ORIGINAL RESEARCH published: 04 November 2020 doi: 10.3389/fpls.2020.572087 Chitosan Induces Plant Hormones and Defenses in Tomato Root Exudates Marta Suarez-Fernandez 1,2* , Frutos Carlos Marhuenda-Egea 3 , Federico Lopez-Moya 2 , Marino B. Arnao 4 , Francisca Cabrera-Escribano 5 , Maria Jose Nueda 6 , Benet Gunsé 7 and Luis Vicente Lopez-Llorca 1,2 1 Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental Studies Ramon Margalef, University of Alicante, Alicante, Spain, 2 Department of Marine Sciences and Applied Biology, Laboratory of Plant Pathology, University of Alicante, Alicante, Spain, 3 Department of Agrochemistry and Biochemistry, Multidisciplinary Institute for Environmental Studies Ramon Margalef, University of Alicante, Alicante, Spain, 4 Department of Plant Biology (Plant Physiology), University of Murcia, Murcia, Spain, 5 Department of Organic Chemistry, Chemistry Faculty, University of Seville, Seville, Spain, 6 Department of Mathematics, University of Alicante, Alicante, Spain, 7 Plant Physiology Laboratory, Faculty of Biosciences, Universidad Autonoma de Barcelona, Bellaterra, Spain Edited by: In this work, we use electrophysiological and metabolomic tools to determine the role Jens Rohloff, Norwegian University of Science of chitosan as plant defense elicitor in soil for preventing or manage root pests and and Technology, Norway diseases sustainably. Root exudates include a wide variety of molecules that plants and Reviewed by: root microbiota use to communicate in the rhizosphere. Tomato plants were treated Dayakar Venkata Badri, Hill’s Pet Nutrition, Inc., United States with chitosan. Root exudates from tomato plants were analyzed at 3, 10, 20, and 30 Berin A. Boughton, days after planting (dap). We found, using high performance liquid chromatography The University of Melbourne, Australia (HPLC) and excitation emission matrix (EEM) fluorescence, that chitosan induces *Correspondence: plant hormones, lipid signaling and defense compounds in tomato root exudates, Marta Suarez-Fernandez marta.suarez@ua.es; including phenolics. High doses of chitosan induce membrane depolarization and affect suarezfernandezmarta@gmail.com membrane integrity. 1 H-NMR showed the dynamic of exudation, detecting the largest number of signals in 20 dap root exudates. Root exudates from plants irrigated with Specialty section: This article was submitted to chitosan inhibit ca. twofold growth kinetics of the tomato root parasitic fungus Fusarium Plant Metabolism oxysporum f. sp. radicis-lycopersici. and reduced ca. 1.5-fold egg hatching of the and Chemodiversity, a section of the journal root-knot nematode Meloidogyne javanica. Frontiers in Plant Science Keywords: chitosan, root exudates, membrane potential, lipid signaling, plant defenses, plant hormones, soil- Received: 12 June 2020 borne pathogens Accepted: 08 October 2020 Published: 04 November 2020 Citation: INTRODUCTION Suarez-Fernandez M, Marhuenda-Egea FC, Lopez-Moya F, Chitosan is a linear polymer of β-(1-4)-linked N-acetyl-2-amino-2-deoxy-D-glucose (acetylated) Arnao MB, Cabrera-Escribano F, and 2-amino-2-deoxy-D-glucose (deacetylated) (Kaur and Dhillon, 2014). It is generally obtained Nueda MJ, Gunsé B and by partial deacetylation of chitin (Kumar, 2000), which is the second most abundant polysaccharide Lopez-Llorca LV (2020) Chitosan in nature after cellulose (Elieh-Ali-Komi and Hamblin, 2016). Chitin is a major component of the Induces Plant Hormones and Defenses in Tomato Root cuticle of insects, exoskeleton of crustaceans and fungal cell walls. Chitosan has been described Exudates. as elicitor of plant defenses (Yin et al., 2016) and hormones of food security crops such as Front. Plant Sci. 11:572087. tomato (Iriti and Faoro, 2008; El-Tantawy, 2009). Chitosan also propitiates accumulation of auxin doi: 10.3389/fpls.2020.572087 [mainly indoleacetic acid (IAA)] in the apex of plant roots (Lopez-Moya et al., 2017). Since Frontiers in Plant Science | www.frontiersin.org 1 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions most studies have been carried out in the phylloplane, MATERIALS AND METHODS we devised experiments to test the effect of chitosan on tomato rhizodeposition in both hydroponic and solid plant Chitosan, Plants, Fungi, and Nematodes substrate systems. Chitosan with 70 kDa molecular weight and 80.5% deacetylation Electrophysiology can monitor the response of plant roots degree was used in all experiments. Chitosan was obtained from to stress (Rodrigo-Moreno et al., 2013). Membrane potential Marine BioProducts GmbH (Bremerhaven, Germany) and made reflects the action of all pumps in the cell membrane to maintain as in Palma-Guerrero et al. (2007). Tomato plants (Solanum ion gradients (Alberts et al., 2002). Root cell membranes detect lycopersicum cv. Marglobe) were used in all experiments. changes in their environment and respond starting metabolic Pochonia chlamydosporia, isolate number 123 (ATCC no. MYA- cascade reactions (Fürstenberg-Hägg et al., 2013; Matzke and 4875; CECT no. 20929; PRJNA68669, NCBI txid1052797) was Matzke, 2013). Those reactions could lead to new compounds of isolated from Heterodera avenae eggs in south west Spain agronomic and ecological interest. (Olivares and López-Llorca, 2002). Fusarium oxysporum f. Rhizodeposition is the process of releasing organic sp. radicis-lycopersici (Strain 4287, CBS 123668, FGSC 9935, compounds from roots to the external medium. Plants exude NRRL 34396) was obtained from CBS-KNAW culture collection. a wide variety of low molecular weight organic compounds Meloidogyne javanica was obtained from a field population (e.g., amino acids and small peptides, organic acids, hormones, and maintained in susceptible tomato plants. Nematode egg sugars, phenolics, and other secondary metabolites) (Flores masses were dissected from root-knot nematode infested roots et al., 1996). Resolving this complex mixture requires the use of and stored at 4◦ C. Egg masses were hand-picked and surface- diverse metabolomics technologies (Escudero et al., 2014; van sterilized as in McClure et al. (1973) with modifications. Dam and Bouwmeester, 2016). Root exudates are paramount in plant–microbe interactions in the rhizosphere, including beneficial and pathogenic microbes and play a key role in Root Electrophysiology Experiments signaling (Hirsch et al., 2003; Bais et al., 2006; Walker et al., Tomato seeds were surface sterilized using 1% sodium 2009; Zhang et al., 2019; Valette et al., 2020). Metabolomics hypochlorite and washed three times 1 min each with sterile can help us to understand the chemical interactions between distilled water (SDW). Tomato seeds were then placed on organisms in the rhizosphere, as well as the importance to germination medium (GM, Glucose 10 g L−1 , Yeast Extract uncover toxic compounds (Baldrian, 2019). Metabolomics 0.1 g L−1 , Bactopeptone 0.1 g L−1 , Technical Agar 12 g L−1 ). allows detection of phenolic compounds by fluorescence GM plates with tomato seeds were placed at 4◦ C for 2 days (Hupp et al., 2019). Phenolics have diverse functions in for stratification and incubated at 24◦ C, 65% relative humidity plants, but one of the most important is their role in plant (RH), in the dark for 5 days and in a photoperiod (16:8) for defense and signaling (Mandal et al., 2010). Hormones further 5 days. Plantlets were then placed individually in a are also signaling molecules that can also be found using holder chamber filled with Gamborgs B5 1:10 (Sigma; Gamborg metabolomics (Street and Schenk, 1981; Li et al., 2009; Yang et al., 1968). Glass microcapillaries filled with 0.5M KCl (tip et al., 2015). Auxin (e.g., IAA), salicylic acid (SA), jasmonic diameter < 1 µm) were inserted into root cortical cells until a acid (JA), and abscisic acid (ABA) are produced in response stable basal potential was obtained (Gunsé et al., 2016). Roots to physiologic or metabolic changes (Asami and Nakagawa, were exposed to increasing concentrations of chitosan (0.1, 1, 2018). Phytomelatonin is considered a master regulator in and 2 mg mL−1 in Gamborgs B5 1:10 medium) and membrane plant stress conditions (Arnao and Hernández-Ruiz, 2019) potentials were recorded. No chitosan was used for control and also involved in the regulation of several plant hormones treatments. Between each change of chitosan concentration, (Arnao and Hernández-Ruiz, 2018). Phytomelatonin promotes root medium was replaced for Gamborgs B5 1:10 to check the growth and root appearance (Hernández-Ruiz et al., 2005; physiological status of the cell. At least three biological replicates Arnao and Hernández-Ruiz, 2007). and four technical replicates were performed per treatment. Fungal (Fusarium oxysporum f. sp. radicis-lycopersici) and nematode (Meloidogyne javanica) root pathogens are used in this Root Vital Staining work. These pathogens threaten food security worldwide (FAO, Tomato plantlets were placed in Gamborgs B5 1:10 liquid 2017). Root-knot nematodes cause 25–100% tomato yield losses medium 1 day for acclimatization. Chitosan was then added (Seid et al., 2015) and Fusarium wilt causes up to 80% of crop at 0.1, 1, and 2 mg mL−1 . Plants exposed only to Gamborgs losses (Singh et al., 2017). B5 1:10 were used as controls. Plantlets with treatments were The aim of this work is to determine the effect of chitosan incubated for 24 h. Roots were then stained with fluorescein on plant rhizodeposition. Root electrophysiology allows us to diacetate (FDA), 5 mg mL−1 in acetone diluted 1:250 in monitor the effect of chitosan on membrane functionality. In Dulbecco’s phosphate-buffered saline (DPBS, Thermo Fisher) addition, metabolomic techniques are used to determine how and propidium iodide (PI, 20 mg mL−1 in DPBS; Jones and Senft, chitosan modulates the composition of root exudates. Finally, 1985). Roots were visualized using a Nikon Optiphot microscope we test these exudates against root pathogens which threaten using 10X objective with an attached Nikon DS–5 M camera food security worldwide. This will allow us to validate the role system using epifluorescence (Ex: 450–490 nm, Em: 520 nm). of chitosan as a plant defense inducer in soil for preventing or Non-damaged cells show green FDA fluorescence and nuclei of managing root pests and diseases sustainably. damaged cells show red PI fluorescence. Frontiers in Plant Science | www.frontiersin.org 2 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions Plant Growth Conditions 220 to 350 nm in 2 nm steps. The slit width was 5 nm and Two experiments were carried out: in vitro and in cups. This the root exudates were placed in a 1 cm path length fused allowed us to progressively approach to a realistic system. quartz cell (Hellma). The UV-visible spectra of samples were Tomato plantlets (Experiment 1) were placed in 200 mL acquired (SHIMADZU UV-160 spectrophotometer, 200–800 nm, expanded polystyrene sterile cups each containing 100 cm3 1 cm quartz cuvette). Absorbance was always lower than 0.1 of sterilized sand. They were then incubated in a culture (ODunits ) at 254 nm in order to reduce the absorbance of chamber (SANYO MLR-351H) at 65% RH, 24◦ C with a the solution to eliminate potential inner filter effects (Mobed 16:8 h (light:dark) photoperiod. Plants were irrigated for 20 et al., 1996). EEM fluorescence spectra of root exudates were days with Gamborgs B5 basal mixture 1:10 keeping moisture analyzed using parallel factor analysis (PARAFAC) as in Ohno to field capacity. Plants were then removed, washed and and Bro (2006). PARAFAC model Components were calculated introduced individually in Magenta Boxes (575 mL, Sigma). for each treatment and time. These analyses have been performed Each Box was filled with 50 mL of Gamborgs B5 1:10 twice with 9 or 10 biological replicates each (Experiment 1 or (control) or Gamborgs B5 1:10 amended with chitosan (0.1, Experiment 2 respectively). 1, 2 mg mL−1 ). Nine biological replicates were made per treatment. Plants were incubated for 3 days as described Plant Hormone Analysis above. Exudates collected in Experiment 1 are root exudates Root exudate pools (Experiment 1) were lyophilized to analyze accumulated over 3 days. This experiment was performed two indoleacetic acid (IAA), abscisic acid (ABA), salicylic acid times independently. (SA) and jasmonic acid (JA) by ultra performance liquid In Experiment 2, tomato plantlets were grown as for chromatography-mass spectrometry (UPLC-MS). Material was Experiment 1. Plants in cups were then irrigated with 1:10 extracted with 80% Methanol-1% Acetic acid. Deuterium- Gamborgs B5 basal mixture on its own or amended with labeled hormones (purchased from Prof. L. Mander-Canberra, 0.1 mg mL−1 chitosan. Plants were incubated as before. Ten OlChemim Ltd. -Olomouc): [2 H5 ] IAA, [2 H4 ] SA, and [2 H6 ] biological replicates per treatment were made. Root Exudates ABA were added as internal standards. For quantification were sampled 10, 20 and 30 days after planting (dap) for of JA, dhJA was used instead. For collecting the fractions further analyses. Exudates collected in Experiment 2 are containing SA, ABA, and JA; extracts were passed consecutively active root exudates (24 h collection, see section “Collection through HLB (reverse phase), MCX (cationic exchange) and of Root Exudates”). This experiment was performed two WAX (ionic exchange) columns (Oasis 30 mg, Waters), as times independently. described in Seo et al. (2011). The final residue was dissolved in 5% Acetonitrile – 1% Acetic acid and separated by Collection of Root Exudates reverse phase UPLC chromatography (2.6 µm Accucore RP- In Experiment 1, root exudates accumulated for 3 days in MS column, 100 mm length × 2.1 mm i.d.; Thermo Fisher Magenta Boxes were collected and filtered through Miracloth Scientific) with a 5 to 50% acetonitrile gradient. Hormones (Calbiochem). Pools were made with the exudates of three plants were analyzed by electrospray ionization and targeted-SIM each, so we worked with three biological replicates per treatment. using a Q-Exactive spectrometer (Orbitrap detector, Thermo This was performed twice. Root exudate pools were filtered by Fisher Scientific). Concentrations of hormones in extracts 0.22 µm (Q-MAX) and frozen at −20◦ C for further use. were determined using embedded calibration curves and In Experiment 2, whole plants were removed, and their the Xcalibur 4.1 SP1 build 48 and TraceFinder programs. root systems washed in SDW. De novo root exudates from We thank Dr. Esther Carrera for hormone quantification these plants were collected by placing individual whole plants carried out at the Plant Hormone Quantification Service, in sterile plastic containers with 20 mL SDW per gram of Valencia, Spain1 . root. Plants were incubated in the dark at 24◦ C, 65% RH for 24 h. Plants were then removed, and root exudates from the Phytomelatonin Detection 10 biological replicates were collected by 0.22 µm (Q-MAX) Phytomelatonin was also tested in both exudates and tissues. filtration and stored frozen at −20◦ C until used. This was Ten-day old seedlings were grown with chitosan (0.1, 1, performed twice. 2 mg mL−1 ) in Gamborgs B5 1:10 for 3 days. Plants exposed to Gamborgs B5 1:10 only were used as control. For each treatment, 0.2–0.3 g of roots were dried onto sterile paper Emission Excitation Matrix (EEM) and placed in a 4 mL polypropylene tube with 3 mL ethyl Fluorescence Analysis acetate. Three biological replicates were made. Samples were Two mL of each root exudate (Experiments 1 and 2) were shaken at 120 rpm and 4◦ C in the dark overnight. Roots collected and excitation emission matrix (EEM) Fluorescence were removed and solvent evaporated under vacuum. The dry spectra were obtained with a spectrofluorometer (Jasco FP- residue was resuspended in 1 mL acetonitrile and 0.22 µm 6500) equipped with a 150W Xenon lamp. Contour maps of filtered. Phytomelatonin from root tissues was quantified by EEM fluorescence spectra were obtained from water extracts HPLC with fluorescence detection with Ex/Em wavelength pair of whole root exudates (10 samples) or pools. The emission of 280/348 nm, as in Hernández-Ruiz and Arnao (2008). For (Em) wavelength range was fixed from 220 to 460 nm in 5 nm steps, whereas the excitation (Ex) wavelength was fixed from 1 www.ibmcp.upv.es Frontiers in Plant Science | www.frontiersin.org 3 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions root exudates, three biological replicates of 1 mL of Experiment 1 Loading values lower than 0 indicate that m/z intensity is higher pools were used. Phytomelatonin was extracted and analyzed as in chitosan-treated plant root exudates. A threshold was set described above. at ± 0.2 to identify m/z with significant variations. Significant m/z values after PLSLDA analyses were selected for Nuclear Magnetic Resonance (1 H NMR) further analysis by HPLC-ESI-MS/MS using a LC-MSD-Trap-SL Root exudates from Experiment 2 were pooled (Agilent). The temperature was 350◦ C and the pressure 70.00 psi (5 replicates/pool), lyophilized and resuspended in 1 mL with a flow of 12 L min−1 . Selected masses are shown in the of D2 O (deuterated water). Two pools were obtained from dataset (Supplementary Material). each treatment and time. Six hundred microliters of filtered After obtaining data on the fragmentation of the ions of pools were placed in a 5 mm NMR tube with 0.75% 3- interest, using MS\MS, we sought to identify the molecules (trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) and that generated the fractionation patterns. We used two different 0.002 g sodium azide. strategies to identify the molecules. In the first strategy, we 1 H NMR experiments were performed on a Bruker AVIII searched the databases by means of the mass of the ions, 700 MHz (CITIUS, University of Sevilla, Spain). The number using the LC–MS search tool, using the positive (adduct type of scans was 256 and the experiments were carried out M+H) or negative (adduct type M-H) mode, with a tolerance at 298 K. 1 H chemical shifts were internally referenced to of 0.1 Da. Once the molecules with masses similar to that the TSP at δ 0.00. 1 H NMR spectra were aligned using searched were obtained, we checked the spectra, both real and TopSpinTM (Bruker). predicted, collected in the databases that were most suitable Sensitivity of NMR is different in each region. For region for the MS\MS spectra we had obtained. The second strategy I (organic-acid and amino-acid region), the threshold was was based on searching different fragments generated by an set in 0.1. For II (sugars/polyalcohols region) and III ion in MS\MS fractionation using the LC–MS\MS search (phenolics/aromatic compounds region) peaks showed less tool and comparing the spectra, in the same way as it was intensity, consequently the threshold was set in 0.01 and done previously. The signals from NMR and HPLC-MS were 0.001 respectively. assigned, putatively, with different databases, such as Human Metabolome Database2 , MassBank3 and Biological Magnetic High Performance Liquid Resonance Data Bank4 . Chromatography Electrospray Ionization Tandem Mass Spectrometry Evaluation of Tomato Root Exudates on (HPLC-ESI-MS) Fungi and Nematode Eggs HPLC-ESI-MS analyses were performed with a High- Bioassays were performed in 96-well plates (Thermo Scientific) Performance Liquid Chromatography system with an Agilent using 20 dap root exudate pools obtained in Experiment 2. 1100 Series model coupled to a UV-visible variable wavelength These exudates were used because higher doses of chitosan detector and a mass spectrometer with ion trap analyser Series would not allow the correct development of the plant and LC/MSD Trap SL (Agilent, Santa Clara, CA, United States). The could not be used in a realistic experiment. Three technical mass spectrometer was operated in the positive and negative replicates of each exudate were made. For each treatment, there ESI modes, and the ion spray voltage was set at 4 kV. Mass were two biological replicates (pools of exudates containing range was set from 50 to 350 atomic mass units. Nitrogen 10 biological replicates initially). For fungal experiments, was used as carrier gas (70 psi), and the ion transfer capillary 200 µL of exudate (pool of 5 plant root exudates each) heated to 350◦ C. Injections were carried out using an HTC Pal and conidia were added per well to reach a concentration autosampler (CTC Analytics, Zwingen, Switzerland) equipped of 106 conidia·mL−1 . For both, FORL and Pc, OD490 was with a 20 µ L sample loop. calculated after 4 and 8-days respectively using a microplate Pools of 20 dap tomato root exudates (Experiment 2) reader (Tecan SPECTRAfluor). Results were handled with XFluor were infused into the flow of the HPLC system (10 µL) SoftwareTM . For time series with FORL and Pc, the relation through a T connection under the following conditions: between two variables is not linear. In both cases, Smoother flow rate, 1 mL min−1 . Ultrapure water with 0.1% Formic Model Lowess (“Locally weighted regression”) was applied Acid was used as Solvent A, whereas MeOH with 0.1% (Cleveland, 1979). Formic Acid was used as Solvent B. From 0 to 15 min To evaluate the effects of root exudates on root-knot nematode Solvent B was kept at 10% and afterward a gradient eggs, experiments were performed with 200 µl of exudate or to 90% was established during 20 min and decreased water (control) containing 100 M. javanica eggs each. Hatching again to 10% until minute 25. The LC separations percentage was scored using an inverted microscope after 72 h were carried out with a Poroshell 120 EC-C18 column incubation at 30◦ C. All experiments were performed twice with (4.6 mm × 100 mm, 2.7 µm -Agilent Technologies-). Runs similar results. were performed at 25◦ C. Raw data were transformed as explained in Marhuenda-Egea et al. (2013). 2 https://hmdb.ca/ Partial least squares regression discriminant analyses 3 https://massbank.eu/MassBank/ (PLSLDA) show that m/z intensities depend on treatment. 4 http://www.bmrb.wisc.edu/metabolomics/ Frontiers in Plant Science | www.frontiersin.org 4 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions FIGURE 1 | Chitosan depolarizes plasma membrane and damages tomato root cells. (A) Variation in membrane potential of root cells with chitosan. High doses (1 and 2 mg mL- 1 ) of chitosan significantly reduce membrane potential. (B) High doses of chitosan damage root cells after 24 h. Red staining labels damaged cells while green staining labels living ones. Three biological replicates were made for each treatment. Multifactorial ANOVA was used to compare treatments [p-values 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****)]. Statistical Analyses to display and group data (Verboven and Hubert, 2005). This data Excitation emission matrix Fluorescence data was analyzed by analysis was carried out using the LIBRA toolbox6 . PARAFAC, as described above, and the contribution of the Components 1, 2, and 3 analyzed by ANOVA tests. The level of significance in all cases was 95%. All statistical analyses RESULTS were performed using GraphPad Prism version 7.00 (GraphPad Software, La Jolla, CA, United States5 ). Chitosan Depolarizes Plasma Membrane HPLC-ESI-MS data were processed using a Partial Least and Reduces Tomato Root Cell Viability Square (PLS) regression model (Verboven and Hubert, 2005; Chitosan (1 and 2 mg mL−1 ) depolarizes (p < 0.001) plasma Marhuenda-Egea et al., 2013). Classical PCA were also performed membrane of tomato root cells (Figure 1A). This is reflected in 5 6 https://www.graphpad.com/ http://wis.kuleuven.be/stat/robust/software Frontiers in Plant Science | www.frontiersin.org 5 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions staining is due to natural senescence of root epidermic cells (Figure 1B, arrow). Chitosan Induces Hormones and Phenolic Compounds in Root Exudates Excitation emission matrix Fluorescence analysis of root exudates resulted in three components (Figure 2 and Supplementary Table 1). Components 1 and 3 are the most induced (p < 0.01) by chitosan. Component 1 includes a putative fluorophore with Ex/Em wavelength pair of 315/430 nm, which could correspond to SA (Street and Schenk, 1981). Component 3 includes Ex/Em wavelength pairs of 245/384 and 265/384 nm, which may correspond to aromatic amino acids and peptides (Yang et al., 2015; Table 1). Chitosan induces other hormones in tomato root exudates (HPLC–MS; Figure 3). High doses of chitosan (1 and 2 mg mL−1 ) induce (p < 0.01) IAA accumulation in root exudates (Figure 3A). Plant defense hormones (SA, JA, and ABA) are also significantly induced (p < 0.05) by chitosan (1 mg mL−1 ) in tomato root exudates (Figures 3B–D). This effect is lost at 2 mg mL−1 chitosan. This could be the result of a root systemic damage caused by large chitosan concentrations. In view of the effect of chitosan on plant hormone homeostasis, endogenous phytomelatonin in roots was evaluated. Chitosan (1 mg mL−1 ) causes a slight rise on phytomelatonin content in tomato root cells (Figure 3E). Phytomelatonin levels detected in tomato root exudates are close to nil (Supplementary Figure 1). Chitosan Induces de novo Exudation of SA and Phenolics in Roots In view of the results obtained with tomato roots elicited for 3 days with chitosan, we irrigated tomato plants with a low dose of chitosan (0.1 mgmL−1 ) during 10, 20, and 30 days and analyzed de novo exudation in roots. Ten days after planting (dap), chitosan increases de novo (p < 0.01) exudation of a fluorescence signature putatively belonging to SA (Figure 4 and Table 1). Twenty dap, chitosan significantly increases (p < 0.001) fluorescence intensity of Component 3, FIGURE 2 | Chitosan increases EEM fluorescence of tomato root exudates. putatively assigned to phenolics (Mostofa et al., 2013; Parri (A) Component 1 (salicylic acid); (B) Component 2 (phenolics and salicylic et al., 2020) and SA derivatives (Street and Schenk, 1981). acid derivatives); (C) Component 3 (aromatic aa and peptides). For Ex/Em This tendency is also found for Components 1 (putatively coordinates of Components see Table 1. UF, fluorescence units; EEM, emission excitation matrix. Mean and Standard Deviation of three biological aromatic amino acids and peptides) and 2 (putatively IAA; replicates (three pools containing root exudates from three individuals each) is Li et al., 2009), although differences are not significant. In represented. Multifactorial ANOVA was used to compare treatments [p-values late root exudates (30 dap), fluorescence intensity decreases 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****)]. respect to early root exudates. This could be due to root aging and lignification. At this time, EEM Fluorescence spectrum is separated into 2 Components only (Supplementary loss of cell viability (PI red staining, Figure 1B). Roots incubated Table 1 and Supplementary Figure 2) with no differences with 2 mg mL−1 chitosan show a curved morphology and dark respect to controls. precipitates. Conversely, a low dose of chitosan (0.1 mg mL−1 ) does not alter plasma membrane potential. However, these Metabolomic Diversity of Tomato Root roots show both viable (FDA green staining) and non-viable Exudates Varies With Time cells, indicating some chitosan damage. Untreated tomato roots Pools were made to concentrate root exudates and avoid show mostly viable cells, stained with FDA. The slight red sample variability (Yuan et al., 2015; Yang et al., 2016). Root Frontiers in Plant Science | www.frontiersin.org 6 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions TABLE 1 | Excitation and emission pairs of coordinates for each PARAFAC model per time. Time Excitation (nm) Emission (nm) Compound 3 dap C1: 240/315 C2: 230/280 C3: C1: 430 C2: 336 C3: 384 C1: SA (Street and Schenk, 1981) C2: Phenolics (Mostofa et al., 2013; Parri et al., 245/265/280 2020) C3: Aromatic amino acids and peptides (Yang et al., 2015) 10 dap C1: 240/310 C2: 285 C3: C1: 436 C2: 364 C3: 312/358 C1: SA (Street and Schenk, 1981) C2: IAA (Li et al., 2009) C3: Aromatic amino acids 230/275 and peptides (Yang et al., 2015), Phenolics (Mostofa et al., 2013; Parri et al., 2020) 20 dap C1: 240/310 C2: 285 C3: C1: 438 C2: 366 C3: 308 C1: SA (Street and Schenk, 1981) C2: IAA (Li et al., 2009) C3: Phenolics (Mostofa 230/270 et al., 2013; Parri et al., 2020) 30 dap C1: 235/310 C2: 230/280 C1: 440 C2: 332 C1: SA (Street and Schenk, 1981) C2: Aromatic amino acids and peptides (Yang et al., 2015), Phenolics (Mostofa et al., 2013; Parri et al., 2020) SA, salicylic acid; IAA, indole acetic acid; dap, days after planting; C1, component 1; C2, component 2; C3, component 3. FIGURE 3 | Chitosan induces plant hormones. (A–D) Figures correspond to hormone quantitation of accumulated tomato root exudates in three biological replicates over 3 days. (A) Indole acetic acid; (B) salicylic acid; (C) jasmonic acid; (D) abscisic acid; (E) Phytomelatonin quantitation in tomato roots. There is a maximum level of hormones present in exudates from plants treated with 1 mg mL−1 chitosan. Multifactorial ANOVA was used to compare treatments [p-values 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****)]. Frontiers in Plant Science | www.frontiersin.org 7 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions FIGURE 4 | Excitation emission matrix fluorescence dynamics of tomato root exudates. For Ex/Em coordinates of Components see Table 1. Ten biological replicates per treatment were used. EEM, emission excitation matrix; dap, days after planting; C1, component 1; C2, component 2; C3, component 3; UF, fluorescence units. Mann–Whitney test was used for not normal distribution treatments. Welch’s parametric test was used for the remaining treatments [p-values 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****)]. exudates from 20 dap tomato plants (chitosan treated and controls) displayed most NMR peaks (Figure 5). This may be because older plants (30 dap) are more lignified (Cervilla et al., 2009) and display less rhizodeposition. Manual curation of1 H NMR profiles of root exudates from plants 10, 20, and 30 dap from chitosan treatments and controls show no qualitative differences and contain 31, 123, and 18, peaks respectively. Ninety-three peaks are specific to 20 dap root exudates. Nine 20 dap specific peaks were identified as (Table 2): leucine/isoleucine (13), acetate (53), raffinose (99, 103), glucose (100), uracil (105 and 120), cinnamic acid (109, 119, 121), fumaric acid (109), p-aminobenzoic acid (112 and 122) and trigonelline (125, 130, 131). Chitosan decreases (p < 0.05) acetate (peak 53) content in 20 dap root exudates. Six peaks, among them methanol (80) and formic acid (129), are found 20 and 30 dap. Lactate (peak 36) and malate (peaks 62 FIGURE 5 | Tomato rhizodeposition varies with time. Venn-diagram of 1 H and 64) have been detected in all time points evaluated. Nuclear Magnetic Resonance analyses peaks detected in tomato root All detected peaks are listed in Supplementary Table 2. exudates at 10, 20, and 30 days after planting. In all times, plants were treated with 0.1 mg mL- 1 chitosan. A representative 1 H NMR profile of tomato root exudates is shown in Supplementary Figure 3. Frontiers in Plant Science | www.frontiersin.org 8 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions TABLE 2 | Peak assignments for 1 H NMR spectra of tomato root exudates. 20 dap [Chitosan] (mg·mL−1 ) Part of the dap Peak number Compound Shift (ppm) H mult. 0 0.1 spectra I 20 13 Leucine/isoleucine 0.94 0.25475 0.17575 I 10-20-30 36 Lactate 1.33 d 0.35685 0.18225 I 20 53 Acetate 1.92 s 4.7895 3.4135 I 10-20-30 62 Malate 2.3 t 0.6217 0.5769 I 10-20-30 64 Malate 2.41 1.725 1.26 II 20-30 80 Methanol 3.36 s 0.34845 0.2379 II 20 99 Raffinose 4.99 d 0.027535 0.03025 II 20 100 Glucose 5.237 d 0.02523 0.035445 II 20 103 Raffinose 5.42 d 0.02462 0.02018 II 20 105 Uracil 5.79 d 0.031275 0.02049 III 20 109 Cinnamic acid/fumaric acid 6.52/6.51 d/s 0.2005 0.15425 III 20 112 p-Aminobenzoic acid 6.82 d 0.0033845 0.0050605 III 20 119 Cinnamic acid 7.43 m 0.049535 0.04216 III 20 120 Uracil 7.52 d 0.00791 0.016235 III 20 121 Cinnamic acid 7.62 dt 0 0.002784 III 20 122 p-Aminobenzoic acid 7.73 d 0.0056825 0.00603 III 20 125 Trigonelline 8.07 m 0.006835 0.01239 III 20-30 129 Formic acid 8.44 m 0.35505 0.1718 III 20 130 Trigonelline 8.82 m 0.028845 0.022265 III 20 131 Trigonelline 9.13 s 0.03144 0.024555 This table includes identified peaks only. Spectra parts I, II, and III are indicated in Supplementary Figure 3. Abbreviations: Dap, days after planting; H mult., H multiplicity; d, doublet; s, singlet; t, triplet; dt, double of triplets; m, multiplet. Treatment abbreviations: 0, Tomato Root Exudates Control; 0.1, Tomato Root Exudates from plants irrigated with 0.1 mg mL−1 chitosan. Numbers in treatments correspond to the mean of maximum intensity of the peaks (arbitrary units). Bold numbers indicate significant differences (ANOVA, p < 0.05). Chitosan Induces Lipid Signaling and (Figure 7A). These exudates also inhibit growth of Fusarium Defense Compounds in Tomato Root oxysporum f.sp. radicis-lycopersici (FORL) ca. twofold respect to controls (Figure 7B). The chitosan resistant fungus Pochonia Exudates chlamydosporia strain 123 (Pc) does not show significant Lipid signaling and defense compounds are putatively identified differences in growth with both exudates over time (Figure 7C). in 20 dap root exudates (Figure 6 and Supplementary Table 4, Supplementary Database HPLC-MS/MS). Putative oxidized fatty acid (FA18:4+1O, m/z 293.5+ ) and other fatty acids (Ethyl DISCUSSION stearate/Arachidic acid, m/z 313.7+ ) are increased with chitosan. On the contrary, FA18:2+3O (m/z 327.3− ) is reduced with Chitosan has a well stablished role as a plant defense activator chitosan (m/z 290.5+ ). Stress response metabolites such as - (El Hadrami et al., 2010). In this study we give evidences that putatively- Atropine / Hyoscyamine (A/H, m/z 290.5+ ) and chitosan induces plant defenses in tomato root exudates. Plasma putative citric acid (CA, m/z 191.0− ) are increased with chitosan. membranes are likely to be a main target of this molecule On the contrary, 3-O0 -Methyladenosine (3OM, m/z 282.7+ ), is (Palma-Guerrero et al., 2010; Meisrimler et al., 2011; Jaime slightly reduced. N-Fructosyl tyrosine (m/z 344.3+ ) is slightly et al., 2012). In this work, we show that increasing chitosan decreased with chitosan. Putative Fructose-1,6-bisphosphate concentrations depolarize tomato root cell plasma membrane. (FB, m/z 327.7− ), involved in the glycolysis pathway, is He et al. (2009) proposed chitosan as a potential cell penetration increased with chitosan. enhancer. This fact has been demonstrated using vital staining in fungi (Palma-Guerrero et al., 2010) and, in this work, in root apices. These effects of chitosan on roots are likely to modify Root Exudates From Plants Treated With rhizodeposition (Pitta-Alvarez and Giulietti, 1999). Chitosan Chitosan Inhibit Soil-Borne Pathogens membrane depolarization may trigger an increase of reactive Soil-borne pathogens (fungi and nematodes) are inhibited by root oxygen species (ROS) in the plant cell, generating secondary exudates from plants treated with chitosan. Root exudates from metabolites (Pandey, 2017). ROS are known to alter chemical chitosan-treated plants cause ca. 1.5-fold reduction (p < 0.05) components of the cell and lipids in particular. We have found on hatching of the root-knot nematode Meloydogine javanica putative FA18:4+1O and Ethyl stearate/Arachidic acid increase eggs after 72 h respect to tomato control root exudates in exudates from roots treated with chitosan. These fatty acids Frontiers in Plant Science | www.frontiersin.org 9 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions FIGURE 6 | Chitosan induces lipid signaling and defense compounds in tomato root exudates. Partial least squares regression discriminant analysis (PLSLDA) of HPLC-ESI-MS analysis of pools of 20 days after planting tomato root exudates in modes positive (A) and negative (B). Four biological replicates per treatment are represented. Blue bars indicate a particular mass that differs from the control treatment. The larger the bar size, the more noticeable the difference in intensity of the mass compared to the control. could have been released from cell membranes by the oxidative shown to mediate stress in plant cell responses (Moharrami stress produced by chitosan in plants (Lopez-Moya et al., 2017). et al., 2017; Mallhi et al., 2019). The increase of fructose-1,6- These long chain fatty acids could be involved in the production bisphosphate in root exudates from plants treated with chitosan of oxylipins (Noverr et al., 2003). Some oxylipins, such as could reflect an extra consumption of glucose in root cells in oxophytodienoic acid (OPDA), are JA precursors (Dave and response to chitosan induced ROS increase (Cao et al., 2020). Graham, 2012). This could explain our finding of JA increase Finally, the slight reduction of Methyladenosine in root exudates in root exudates from chitosan treated plants. Oxylipins other from chitosan-treated plants, could indicate that this polymer is than jasmonates are probably also essential for the resistance perhaps affecting gene expression in roots (Zheng et al., 2020). of plants to pathogens (Blée, 2002). Putative alkaloids and CA Importance of chitosan in plant hormone production and are also increased with chitosan. These molecules have been systemic acquired resistance has been widely demonstrated Frontiers in Plant Science | www.frontiersin.org 10 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions FIGURE 7 | Root exudates from plants treated with chitosan inhibit soil-borne pathogens. (A) Effect on hatching of Meloidogyne javanica eggs in tomato root exudates. Egg hatching is reduced ca. 1.5-fold. Multifactorial analysis Kruskal–Wallis was performed to compare treatments [p-values 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****)] Control is egg hatching in water, 0 corresponds to untreated tomato root exudates. (B) Growth kinetics of FORL in root exudates, chitosan inhibits ca. twofold growth. C, Growth kinetics of P. chlamydosporia strain 123 in tomato root exudates. Smoother Models were adjusted with band 0.75 and degree 2. Shadow area stablishes the prediction confidence intervals at 95%. (Mika et al., 2010; Colman et al., 2019; Fooladi vanda et al., Jaisi and Panichayupakaranant, 2020; Samari et al., 2020). 2019; Iglesias et al., 2019; Ma et al., 2019). We have found that Chitosan enhances metabolic pathways (e.g., phenylpropanoid) increasing chitosan leads to the accumulation of fluorescence involved in the biosynthesis of phenolic compounds (Asgari- compounds corresponding to phenolics, as well as hormones Targhi et al., 2018; Fooladi vanda et al., 2019; Singh et al., 2020). related to plant growth and defense (JA, SA, ABA, and IAA). This A low dose of chitosan enhances plant immunity via plant correlation between chitosan and phenolics has been previously hormone (JA and SA, mainly) accumulation in root tissues studied (Pitta-Alvarez and Giulietti, 1999; Park et al., 2019; (Lopez-Moya et al., 2017; Iglesias et al., 2019; Singh et al., 2020). Frontiers in Plant Science | www.frontiersin.org 11 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions However, this effect has not yet been detected in tomato root DATA AVAILABILITY STATEMENT exudates. In addition, there is evidence that chitosan could be used as a substitute for commonly used growth factors The datasets presented in this study can be found in such as methyl jasmonate, auxins, or cytokinins (Cui et al., online repositories. The names of the repository/repositories 2012; Sivanandhan et al., 2012; Ahmad et al., 2019; Acemi, and accession number(s) can be found below: https://datadryad. 2020) due to its elicitor effects (Salachna and Zawadzińska, org/stash/share/iQ4Fh9XTzpF5Rbsgz9dakjCP2BfvpSigHx- 2014; Malerba and Cerana, 2016). Phytomelatonin has been hhuy57GY. recently considered a plant master regulator involved in abiotic and biotic stress responses. In our work, exposure of tomato roots to increasing chitosan doses accumulates AUTHOR CONTRIBUTIONS phytomelatonin and hormones in roots and root exudates in a typical biotic stress response (Arnao and Hernández-Ruiz, LVL-L conceived the original screening and research plans 2019; Moustafa-Farag et al., 2019). The decrease in levels of and wrote the manuscript. MS-F performed the main some plant hormones at toxic chitosan doses has also been experiments and wrote the manuscript. FCM-E analyzed the found for phenolics (Park et al., 2019). EEM Fluorescence fluorescence, 1H NMR and HPLC-MS data. FL-M supervised signatures corresponding to IAA (Li et al., 2009) and SA the experiments and writing. MBA provided technical assistance (Miles and Schenk, 1970) are increased by chitosan in tomato to phytomelatonin detection experiments. FC-E provided root exudates. These hormones are known to be increased technical assistance to 1H NMR experiments. MJN performed by chitosan (Lopez-Moya et al., 2017; Fooladi vanda et al., the time series analyses. BG performed electrophysiological 2019). SA and JA are related to plant responses to stress and vital staining experiments. MS-F agrees to serve as the (War et al., 2011). Therefore, chitosan can be an elicitor author responsible for contact and ensures communication. of plant defenses (Benhamou, 1996) in root exudates. Our All authors contributed to the article and approved the bioassays show that chitosan induces root exudates inhibitory submitted version. to root pathogenic fungus FORL and root-knot nematode eggs without significantly affecting the growth and development of a biocontrol fungus (Pc). The toxic effect of chitosan-derived FUNDING exudates may be related to the overproduction of SA which, in combination with chitosan induce systemic acquired resistance This work was supported by AGL 2015 66833-R Grant from the and reduce infection by root-knot nematodes (Vasyukova et al., Spanish Ministry of Economy and Competitiveness and H2020 2003; Singh et al., 2020). Chitosan, moreover, by itself, is MUSA 727624 European Project. toxic to fungi such as Fusarium spp. (Palma-Guerrero et al., 2010; Al-Hetar et al., 2011). Other studies show that plants treated with chitosan are protected from FORL infection ACKNOWLEDGMENTS (Benhamou and Thériault, 1992). Chitosan depolarizes plasma membrane of tomato root Thanks are due to members of the Plant Pathology Laboratory of cells, causing the secretion of hormones, lipid signaling and the University of Alicante, for their help and support. Authors plant defense compounds, including phenolics. This process also wish to thank Mr. Arnau Hernández Royo for his help affects cell viability and rhizodeposition, so plant age must be conducting electrophysiological and microscopy experiments. considered before applying chitosan, as well as the duration This manuscript has been released as a pre-print at bioRXiv of applications. Further studies should test chitosan derived (Suarez-Fernandez et al., 2020). exudates under in vivo conditions to determine their efficacy to prevent the proliferation of root pathogens and subsequently reduce disease severity. Overall, our results have proven that SUPPLEMENTARY MATERIAL root exudates of chitosan-treated plants are capable to reduce soil-borne pathogens growth by in vitro. This makes chitosan a The Supplementary Material for this article can be found promising agent to prevent and manage pests and diseases in a online at: https://www.frontiersin.org/articles/10.3389/fpls.2020. sustainable way. 572087/full#supplementary-material REFERENCES salicifolia (Bedd. ex Hook.f.) Venter. Plant Cell Tiss Organ. Cult. 136, 29–40. doi: 10.1007/s11240-018-1488-4 Acemi, A. (2020). Chitosan versus plant growth regulators: a comparative analysis Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). of their effects on in vitro development of Serapias vomeracea (Burm.f.) Briq. Ion Channels and the Electrical Properties of Membranes. Molecular Biology Plant Cell Tiss Organ. Cult. 141, 327–338. doi: 10.1007/s11240-020-01789-3 of the Cell, 4th Edn. Available online at: https://www.ncbi.nlm.nih.gov/books/ Ahmad, Z., Shahzad, A., and Sharma, S. (2019). Chitosan versus yeast extract NBK26910/ (accessed March 19, 2020) driven elicitation for enhanced production of fragrant compound 2-hydroxy- Al-Hetar, M. Y., Zainal Abidin, M. A., Sariah, M., and Wong, M. Y. 4-methoxybenzaldehyde (2H4MB) in root tuber derived callus of Decalepis (2011). Antifungal activity of chitosan against Fusarium oxysporum Frontiers in Plant Science | www.frontiersin.org 12 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions f. sp. cubense. J. Appl. Polym. Sci. 120, 2434–2439. doi: 10.1002/app. FAO (2017). Good Agricultural Practices for Greenhouse Vegetable Production in the 33455 South East European Countries. Rome: FAO. ISSN 2070-2515. Plant Production Arnao, M. B., and Hernández-Ruiz, J. (2018). Melatonin and its relationship and Protection Paper 230. to plant hormones. Ann. Bot. 121, 195–207. doi: 10.1093/aob/ Flores, H. E., Weber, C., and Puffett, J. (1996). “Underground metabolism: the mcx114 biosynthetic potential of roots,” in Roots: The Hidden Half 2nd edn., ed. Y. Arnao, M. B., and Hernández-Ruiz, J. (2019). Melatonin: a new plant hormone Waisel (New York: Marcel Dekker Inc.), 931–956. and/or a plant master regulator? Trends Plant Sci. 24, 38–48. doi: 10.1016/j. Fooladi vanda, G., Shabani, L., and Razavizadeh, R. (2019). Chitosan enhances tplants.2018.10.010 rosmarinic acid production in shoot cultures of Melissa officinalis L. through Arnao, M. B., and Hernández-Ruiz, J. (2007). Melatonin promotes the induction of methyl jasmonate. Bot. Stud. 60:26. doi: 10.1186/s40529-019- adventitious- and lateral root regeneration in etiolated hypocotyls of 0274-x Lupinus albus L. J. Pineal Res. 42, 147–152. doi: 10.1111/j.1600-079X.2006. Fürstenberg-Hägg, J., Zagrobelny, M., and Bak, S. (2013). Plant defense against 00396.x insect herbivores. Int. J. Mol. Sci. 14, 10242–10297. doi: 10.3390/ijms140510242 Asami, T., and Nakagawa, Y. (2018). Preface to the special issue: brief review of Gamborg, O. L., Miller, R. A., and Ojima, K. (1968). Nutrient requirements of plant hormones and their utilization in agriculture. Pestic. Sci. 43, 154–158. suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158. doi: 10. doi: 10.1584/jpestics.M18-02 1016/0014-4827(68)90403-5 Asgari-Targhi, G., Iranbakhsh, A., and Ardebili, Z. O. (2018). Potential benefits Gunsé, B., Poschenrieder, C., Rankl, S., Schröeder, P., Rodrigo-Moreno, A., and and phytotoxicity of bulk and nano-chitosan on the growth, morphogenesis, Barceló, J. (2016). A highly versatile and easily configurable system for plant physiology, and micropropagation of Capsicum annuum. Plant Physiol. electrophysiology. MethodsX 3, 436–451. doi: 10.1016/j.mex.2016.05.007 Biochem. 127, 393–402. doi: 10.1016/j.plaphy.2018.04.013 He, W., Guo, X., Xiao, L., and Feng, M. (2009). Study on the mechanisms of Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., and Vivanco, J. M. (2006). The role chitosan and its derivatives used as transdermal penetration enhancers. Int. J. of root exudates in rhizosphere interactions with plants and other organisms. Pharm. 382, 234–243. doi: 10.1016/j.ijpharm.2009.07.038 Annu. Rev. Plant Biol. 57, 233–266. doi: 10.1146/annurev.arplant.57.032905. Hernández-Ruiz, J., and Arnao, M. B. (2008). Distribution of melatonin in different 105159 zones of lupin and barley plants at different ages in the presence and absence of Baldrian, P. (2019). The known and the unknown in soil microbial ecology. FEMS light. J. Agric. Food Chem. 56, 10567–10573. doi: 10.1021/jf8022063 Microbiol. Ecol. 95:fiz005. doi: 10.1093/femsec/fiz005 Hernández-Ruiz, J., Cano, A., and Arnao, M. B. (2005). Melatonin acts as a Benhamou, N. (1996). Elicitor-induced plant defence pathways. Trends Plant Sci. growth-stimulating compound in some monocot species: melatonin as growth 1, 233–240. doi: 10.1016/1360-1385(96)86901-9 promoter in plants. J. Pineal Res. 39, 137–142. doi: 10.1111/j.1600-079X.2005. Benhamou, N., and Thériault, G. (1992). Treatment with chitosan enhances 00226.x resistance of tomato plants to the crown and root rot pathogen Fusarium Hirsch, A. M., Bauer, W. D., Bird, D. M., Cullimore, J., Tyler, B., and Yoder, J. I. oxysporum f. sp. radicis-lycopersici. Physiol. Mol. Plant Pathol. 41, 33–52. doi: (2003). Molecular signals and receptors: controlling rhizosphere interactions 10.1016/0885-5765(92)90047-Y between plants and other organisms. Ecology 84, 858–868. Blée, E. (2002). Impact of phyto-oxylipins in plant defense. Trends Plant Sci. 7, Hupp, S., Rosenkranz, M., Bonfig, K., Pandey, C., and Roitsch, T. (2019). 315–322. doi: 10.1016/S1360-1385(02)02290-2 Noninvasive phenotyping of plant–pathogen interaction: consecutive In Situ Cao, B., Ma, Q., and Xu, K. (2020). Silicon restrains drought-induced ROS Imaging of Fluorescing Pseudomonas syringae, plant phenolic fluorescence, accumulation by promoting energy dissipation in leaves of tomato. Protoplasma and chlorophyll fluorescence in Arabidopsis leaves. Front. Plant Sci. 10:1239. 257, 537–547. doi: 10.1007/s00709-019-01449-0 doi: 10.3389/fpls.2019.01239 Cervilla, L. M., Rosales, M. A., Rubio-Wilhelmi, M. M., Sánchez-Rodríguez, E., Iglesias, M. J., Colman, S. L., Terrile, M. C., París, R., Martín-Saldaña, S., Chevalier, Blasco, B., Ríos, J. J., et al. (2009). Involvement of lignification and membrane A. A., et al. (2019). Enhanced properties of chitosan microparticles over bulk permeability in the tomato root response to boron toxicity. Plant Sci. 176, chitosan on the modulation of the auxin signaling pathway with beneficial 545–552. doi: 10.1016/j.plantsci.2009.01.008 impacts on root architecture in plants. J. Agric. Food Chem. 67, 6911–6920. Cleveland, W. S. (1979). Robust locally weighted regression and smoothing doi: 10.1021/acs.jafc.9b00907 scatterplots. J. Am. Stat. Assoc. 74, 829–836. doi: 10.1080/01621459.1979. Iriti, M., and Faoro, F. (2008). Abscisic acid is involved in chitosan-induced 10481038 resistance to tobacco necrosis virus (TNV). Plant Physiol. Biochem. 46, 1106– Colman, S. L., Salcedo, M. F., Mansilla, A. Y., Iglesias, M. J., Fiol, D. F., Martín- 1111. doi: 10.1016/j.plaphy.2008.08.002 Saldaña, S., et al. (2019). Chitosan microparticles improve tomato seedling Jaime, M. D., Lopez-Llorca, L., Conesa, A., Lee, A. Y., Proctor, M., Heisler, L. E., biomass and modulate hormonal, redox and defense pathways. Plant Physiol. et al. (2012). Identification of yeast genes that confer resistance to chitosan Biochem. 143, 203–211. doi: 10.1016/j.plaphy.2019.09.002 oligosaccharide (COS) using chemogenomics. BMC Genomics 13:267. doi: 10. Cui, L., Wang, Z.-Y., and Zhou, X.-H. (2012). Optimization of elicitors and 1186/1471-2164-13-267 precursors to enhance valtrate production in adventitious roots of Valeriana Jaisi, A., and Panichayupakaranant, P. (2020). Enhanced plumbagin production in amurensis Smir. ex Kom. Plant Cell Tiss. Organ. Cult. 108, 411–420. doi: 10. Plumbago indica root culture by simultaneous and sequential dual elicitations 1007/s11240-011-0052-2 using chitosan with L-alanine and methyl-β-cyclodextrin. Bioresour. Bioprocess. Dave, A., and Graham, I. A. (2012). Oxylipin signaling: a distinct role for the 7:10. doi: 10.1186/s40643-020-0298-9 jasmonic acid precursor cis-(+)-12-Oxo-phytodienoic acid (cis-OPDA). Front. Jones, K. H., and Senft, J. A. (1985). An improved method to determine cell Plant Sci. 3:42. doi: 10.3389/fpls.2012.00042 viability by simultaneous staining with fluorescein diacetate-propidium iodide. El Hadrami, A., Adam, L. R., El Hadrami, I., and Daayf, F. (2010). Chitosan in plant J. Histochem Cytochem. 33, 77–79. doi: 10.1177/33.1.2578146 protection. Mar. Drugs 8, 968–987. doi: 10.3390/md8040968 Kaur, S., and Dhillon, G. S. (2014). The versatile biopolymer chitosan: potential Elieh-Ali-Komi, D., and Hamblin, M. R. (2016). Chitin and chitosan: production sources, evaluation of extraction methods and applications. Crit. Rev. Microbiol. and application of versatile biomedical nanomaterials. Int. J. Adv. Res. 4, 40, 155–175. doi: 10.3109/1040841X.2013.770385 411–427. Kumar, M. N. V. (2000). A review of chitin and chitosan applications. React. Funct. El-Tantawy, E. M. (2009). Behavior of tomato plants as affected by spraying with Polym. 46, 1–27. doi: 10.1016/S1381-5148(00)00038-9 chitosan and aminofort as natural stimulator substances under application of Li, Y.-N., Wu, H.-L., Zhu, S.-H., Nie, J.-F., Yu, Y.-J., Wang, X.-M., et al. (2009). soil organic amendments. Pak. J. Biol. Sci. 12, 1164–1173. doi: 10.3923/pjbs. Determination of indole-3-acetic acid in soil using excitation-emission matrix 2009.1164.1173 fluorescence with trilinear decomposition-based calibration methods. Anal. Sci. Escudero, N., Marhuenda-Egea, F. C., Ibanco-Cañete, R., Zavala-Gonzalez, E. A., 25, 83–88. doi: 10.2116/analsci.25.83 and Lopez-Llorca, L. V. (2014). A metabolomic approach to study the Lopez-Moya, F., Escudero, N., Zavala-Gonzalez, E. A., Esteve-Bruna, D., Blázquez, rhizodeposition in the tritrophic interaction: tomato, Pochonia chlamydosporia M. A., Alabadí, D., et al. (2017). Induction of auxin biosynthesis and WOX5 and Meloidogyne javanica. Metabolomics 10, 788–804. doi: 10.1007/s11306- repression mediate changes in root development in Arabidopsis exposed to 014-0632-3 chitosan. Sci. Rep. 7:16813. doi: 10.1038/s41598-017-16874-5 Frontiers in Plant Science | www.frontiersin.org 13 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions Ma, B., Wang, J., Liu, C., Hu, J., Tan, K., Zhao, F., et al. (2019). Preventive effects Pitta-Alvarez, S. I., and Giulietti, A. M. (1999). Influence of chitosan, acetic acid an of fluoro-substituted benzothiadiazole derivatives and chitosan oligosaccharide citric acid on growth and tropane alkaloid production in transformed roots of against the rice seedling blight induced by Fusarium oxysporum. Plants 8:538. Brugmansia candida – Effect of medium pH and growth phase. Plant Cell Tiss. doi: 10.3390/plants8120538 Org. 59, 31–38. doi: 10.1023/A:1006359429830 Malerba, M., and Cerana, R. (2016). Chitosan effects on plant systems. Int. J. Mol. Rodrigo-Moreno, A., Andrés-Colás, N., Poschenrieder, C., Gunsé, B., Peñarrubia, Sci. 17:996. doi: 10.3390/ijms17070996 L., and Shabala, S. (2013). Calcium- and potassium-permeable plasma Mallhi, Z. I., Rizwan, M., Mansha, A., Ali, Q., Asim, S., Ali, S., et al. (2019). membrane transporters are activated by copper in Arabidopsis root tips: linking Citric acid enhances plant growth, photosynthesis, and phytoextraction of lead copper transport with cytosolic hydroxyl radical production: copper transport by alleviating the oxidative stress in castor beans. Plants 8:525. doi: 10.3390/ and hydroxyl radical production. Plant Cell Environ. 36, 844–855. doi: 10.1111/ plants8110525 pce.12020 Mandal, S. M., Chakraborty, D., and Dey, S. (2010). Phenolic acids act as signaling Salachna, P., and Zawadzińska, A. (2014). Effect of chitosan on plant growth, molecules in plant-microbe symbioses. Plant Signal. Behav. 5, 359–368. flowering and corms yield of potted. J. Ecol. Eng. 15, 97–102. doi: 10.12911/ Marhuenda-Egea, F. C., Gonsálvez-Álvarez, R. D., Lledó-Bosch, B., Ten, J., 22998993.1110223 and Bernabeu, R. (2013). New approach for chemometric analysis of mass Samari, E., Sharifi, M., Ghanati, F., Fuss, E., and Ahmadian Chashmi, N. (2020). spectrometry data. Anal. Chem. 85, 3053–3058. doi: 10.1021/ac303255h Chitosan-induced phenolics production is mediated by nitrogenous regulatory Matzke, A. J. M., and Matzke, M. (2013). Membrane “potential-omics”: toward molecules: NO and PAs in Linum album hairy roots. Plant Cell Tiss. Organ. Cult. voltage imaging at the cell population level in roots of living plants. Front. Plant 140, 563–576. doi: 10.1007/s11240-019-01753-w Sci. 4:311. doi: 10.3389/fpls.2013.00311 Seid, A., Fininsa, C., Mekete, T., Decraemer, W., and Wesemael, W. M. L. (2015). McClure, M. A., Kruk, T. H., and Misaghi, I. (1973). A method for obtaining Tomato (Solanum lycopersicum) and root-knot nematodes (Meloidogyne quantities of clean meloidogyne eggs. J. Nematol. 5:230. spp.) – a century-old battle. Nematology 17, 995–1009. doi: 10.1163/15685411- Meisrimler, C.-N., Planchon, S., Renaut, J., Sergeant, K., and Lüthje, S. (2011). 00002935 Alteration of plasma membrane-bound redox systems of iron deficient pea Seo, M., Jikumaru, Y., and Kamiya, Y. (2011). “Profiling of hormones and related roots by chitosan. J. Proteomics 74, 1437–1449. doi: 10.1016/j.jprot.2011.01.012 metabolites in seed dormancy and germination studies,” in Seed Dormancy Mika, A., Boenisch, M. J., Hopff, D., and Lüthje, S. (2010). Membrane-bound Methods in Molecular Biology, ed. A. R. Kermode (Totowa, NJ: Humana Press), guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl 99–111. doi: 10.1007/978-1-61779-231-1_7 jasmonate, salicylic acid, and pathogen elicitors. J. Exp. Bot. 61, 831–841. doi: Singh, M., Poddar, N. K., Singh, D., and Agrawal, S. (2020). Foliar application 10.1093/jxb/erp353 of elicitors enhanced the yield of withanolide contents in Withania somnifera Miles, C. I., and Schenk, G. H. (1970). Fluorescence of acetylsalicylic acid in (L.) Dunal (variety. Poshita). 3 Biotech 10:157. doi: 10.1007/s13205-020- solution and its measurement in presence of salicylic acid. Anal. Chem. 42, 2153-2 656–659. doi: 10.1021/ac60288a032 Singh, V. K., Singh, A. K., and Kumar, A. (2017). Disease management of tomato Mobed, J. J., Hemmingsen, S. L., Autry, J. L., and McGown, L. B. (1996). through PGPB: current trends and future perspective. 3 Biotech 7:255. doi: Fluorescence characterization of IHSS humic substances: total luminescence 10.1007/s13205-017-0896-1 spectra with absorbance correction. Environ. Sci. Technol. 30, 3061–3065. doi: Sivanandhan, G., Arun, M., Mayavan, S., Rajesh, M., Mariashibu, T. S., 10.1021/es960132l Manickavasagam, M., et al. (2012). Chitosan enhances withanolides Moharrami, F., Hosseini, B., Sharafi, A., and Farjaminezhad, M. (2017). Enhanced production in adventitious root cultures of Withania somnifera (L.) production of hyoscyamine and scopolamine from genetically transformed root Dunal. Ind. Crop. Prod. 37, 124–129. doi: 10.1016/j.indcrop.2011. culture of Hyoscyamus reticulatus L. elicited by iron oxide nanoparticles. In 11.022 Vitro Cell Dev. Biol. Plant 53, 104–111. doi: 10.1007/s11627-017-9802-0 Street, K. W., and Schenk, G. H. (1981). Spectrofluorometric determination Mostofa, K. M. G., Yoshioka, T., Mottaleb, A., and Vione, D. (eds) (2013). of acetylsalicylic acid, salicylamide, and salicyclic acid as an impurity in Photobiogeochemistry of Organic Matter: Principles and Practices in Water pharmaceutical preparations. J. Pharm. Sci. 70, 641–646. doi: 10.1002/jps. Environments. Berlin: Springer-Verlag, doi: 10.1007/978-3-642-32223-5 2600700617 Moustafa-Farag, M., Almoneafy, A., Mahmoud, A., Elkelish, A., Arnao, M. B., Li, Suarez-Fernandez, M., Marhuenda-Egea, F. C., Lopez-Moya, F., Arnao, M. B., L., et al. (2019). Melatonin and its protective role against biotic stress impacts Cabrera-Escribano, F., Nueda, M. J., et al. (2020). Chitosan induces plant on plants. Biomolecules 10:54. doi: 10.3390/biom10010054 hormones and defences in tomato root exudates. bioRxiv [Preprint]. doi: 10. Noverr, M. C., Erb-Downward, J. R., and Huffnagle, G. B. (2003). Production 1101/2020.06.09.142653 of eicosanoids and other oxylipins by pathogenic eukaryotic microbes. Clin. Valette, M., Rey, M., Gerin, F., Comte, G., and Wisniewski-Dyé, F. (2020). ). Microbiol. Rev. 16, 517–533. doi: 10.1128/cmr.16.3.517-533.2003 A common metabolomic signature is observed upon inoculation of rice roots Ohno, T., and Bro, R. (2006). Dissolved organic matter characterization using with various rhizobacteria. J. Integr. Plant Biol. 62, 228–246. doi: 10.1111/jipb. multiway spectral decomposition of fluorescence landscapes. Soil Sci. Soc. Am. 12810 J. 70, 2028–2037. doi: 10.2136/sssaj2006.0005 van Dam, N. M., and Bouwmeester, H. J. (2016). Metabolomics in the rhizosphere: Olivares, C. M., and López-Llorca, L. V. (2002). Fungal egg-parasites of plant- tapping into belowground chemical communication. Trends Plant Sci. 21, parasitic nematodes from Spanish soils. Rev. Iberoam. Micol. 19, 104–110. 256–265. doi: 10.1016/j.tplants.2016.01.008 Palma-Guerrero, J., Jansson, H.-B., Salinas, J., and Lopez-Llorca, L. V. (2007). Effect Vasyukova, N. I., Zinov’eva, S. V., Udalova, Z. V., Panina, Y. S., Ozeretskovskaya, of chitosan on hyphal growth and spore germination of plant pathogenic and O. L., and Sonin, M. D. (2003). The role of salicylic acid in systemic biocontrol fungi. J. Appl. Microbiol 104, 541–553. doi: 10.1111/j.1365-2672. resistance of tomato to nematodes. Dokl. Biol. Sci. 391, 343–345. doi: 10.1023/A: 2007.03567.x 1025158702692 Palma-Guerrero, J., Lopez-Jimenez, J. A., Pérez-Berná, A. J., Huang, I.-C., Jansson, Verboven, S., and Hubert, M. (2005). LIBRA: a MATLAB library for robust H.-B., Salinas, J., et al. (2010). Membrane fluidity determines sensitivity of analysis. Chemom. Intell. Lab. Syst. 75, 127–136. doi: 10.1016/j.chemolab.2004. filamentous fungi to chitosan. Mol. Microbiol. 75, 1021–1032. doi: 10.1111/j. 06.003 1365-2958.2009.07039.x Walker, T. S., Bais, H. P., Halligan, K. M., Stermitz, F. R., and Vivanco, Pandey, G. K. (2017). Mechanism of Plant Hormone Signaling Under Stress. J. M. (2009). Correction to metabolic profiling of root exudates of Hoboken, NJ: John Wiley & Sons. Arabidopsis thaliana. J. Agric. Food Chem. 57, 9346–9346. doi: 10.1021/jf90 Park, C. H., Yeo, H. J., Park, Y. E., Chun, S. W., Chung, Y. S., Lee, S. Y., et al. (2019). 2670b Influence of chitosan, salicylic acid and jasmonic acid on phenylpropanoid War, A. R., Paulraj, M. G., War, M. Y., and Ignacimuthu, S. (2011). Role of salicylic accumulation in germinated buckwheat (Fagopyrum esculentum Moench). acid in induction of plant defense system in chickpea (Cicer arietinum L.). Plant Foods 8:153. doi: 10.3390/foods8050153 Signal. Behav. 6, 1787–1792. doi: 10.4161/psb.6.11.17685 Parri, E., Santinami, G., and Domenici, V. (2020). Front-face fluorescence of honey Yang, G., Zhou, B., Zhang, X., Zhang, Z., Wu, Y., Zhang, Y., et al. (2016). Effects of different botanic origin: a case study from tuscany (Italy). Appl. Sci. 10:1776. of tomato root exudates on Meloidogyne incognita. PLoS One 11:e0154675. doi: 10.3390/app10051776 doi: 10.1371/journal.pone.0154675 Frontiers in Plant Science | www.frontiersin.org 14 November 2020 | Volume 11 | Article 572087 Suarez-Fernandez et al. Chitosan Induces Defenses in Rhizodepositions Yang, H., Xiao, X., Zhao, X., and Wu, Y. (2015). “Intrinsic fluorescence spectra of Zheng, H., Li, S., Zhang, X., and Sui, N. (2020). Functional implications of active tryptophan, tyrosine and phenyloalanine,” in 5th International Conference on N6-Methyladenosine in plants. Front. Cell Dev. Biol. 8:291. doi: 10.3389/fcell. Advanced Design and Manufacturing Engineering, Amsterdam: Atlantis Press, 2020.00291 doi: 10.2991/icadme-15.2015.46 Yin, H., Du, Y., and Dong, Z. (2016). Chitin oligosaccharide and chitosan Conflict of Interest: The authors declare that the research was conducted in the oligosaccharide: two similar but different plant elicitors. Front. Plant Sci. 7:522. absence of any commercial or financial relationships that could be construed as a doi: 10.3389/fpls.2016.00522 potential conflict of interest. Yuan, J., Zhang, N., Huang, Q., Raza, W., Li, R., Vivanco, J. M., et al. (2015). Organic acids from root exudates of banana help root colonization of PGPR Copyright © 2020 Suarez-Fernandez, Marhuenda-Egea, Lopez-Moya, Arnao, strain Bacillus amyloliquefaciens NJN-6. Sci. Rep. 5:13438. doi: 10.1038/ Cabrera-Escribano, Nueda, Gunsé and Lopez-Llorca. This is an open-access article srep13438 distributed under the terms of the Creative Commons Attribution License (CC BY). Zhang, B., Weston, P. A., Gu, L., Zhang, B., Li, M., Wang, F., et al. (2019). The use, distribution or reproduction in other forums is permitted, provided the Identification of phytotoxic metabolites released from Rehmannia glutinosa original author(s) and the copyright owner(s) are credited and that the original suggest their importance in the formation of its replant problem. Plant Soil 441, publication in this journal is cited, in accordance with accepted academic practice. No 439–454. doi: 10.1007/s11104-019-04136-4 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 15 November 2020 | Volume 11 | Article 572087