Phosphorus availability drives mycorrhiza induced resistance in tomato

Phosphorus availability drives mycorrhiza induced resistance in tomato Laura Dejana 1 , Beatriz Ramı ´rez-Serrano 2 , Javier Rivero 1 , Jordi Gamir 3 , Juan A. Lo ´pez-Ra ´ez 1 and Marı ´aJ.Pozo 1 * 1 Department of Soil Microbiology and Symbiotic Systems, Estacio ´n Experimental del Zaidı ´n, Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Granada, Spain, 2 Institut de Recherche sur la Biologie de l’Insecte (IRBI), UMR 7261, /Universite de Tours Centre National de la Recherche Scientifique (CNRS), Tours, France, 3 Plant Immunity and Biochemistry Group, Department of Biology Biochemistry and Natural Sciences, Universitat Jaume I, Avd. Vicente Sos Baynat s/n, Castello ´n, Spain Arbuscular mycorrhizal (AM) symbiosis can provide multiple benefits to the host plant, including improved nutrition and protection against biotic stress. Mycorrhiza induced resistance (MIR) against pathogens and insect herbivores has been reported in different plant systems, but nutrient availability may influence the outcome of the interaction. Phosphorus (P) is a key nutrient for plants and insects, but also a regulatory factor for AM establishment and functioning. However, little is known about how AM symbiosis and P interact to regulate plant resistance to pests. Here, using the tomato-Funneliformis mosseae mycorrhizal system, we analyzed the effect of moderate differences in P fertilization on plant and pest performance, and on MIR against biotic stressors including the fungal pathogen Botrytis cinerea and the insect herbivore Spodoperta exigua. P fertilization impacted plant nutritional value, plant defenses, disease development and caterpillar survival, but these effects were modulated by the mycorrhizal status of the plant. Enhanced resistance of F. mosseae-inoculated plants against B. cinerea and S. exigua depended on P availability, as no protection was observed under the most P-limiting conditions. MIR was not directly explained by changes in the plant nutritional status nor to basal differences in defense-related phytohormones. Analysis of early plant defense responses to the damage associated molecules oligogalacturonides showed primed transcriptional activation of plant defenses occurring at intermediate P levels, but not under severe P limitation. The results show that P influences mycorrhizal priming of plant defenses and the resulting induced-resistance is dependent on P availability, and suggest that mycorrhiza fine-tunes the plant growth vs defense prioritization depending on P availability. Our results highlight how MIR is context dependent, thus unravel molecular mechanism based on plant defence in will contribute to improve the efficacy of mycorrhizal inoculants in crop protection. KEYWORDS DAMPs (damage-associated molecular patterns), defense priming, jasmonate signalling, plant immunity, plant nutrition, oligogalacturonides (OGs), herbivory, pathogen Frontiers in Plant Science frontiersin.org01 OPEN ACCESS EDITED BY Artemio Mendoza-Mendoza, Lincoln University, New Zealand REVIEWED BY Wenwu Zhou, Zhejiang University, China Karin E. Groten, Max Planck Institute for Chemical Ecology, Germany *CORRESPONDENCE Marı ´a J. Pozo [email protected] SPECIALTY SECTION This article was submitted to Plant Pathogen Interactions, a section of the journal Frontiers in Plant Science RECEIVED 03 October 2022 ACCEPTED 24 November 2022 PUBLISHED 19 December 2022 CITATION Dejana L, Ramı ´rez-Serrano B, Rivero J, Gamir J, Lo ´pez-Ra ´ez JA and Pozo MJ (2022) Phosphorus availability drives mycorrhiza induced resistance in tomato. Front. Plant Sci. 13:1060926. doi: 10.3389/fpls.2022.1060926 COPYRIGHT © 2022 Dejana, Ramı ´rez-Serrano, Rivero, Gamir, Lo ´pez-Ra ´ez and Pozo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 19 December 2022 DOI 10.3389/fpls.2022.1060926 Introduction The development of sustainable technologies for crop management aiming to reduce the use of fertilizers and pesticides is an ongoing global challenge in agriculture (Pretty and Bharucha, 2018). In this scenario, the potential of beneficial symbiotic microbes to improve plant nutrition and stress resistance/tolerance is well established (Berendsen et al., 2012; Pieterse et al., 2014;Pozo et al, 2020). However, their performance under agronomic conditions is not optimal, as the outcomes of plant-microbe interactions are highly context-dependent and may vary according to the plant growing conditions (van der Heyde et al., 2017;Lee Dı  az et al., 2021). Among the beneficial organisms used as inoculants in agriculture, fungi, and particularly arbuscular mycorrhizal fungi (AMF), are receiving increasing interest (Pozo et al., 2021). These soil-borne microorganisms, belonging to the phylum Glomeromycota, are widespread in natural and agricultural ecosystems and colonize the roots of more than 80% of terrestrial plant species, including major crops (Smith and Smith, 2011). This mutualistic association, known as mycorrhiza, has major benefits for both partners: AMF receive plant organic carbon in the form of carbohydrates and lipids (Keymer et al., 2017;Salmeron-Santiago et al, 2021). In return, AMF improve water and nutrients uptake by the plant, especially phosphorus (P) (Chen et al., 2018;Ferrol et al, 2019). In addition, they also increase plant phenotypic and metabolic plasticity to cope with biotic and abiotic stressors (Rivero et al., 2018;SanchezBel et al, 2018;Campo et al, 2020;Mitra et al., 2021;Rivero et al., 2021;Pozo de la Hoz et al., 2021). AM symbiosis usually renders the plant more resistant to certain soil-borne and aboveground pathogens and chewing herbivores, as shown in multiple systems (Jung et al., 2012; Song et al., 2013;Song et al., 2015;Selvaraj and Thangavel, 2021; Dowarah et al., 2022). This Mycorrhiza-Induced Resistance (MIR) is related to an improved ability of mycorrhizal plants to trigger defense responses upon challenge, a cost-efficient strategy known as defense priming (Pozo and Azcon-Aguilar, 2007;Martinez-Medina et al., 2016;Mauch-Mani et al., 2017). Priming is a common mechanism during induced systemic resistance triggered by beneficial microbes, consisting of a stronger and faster activation of plant defense mechanisms, usually dependent on jasmonate signaling (Pieterse et al., 2014; Mora-Romero et al., 2014;Gruden et al., 2020). Despite priming of plant defenses in response to pathogens and herbivores in mycorrhizal plants is well documented (Campos-Soriano et al., 2012;Song et al., 2013;Song et al., 2015;Sanchez-Bel et al., 2016; Fiorilli et al., 2018;Schoenherr et al., 2019;Sanmartı  n et al., 2020;Rivero et al., 2021;Manresa-Grao et al., 2022) consequences of AM symbiosis for insect herbivores are complex to predict. Indeed, the nutritional benefits of the symbiosis may increase food availability or improve the diet quality for the pest, depending on the fungal and insect identity and growing conditions (Koricheva et al., 2009;Frew and Price, 2019). Thus, the interaction outcome results from the interplay of opposite effects: nutritional improvement, benefiting the herbivore, and defense priming, potentially reducing herbivore performance (Pozo et al., 2020). Indeed, plant-microbe-insect interactions are very complex, with plants coordinating their responses according to multiple external and internal cues through a precise regulation orchestrated by phytohormone networks (Pozo et al., 2015;Gruden et al., 2020). Many biotic and abiotic factors influence mycorrhizal colonization (Diagne et al., 2020;Mitra et al., 2021). Indeed, the plant-AMF interaction is finely regulated by several factors, including the plant and fungal genotypes and environmental conditions, with nutrients availability as one of the most influential aspects (Pozo et al., 2015;Lee Dı  az et al., 2021). P is a central regulator of mycorrhizal establishment, root colonization and fungal growth within the host plant. Thus, the abuse of P fertilizers inhibits the establishment of mycorrhizal symbiosis in different plants (Smith and Smith, 2011;Smith et al., 2011;Higo et al., 2020). P, an essential nutrient for plants, is a non-renewable resource and poorly available in the field, being a limiting factor for plant growth (Wang et al., 2021). This element is taken up from the soil in the form of inorganic P by the roots (Rouached et al., 2010), and its deficiency triggers important changes in plant physiology and biochemistry including changes in plant growth, metabolism, hormonal balance, gene expression and root architecture. P deficiency further promotes the production of the phytohormones strigolactones, accumulation of anthocyanins and other phenolic compounds (Ha and Tran, 2014;Vysotskaya et al., 2016;Marro et al., 2022). In agriculture, chemical and organic fertilizers are strongly and widely applied to solve P soil deficiency, causing contamination of aquifers and altering plant interactions with beneficial microbes, including AMF (Smith and Smith, 2011; Cordell and White, 2015). P levels also impact plant interactions with other organisms by altering plant tissues’nutritional value for organisms feeding on them (pathogens and herbivores) and by modulating plant defenses (Breuillin et al, 2010;Castrillo et al, 2017;Chan et al., 2021). The role of P in plant immunity is currently under intense scrutiny, and the data reveal a very complex scenario (Chan et al., 2021;Qu et al., 2021;ValTorregrosa et al, 2022). The phosphate starvation response in plants has been suggested to increase plant resistance to necrotrophic pathogens and leaf chewing herbivores in several plant species (Arabidopsis, tomato, tobacco) pointing to a positive cross-talk between the JAand Pstarvation signaling pathways that activates plant immunity (Khan et al., 2016). However, opposite results have been also found in different plant species facing different aggressors (Campo et al., 2020;ValTorregrosa et al, 2022). It should be noted that most basic studies dealing with the molecular mechanisms mediating P effects on plant immunity compare very contrasting P levels, usually far from those used in agricultural settings. Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org02 Despite the increasing number of studies addressing P influence on plant defenses, only a few have investigated the relationship between P and MIR, suggesting a complex interaction with contrasting results (Wang et al., 2020;Qu et al., 2021). While evidence of the role of P in regulating mycorrhizal colonization, plant growth and immunity responses exist, the interplay among the different effects and mechanisms behind this interplay remain poorly studied (Wang et al., 2020;Qu et al., 2021). Our study aims to elucidate whether the mycorrhizal effect on tomato resistance to biotic stressors is regulated by P availability. For that, we evaluated the impact of different P fertilization regimes on tomato growth, mycorrhizal colonization and herbivore resistance using the tomato/ Funneliformis mosseae system and the necrotrophic pathogen Botrytis cinerea and the generalist chewing herbivore Spodoptera exigua. We found that P availability strongly influences plant growth and resistance to biotic stresses, and that these effects are modulated by the AM symbiosis, showing that MIR is indeed dependent on P availability. By analyzing plant nutrients, defense-related phytohormones and transcriptional regulation of defenses in response to the damage-associated signals oligogalacturonides (OGs), we aimed to uncover the mechanistic basis of the impact of P availability on MIR. This knowledge will contribute to improve AMF application and management for sustainable crop protection. Material and methods Biological material and mycorrhizal inoculation The AMF Funneliformis mosseae BEG12 (Young, 2015) obtained from the International Bank of Glomeromycota (http://www.i-beg.eu), was maintained at the EEZ-CSIC greenhouse as open-pot cultures of Trifolium repens mixed with Sorghum vulgare plants using vermiculite-sepiolite substrate. The inoculum consisted in the substrate containing colonized root fragments, fungal mycelia and spores. Botrytis cinerea was cultivated in potato dextrose agar plates, supplemented with freeze-dried tomato leaves. Three weeks later, B. cinerea spores were collected from plates in 0.5X potato dextrose broth as previously described (Sanmartı n et al., 2020). Spodoptera exigua (Lepidoptera: Noctuidae) eggs were provided by Dr. S. Herrero lab (ERI-BIOTECMED, Universitat de Valencia, Spain). Larvae were reared on artificial diet (Greene et al., 1976) at 25°C with 16:8h light: dark regime and 70% relative humidity until L2-L3 larval stage, for their application. Tomato seeds (Solanum lycopersicum L. cv. Moneymaker) were surface disinfected by immersion in 4% NaHClO (10 min), rinsed thoroughly with sterile water and incubated for 10 days in an open container with sterile vermiculite at 25°C. Plantlets were transferred to 100 mL pots containing a sterile sand:vermiculite (1:1) mixture. Pots for mycorrhizal treatments were inoculated by adding 10% (v/v) F. mosseae inoculum. All plants, including non-inoculated ones, received a 3 ml aliquot of a filtrate (<20 mm) of the inoculum, in order to provide the general microbial population but free of AMF spores. Experimental design Different experiments were carried out to elucidate the effect of P nutrition on plant performance and herbivore resistance in mycorrhizal and non-mycorrhizal plants. All experiments included tomato plants inoculated with F. mosseae (Fm) or not (Nm). We performed a first screening using three P concentrations: 0.3 mM, 0.6 mM and 1.0 mM (10 plants per treatment). For this, plants were watered with Hewitt nutrient solution (Hewitt, 1996), modified in the P (H 2 NaPO4) concentration as described in Table S1. Plants were harvested after 8 weeks of growth and the performance of S. exigua fed on detached leaves was evaluated (see herbivore bioassays below). Plant biomass, mycorrhizal colonization, plant nutrients, anthocyanin content and phytohormone levels were also determined. Based on the results obtained, 0.3 or 0.7 mM P concentrations were selected for follow-up experiments. In a second experiment, we assessed P effects on Botrytis cinerea lesion development through a detached leaf assay as described below, and we evaluated the performance of S. exigua larvae directly fed on Fm and Nm plants (whole plant bioassay). Sixweeks-old plants were infested with S. exigua larvae and caterpillar performance (weight, survival and pupation) was steadily monitored for 3 weeks (9 plants per treatment). Finally, early plant defense responses were compared in mycorrhizal and non-mycorrhizal plants growing at these two P levels (0.3 and 0.7 mM; 6 plants per treatment). For that, we assessed the plant response to damage by using oligogalacturonides (OGs), well characterized damageassociated molecular patterns (DAMPs) (see Plant treatment with oligogalacturonides below). The levels of defense-related phytohormones and defense-related gene expression were determined 6 hours post treatment (hpt) as described below. Thus, the factors considered in this study were M: mycorrhizal inoculation (levels: Nm, Fm), P: P fertilization regimes (levels 0.3, 0.7 and 1.0 mM), and OG: Oligogalacturonide treatment (levels: -OG, +OG). Plant growing conditions For the different experiments, plants were randomly distributed and grown in a greenhouse at 24/16°C with a 16/ Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org03 8 h diurnal photoperiod and 70% humidity. Plants were watered twice a week with half strength Hewitt nutrient solution (Hewitt, 1996)modified in the P content as described above, and water was supplied as needed. Upon harvesting shoots and roots, fresh weight was determined and the material was immediately frozen in liquid N and stored at -80°C for further analyses. An aliquot of each individual root system was preserved for mycorrhizal quantification. Botrytis cinerea infection The fourth leaf of tomato plants was detached for pathogen bioassays. Pathogen infection was performed by applying 10 mL drops containing a conidia suspension (1 × 10 6 spores/ml) to the detached leaves. One leaf per plant was inoculated by adding two drops per leaflet, five leaflets per leaf, with a total of 90 lesions. The infected leaves were maintained in 15cm Petri dishes on wet filter paper and incubated in a phytotron chamber at 80% of humidity and 22°C. Necrotic lesions were measured 3 days post-inoculation. Herbivore performance bioassays Insect performance was evaluated in two different bioassays, using detached leaves or whole plants. For the detached leaves assay, two second-instar S. exigua larvae were placed on one detached leaf of each tomato plant, placed on wet filter paper in 15 cm Petri dishes. Plates were then incubated in a phytotron at 26-24°C, 16:8h day/night and 60% relative humidity. Larval mortality and weight was evaluated every 2 days for 8 days. For whole plants assay, two second-instar larvae were placed in a leaflet of the fourth true leaf of each tomato plant, using clipcages to avoid their escape, as described in Rivero et al. (2021). Every two days, clip-cages were moved to new fresh leaflets and caterpillar biomass, mortality and pupation were monitored. Plant treatment with oligogalacturonides For the analysis of early plant responses, the damage associated molecules oligogalacturonides (OGs) were used to elicit plant defense responses. The OGs (DP 10-15) were prepared as described in Benedetti et al. (2017) and provided by Dr. De Lorenzo lab (Department of Biology and Biotechnology “Charles Darwin”BBCD, La Sapienza University, Rome, Italy). Six weeks post AMF inoculation tomato plants grown at 0.3 or 0.7 mM P fertilization regimes were treated with an aqueous solution of OGs (50 mg/ml in milliQ water) as described in Gamir et al. (2020). The fourth true leaf of each plant was sprayed with the OG solution or milliQ water for control plants using an aerograph until running off. Treated leaves were harvested after 6 hours to study early plant defense responses, as this time was the most appropriate to identify changes in hormone contents and in the expression levels of OG responsive, defense related (Gamir et al., 2020). Determination of mycorrhizal colonization AM colonization was measured after clearing washed roots in KOH (10%) and staining fungal structures with 5% ink in 2% acetic acid (Vierheilig et al., 2005). The percentage of total root length colonized by F. mosseae was estimated according to the gridline intersection method (Giovannetti and Mosse, 1980) using a BOECO zoom stereo microscope Model BST-606. Anthocyanin content Anthocyanin content, as P starvation indicator, was evaluated by adapting the protocol established in Rabino and Mancinelli (1986). Frozen leaves were grinded and lyophilized, and 5 mg aliquot of dry tissue was used per sample. The pigments were extracted by shaking the plant freeze-dried leaf powder in acidic (1% HCI, w/v) methanol in dark overnight. Extracts were centrifuged for 10 min at 13000 rpm. The amount of anthocyanin was calculated measuring absorbance at 530 and 657 nm of crude extract, using the formula A530 - 0.25 A657 to compensate for the contribution of chlorophyll and its degradation products to the absorption at 530 nm. Six independent biological replicates were analyzed per treatment. Determination of mineral nutrients in leaves Nutrient analyses were performed at the Technical Services of the Estacion Experimental del Zaidı  n (CSIC). Frozen leaves were grinded and lyophilized, and 50-100 mg aliquot of dry tissue was used per sample. The concentration of P and micronutrients were determined after acid digestion of samples, by inductively coupled plasma optical emission spectrometry (ICP‐OES; Varian ICP 720‐ES). Total C and N contents were analyzed using an Elemental Analyzer (LECO TruSpec CN), according to standard procedures. Six independent biological replicates were analyzed per treatment. Targeted hormonal extraction and quantification Hormone extraction was performed from freeze-dried powdered plant leaves as described in Sanchez-Bel et al. Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org04 (2016). Six independent biological replicates per treatment were analyzed. Briefly, 30 mg of plant dry tissue was extracted with 1mlofH 2 O:MeOH (9:1) containing 0.001% of HCOOH and 100 ng/ml of internal standards. After different centrifugations and resuspensions, an aliquot of the extract was injected into an Acquity Ultra Performance Liquid Chromatography system (UPLC) (Waters, Mildford, MA, USA). Hormones were chromatographically separated using an HPLC Kinetex C18 analytical column (Phenomenex) connected to a triple quadrupole mass spectrometer (TQD, Waters, Manchester, UK). The chromatographic and mass spectrometry conditions were those used by Gamir et al. (2012). Hormone quantification (ng/g dry weight) was performed using calibration curves with each pure chemical standard. The plant hormones abscisic acid (ABA), indolacetic acid (IAA), jasmonic acid (JA), its precursor (+)-12-oxo-phytodienoic acid (OPDA) and salicylic acid (SA) were determined. Analysis of gene expression by qPCR The expression of marker genes from different metabolic pathways was analyzed by real time quantitative PCR (qPCR). Six independent biological replicates per treatment were used. Total RNA from tomato leaves was extracted and treated with DNase using the Direct-zol RNA MiniPrep kit (Zymo Research). Subsequently, the RNA was purified through a column using the RNA Clean and Concentrator-5 kit (Zymo Research), and stored at -80°C until use. The first-strand cDNA was synthesized with 1 mg of purified total RNA using the iScript cDNA Synthesis kit (Bio-Rad). All kits were used according to the manufacturer’s suggested protocols. The expression of three different housekeeping genes, actin (Solyc03g078400), elongation factor 1‐a(Solyc06g005060) and b‐tubulin (Solyc04g081490) was measured to find the optimal normalization gene, using the Normfinder software (https:// moma.dk/normfinder-software)(Andersen et al., 2004). According to the results, expression values were normalized using the housekeeping gene b-tubulin and relative quantification of specific mRNA levels was performed using the comparative 2–D(DCt) method (Livak and Schmittgen, 2001). The sequences of the specific primers used are shown in Table S2. Statistical analyses All statistical analyses (multi-way ANOVAs and post hoc tests applied when appropriated, as indicated in the corresponding figure legends) were conducted using Statgraphics Plus 3.1 (Rockville, MD, USA) or ‘R’software v.3.5.2 (R Development Core Team). Figures were obtained using ggplot2 R package (Wickham et al, 2016). Treatment effects on larval survival were assessed by comparing the survival curves using the Kaplan-Meier estimator (Kaplan and Meier, 1958). Survival distribution comparison between treatments was performed using the non-parametric Logrank test (Mantel-Cox). Survival analyses were performed using survival and survminer R packages. Model validations were performed using Shapiro-Wilk and Levene’s tests. Results P fertilization levels impact mycorrhizal colonization, plant growth and herbivore performance in tomato To explore how mycorrhizal development and its effects on plant and caterpillar performance are affected by P fertilization, we compared 3 fertilization regimes differing only in the P content, ranging from limiting to sufficient P (0.3, 0.7 and 1.0 mM). Analysis of the plant biomass confirmed that P levels had a significant impact on plant growth (p<0.001) (Figures 1A,B). Plants grown at 0.3 mM P showed about 50% reduced root and shoot weights. Significant differences between 0.7 and 1.0 mM P were also observed; however, these were mild compared to the most P limiting conditions (Figures 1A,B). Interestingly, mycorrhization did not have a global significant effect on plant fresh weight, but there was a significant interaction between the P and mycorrhizal treatments, with mycorrhiza promoting plant growth only at the intermediate (0.7 mM) P level and repressing root biomass at the highest P level (Two way ANOVA, Figures 1A,B). The evaluation of anthocyanin accumulation in leaves, as an indicator of plant P-starvation response, also confirmed the dose-dependent effects of P fertilization on the plants. Plants grown under low P (0.3 mM) showed the highest anthocyanin levels, while the levels in plants growing at 0.7 and 1.0mMwerenotsignificantly different (Figure 1C). Mycorrhization also had a significant effect reducing the anthocyanin levels (two-way ANOVA; p<0.05). Mycorrhizal colonization was also significantly impacted by P fertilization, with increasing P concentrations leading to a reduction in colonization (Figure 1D). Differences were significant already after 4 weeks of growth, with mycorrhizal colonization in the moderate and high P conditions being half of those at low P. The effect was more pronounced at the later time point (final harvest, 8 weeks), as root colonization continued to increase in plants fertilized with the lowest P concentration (0.3 mM), but not with the other P levels (Figure 1D). The effect of plant P fertilization on the performance of S. exigua larvae fed on leaves of those plants was also evaluated by using detached leaves. Larval weight was influenced by P levels, being significantly lower at 0.3 mM P (Figure 1E). No differences between larvae fed in 0.7 or 1.0 mM were found, regardless of the mycorrhizal status of the plant (Figure 1E). P levels also had a Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org05 B CD EF A FIGURE 1 Impact of P levels on plant growth, AM colonization and herbivore performance. Plant growth parameters, fungal colonization and herbivore performance on non-mycorrhizal (Nm) and mycorrhizal tomato plants colonized by Funneliformis mosseae (Fm). Plants were fertilized with different P concentrations: 0.3 mM, 0.7 mM and 1mM of H 2 NaPO₄.(A) Shoot and (B) root fresh weight (n=10), and (C) anthocyanin content (n=6) were determined in tomato plants at harvest, 8 weeks post mycorrhizal inoculation (pmi). (D) Percentage of root length colonized by the mycorrhizal fungi at 4 and 8 weeks pmi (n=10). One leaf per plant was detached 8 weeks pmi and infested with 2 second instar S. exigua larvae (n=20), and (E) weight of the larvae was determined after 6 days of feeding. (F) Larval mortality was monitored during 8 days of continuous feeding on the detached tomato leaves. Data from A to E represent means of the n independent biological replicates ± SD. Two-way factorial ANOVA (A–C, E, F) using AM symbiosis (M) and P treatments (P) as factors, or (D) using time (T) and P treatments (P) as factors were performed, and significance values of each factor and their interactions are indicated in the upper right corner of each graph. Asterisks denote significant effect of a factor and their interaction. ns: no significant; *: p < 0.5; **:p < 0.01; ***:p < 0.001; ****:p < 0.0001. Different letters represent statistically significant differences (ANOVA, Fisher’s Least Significant Difference (LSD) test; p<0.05). For (F), data represent the percentage of mortality at the different time points, and the differences in the survival distribution according to P, M and MxP were performed using the nonparametric Log-rank (Mantel-Cox) test. Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org06 significant impact on S. exigua mortality, showing the highest mortality at the low 0.3 P level (ranging between 50-60%) (Figure 1F). At higher P levels (0.7 and 1.0 mM) larvae mortality ranged from 6 to 21% (Figure 1F). Although higher mortality was found in Fm compared to Nm plants at these medium and high P levels (21% Fm vs 6% Nm for 0.7mM, and 16%Fm vs 6%Nm for 1.0 mM), the differences were not significant. Noteworthy, when analyzing the data separately according to the mycorrhizal status, P effect on larval survival was more pronounced in Nm than in Fm plants: P levels had a very significant impact on larval survival when feeding on Nm plants (p<0.0001), but not on those feeding in mycorrhizal plants (p=0.052) (Figure S1A). P effects on herbivore performance depends on the mycorrhizal status of the plant and determine MIR Overall, the results of the P dose screening revealed that most differences occur between the low (0.3mM) and the higher P regimes (0.7 and 1.0 mM) that showed similar values for most parameters. Accordingly, 0.3 and 0.7 mM levels were selected for further experiments as the closest doses with contrasting effects. Bioassays on detached leaves are useful for quick screenings of major effects, but these effects are usually weaker than those using whole plant bioassays. The latter allow a more realistic set up and longer evaluation periods. Therefore, we performed a second experiment focused on the selected P levels (0.3 and 0.7 mM) to better address the effect of mycorrhization on larval performance under different P fertilization. Mycorrhizal colonization by F. mosseae was 14% and 6% for the 0.3 and 0.7 mM P levels, respectively. Again, P effect on S. exigua mortality was significant for larvae feeding in Nm plants (p<0.0001), but not for those feeding on mycorrhizal (Fm) ones (p=0.27) (Figure S1B). Thus, mycorrhizal colonization seems to buffer the strong effect of P on plant resistance to the pest. As in the previous experiment using detached leaves, larvae performed worst in the lowest P fertilized plants, showing higher mortality levels (Figures 2A,B), lower weight (Figures 2C,D) and worst development –evaluated as the percentage of individuals reaching the pupal stage- (Figures 2E,F). Regarding the effect of mycorrhization on S. exigua performance, under low P no significant changes were found in mortality nor development between Fm and Nm plants (Figures 2A,E), and larval weight was even higher at some time points in mycorrhizal plants (Figure 2C). In contrast, under moderate P levels (0.7 mM), larvae fed on mycorrhizal Fm plants performed worse than those fed on Nm, showing higher mortality, lower weight and impaired development (Figures 2B,D,F). The results reveal that the effect of mycorrhization on larval performance depends on P availability, as MIR was observed at the moderate (0.7 mM) P levels, but not at the low (0.3 mM) one. Remarkably, a similar pattern was observed in the interaction with B. cinerea. Again, MIR was only observed at 0.7 mM P, but not at 0.3mM P, and while the effect of P was significant in Nm plants (Nm 0.3 vs Nm 0.7 t-test, p<0.0001), it was not significant in Fm plants (Fm 0.3 vs Fm 0.7 t-test, p= 0.24) (Figure 3A). Differences in MIR are not directly related to changes in the nutritional status of the plant To address whether the P-dependent effects of mycorrhiza on larval performance were due to nutritional aspects, we evaluated plant biomass, anthocyanin and nutrient contents. Shoot biomass was dependent on P fertilization, and growth promotion by mycorrhiza depended on P levels: Fm promoted plant growth at low P, while no plant growth promotion was observed at 0.7 mM P (Figure 3B). Anthocyanins were only influenced by P levels (Figure 3C). Regarding the nutritional value of the leaves, P content in leaves increased with P fertilization, and it was significantly higher in mycorrhizal plants (Figure 3D). P fertilization also significantly influenced carbon (C) and nitrogen (N) levels in leaves, increasing C and reducing N concentration at 0.7mM as compared to 0.3 mM. While mycorrhization did not have a global effect on their content, the interaction between P and M was significant for C content, with mycorrhiza displaying higher C levels than Nm plants under the low P fertilization (Figures 3E,F), while the interaction between P and M was significant in C content. As mycorrhization had the same effect on the nutrient content of leaves at the highest P level (higher P values, no changes in C nor N as compared to Nm plants), mycorrhiza-related changes in the nutritional value of leaves do not seem to explain the differential impact of mycorrhization on larval performance under the different P fertilization levels. Most micronutrients were significantly influenced by P (Ca, Cr, Cu, Fe, Mg, Mn, Mo, Na, Ni, S, Sr and Zn), while M influenced only some of them (as Cu, Fe, K, Li, Mn, Sr, Zn) (Table S3). Only K was significantly regulated by the interaction of the two factors (Table S3). P availability impacts the levels of defense-related phytohormones and gene expression We explored whether the impact of P levels on MIR was related to differential activation of plant defense responses. In order to monitor early defense responses, and aiming to reduce the variability associated with pathogen development or differential feeding by the larvae, we used OGs as elicitors. Plants grown in parallel to those in the whole plant herbivory Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org07 assay (thus same age, growing conditions and mycorrhizal colonization levels) were challenged by spraying a fully expanded leaf with an OG solution, as described in Gamir et al. (2020). Treated leaves were analyzed for hormone content and defense-related gene expression 6 hours after OG application. The levels of jasmonic acid (JA), its precursor OPDA, auxin (IAA) and abscisic acid (ABA), as the major hormones involved in plant responses to chewing herbivores and necrotrophic pathogens, and described to be regulated by OGs, were evaluated (Figure 4). Multiway ANOVA confirmed a significant impact of P fertilization on the OPDA, ABA and IAA levels, but not on the JA content. AM symbiosis only impacted B CD EF A FIGURE 2 Impact of mycorrhiza on herbivore performance under different P availability. S. exigua performance of larvae fed on leaves of mycorrhizal (Fm, dotted line) and non-mycorrhizal (Nm, continuous line) plants fertilized with 0.3 or 0.7 mM of P, light and dark grey, respectively; (n=9). Sixweeks post-inoculation with F. mosseae (Fm), plants were infested with second instar S. exigua larvae (two per plant, n=18 per treatment) using a clip-cage to confine the larvae to a leaflet. Infestation was maintained for three weeks by moving the clip-cage every two days. (A, B) S. exigua mortality (C, D) weight and (E, F) individuals reaching pupa stage. Statistical analyses were performed independently for each P fertilization level: (A, E) 0.3 mM and (B, F) 0.7 mM. (A–F) P values in the upper right corner of each graph indicate statistically differences in mortality and pupation between Nm and Fm, according to Log-rank (Mantel-Cox) test. For larval biomass (C, D), values are the weight mean of survived larvae ± SD. Asterisks indicate significant differences between Nm and Fm treatments at given time point according to t-test. ns: no significant; +:p < 0.1; *:p < 0.05; **:p < 0.01; ***:p < 0.001; ****:p<0.0001. Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org08 the IAA levels, while OG treatment significantly affected all hormone levels except for ABA. Noteworthy, no significant interaction between the different factors was observed for hormones except for ABA levels. Overall, P deficiency significantly reduced OPDA and IAA levels, but increased ABA. OGs enhanced JA, OPDA and IAA levels regardless of the mycorrhizal status of the plant (Figure 4). Mycorrhization only had a significant effect on IAA levels, although it modulated the impact of P and OG on ABA levels (Figure 4). Salicylic acid (SA) levels were also determined, showing a reduction by increasing P or OG treatment, but no significant effect of mycorrhiza or factors interaction was observed (Figure S2). As hormone levels were not different in leaves of mycorrhizal plants, we hypothesized that mycorrhizal plants could prime downstream defense responses, but that this effect was dependent on P levels. We analyzed the gene expression of well characterized JA regulated anti-herbivore defense markers, including the Leucyl aminopeptidase A (LapA), Proteinase inhibitor II (PinII), Threonine deaminase (TD)andMulticystatin (MC)(Uppalapati et al, 2005); Yan et al, 2013. Surprisingly, in non-challenged plants (-OG) all these defense genes (except MC) were up-regulated in Nm plants grown under low P levels as compared to Nm grown under moderate P levels, but this upregulation was not observed in mycorrhizal Fm plants (Figures 5A–E;Tables S4 and S5). OG treatmentinplantsgrownunderlowPresultedinareduced expression of these genes in Nm plants, while they showed a slight induction in Fm plants (see fold changes in Table S5). In contrast, under moderate (0.7 mM) P levels, these genes showed similar expression levels in mycorrhizal and non-mycorrhizal plants in the absence of challenge; however they were significantly induced by OG treatment only in Fm plants. Thus, the expression analyses confirmed a primed response of mycorrhizal plants to the OG treatment under sufficient P (Figures 5A–D;Table S5). A similar primed response was found for the gene encoding a defense polygalacturonase inhibiting protein (LePGIP), also related to defense responses (Baroncelli et al., 2016)(Figure 5E). The reduced levels of defense genes in non-challenged (-OG) mycorrhizal plants under P starvation, led us to explore the expression of regulators of JA-dependent defense responses. JAZ proteins are key negative regulators of JA signaling, repressing BC DEF A FIGURE 3 Impact of P fertilization and mycorrhization on the nutritional value of tomato leaves. Botrytis cinerea infection, shoot biomass, anthocyanin, and nutrient contents in tomato leaves of non-mycorrhizal (Nm) and mycorrhizal tomato plants colonized by (F)mosseae (Fm). Plants were fertilized by two P concentrations: 0.3 mM and 0.7 mM P. (A) Diameter of necrotic lesions 3 days post inoculation with (B) cinerea in detached adult leaves from tomato (n=9). (B) Shoot fresh weight (n=9), (C) anthocyanin (n=6), (D) phosphorus (n=6), (E) carbon (n=6) and (F) nitrogen (n=6) content were measured in tomato plants 6 weeks post mycorrhizal inoculation. Data represent the means of n independent biological replicates ± SD. Two-way ANOVA with mycorrhizal (M) and P treatments (P) as factors, was performed, and the significance of the factors and their interaction is indicated in the upper right corner of each graph. Different letters represent statistically significant differences (ANOVA, Fisher’s Least Significant Difference (LSD) test; p<0.05) where the interaction between factors was observed. Otherwise, asterisks denote significant effect of a factor and their interaction. ns: no significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Dejana et al. 10.3389/fpls.2022.1060926 Frontiers in Plant Science frontiersin.org09 SUPPLEMENTARY TABLE 5 Fold changes in gene expression. Colors indicate up-regulation (red) and down-regulation (blue) and intensities are determined by the intensity of the changes: light color >1.5 or <0.75 and dark color >2 or <0.5 and significant effects are highlighted in bold. (A) Effect of P starvation on gene expression in Nm plants (fold change in Nm and Fm plants fertilized at 0.3 vs 0.7 mM). (B) Effect of OG elicitation on gene expression in Fm or Nm plants grown at 0.3 mM and 0.7 mM P (fold change +OG/-OG). (C) Effect of AM symbiosis on gene expression at 0.3 mM and 0.7 mM P (fold change Fm/Nm). Bold numbers and asterisks indicate significant differences (ttest, p<0.05) between (A) 0.3 vs 0.7 mM, (B) +OG vs -OG and (C) Fm vs Nm. +: p<0.1; *: p<0.05; **: p<0.01; ***: p<0.001. SUPPLEMENTARY TABLE 6 Multiway ANOVA of gene expression data. Effects of the different factors: AM symbiosis (M), P fertilization (P) and elicitation (OG treatment, OG) and their interactions were analyzed. 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