC4/EDexa5GekPzjYU2GTO39G8jOJHwZ28vz9hw9+/PHHTo6/H7vDYJYhcyDxxINP/SgYBaNgFIwC/AAAB6xVsb9Tv9AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0004-0170-7881","institution":"Estacion Experimental del Zaidín","correspondingAuthor":true,"prefix":"","firstName":"Zhivko","middleName":"","lastName":"Minchev","suffix":""},{"id":276466504,"identity":"29962ac8-ec6c-41d7-baac-86edf79c0d7d","order_by":1,"name":"Beatriz Ramírez-Serrano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Ramírez-Serrano","suffix":""},{"id":276466505,"identity":"ace195fb-e91a-4b8f-bc46-bb03fa163e86","order_by":2,"name":"Laura Dejana","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Dejana","suffix":""},{"id":276466506,"identity":"c088cc7c-9104-46b4-8b08-740563a70545","order_by":3,"name":"Ana S. Lee Díaz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"S. Lee","lastName":"Díaz","suffix":""},{"id":276466507,"identity":"033c14a9-c29e-4629-a91e-bc9fa608bb81","order_by":4,"name":"Guadalupe Zitlalpopoca-Hernandez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Guadalupe","middleName":"","lastName":"Zitlalpopoca-Hernandez","suffix":""},{"id":276466508,"identity":"bb7f61d9-cdd7-4ba4-891a-3c81e2cfa851","order_by":5,"name":"Dimitri Orine","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dimitri","middleName":"","lastName":"Orine","suffix":""},{"id":276466509,"identity":"727014ab-a872-429f-99d2-d5dc94157551","order_by":6,"name":"Haymanti Saha","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haymanti","middleName":"","lastName":"Saha","suffix":""},{"id":276466510,"identity":"8efa6e7e-9592-4aa6-95f5-dcea4899bf97","order_by":7,"name":"Dimitra Papantoniou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dimitra","middleName":"","lastName":"Papantoniou","suffix":""},{"id":276466511,"identity":"a39422eb-0093-4897-92db-d1ebccb11759","order_by":8,"name":"Juan M. García","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"M.","lastName":"García","suffix":""},{"id":276466512,"identity":"1ea28f6c-28ce-4df0-9cc8-8684cc421284","order_by":9,"name":"Alicia González-Céspedes","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alicia","middleName":"","lastName":"González-Céspedes","suffix":""},{"id":276466513,"identity":"345de872-822a-46df-9200-364e9f8c1c40","order_by":10,"name":"Paolina Garbeva","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Paolina","middleName":"","lastName":"Garbeva","suffix":""},{"id":276466514,"identity":"d7b1ce2b-dc55-437c-8c80-10a9f0bc56ed","order_by":11,"name":"Nicole M. van Dam","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"M. van","lastName":"Dam","suffix":""},{"id":276466515,"identity":"adca38ef-3316-498f-9a08-38cff83c779e","order_by":12,"name":"Roxina Soler","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Roxina","middleName":"","lastName":"Soler","suffix":""},{"id":276466516,"identity":"8168ec34-8f56-469d-a601-5b46d83063eb","order_by":13,"name":"David Giron","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Giron","suffix":""},{"id":276466517,"identity":"67450a7b-f90c-490e-8ad9-6e94881bce16","order_by":14,"name":"Ainhoa Martínez-Medina","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ainhoa","middleName":"","lastName":"Martínez-Medina","suffix":""},{"id":276466518,"identity":"7c289320-30e8-4693-bd83-bbd1cbf8f57c","order_by":15,"name":"Arjen Biere","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Arjen","middleName":"","lastName":"Biere","suffix":""},{"id":276466519,"identity":"e83ae533-7553-46b8-89d8-cb5be8cd3ca5","order_by":16,"name":"Thure Hauser","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thure","middleName":"","lastName":"Hauser","suffix":""},{"id":276466520,"identity":"b6f2277b-15db-4781-a655-07dffb31652e","order_by":17,"name":"Nicolai V. Meyling","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nicolai","middleName":"V.","lastName":"Meyling","suffix":""},{"id":276466521,"identity":"c5374335-45e4-456b-b14b-b2f38e861f7b","order_by":18,"name":"Sergio Rasmann","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Rasmann","suffix":""},{"id":276466522,"identity":"69883d64-8ba1-49f8-abaf-c4857e548588","order_by":19,"name":"María J. Pozo","email":"","orcid":"https://orcid.org/0000-0003-2780-9793","institution":"","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"J.","lastName":"Pozo","suffix":""}],"badges":[],"createdAt":"2024-02-13 09:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3953202/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3953202/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52142164,"identity":"e1abb64c-de43-4c2e-b6b4-d8e7ffea8578","added_by":"auto","created_at":"2024-03-07 11:28:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2861327,"visible":true,"origin":"","legend":"\u003cp\u003ePicture showing tomato plants from the experiment performed under commercial production conditions including common tomato crop management practices.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/485fd371b9208b8588bb60fe.png"},{"id":52142166,"identity":"949b70f3-dfae-4a0b-873a-58406f5c60f1","added_by":"auto","created_at":"2024-03-07 11:28:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42408,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of microbial inoculation on \u003cstrong\u003e(A)\u003c/strong\u003e plant height, \u003cstrong\u003e(B)\u003c/strong\u003e leaf carbon-nitrogen ratio, and \u003cstrong\u003e(C)\u003c/strong\u003e number of inflorescences. Plants were inoculated with: \u003cem\u003eR. irregularis\u003c/em\u003e (RI), \u003cem\u003eF. mosseae\u003c/em\u003e (FM), \u003cem\u003eC. etunicatum\u003c/em\u003e (CE), \u003cem\u003eP. azotoformans\u003c/em\u003e (PA), \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e(BA), \u003cem\u003eT. afroharzianum\u003c/em\u003e T22 (T2), \u003cem\u003eT. harzianum\u003c/em\u003e T78 (T7), \u003cem\u003eB. bassiana\u003c/em\u003e (BB), \u003cem\u003eM. robertsii\u003c/em\u003e (MR), consortium 1 (M1) including RI+PA+BA+T2 and consortium 2 (M2) including RI+BB+MR. Non-inoculated plants were included as a control (NI). Boxes represent the interquartile range, black lines represent the median, whiskers represent maximum and minimum within 1.5 times the interquartile range, and black dots represent outliers. The asterisk indicates statistically significant differences compared to the control (*p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/52db3698fab7c57c732f5c65.png"},{"id":52142955,"identity":"97c570a9-ddfd-4328-9655-9f8e8d030e6b","added_by":"auto","created_at":"2024-03-07 11:36:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":53691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of microbial inoculants on tomato production. \u003c/strong\u003eA and C, total tomato production; B and D, commercial quality tomato production. Plants were inoculated with: \u003cem\u003eR. irregularis\u003c/em\u003e (RI), \u003cem\u003eF. mosseae\u003c/em\u003e (FM), \u003cem\u003eC. etunicatum\u003c/em\u003e (CE), \u003cem\u003eP. azotoformans\u003c/em\u003e (PA), \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (BA), \u003cem\u003eT. afroharzianum\u003c/em\u003eT22 (T2), \u003cem\u003eT. harzianum\u003c/em\u003e T78 (T7), \u003cem\u003eB. bassiana\u003c/em\u003e (BB), \u003cem\u003eM. robertsii\u003c/em\u003e (MR), consortium 1 (M1) including RI+PA+BA+T2 and consortium 2 (M2) including RI+BB+MR. Non-inoculated plants were included as a control (NI). Lines represent the average yield increase across time, dots represent the mean tomato biomass, and error bars represent ± the standard deviation. Asterisks indicate statistically significant difference compared to the control (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/b809c8a5042e6446fc5b5b95.png"},{"id":52142163,"identity":"a3602be1-e5aa-4ef2-ba22-5d961a5b5704","added_by":"auto","created_at":"2024-03-07 11:28:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64350,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of microbial inoculation on \u003cstrong\u003e(A)\u003c/strong\u003e percent of plants infested by \u003cem\u003eT. absoluta\u003c/em\u003e, \u003cstrong\u003e(B)\u003c/strong\u003e number of damaged leaves per plant by thrips, \u003cstrong\u003e(C)\u003c/strong\u003enumber of whiteflies per trap, and \u003cstrong\u003e(D)\u003c/strong\u003epercent of plants diseased by powdery mildew. Plants were inoculated with: \u003cem\u003eR. irregularis\u003c/em\u003e (RI), \u003cem\u003eF. mosseae\u003c/em\u003e (FM), \u003cem\u003eC. etunicatum\u003c/em\u003e (CE), \u003cem\u003eP. azotoformans\u003c/em\u003e (PA), \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (BA), \u003cem\u003eT. afroharzianum\u003c/em\u003eT22 (T2), \u003cem\u003eT. harzianum\u003c/em\u003e T78 (T7), \u003cem\u003eB. bassiana\u003c/em\u003e (BB), \u003cem\u003eM. robertsii\u003c/em\u003e (MR), consortium 1 (M1) including RI+PA+BA+T2 and consortium 2 (M2) including RI+BB+MR. Non-inoculated plants were included as a control (NI). Boxes represent the interquartile range, black lines represent the median, whiskers represent maximum and minimum within 1.5 times the interquartile range, and black dots represent outliers. Asterisks indicate statistically significant difference compared to the control (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/21bf3eff7f68e620b1d8420f.png"},{"id":52142167,"identity":"3bc54e4e-2150-4dc7-9b5b-50f71e02615c","added_by":"auto","created_at":"2024-03-07 11:28:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSoil microbial effect on overall plant resistance against pests and pathogens. \u003c/strong\u003eRadial plots show the effect of each of the different soil microbial treatments (i.e, plants were inoculated with: \u003cem\u003eR. irregularis\u003c/em\u003e (RI), \u003cem\u003eF. mosseae\u003c/em\u003e (FM), \u003cem\u003eC. etunicatum\u003c/em\u003e (CE), \u003cem\u003eP. azotoformans\u003c/em\u003e (PA), \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (BA), \u003cem\u003eT. afroharzianum\u003c/em\u003eT22 (T2), \u003cem\u003e\u0026nbsp;T. harzianum\u003c/em\u003e T78 (T7), \u003cem\u003eB. bassiana\u003c/em\u003e (BB), \u003cem\u003eM. robertsii\u003c/em\u003e (MR), consortium 1 (M1) including RI+PA+BA+T2 and consortium 2 (M2) including RI+BB+MR) when compared to the non-inoculated plants (red polygons). Radar plots show the scaled values for the percent damage by \u003cem\u003eT. absoluta\u003c/em\u003e per plant, thenumber of damaged leaves per plant by thrips, the number of whiteflies per trap, and the average percent leaves per plant infested by powdery mildew. Bigger values mean that the attack is stronger, and plant resistance is low (and \u003cem\u003evice-versa\u003c/em\u003e). The table shows the scaled area of each polygon.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/34c16abb9258a0ed08b88db8.png"},{"id":52143255,"identity":"f623a55a-ffa8-4c7b-b42d-cd044bdc82f8","added_by":"auto","created_at":"2024-03-07 11:44:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3657710,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/25d44714-d3ff-40f8-a0c5-310a242ae390.pdf"},{"id":52142168,"identity":"9138925d-7533-4721-bf5b-a4471cbe0433","added_by":"auto","created_at":"2024-03-07 11:28:50","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":518586,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3953202/v1/517e5d9b7b9650fa45b900ef.docx"}],"financialInterests":"","formattedTitle":"On testing the effectiveness of soil microbial inoculants in integrated pest management for commercial tomato production","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe urgent need to enhance agricultural sustainability has compelled scientists, agroindustry, growers, and consumers to seek for innovative approaches to mitigate the reliance on agrochemicals, all while ensuring optimal crop yields (Arora, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One emerging environmentally friendly biotechnology is the use of soil-beneficial microbes, applied as bioinoculants, to improve plant growth and productivity across a variety of systems (Berg, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Barea, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Trivedi et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ab Rahman et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Compant et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, several of these microbes have been shown to antagonize soil pathogens (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and boost the resistance of crops to a broad spectrum of pests and diseases, a phenomenon known as induced resistance (IR) (Pieterse et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; De Kesel et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the multifaceted functionality of soil beneficial microbes could be harnessed to potentially increase crop productivity via direct boosting of resource acquisition, and indirectly, by reducing yield losses due to pest and pathogen attack (Li et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Yet, soil beneficial microbes constitute a complex and diverse community of bacteria or fungi (Bakker et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), with their cumulative effect shown to be context and crop dependent (Lee D\u0026iacute;az et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), thereby currently limiting their competitive commercial exploitation against agrochemicals. Consequently, more studies in proper commercial settings are needed to test the efficacy of soil microbes for more sustainable agriculture practices.\u003c/p\u003e \u003cp\u003eBeneficial soil microbes used as bioinoculants include several functional groups, such as the plant growth-promoting rhizobacteria (PGPR), the plant growth-promoting fungi (PGPF), and the arbuscular mycorrhizal fungi (AMF) (Woo et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Aamir et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bitterlich et al., 2020; Bamisile et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides their growth promoting properties, PGPR have been shown to act as efficient biological control agents, either by direct pathogen or disease suppression or through IR (Orozco-Mosqueda et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bacteria from the genera \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e are among the most studied and best characterized PGPR (Santoyo et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Orozco-Mosqueda et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Elnahal et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as evidenced by the high number of commercial biofertilizer and biocontrol products in the market containing them (Aamir et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, the ability of some PGPF species from the genus \u003cem\u003eTrichoderma\u003c/em\u003e to promote plant growth, and resistance to pests and pathogens has been widely acknowledged during the last decades (Harman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Guzm\u0026aacute;n-Guzm\u0026aacute;n et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Poveda, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Papantoniou et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Modrzewska et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, their current success in the market as bioinoculants is primarily attributed to their mycoparasitic capacity, constituting 64.8% of available products claiming to be fungicidal (Woo et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). AMF are obligate biotrophs that establish symbiotic associations with the roots of most terrestrial plants, constituting one of the most studied plant-fungal interactions (Pozo et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This symbiosis has been shown to improve plant nutrient uptake (van der Heijden et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sardans et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and increases plant tolerance to biotic and abiotic stresses (Pozo \u0026amp; Azc\u0026oacute;n-Aguilar, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rivero et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rivero et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dejana et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ram\u0026iacute;rez-Serrano et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Accordingly, the use of AMF has been amply proposed in sustainable agriculture (Smith \u0026amp; Smith, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jeffries \u0026amp; Barea, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Barea, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Salomon et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Martin \u0026amp; van der Heijden \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). AMF-based inoculants are commercially available in the market and the number of companies selling them has steadily increased (Bitterlich et al., 2020). The commercial products containing AMF are mostly used in agriculture as biofertilizers, mainly for nutrient and growth promotion benefits, but also for stress alleviation (Basiru et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Finally, EPF constitute another important group of fungi in agroecosystems because of their well-known ability to infect and kill insect and mite pests (Quesada-Moraga, 2020). Besides this direct antagonism, EPF can interact and colonize plants endophytically, promoting plant growth and negatively affecting pathogens and phytophagous insects without direct contact with them (Gange et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Quesada-Moraga, 2020; Rasool et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bamisile et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). EPF have been used in biological control of insects for more than 150 years, and several products are currently commercially available, with more than 170 species formulated as biopesticides (Bamisile et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides single microbe applications, the design of synthetic microbial communities (SynComs) for improving plant growth and health is receiving increasing interest within the scientific community and on the market (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Trivedi et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Batista \u0026amp; Singh, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). SynComs can offer extended functionality compared to single strain inoculations for the biocontrol of foliar and soil pathogens, as they can simultaneously combine different modes of action (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, the combined application of EPF and AMF showed functional complementarity for plant protection and growth promotion (Zitlalpopoca-Hernandez et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNonetheless, despite the widespread optimism for using these microbial inoculants to improve plant growth and health, most of the research has been performed under highly controlled conditions, posing challenges to the successful transfer and adoption of this technology in agriculture (Mitter et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Saad et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, plant-microbe interactions and their effect on plant growth and health are often conditioned by environmental factors (Saad et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee D\u0026iacute;az et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, temperature (Di Lelio et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), nitrogen or phosphorous fertilization (Ram\u0026iacute;rez-Serrano et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dejana et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), soil water content (Orine et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and light intensity (de La Hoz et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and quality (Saha et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have all been reported to impact plant-microbe interactions and microbial benefits for host plants. In other words, the intricate nature and context dependency of these interactions emphasizes the need to consider how variable environmental conditions and agricultural practices can potentially impede the success and reproducibility of microbial inoculation results under field conditions (Compant et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Consequently, it is essential to perform rigorous assessments of previously characterized plant beneficial microorganisms within real production systems. Such evaluations serve not only to gauge microbial impacts on the target crop under commercial production conditions, but also to ascertain their compatibility with commonly used crop management practices.\u003c/p\u003e \u003cp\u003eOur aim here was to address this gap by investigating how various microbial inoculations impact plant productivity and the interaction between plants and pests and pathogens under commercial production conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We used tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the second most produced vegetable crop worldwide, as model system. We selected well characterized strains of bacteria and fungi, and previously designed SynComs to test their impact on yield and pest resistance in a commercial greenhouse that uses standard tomato management practices, including integrated pest management (IPM) methods (Acebedo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We hypothesized that microbial inoculations would benefit plant performance by reducing susceptibility to biotic stressors without major costs in plant growth or production even under standard crop management practices. We show that microbial inoculation can increase plant resistance to pests without compromising yield, thus supporting the inclusion of microbe-induced resistance in IPM programs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial treatments\u003c/h2\u003e \u003cp\u003eWe performed a large-scale experiment in a commercial greenhouse with a total of 12 microbial treatments, including two bacteria: \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e CECT8238 (BA) and \u003cem\u003ePseudomonas azotoformans\u003c/em\u003e F30A (PA), two strains of \u003cem\u003eTrichoderma afroharzianum\u003c/em\u003e: T22 (T2) and \u003cem\u003eT. harzianum\u003c/em\u003e T78 (T7), two EPF: \u003cem\u003eBeauveria bassiana\u003c/em\u003e KVL 13\u0026ndash;39 (BB) and \u003cem\u003eMetarhizium robertsii\u003c/em\u003e KVL 12\u0026ndash;35 (MR), and three AMF: \u003cem\u003eRhizophagus irregularis\u003c/em\u003e MUCL57021 (RI), \u003cem\u003eFunneliformis mosseae\u003c/em\u003e BEG12 (FM) and \u003cem\u003eClaroideoglomus etunicatum\u003c/em\u003e EEZ163 (CE). In addition, two SynComs (M1, M2), described below, and a control treatment without soil microbe addition (non-inoculated, NI) were included.\u003c/p\u003e \u003cp\u003eThe bacterium \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e was cultured on tryptone soy agar (TSA) and grown at 28\u0026deg;C for 24 hours. For spore production, liquid Difco sporulation medium (Nicholson \u0026amp; Setlow, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) was inoculated with a single bacterial colony and incubated at 28\u0026deg;C for 48 hours with a rotary shaking at 200 rpm. Spore concentration of the liquid culture was quantified using a Neubauer hemocytometer, then the culture was centrifuged for 15 min at 5000 rpm to separate the spores from the growing medium. Finally, the recovered spores were resuspended in sterile water to a concentration of 1 x 10⁷ spores/mL. For inoculation, 1 mL of spore solution was applied to each plant in the root system during transplanting (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The bacterium \u003cem\u003eP. azotoformans\u003c/em\u003e was cultured on TSA and grown at 28\u0026deg;C for 24 hours. A pre-culture was prepared in tryptone soya broth (TSB) inoculated with a single colony and incubated overnight at 28\u0026deg;C with rotary shaking at 200 rpm. Next, 1 mL of pre-culture was added to 25 mL of TSB and incubated at 28\u0026deg;C for 2h 30 min with rotary shaking at 200 rpm to reach the exponential growth phase. Then, the cell concentration was quantified measuring the optical density (620 nm) of the bacterial culture using a spectrophotometer. The bacterial culture was centrifuged for 15 min at 5000 rpm to separate the bacterial cells from the growing medium. Finally, the obtained cells were resuspended in sterile water to a concentration of 1 x 10⁷ CFU/mL. For inoculation, 1 mL of bacterial solution per plant was applied to the root system during transplanting (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The fungus \u003cem\u003eT. afroharzianum\u003c/em\u003e strain T22 was cultured on potato dextrose agar (PDA) and grown at room temperature for seven days. The sporulated plates were scraped using a sterile spatula and sterile water. The resulting spore suspension was filtered using a sterile miracloth filter to remove remaining mycelia and the spore concentration was quantified using a Neubauer hemocytometer and adjusted to 1 x 10⁷ spores/mL. For inoculation 1 mL of spore suspension was added to the root system of each plant during transplanting (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The fungus \u003cem\u003eT. harzianum\u003c/em\u003e strain T78 was cultured on PDA. The fungal inoculum was prepared by adding aseptically a square piece of the fungal culture on a sterile mix of vermiculite and oat (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and incubated at 28\u0026deg;C in the dark for five days. The inoculum, containing 1x10\u003csup\u003e9\u003c/sup\u003e spores/g, was mixed with the substrate in a proportion of 1 g per Kg of substrate (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The EPF \u003cem\u003eB. bassiana\u003c/em\u003e and \u003cem\u003eM. robertsii\u003c/em\u003e were cultured in Sabouraud dextrose agar (SDA) and grown at 24\u0026deg; C in darkness for three weeks. The sporulated plates were scraped using a sterile spatula and the spores were recovered in a sterile solution of Triton X (0.05%). The spore concentration was quantified using a Neubauer hemocytometer and adjusted to 1 x 10\u003csup\u003e8\u003c/sup\u003e spores/mL. Inoculation was done by adding 1 mL of spore suspension per plant directly to the roots during transplanting (Zitlalpopoca-Hernandez et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The AMF \u003cem\u003eR. irregularis\u003c/em\u003e was grown \u003cem\u003ein vitro\u003c/em\u003e on a minimal (M) medium with \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e-transformed carrot (\u003cem\u003eDaucus carota\u003c/em\u003e) roots as host (St-Arnaud et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Spore extraction was performed by adding citrate buffer (0.01 M, pH\u0026thinsp;=\u0026thinsp;6) to the AMF culture in a proportion of 3:1 (v/v) and maintained for 1 hour on a rotary shaker to dissolve the agar. The spores were recollected using sieves with mesh size of 250 and 53 \u0026micro;m and resuspended in sterile water at 1000 spores/mL. For inoculation 1 mL of spore solution was applied to the root system of each plant (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The AMF \u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eC. etunicatum\u003c/em\u003e were maintained as living inocula on mixed cultures of \u003cem\u003eTrifolium repens\u003c/em\u003e and \u003cem\u003eSorghum vulgare\u003c/em\u003e in vermiculite-sepiolite substrate. The inoculants consisted of substrate containing colonized root fragments, mycelia and spores. For inoculation, 10% (v/v) of mycorrhizal inocula were mixed with the substrate at transplanting (Rivero et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther, two SynComs were used. The M1 inoculum included \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e, \u003cem\u003eP. azotoformans\u003c/em\u003e and \u003cem\u003eT. afroharzianum\u003c/em\u003e T22 at concentration of 1 x 10⁷ CFU/mL each, and \u003cem\u003eR. irregularis\u003c/em\u003e at a concentration of 1000 spores/mL (Minchev et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The M2 inoculum included \u003cem\u003eM. robertsii\u003c/em\u003e and \u003cem\u003eB. bassiana\u003c/em\u003e both inoculated at a concentration of 1 x 10\u003csup\u003e8\u003c/sup\u003e spores/mL, and \u003cem\u003eR. irregularis\u003c/em\u003e at a concentration of 1000 spores/mL. For both SynComs 1mL/plant was applied to the root system during transplanting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growing conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSolanum lycopersicum\u003c/em\u003e cv Money maker seeds (Vreeken\u0026rsquo;s Zaden, The Netherlands) were surface sterilized by immersion in 5% Sodium hypochlorite solution for 10 min and rinse three times in sterile water for 10 min each. The surface sterilized seeds were sown in sterile vermiculite and incubated for seven days in a greenhouse at 24\u0026deg;C : 16\u0026deg;C day : night with a photoperiod 16 h: 8 h light : dark and 70% of relative humidity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental set up\u003c/h3\u003e\n\u003cp\u003eOne-week old tomato seedlings were transferred to starting trays, with cell dimensions 2.9 x 2.9 x 6.8 cm- containing blond seedling peat (Kekkil\u0026auml; LSM 0 R8406, Projar, Valencia, Spain): zeolite: perlite (1:1:1) mixture and inoculated with the microbial treatments described previously. Inoculated seedlings were grown in a commercial nursery (ACRENA SAT 251, El Ejido, Spain; 36\u0026deg;, 47', 52.9''N; 2\u0026deg;, 43', 36.3''W) for four weeks. On September 3rd, 2020 the plants were transplanted to a commercial production greenhouse (Estaci\u0026oacute;n experimental Cajamar, Paraje las Palmerillas, El Ejido, Almer\u0026iacute;a; 36\u0026deg;, 47', 36.3\"N; 2\u0026deg;, 43', 15.2\"W) and maintained during the whole crop cycle from September 2020 to March 2021. The greenhouse consisted of a typical \"raspa y amagado\" type (\u0026Aacute;valos-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), 37.8 m long and 23.2 m wide with a total area of 877 m\u003csup\u003e2\u003c/sup\u003e and usable area of 720 m\u003csup\u003e2\u003c/sup\u003e, passive ventilation (25.0% window surface) with side windows (north and south sides) and zeniths, covered with anti-trip mesh. The microbial inoculation treatments were organized following a randomized complete block design, with four blocks. Each block contained all 12 treatments, and each treatment in all blocks was replicated with six plants (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e;\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;12 treatments x 4 blocks x 6 replicates\u0026thinsp;=\u0026thinsp;288 plants).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiological control, pheromone and pollinator application\u003c/h2\u003e \u003cp\u003eTwo weeks after transplanting, the predatory mirid bug \u003cem\u003eNesidiocoris tenuis\u003c/em\u003e (Hemiptera: Miridae) (NESIDIOcontrol, Agrobio, Spain) was released in the greenhouse with a density of 0.5\u0026ndash;1.5 individuals/m2 following the product label recommendation, to reduce incidence of whiteflies (Hemiptera: Sternorrhyncha) and \u003cem\u003eTuta absoluta\u003c/em\u003e (Lepidoptera: Gelechiidae) on tomato plants. In addition, pheromones for the mating disruption of \u003cem\u003eT. absoluta\u003c/em\u003e were released during the whole cropping season. For ensuring pollination of tomato flowers from the start of flowering, \u003cem\u003eBombus terrestris\u003c/em\u003e bumblebees (Hymenoptera: Apidae) were released three weeks post transplantation (wpt), placing one hive (Agrobio, Spain) in the middle of the greenhouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIrrigation and fertilization\u003c/h2\u003e \u003cp\u003eThe irrigation scheme during the whole cropping season and nutrient supply are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Nutrient content in soil and irrigation water (nutrient solution) was evaluated periodically to adjust to the crop needs for nutrient supply. Specifically, phosphorus was measured by visible spectrophotometry using the compound phosphorous vanadate molybdate (Tandon et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). Nitrates were measured spectrophotometrically at 220 and 275 nm (Norman \u0026amp; Stucki, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Ammonia was measured by the Nessler reagent method (Yuen \u0026amp; Pollard, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). Sodium, calcium, potassium, magnesium, iron, copper, manganese and zinc, were determined by atomic absorption / emission (Isaac \u0026amp; Kerber, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Carbonates and bicarbonates were measured by titration with 0.01 N sulfuric acid (Allison et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). Chlorides were also measured by volumetry with silver nitrate between 0.01 and 1 N using potassium chromate as an indicator (Mohr's titration). Boron was determined by spectrophotometry with the azomethine reaction (John et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Sulfates were measured by precipitation of barium sulfate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eResponse variables and data collection\u003c/h2\u003e \u003cp\u003eIn total we measured 17 response variables related to plant growth and yield, and plant resistance to pathogens and insect pests (See below, and \u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth, nutritional status and yield\u003c/h2\u003e \u003cp\u003eAs a proxy of plant growth, plant height from soil surface to the top of the shoot of each plant (6 plants per treatment per block) was measured on December 3rd, 2020 (12 wpt). As a proxy for plant productivity, we quantified the number of inflorescences per plant (6 plants per treatment per block) on October 26th, 2020 (8 wpt). Total leaf carbon and nitrogen content was measured on the January 21st, 2021 (19 wpt), evaluating 3 plants per treatment per block. Leaves were sampled, immediately frozen in liquid nitrogen and lyophilized. Then lyophilized leaves were ground in a Tissue Lyser II (Qiagen, Germany) using metal beads at a maximum speed for 3 min. Two milligrams were weighed from each sample for analysis of carbon (C) and nitrogen (N) content in the Flash 1112 Elemental Analyzer (Thermo Scientific, MA, USA).\u003c/p\u003e \u003cp\u003eFruit productivity (average g/plant) and quality were evaluated every week between November 12th, 2020 (10 wpt) and February 4th, 2021 (21 wpt) on 6 plants per treatment per block. Tomato fruits were classified by size (size GG 82-102 mm; size G 67‐82 mm; size M 57‐67 mm; size MM 47‐57 mm) and by categories (first, second and non‐commercial). Fruits were considered non-commercial when their size was too small (\u0026lt;\u0026thinsp;45 mm of diameter), when they showed presence of pathogen damage, cracks, blossom-end rot or blotchy ripening, or when they were misshapen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFruit quality and nutraceutical value\u003c/h2\u003e \u003cp\u003eParameters such as fruit dry weight (determined after drying the fruits in a forced air stove at 70 \u0026ordm;C for 48 hours), acidity % (Acid-base volumetry using 1 N NaOH as base and phenolphthalein indicator), \u0026ordm;Brix or total soluble solids (manual refractometer), maturity index (the ratio between the content of total soluble solids and assessable acidity) were assessed on December 16th, 2020 (14 wpt) on one fruit per treatment per block (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003ePolyphenol and carotenoid content in fruits were evaluated on February 25th, 2021 (23 wpt) on one fruit per treatment per block (n\u0026thinsp;=\u0026thinsp;4). Polyphenols were measured by the spectrophotometric method of Folin-Ciocalteau (Georg\u0026eacute; et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) using a standard curve of gallic acid from 0 to 1000 ppm at 760 nm (double ultraviolet‐visible beam, Unicam Helios Alpha) and expressed as mg of gallic acid/100g dry fruit biomass. Lycopene and beta-carotene content of fruits was measured with an acetone‐hexane extraction and spectrophotometric determination at 487.5 nm (Sadler et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) with modifications (Rousseaux et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and expressed as mg/100g fresh fruit.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePest and disease incidence.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe incidence of thrips, \u003cem\u003eT. absoluta\u003c/em\u003e, whiteflies and powdery mildew was evaluated on December 3rd, 2020 (12 wpt). For thrips, occurrence was assessed by counting the number of leaves per plant presenting lacerations caused by thrips. The incidence of \u003cem\u003eT. absoluta\u003c/em\u003e was estimated as the percentage of plants per treatment displaying mines (the typical lesions caused by the larvae of this species). Whiteflies were evaluated using yellow sticky traps for a period of 4 weeks until December 3rd, 2020 (12 wpt), placing one sticky trap per treatment per block and assessing the number of whiteflies per trap. Finally, powdery mildew prevalence was measured as the percentage of infected plants per treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAbundance of natural enemies\u003c/h2\u003e \u003cp\u003eThe abundance of the predatory mirid bug \u003cem\u003eNesidiocoris tenuis\u003c/em\u003e, released in the greenhouse at the beginning of the cropping season for the control of whiteflies and \u003cem\u003eT. absoluta\u003c/em\u003e, was evaluated on December 3rd, 2020 (12 wpt), based on evaluation of the same yellow sticky traps as for whitefly incidence (see above) and counting the number of \u003cem\u003eN. tenuis\u003c/em\u003e per trap.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analysed using R statistical language, version 4.1.1 (R Development Core Team 2021) and figures were produced using the package ggplot2 (Wickham, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The effects of the 12 microbial treatments (including the control) on the 17 different response variables were analyzed using linear (lm) or generalized linear models (glm) following the details as shown \u003cb\u003eTable S2\u003c/b\u003e. Treatment effects were always compared to the control (non-inoculated) treatment, as shown by the asterisks in Figures presented in the \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section. Moreover, to measure the overall effect of soil microbial inoculation on plant resistance, we produced radar plots using scaled values for each of the four pests and pathogens studied (function \u003cem\u003eggradar\u003c/em\u003e in ggplot2), and calculated the area of each polygon generated for each treatment using R.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of soil beneficial microbes on plant growth, nutritional status and flowering\u003c/h2\u003e \u003cp\u003eTo assess the effect of soil beneficial microbes on tomato plants\u0026rsquo; growth, nutritional status and reproduction potential, we measured plant height, leaf carbon and nitrogen content and their ratio (C/N) and counted the number of inflorescences. For plant height, we found weak negative effects of the soil microbial treatments, in which only the AMF \u003cem\u003eC. etunicatum\u003c/em\u003e decreased tomato plant height on average by 12.96 cm compared to the control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, treatment effect; F\u003csub\u003e11,264\u003c/sub\u003e = 2.29, p\u0026thinsp;=\u0026thinsp;0.01). We however found no effect of soil microbial treatments on C and N leaf content, resulting in an unaltered C/N (F\u003csub\u003e11,116\u003c/sub\u003e = 1.3, p\u0026thinsp;=\u0026thinsp;0.23; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), nor on the number of inflorescences (F\u003csub\u003e11,130\u003c/sub\u003e = 1, p\u0026thinsp;=\u0026thinsp;0.45; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of soil beneficial microbes on fruit yield\u003c/h2\u003e \u003cp\u003eTo evaluate the impact of the microbial inoculants on fruit production, we assessed the fruit yield during the whole fruit production period, quantifying the total production as well as the commercial quality production. Soil microbial inoculations had a prominent effect on total tomato production (Chisq\u003csub\u003e11,464\u003c/sub\u003e = 23.1, p\u0026thinsp;=\u0026thinsp;0.017; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), as well as on commercial quality tomato production (Chisq\u003csub\u003e11,464\u003c/sub\u003e = 25.1, p\u0026thinsp;=\u0026thinsp;0.009; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Specifically, \u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eT. afroharzianum\u003c/em\u003e T22 inoculated plants showed a 13% and 15% higher total productivity than control plants, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Even more so, the same soil microbes increased commercial quality fruit production by 12% and 14% at the end of the experiment as compared to the non-inoculated control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of soil beneficial microbes on fruit quality and nutraceutical value\u003c/h2\u003e \u003cp\u003eFruit quality was evaluated by assessing different parameters: \u0026ordm;Brix, %Acidity, Maturity index and %Dry weight. We did not observe any effect of the microbial inoculations on \u0026ordm;Brix (F\u003csub\u003e11,33\u003c/sub\u003e = 1.00, p\u0026thinsp;=\u0026thinsp;0.47), Maturity index (F\u003csub\u003e11,33\u003c/sub\u003e = 1.19, p\u0026thinsp;=\u0026thinsp;0.31) and %Dry weight (F\u003csub\u003e11,33\u003c/sub\u003e = 0.86, p\u0026thinsp;=\u0026thinsp;0.59). The only fruit quality parameter significantly affected by the microbial treatments was the %Acidity (F\u003csub\u003e11,33\u003c/sub\u003e = 2.46, p\u0026thinsp;=\u0026thinsp;0.02), in which fruits from \u003cem\u003eC. etunicatum\u003c/em\u003e inoculated plants showed an increase of 0.04% in acidity compared to the control treatment (t\u0026thinsp;=\u0026thinsp;2.25, p\u0026thinsp;=\u0026thinsp;0.03; \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, regarding fruit nutraceutical value, we found that fruit polyphenols\u0026rsquo; content was not significantly affected by the soil microbial treatment (F\u003csub\u003e11,36\u003c/sub\u003e = 0.61, p\u0026thinsp;=\u0026thinsp;0.81, \u003cb\u003eFigure S2A\u003c/b\u003e), as well as carotenoids\u0026rsquo; content of fruits (lycopene, beta-carotene and total carotenoids) were also not significantly altered by soil microbes (F\u003csub\u003e11,36\u003c/sub\u003e = 1.25, p\u0026thinsp;=\u0026thinsp;0.29, \u003cb\u003eFigure S2B;\u003c/b\u003e F\u003csub\u003e11,36\u003c/sub\u003e = 0.66, p\u0026thinsp;=\u0026thinsp;0.77, \u003cb\u003eFigure S2C and\u003c/b\u003e F\u003csub\u003e11,36\u003c/sub\u003e = 1.18, p\u0026thinsp;=\u0026thinsp;0.34, \u003cb\u003eFigure S2D\u003c/b\u003e, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of soil beneficial microbes on natural enemies\u003c/h2\u003e \u003cp\u003eTo evaluate any potential impact of the microbial inoculants on applied beneficial insects, we evaluated the abundance of the predatory mirid bug \u003cem\u003eN. tenuis\u003c/em\u003e, released in the greenhouse at the beginning of the cropping season for the control of whiteflies and \u003cem\u003eT. absoluta\u003c/em\u003e. We did not find significant differences in \u003cem\u003eN. tenuis\u003c/em\u003e abundance among the different microbial treatments, indicating no negative effect of the microbial inoculants on the predator (F\u003csub\u003e11,36\u003c/sub\u003e = 0.55, p\u0026thinsp;=\u0026thinsp;0.86; \u003cb\u003eFigure S3\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of soil beneficial microbes on pest and disease incidence\u003c/h2\u003e \u003cp\u003eWe next assessed the natural incidence of different pests and diseases appearing during the cropping season. We found that the incidence of \u003cem\u003eT. absoluta\u003c/em\u003e was significantly impacted by microbial inoculation (Chisq\u003csub\u003e11,273\u003c/sub\u003e = 24.37, p\u0026thinsp;=\u0026thinsp;0.01). In particular \u003cem\u003eR. irregularis\u003c/em\u003e (z = -2.698, p\u0026thinsp;=\u0026thinsp;0.007), \u003cem\u003eF. mosseae\u003c/em\u003e (z = -2.31, p\u0026thinsp;=\u0026thinsp;0.02), \u003cem\u003eC. etunicatum\u003c/em\u003e (z = -2.31, p\u0026thinsp;=\u0026thinsp;0.02), \u003cem\u003eT. afroharzianum\u003c/em\u003e T22 (z = -2.31, p\u0026thinsp;=\u0026thinsp;0.02), \u003cem\u003eT. harzianum\u003c/em\u003e T78 (z = -1.98, p\u0026thinsp;=\u0026thinsp;0.05), \u003cem\u003eM. robertsii\u003c/em\u003e (z = -2.69, p\u0026thinsp;=\u0026thinsp;0.007) and the consortium M2 (z = -1.98, p\u0026thinsp;=\u0026thinsp;0.05) treatments significantly decreased the percentage of plants damaged by the leaf miner as compared to the control treatment, with average reductions ranging from 60% for \u003cem\u003eT. harzianum\u003c/em\u003e T78 up to 90% for \u003cem\u003eR. irregularis\u003c/em\u003e and \u003cem\u003eM. robertsii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Thrips and whitefly incidences were not significantly affected by the microbial treatments (Chisq\u003csub\u003e11,131\u003c/sub\u003e = 15.82, p\u0026thinsp;=\u0026thinsp;0.15; \u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e and F\u003csub\u003e11,36\u003c/sub\u003e = 1, p\u0026thinsp;=\u0026thinsp;0.46; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Regarding diseases, the powdery mildew was the only pathogen that naturally appeared on the crop. No significant effect of the microbial treatments was observed on the incidence of powdery mildew (Chisq\u003csub\u003e11,132\u003c/sub\u003e = 9.01, p\u0026thinsp;=\u0026thinsp;0.62; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCompound effect of soil beneficial microbes on plant resistance\u003c/h2\u003e \u003cp\u003eWe visualized the overall effect of each different soil microbial inoculation with radar plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For this analysis, smaller areas indicate higher resistance induced by the soil microbial inoculations. Notably, the polygon for the control (0.8; red polygons of Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) show the highest area compared to rest of treatments (expect for \u003cem\u003eC. etunicatum\u003c/em\u003e; see area values in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Of those, the smallest area is with \u003cem\u003eR. irregularis\u003c/em\u003e, with a decrease of 95% from the control treatment. Those results suggest that besides \u003cem\u003eC. etunicatum\u003c/em\u003e, soil microbial inoculations increase general resistance of tomato plants (smaller areas in our model) against the four pest and pathogens studied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, by testing diverse plant beneficial microorganisms under commercial settings, we demonstrated the viability of using microbial inoculants for crop protection and yield improvement for a commercial crop production system. We have identified microbial strains that can be used as biostimulants and bioprotectors under real tomato production conditions, thus confirming their potential as bioinoculants to improve agricultural sustainability. Considering all measured traits, our results point out a prominent effect of fungal inoculants (including different AMF, EPF and \u003cem\u003eTrichoderma\u003c/em\u003e strains) in promoting plant resistance, and in some cases, improving crop yield, while the tested bacterial inoculants did not show any significant effects on the evaluated parameters.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSoil beneficial microbe effect on tomato growth and yield\u003c/h2\u003e \u003cp\u003ePlant beneficial microbes such as PGPR, \u003cem\u003eTrichoderma\u003c/em\u003e, AMF and EPF have been widely reported to improve plant growth and nutritional status (Quesada Moraga, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Orozco-Mosqueda et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Salomon et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Contrary to previous observations, the microbial inoculation did not impact plant height or leaf C/N under commercial production conditions. Although plants inoculated with the AMF \u003cem\u003eC. etunicatum\u003c/em\u003e were significantly smaller than control plants, this reduction did not negatively affect tomato yield, which is the most relevant parameter for tomato producers. The lack of microbial effects on plant nutritional levels can be related to the standard periodic application of fertilization, so that the plant nutritional needs were sufficiently covered, making the role of nutrient acquisition by the beneficial microbes redundant. Indeed, nutritional benefits by interaction with beneficial microorganisms are usually visible only under limiting conditions (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Specifically, under reduced fertilization dosage, some of the tested microbes such as \u003cem\u003eT. harzianum\u003c/em\u003e T78, \u003cem\u003eR. irregularis\u003c/em\u003e and \u003cem\u003eF. mosseae\u003c/em\u003e, has shown to improve plant growth, nutrient acquisition and fruit production in melon plants under field conditions, while such effects were absent under conventional fertilization conditions (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYet, when evaluating the potential of microbial inoculants as biostimulants, crop yield and fruit quality are the most relevant parameters, particularly for the economy of producers. A recent meta-analysis conducted on 97 peer-reviewed articles (69% conducted under greenhouse and 31% under field conditions) that examined the effect of different microbial inoculants -mostly PGPR- on crop productivity concluded that microbial inoculants can overall improve crop productivity, mainly by stress alleviation or by improving nutrient availability for plants (Li et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our findings demonstrate that while none of the treatments influenced flower production, two fungal treatments, the AMF \u003cem\u003eF. mosseae\u003c/em\u003e and the fungus \u003cem\u003eT. afroharzianum\u003c/em\u003e T22, increased the total and marketable tomato yield during the cropping season. Increased tomato yield after microbial inoculants application can derive from a better alleviation of stress (Li et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In agreement with this hypothesis, we observed that the two fungal treatments that increased yield also caused significant pest reduction of the leaf miner \u003cem\u003eT. absoluta\u003c/em\u003e (see below). Of particular interest is the fact that tomato plants with increased yield did not show a trade-off by reducing fruit quality, suggesting a net benefit for the farmers under these conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSoil microbial effect on tomato resistance against pests\u003c/h2\u003e \u003cp\u003eSoil-borne beneficial microbes are widely reported to improve plant resistance by triggering defenses against a broad range of attackers, including pathogens and herbivorous insects (Pieterse et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Here, we evaluated the impact of microbial inoculation on the incidences of powdery mildew, the phloem and cell-content feeders whiteflies and thrips respectively, and the leaf miner \u003cem\u003eT. absoluta\u003c/em\u003e. While we found no effect of soil microbes on powdery mildew, thrips or whiteflies, the percentage of plants damaged by the leafminer \u003cem\u003eT. absoluta\u003c/em\u003e was significantly reduced by most of the fungal inocula. This benefit was not observed upon inoculation with PGPR, underscoring the positive outcomes associated with fungal inoculations. All three mycorrhizal strains, both \u003cem\u003eTrichoderma\u003c/em\u003e strains, the EPF \u003cem\u003eM. robertsii\u003c/em\u003e and the M2 SynCom (a fungal consortia including the EPF \u003cem\u003eB. bassiana\u003c/em\u003e and \u003cem\u003eM. robertsii\u003c/em\u003e, and the AMF \u003cem\u003eR. irregularis\u003c/em\u003e) reduced the natural incidence of \u003cem\u003eT. absoluta\u003c/em\u003e, in some cases (\u003cem\u003eR. irregularis\u003c/em\u003e and \u003cem\u003eM. robertsii\u003c/em\u003e) up to 90%. These results agree with recent studies showing induced resistance against \u003cem\u003eT. absoluta\u003c/em\u003e under controlled conditions by strains of AMF (Shafiei et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), T. \u003cem\u003eafroharzianum\u003c/em\u003e (Aprile et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and by the EPF \u003cem\u003eB. bassiana\u003c/em\u003e and \u003cem\u003eM. anisopliae\u003c/em\u003e (Giannoulakis et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile AMF and \u003cem\u003eTrichoderma\u003c/em\u003e are widely documented to induce plant resistance against very diverse pathogens and pests (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Coppola et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sanmart\u0026iacute;n et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Di Lelio et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rivero et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dejana et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), only few recent studies have demonstrated their negative impact on the performance of the leafminer \u003cem\u003eT. absoluta\u003c/em\u003e (Aprile et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shafiei et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For EPF, most studies are focused on the direct biological control action of fungal conidia resulting in the infection or reduction of the performance and fitness of \u003cem\u003eT. absoluta\u003c/em\u003e (Chouikhi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). More recently, evidence on the induction of plant resistance by EPF against insects and pathogens is increasing (Raad et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rivas-Franco et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rasool et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zitlalpopoca-Hernandez et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This effect is particularly important for concealed pest stages such as mining larvae, which are hidden for contact with sprayed biopesticides. Thus, the results illustrate the ability of AMF, \u003cem\u003eTrichoderma\u003c/em\u003e and EPF to enhance plant resistance and reduce \u003cem\u003eT. absoluta\u003c/em\u003e incidence in commercial production conditions, and thus, to improve or complement current IPM practices in tomato crop protection.\u003c/p\u003e \u003cp\u003eWorldwide, \u003cem\u003eT. absoluta\u003c/em\u003e is a major pest of tomato (Biondi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) so the present results are encouraging for its management. The high reproductive capacity of \u003cem\u003eT. absoluta\u003c/em\u003e allows for rapid population growth and widespread infestation, and its miner lifestyle difficult the use of surface applied (bio)pesticides (Abd El-Ghany et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, \u003cem\u003eT. absoluta\u003c/em\u003e is known for its ability to develop resistance to chemical pesticides, making control measures against this pest even more challenging (Guedes et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hence, applying effective soil microbes that can hinder the development of this pest, without reducing fruit quality nor yield -and even improving it-, can be an efficient and ecologically-sound solution for tomato crop protection.\u003c/p\u003e \u003cp\u003eHowever, in real crop production several different pests and diseases could emerge at the same time or sequentially during the cropping season, challenging the crop performance and productivity. Thus, we explored the impact of each microbial inoculant on the global pest and disease incidence, considering all four aggressors, to get an insight on the impact of microbial inoculations on the overall plant resistance to pests and pathogens. We found that most of the beneficial microbes used in our study increased the global plant resistance, prominently reducing the overall pest and disease incidence in the crop. Remarkably, for some microbial strains as the AMF \u003cem\u003eR. irregularis\u003c/em\u003e and the PGPF \u003cem\u003eT. harzianum\u003c/em\u003e the reduction was around 95% as compared to the non-inoculated control plants. These finding are in agreement with the general idea that IR by beneficial microbes such as AMF and \u003cem\u003eTrichoderma\u003c/em\u003e could be effective against a broad range of pest and pathogens (Mart\u0026iacute;nez-Medina et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sanmart\u0026iacute;n et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Di Lelio et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rivero et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe successful implementation of microbe-induced resistance in agriculture does not only rely on its effectiveness to control pests and pathogens but also on its compatibility with other strategies regularly used in IPM (Stenberg, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). One point of caution would be if the microbes, by modifying plant defenses, may negatively impact auxiliary fauna, as for example biocontrol insects. As this study was performed under common pest management practices in Spanish tomato production based on IPM (Acebedo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we also evaluated the effect of the microbial inoculation on the pest predator \u003cem\u003eN. tenuis\u003c/em\u003e. Our results did not show any negative effects of the inoculations on the abundance of the predator, suggesting that microbe-induced resistance is compatible with the release of this predator, a generalist biocontrol agent commonly used in IPM programs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOverall, this study highlights the potential of soil-inoculated microorganisms, particularly fungi, to improve tomato crop productivity and resistance to important pests such as the devastating leaf miner \u003cem\u003eT. absoluta\u003c/em\u003e. By testing microbial strains -previously characterized under controlled lab conditions- in agronomic settings we identified beneficial microbes that are competent and functional under commercial growing conditions. The identification of microbes that effectively improve plant health and productivity in real crop production system will contribute to a faster and wider adoption of microbial inoculants for environmentally safe crop protection and to improve future agricultural sustainability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the European Union\u0026rsquo;s Horizon 2020 Research and Innovation Programme under grant agreement No 765290 in the project \u0026ldquo;MiRA \u0026ndash; Microbe-induced Resistance to Agricultural Pests\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026quot; Conceptualization, M.J.P, S.R, A.B., T.H., N.V.M and Z.M; Investigation, Z.M., B.R.S., L.D, A.L.D, G.Z.H., D.O., H.S., D.P., J.M.G., A.G.; Formal analysis, S.R. and Z.M; Writing \u0026ndash; Original Draft, Z.M. and M.J.P.; Writing \u0026ndash;Review \u0026amp; Editing, M.J.P., S.R., A.B., A.M.M., N.V.M., D.O., B.R.S. and Z.M; Visualization, S.R. and Z.M; Funding Acquisition, T.H., A.B., M.J.P., S.R., A.M.M, N.V.D. , N.V.M, R.S., D.G., P.G; Project administration, M.J.P. and Z.M.; Supervision, M.J.P.\u0026quot;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAamir M, Rai KK, Zehra A, Dubey MK, Kumar S, Shukla V, Upadhyay RS (2020) Microbial bioformulation-based plant biostimulants: a plausible approach toward next generation of sustainable agriculture. 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Biol Control 175:1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biocontrol.2022.105034\u003c/span\u003e\u003cspan address=\"10.1016/j.biocontrol.2022.105034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"agronomy-for-sustainable-development","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ASDE","sideBox":"Learn more about [Agronomy for Sustainable Development](https://www.springer.com/journal/13593)","snPcode":"13593","submissionUrl":"https://www2.cloud.editorialmanager.com/asde/default2.aspx","title":"Agronomy for Sustainable Development","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Beneficial soil-borne microorganisms, bioinoculants, crop protection, field research, microbe-induced resistance, plant-microbe-insect interaction, Tuta absoluta, yield improvement","lastPublishedDoi":"10.21203/rs.3.rs-3953202/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3953202/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResearch is showing that soil-borne beneficial microorganisms can enhance plant growth, productivity, and resistance against pests and pathogens, and could thus serve as a sustainable alternative to agrochemicals. To date, however, the effect of soil beneficial microbes under commercial crop production has not been fully assessed. We here investigated the effect of root inoculation with 11 well-characterized bacterial and fungal strains on tomato performance under intensive tomato crop management practices. We measured the impact of these strains on plant growth, fruit quality, yield, and pest and pathogen incidence. While most microbial strains showed weak effects, we found that the fungal strains \u003cem\u003eTrichoderma afroharzianum\u003c/em\u003e T22 and \u003cem\u003eFunneliformis mosseae\u003c/em\u003e significantly increased marketable tomato yield. Moreover, we found that inoculation with most of the fungal strains led to a significant reduction in the incidence of the devastating leaf mining pest \u003cem\u003eTuta absoluta\u003c/em\u003e, while this effect was not observed for bacterial inoculants. In addition, we found that microbial inoculations did not impact the incidence of introduced natural enemies, supporting their compatibility with well-established integrated pest management strategies in horticulture. In sum, the observed general positive effects of soil microbes on tomato yield and resistance reinforce the move toward a broader adoption of microbial inoculants in future crop production, ultimately improving agricultural sustainability.\u003c/p\u003e","manuscriptTitle":"On testing the effectiveness of soil microbial inoculants in integrated pest management for commercial tomato production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 11:28:45","doi":"10.21203/rs.3.rs-3953202/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-03-07T12:44:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-05T09:58:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Agronomy for Sustainable Development","date":"2024-02-22T19:14:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-15T09:01:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Agronomy for Sustainable Development","date":"2024-02-13T04:02:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"agronomy-for-sustainable-development","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ASDE","sideBox":"Learn more about [Agronomy for Sustainable Development](https://www.springer.com/journal/13593)","snPcode":"13593","submissionUrl":"https://www2.cloud.editorialmanager.com/asde/default2.aspx","title":"Agronomy for Sustainable Development","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7058aaf4-7e69-42e3-8e04-cb8eb33d0b36","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-08T08:31:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 11:28:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3953202","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3953202","identity":"rs-3953202","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}