Halotolerant Endophytes Promote Grapevine Regrowth After Salt-induced Defoliation

PLANT-MICROORGANISM INTERACTIONS Halotolerant endophytes promote grapevine regrowth after salt-induced defoliation Salvadora Navarro-Torre a,b *, Sara Ferrario c *, Ana D. Caperta b , Gonçalo Victorino b , Marion Bailly c , Vicelina Sousa d , Wanda Viegas b and Amaia Nogales b a Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain; b Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal; c Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal; d Forest Research Centre (CEF), Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal ABSTRACT Salinity is an important problem for agriculture in the Mediterranean area, and thus, it is essential to develop mitigation strategies to reduce its impact. The main objective of this study was to test the effectiveness of halotolerant plant growth-promoting bacteria (H-PGPB) in improving grapevine salt stress tolerance. Grapevines grafted onto a salt-sensitive rootstock were inoculated with a consortium of H-PGPB. The substrate of half of the plants of each treatment was salinized up to 2dSm −1 . Plants grew for six days under these conditions, and afterward, NaCl was removed to assess plant recovery through growth, physiology, and canopy temperature measurements. Inoculation with H-PGPB had a positive effect on plant physiology, but after salt treatment, grapevines stopped their photosynthetic metabolism, leading to severe defoliation. Remarkably, after salt stress removal, inoculated plants re-sprouted faster, demonstrating that H-PGPB inoculation could be a good practice to increase vineyard resilience to salt stress. ARTICLE HISTORY Received 5 December 2022 Accepted 13 May 2023 KEYWORDS Halophytes; plant growth promoting bacteria; soil salinization; stress recovery; symbiosis; Vitis vinifera 1. Introduction Grapevine (Vitis vinifera L.)isoneofthelargestcropsworldwide with a total coverage of 7.3 million hectares (ha) (OIV 2020). Total wine production, including sparkling and specialty wines, was estimated at 260 million hectoliters (hL) in 2020 (OIV 2021). However, grapevine cultivation may be at risk in several regions of the world due to climate change and soil salinization (Cook et al. 2014; Fraga et al. 2016;Corwin 2021;Stavietal.2021), since more frequent alternating periods of droughts and sudden strong rainfalls usually lead to an accumulation of salts in ground-waters (Tiam-Ren 1985; Kalra et al. 2002). In semi-arid and hot-summer Mediterranean climates, where the application of irrigation techniques is essential to maintain an optimal level of production and grapequalityinvineyards,theuseofsalineground-water sources for irrigation can expose grapevine plants to salt stress (Rengasamy 2006;Chavesetal.2010;Jamiletal.2011). Since grapevines are classified as a relatively salinity-sensitive species, salt stress is one of the most important abiotic stressors affecting its growth, physiology, yield and quality in hot and dry climate conditions (Shani and Ben-Gal 2005). Salinity induces osmotic stress and ion toxicity, as well as oxidative stress in grapevines (Urdanoz and Aragüés 2009). Osmotic stress translates into a reduction in leaf water potential, photosynthesis and transpiration, which ultimately lead to plant growth decrease (Urdanoz and Aragüés 2009). On the other hand, ionic toxicity caused by the accumulation of some ions such as Cl − and Na + can induce toxicity and nutrient imbalances. For example, a decrease in K uptake by root systems commonly occurs under saline conditions (Blumwald 2000; Shabala et al. 2003; Sairam and Tyagi 2004). Ionic cell imbalance due to the accumulation of Na + is mainly observed in mature leaves, disrupting metabolic processes such as ion transport and respiration (Allen et al. 1994), and protein synthesis (Hasegawa et al. 2000; Mäser et al. 2002; Munns and Tester 2008). This leads to leaf marginal necrosis, decreased leaf growth, and ultimately premature senescence (Downton and Millhouse 1983). Chloride appears to be the main ion with toxic action for vine growth under saline conditions, and several studies clearly showed the injurious effects of Cl − at concentrations of 300 mmol kg −1 in leaves (Bernstein et al. 1969; Maas and Grattan 1999; Fisarakis et al. 2001; Sinclair and Hoffmann 2003). Grapevines respond to salt stress conditions by activating a series of tolerance mechanisms. The main adaptive stress responses are the increase in osmolyte production (e.g. carbohydrates, amino acids and proteins with osmolyte action), vacuolar sequestration of intracellular ions (Serraj and Sinclair 2002; Yokoi et al. 2002), as well as the activation of antioxidant machinery for reactive oxygen species (ROS) detoxification (Ozden et al. 2009; Subramanyam et al. 2011; Sadak 2019). Salt exclusion is another tolerance mechanism mediated by plant’s ability to limit Na + and Cl − uptake, preventing their translocation from roots to shoots (Munns and Gilliham 2015), and the undesirable © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. CONTACT Amaia Nogales [email protected] Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon 1349-017, Portugal; Salvadora Navarro-Torre [email protected] Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41012, Spain *These authors contributed equally to this study. Supplemental data for this article can be accessed at https://doi.org/10.1080/17429145.2023.2215235. JOURNAL OF PLANT INTERACTIONS 2023, VOL. 18, NO. 1, 2215235 https://doi.org/10.1080/17429145.2023.2215235 accumulation in berries (Walker et al. 2014). The use of salinity tolerant rootstocks can therefore be a useful strategy in vineyards affected by salinity (Walker et al. 2019). For instance, Dogridge, SO4, 5C, 420 and Ramsey rootstocks are considered as salt tolerant (Hao et al. 2013; Zhou- Tsang et al. 2021), as well as 1103 Paulsen, Ramsey and 140 Ruggeri, due to their positive effects in reducing salt accumulation in scion’s leaves and grape bunches (Ollat et al. 2016). Some vineyard management strategies can also help counteract salinity effects in grapevines, such as mulching, that can reduce the evaporation of water from the soil, prolonged irrigation with fresh water to reduce Na + and Cl − accumulation at the root system level, or rainwater recovery to increase soil drainage (Stevens et al. 2011). In addition, some current studies about salinity mitigation strategies in crops are also focused on the use of salinity-tolerant or halophilic microorganisms as inoculants, since they are environmentally friendly and cost-effective solutions (Teo et al. 2022). Plant growth promoting bacteria (PGPB) are mutualistic bacteria that promote plant growth and nutrition through direct (e.g. phytohormone production, N fixation, phosphate solubilization, siderophore production) or indirect mechanisms (e.g. protection against plant pathogens, enhancement of abiotic stress tolerance) (Navarro-Torre et al. 2020). Among them, halotolerant bacteria are the ones able to survive at high salt concentrations through various mechanisms such as accumulation of osmolytes, production of extracellular proteases, and activation of Na + /H + antiporters. Many halotolerant and halophilic bacteria can establish symbiotic associations with halophytes, which are salt tolerant species that can survive at 200 mM NaCl or approximately 20 dS m −1 (Flowers and Colmer 2008). Therefore, halophytes, as species from the genus Limonium (Caperta et al. 2020) can be an excellent source of halotolerant/halophilic PGPB to potentially improve crop performance under saline stress conditions (Mesa et al. 2015a;2015b; Navarro-Torre et al. 2016;2017a;2017b; Shurigin et al. 2020; Teo et al. 2022) (Supplementary Figure S1). However, studies on the use of halotolerant PGPB in grapevines are very scarce (Jiao et al. 2016; Ma et al. 2017), and are based on single-species inoculation, although several works suggest inoculations with bacterial consortia are more beneficial due to potential complementary effects of different strains in plants under stressful conditions (Glick and Gamalero 2021). The hypothesis of our study was that endophytic bacteria isolated from autochthonous halophyte (salt tolerant) Limonium species (sea-lavender) could promote grapevine tolerance to salt stress, and the goals of this study were to (1) isolate and characterize halophilic/halotolerant bacteria from distinct species of Limonium plants from different saline environments; (2) select the best performing bacteria based on their plant growth promoting traits and NaCl tolerance; and (3) test the selected bacteria in grapevines exposed to saline stress, and assess their potential benefits in improving host’s salt stress tolerance. 2. Materials and methods 2.1. Isolation of culturable bacterial endophytes Three entire plants of similar age and size of three different Portuguese autochthonous Limonium species (all three considered halophytes) were collected in April 2021. Limonium vulgare Mill. plants were collected in a salt marsh (Sapal das Hortas, Alcochete, Portugal) and L. daveaui Erben in sand dunes (Praia do Samouco, Alcochete, Portugal), both in the Tagus estuary surroundings. Limonium multiflorum Erben in a rocky salt spray environment (Cabo Raso, Cascais, Portugal) (Supplementary Figure S2). Plants were transported to the laboratory in individual bags and stored at 4°C, until utilization. Root endophytic bacteria were isolated from those plants. Since those plants live in saline soils, their symbiotic microorganisms, including bacteria, are expected to be halotolerant or even halophilic. Roots from each plant were separated from the aerial part and surface-disinfected using 70% ethanol and 6% sodium hypochlorite (Navarro-Torre et al. 2016). Then, roots were cut with a sterile scalpel into small pieces (2 cm of length approximately) and crushed in a sterile mortar containing 1 ml of sterile saline solution (0.9% NaCl). The molded paste was diluted to 10 −1 ,10 −2 and 10 −3 in saline solution (0.9% NaCl) and plated in three TSA (tryptic soy agar) plates, supplemented or not with 0.3 M NaCl (w/v). All plates were incubated at 28°C for 72 h. In parallel, negative controls were done at each isolation step to ensure that the isolated bacteria were root endophytes and not potential contaminants from the air of the flow chamber, the distilled water, the saline solution, or even epiphytic bacteria remaining in the disinfected roots. After colony observation, the ones presenting different sizes, colors or morphologies were individually sub-cultured in new TSA plates. A pure culture of each isolate was stored at −80°C in 30% glycerol. 2.2. Taxonomic identification of culturable bacterial endophytes For the taxonomic identification of the bacterial isolates, each culture was grown in TSB (tryptic soy broth) for 24 h at 28°C at constant shaking (150 rpm). After incubation, cultures were centrifuged at 13,000 rpm for 3 min and the pellets containing the bacteria were used to extract DNA using innuPREP Plant DNA kit (Analytik Jena, Germany), following manufacturer’instructions. The amplification of the 16S rRNA gene was done using NZYTaq II DNA polymerase (Nzytech, Portugal) and 16SF27 and 16SR1488 as primers (Navarro-Torre et al. 2016) with the following PCR conditions: initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a final extension step at 72°C for 5 min. The PCR products were purified and sequenced by StabVida company (Portugal). Each bacterial strain was taxonomically assessed by performing a BLAST analysis of the obtained 16S rRNA gene sequence on the EzBioCloud database using the Ez-Taxon server (Yoon et al. 2017). Finally, 16S rRNA gene sequences were deposited in the NCBI GenBank and an accession number was assigned to each sequence (Table 1). 2.3. Characterization of bacterial plant growth promoting traits and salt tolerance To select the halophilic bacterial species or strains with the highest potential to establish mutualistic relationships with 2S. NAVARRO-TORRE ET AL. plants, their plant growth promoting (PGP) traits were studied. In each bacterial strain PGP traits were studied under control conditions (without NaCl) and in the presence of 0.2 M NaCl (w/v) in the media. The tested PGP properties included indole-3-acetic acid (IAA) and siderophore production, phosphate solubilization, nitrogen fixation, biofilm formation, and the presence of 1-aminocyclopropane-1-car- boxylic acid (ACC) deaminase gene. Auxins, among which IAA, are plant phytohormones produced also by bacteria, that promote root elongation and ramification (Turan et al. 2016), and siderophores are Fe 3+ chelators that improve Fe availability for plants (Alori and Babalola 2018). For testing IAA production, bacteria were cultivated in TSB medium supplemented with 100 mg L −1 of L-trypto- phan at 28°C for three days at constant shaking. Then, cultures were centrifuged and Salkowski reagent (Gordon and Table 1. Identification of culturable bacterial endophytes isolated from roots of Limonium daveaui (LDR), L. vulgare (LVR) and L. multiflorum (LMR) using EzBiocloud database. Strain Sequence size (bp) Related species Similarity (%) Accession number LVR1 1424 Pantoea allii 99.30% OM471718 LVR2 1430 Pseudomonas rhodesiae 99.23% OM471719 LVR3 1004 Pseudomonas rhodesiae 100% OM471720 LVR4 986 Pantoea allii 99.80% OM471721 LVR6 1345 Pantoea hericii 94.65% OM471722 LVR7 1010 Pantoea hericii 93.84% OM471723 LVR9 1008 Erwinia persicina 99.90% OM471724 LVR10 1020 Staphylococcus epidermidis 100% OM471725 LVR11 1422 Acinetobacter lwoffii95.84% OM471726 LVR12 1004 Prolinoborus fasciculus 91.61% OM471727 LVR13 1425 Pantoea hericii 94.86% OM471728 LVR14 1021 Staphylococcus pettenkoferi 100% OM471729 LVR15 1013 Pantoea hericii 92.86% OM471730 LVR16 1347 Pantoea hericii 94.14% OM471731 LVR17 1004 Pseudomonas rhodesiae 100% OM471732 LVR18 1009 Pantoea hericii 92.96% OM471733 LVR19 929 Pantoea conspicua 100% OM471734 LVR20 1202 Pseudomonas rhodesiae 99.25% OM471735 LVR21 1020 Staphylococcus warneri 100% OM471736 LVR22 1006 Prolinoborus fasciculus 99.90% OM471737 LVR23 1347 Pantoea hericii 94.65% OM471738 LVR24 1088 Pantoea agglomerans 74% OM471739 LVR25 1394 Pantoea allii 98.99% OM471740 LVR26 1006 Acinetobacter lwoffii99.90% OM471741 LDR1 1428 Erwinia endophytica 97.60% OM471742 LDR2 1388 Pantoea anthophila 98.05% OM471743 LDR3 1010 Erwinia endophytica 97.91% OM471744 LDR4 1009 Raoultella planticola 100% OM471745 LDR5 1011 Erwinia endophytica 97.72% OM471746 LDR6 1010 Erwinia persicina 98.51% OM471747 LDR7 1425 Pantoea hericii 93.74% OM471748 LDR8 1023 Weizmannia ginsengihumi 99.41% OM471749 LDR9 1009 Pantoea conspicua 94.74% OM471750 LDR10 1011 Pantoea dispersa 98.01% OM471751 LDR11 801 Bacillus tequilensis 98.75% OM471752 LDR12 1009 Pantoea conspicua 91.56% OM471753 LDR13 1016 Pantoea dispersa 96.86% OM471754 LDR14 1005 Pseudomonas rhodesiae 100% OM471755 LDR15 1347 Pantoea conspicua 95.32% OM471756 LDR16 1007 Acinetobacter lwoffii99.11% OM471757 LDR17 1370 Pantoea agglomerans 98.25% OM471759 LDR18 1425 Acinetobacter lwoffii99.30% OM471760 LDR19 1010 Erwinia endophytica 98.11% LDR20 1346 Pantoea conspicua 96.35% OM471761 LDR21 1005 Pseudomonas rhodesiae 99.20% OM471762 LDR22 1009 Pseudomonas marginalis 99.35% OM471763 LDR23 1424 Acinetobacter lwoffii99.16% OM471764 LDR24 752 Erwinia endophytica 100% OM471765 LDR25 1394 Pantoea agglomerans 97.76% OM471766 LDR26 1006 Prolinoborus fasciculus 99.90% OM471767 LDR27 1009 Erwinia persicina 99.60% OM471768 LDR28 1016 Cytobacillus horneckiae 98.62% OM471769 LDR29 1346 Pantoea septica 98.29% OM471770 LMR1 1021 Bacillus toyonensis 98.43% OM471771 LMR2 1018 Bacillus siamensis 99.61% OM471772 LMR3 1009 Erwinia persicina 99.60% OM471773 LMR4 1009 Erwinia persicina 99.70% OM471774 LMR5 1192 Kluyvera cryocrescens 100% OM471775 LMR6 1009 Erwinia persicina 99.90% OM471776 LMR7 1009 Erwinia persicina 99.60% OM471777 LMR8 1009 Erwinia persicina 99.90% OM471778 LMR9 1009 Erwinia persicina 99.90% OM471779 LMR10 1011 Erwinia rhapontici 99% OM471780 LMR11 1010 Erwinia rhapontici 99% OM471781 LMR12 1010 Erwinia persicina 98.32% OM471782 LMR13 1009 Erwinia persicina 99.90% OM471783 The species selected for the greenhouse experiment are marked in bold. JOURNAL OF PLANT INTERACTIONS 3 Weber 1951) added to the supernatant. The absorbance of the solution was measured at 535 nm and IAA concentration was determined using a IAA pattern curve (Gordon and Weber 1951). For the analysis of siderophore production and phosphate solubilization activity, bacteria were initially cultured on TSB for 24 h at 28°C with constant shaking, and then, 100 μLof each culture was inoculated on TSA plates containing CAS medium (chrome azurol S) (Schwyn and Neilands 1987)or on plates containing NBRIP (National Botanical Research Institute’s phosphate growth medium) medium (Nautiyal 1999), respectively. Plates were incubated for five days at 28°C and were considered positive when a halo appeared around the bacterial growth, which was orange in the case of the CAS medium and transparent in the case of NBRIP medium, due to the solubilization of Ca 3 PO 4 . To determine if bacteria could fix atmospheric nitrogen, they were incubated at 28°C for five days in NFB (nitrogen free broth) solid medium (Ji et al. 2014) and those able to grow in such medium were identified with the capacity to fix nitrogen. Biofilm formation was also tested in all bacterial strains individually cultured in 24-well-plates filled with TSB medium for four days at 28°C. After this period, biofilm position in the well was determined (surface or bottom) and then, biofilms were stained with 0.01% crystal violet (w/v) (del Castillo et al. 2012) to see if the bacterial biomass adhered to the wall of the wells (ring formation). Finally, the presence of the enzyme ACC deaminase was determined by the amplification of the acdS gene. The ACC deaminase enzyme degrades a precursor on the ethylene formation, contributing to reduce plant stress (Glick 2014). The PCR was conducted using NZYTaq II DNA polymerase (Nzytech, Portugal) and the primers acdSf3 and acdSr3, as described in Li et al. (2015). The PCR conditions used were the following: an initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. Electrophoresis was run for 30 min at 120 V in TAE 1X, and then, the gel was stained with EtBr solution and visualized under UV light. In addition, bacterial tolerance to high NaCl concentrations was evaluated. Each isolate was cultured in TSA plates supplemented with 0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 M NaCl (w/v) and incubated for five days at 28°C. Tolerance to NaCl was expressed as the maximum tolerable concentration (MTC) i.e. the highest NaCl concentration at which bacterial growth was observed. Based on the results obtained, a consortium with the best three bacteria in terms of IAA and siderophore production, phosphate solubilization, N fixation and biofilm production both under saline and non-saline conditions were selected for subsequent analyses: LDR2, LDR25, and LVR13. Their compatibility was tested by culturing them together on the same TSA plate, ensuring contact among their colonies. 2.4. Bacterial transformation with mCherrycontaining plasmid The selected bacterial strains LDR2, LDR25 and LVR13 were transformed with the plasmid pMP7604 by bacterial conjugation. Bacterial strains and plasmids used for this purpose are described in Supplementary Table S1. First, spontaneous antibiotic (ampicillin or rifampicin) resistant mutants were isolated from each strain and incubated overnight at 28°C in TSB under agitation (115 rpm). The next day those cultures were centrifuged at 13,000 rpm for 3 min and the pellets resuspended in 100 μL TSB. Then, each bacterial culture was plated in TSA medium supplemented with the corresponding antibiotic (100 μgmL −1 ampicillin or rifampicin). Plates were incubated for 48 h at 28°C and the newly formed colonies were considered spontaneous mutants to the corresponding antibiotic. Conjugation was performed by culturing together in TSB the corresponding ampicillin- or rifampicin-resistant bacterial strain (LDR2, LDR25, and LVR13), the helper Escherichia coli DH5αbacteria (containing the plasmid pRK2013), and the donor E. coli DH5αbacteria (containing the pMP7604 plasmid with the tetracyclin resistance gene). The bacterial cultures were incubated for 24 h at 28°C in agitation, and were plated in TSA supplemented with 100 μgmL −1 ampicillin or rifampicin, and tetracycline (40 μgmL −1 ). Plates were incubated 48 h at 28°C and colonies were selected. The presence of the plasmid was checked by PCR amplification of the mCherry gene using the primers mCherryFw (5′-ATGGTGAGCAAGGGCGAG-3′) and mCherryRv (5′-TTACTTGTACAGCTCGTC-3′). 2.5. Inoculum preparation Liquid bacterial cultures of LDR2, LDR25 and LVR13 carrying the plasmid with the mCherry gene were used as inoculum for grapevine plants. For this, an individual colony of each bacterium was placed in 50 mL of TSB and was left overnight at 28°C under agitation at 115 rpm. The next day the three bacterial suspensions were processed as in Navarro-Torre et al. (2017a) and were finally mixed in a volume of 800 mL of irrigation water. The optical density (OD 600nm ) of the final bacterial suspension was 1.374. 2.6. Study of grapevine response to the inoculation with halotolerant bacteria and salt stress 2.6.1. Experimental set up Thirty-two dormant vines of Aragonez-Tempranillo grafted onto Richter 110 rootstock obtained from Viveiros Vitioeste (Bombarral, Portugal) were used. Richter 110 has excellent adaptability to dry soils but is sensitive/moderately sensitive to salinity (Troncoso de Arce et al. 1999; Corso and Bonghi 2014; Walker et al. 2019; Zhou-Tsang et al. 2021). Plants were rooted under greenhouse conditions in rooting beds filled with sterile perlite. When plants presented a well-devel- oped root system, they were transplanted to 3 L square pots filled with a mixture of autoclave-sterilized (1 h at 120°C) peat and perlite (1:2 v/v). Grapevines were kept under greenhouse conditions and watered daily according to their needs. Every week 50 ml of Hoagland’s nutrient solution (Hoaglands and Arnon 1972) was applied in each plant. Three weeks after transplant, half of the plants were inoculated with 50 ml of a bacterial suspension consisting of a consortium of the three previously selected bacterial strains carrying the mCherry gene. This inoculation procedure was repeated every 10 days. Three months later, half of the plants of each treatment were watered with a saline solution (75 mM) to achieve a final soil electric conductivity (EC) of approximately 4S. NAVARRO-TORRE ET AL. 2dSm −1 . In non-salinized substrates the EC was 0.3 dS m −1 , and was measured with an Orion Star A212 Conductivity Benchtop Meter (Thermofisher Scientific, USA). For six days, grapevines were watered daily to maintain plants under water comfort conditions. After that period, to favor plant recovery, for two days, plants were watered in excess to promote salt leaching and eliminate NaCl present in the substrate, until EC decreased to 0.6 dS m −1 in salinized pots (Supplementary Figure S3). Afterwards, irrigation was re-adjusted to maintain water comfort conditions in grapevines. To avoid potential differences in soil nutrient content between salt-treated and non-treated plants, non-salinized plants were also watered with the same amounts of water. In the weeks following NaCl elimination, Hoagland’s solution dosage was doubled for treated and untreated plants. 2.6.2. Plant physiology and growth monitorization Plant performance was monitored by reflectance index assessment (Normalized Difference Vegetation Index- NDVI and Photochemical Reflectance Index-PRI) and by foliar gas exchange parameter measurements. The NDVI and PRI were measured using PlantPen NDVI 300 (PSI, Czech Republic) and PlantPen PRI 200 (PSI, Czech Republic) portable devices, respectively. Photochemical Reflectance Index is indicative of photosynthesis efficiency and plant stress levels (Garbulsky et al. 2011) while NDVI reflects plant vigor and, indirectly, chlorophyll status, phosphorus, and nitrogen nutrition (Sembiring et al. 1998). Measurements were performed in two mature leaves per plant before salt stress application (i.e. four months after the first bacterial inoculation was done), two days after salt stress application, and during the recovery phase after salt stress elimination (July 28th, August 16th and August 31st) (Supplementary Figure S3). Measurements were performed always at the same time, at 11 am. Foliar gas exchange parameters (net photosynthesis rate- A, stomatal conductance-g s and transpiration-E) were measured with a CIRAS 3 infrared gas exchange analyzer- IRGA (PP Systems, USA) in two mature leaves per plant. Measurements were performed always at the same time, at 12 am, on the same dates as the reflectance indexes. Since canopy temperature is commonly used as an indicator of plant stress (Jones 2002), thermal images were collected two days after salt stress exposure and during the recovery phase after salt stress elimination. For that purpose, a FLIR A35 thermal camera (Teledyne FLIR ® , Germany) equipped with an infrared temperature sensor with manual focus and a spectral range between 7.5 and 14 µm and a 320 × 256 pixel resolution was used. The camera was mounted on a tripod to stably stand approximately 1.5 m above the ground, right in front of the plants. A board was placed behind the grapevines to homogenize the background as much as possible. Data were collected always at 13 pm. Thermal images were acquired using FLIR Tools ® v6.4 and analyzed using ImageJ ® software v1.53 (National Institutes of Health, USA). For this, images were converted to 8 bit and calibrated so that the gray scale corresponded to the temperature range recorded by the thermal camera. Then, images of individual plants were manually segmented to ensure accuracy, rather than relying solely on automatic image segmentation methods. To determine the average temperature of the entire plant leaf canopy area, the selected pixels corresponding to the plant canopy were analyzed. Canopy temperature values were not normalized according to dry and wet leaf references (Jones 2004; Costa et al. 2010) since the aim of the trial was to compare differences between experimental treatments at each timepoint. Plant growth was monitored by counting the number of green leaves per plant, regardless of their size, and by measuring grapevine shoot length (shoot length was considered 0 when it was dry and with no leaves attached). By the end of the experiment, shoot and root dry biomass were recorded. 2.6.3. Bacterial colonization Sixteen days after NaCl was eliminated from the substrate, root samples were collected from two non NaCl-treated plants (one inoculated and one non-inoculated), and from one bacteria-inoculated plant previously treated with NaCl. Roots were fixed using a 4% formaldehyde acetic solution in 70% ethanol. To avoid ethanol-induced tissue dehydration, glacial acetic acid (∼5% of total volume) was added. After 48 h in this fixing solution, roots were washed in a 70% ethanol solution and stored at 4°C until used. Afterwards, samples were impregnated with DP1500 polyethylene glycol (Barbosa et al. 2010). Transverse sections of approximately 30 μm thick were prepared with a sliding microtome (Leica SM 2400), were double stained with Safranin/Astrablue (1% aqueous solution) and then rinsed with distilled water. Slides were mounted in a glycerine/water solution and observed on a light microscope (Leitz Dialux 20EB). Images were captured using a digital camera (Leica EC3) and an image software (Leica Application Suite, v.4.0). For visualizing root bacteria colonization, the slides were observed under a fluorescence microscope (Zeiss Axioskop 2), equipped with a digital camera (Zeiss AxioCam) with a 10× objective. Image acquisition was performed using the Carl Zeiss/AxioVision 4.8 software. 2.6.4. Statistical analysis Differences in shoot length, number of leaves, NDVI, PRI, A, g s and E before NaCl treatment application were evaluated by a t-student test, after data normality was assessed by a Shapiro–Wilk test. After salinity stress and during plant recovery phase, different experimental treatments were compared by a oneway ANOVA test followed by a Duncan post hoc test. At harvest, A, g s , E, NDVI, PRI, canopy temperature, dry shoot and root biomass were analyzed by a two-way ANOVA with interaction where inoculation and salt stress were considered as main factors. Differences among the experimental treatments were further assessed by a Duncan post hoc test. All analyses were performed in SPSS Statistics vs. 23 (IBM) software. 3. Results 3.1. Taxonomic identification of culturable bacterial endophytes isolated from Limonium species A total of 66 endophytic bacterial strains differing in colony size, color, and morphology were isolated from the three Limonium species growing in different saline environments. Of these, 24 endophytes belonged to L. vulgare roots (LVR strains) being 96% Gram-negative and 4% Gram-positive JOURNAL OF PLANT INTERACTIONS 5 bacteria. Other 29 bacteria were isolated from L. daveaui roots (LDR strains), of which 90% were Gram-negative and 10% Gram-positive. Finally, from L. multiflorum plants only 13 endophytes (LMR strains) were isolated, of which 85% were Gram-negative and 15% Gram-positive bacteria. The most represented genera were Pantoea (35%) and Erwinia (27%) (Table 1). The Pantoea genus was present in strains isolated from L. vulgare and L. daveaui, being more frequent in L. vulgare (50% of its endophytes) with Pantoea hericii as the most represented species (Table 1). On the other hand, the Erwinia genus was present in the three plant species, being especially abundant in L. multiflorum (77% of its endophytes) with Erwinia persicina as the most frequent species (Table 1). Other frequent genera such as Pseudomonas,Acinetobacter,Bacillus,Prolinoborus,Staphylococcus,Kluyvera,Cytobacillus,Raoultella, and Weizmannia were also found among the isolated bacterial endophytes (Table 1). Erwinia was the sole genus common to the three Limonium species. By contrast, the genus Kluyvera was exclusive of L. multiflorum, while the genera Cytobacillus,Raoultella, and Weizmannia were only present in L. daveaui, and the genus Staphylococcus was exclusive of the root endosphere of L. vulgare (Table 1). 3.2. Characterization of the isolated bacterial endophytes All endophytes showed at least one of the tested PGP properties and 46% of them showed at least four (Figure 1). The most common PGP property was biofilm formation, present in 94% of the endophytes. Biofilm formation was observed mainly at the bottom of the wells, although some endophytes were also able to adhere to the wall, showing a characteristic ring. Only 2% of the isolates produced biofilm on the surface of the medium (Supplementary Table S2). The second and third most frequent properties were siderophore and IAA production (present in 83% and 76% of the isolates, respectively). The largest halos, indicative of higher siderophore production, were observed in strains LVR21, LDR21, LVR4, LVR20, LDR11, LVR1, LDR29 and LDR25, in descending order, showing halo diameters from 5 to 6.1 cm (Supplementary Table S2). The best IAA producers were LDR4 and LDR2 with 12.03 and 11.78 mg·L −1 of IAA, respectively (Supplementary Table S2). The presence of bacterial growth in a minimal medium without nitrogen source was observed in 42% of the isolates, which suggests that they have the capacity of fixing nitrogen from the air (Supplementary Table S2). Phosphate solubilization (Ca 3 (PO 4 ) 2 ) was the less frequently observed PGP property across isolates, with strain LVR10 as the best phosphate solubilizer producing a halo of 4.6 cm (Supplementary Table S2). Concerning the ACC deaminase gene acdS,no PCR amplification was observed with the primers used in any endophytic bacteria, and therefore ACC deaminase activity was considered absent in all of them. All isolated bacteria tolerated up to 0.5 M NaCl (Supplementary Table S2). Fifty percent of the isolated endophytes grew in the presence of 1 M NaCl in the medium while only 12% did it in the presence of 3.5 M NaCl. Endophytes from L. vulgare presented the highest halotolerance, with 29% able to grow at 3.5 M NaCl. Contrastingly, only 15% of bacteria isolated from L. multiflorum tolerated a maximum of 2 M NaCl, being such conditions lethal for all the other bacteria isolated from this plant species. 3.4. The effect of salt on bacterial plant growth promoting traits In the presence of 0.2 M NaCl, all bacterial strains formed biofilms and the amount of IAA produced also increased in most strains (Figure 2). The highest increase was found Figure 1. Representation of the PGP properties expressed by endophytes isolated from roots of (A) Limonium vulgare (LVR), (B) L. multiflorum (LMR), and (C) L. daveaui (LDR). 6S. NAVARRO-TORRE ET AL. in strain LDR15, since IAA production increased in 57% in the presence of NaCl. Overall, the best IAA producers in the presence of 0.2 M NaCl were LDR25 (22.68 mg L −1 ), LDR5 (21.87 mg L −1 ), LDR2 (20.62 mg L −1 ), and LDR1 (19.81 mg L −1 ) (Supplementary Table S3). However, decreases in IAA production were also observed in 34% of the bacterial strains. For example, in strain LDR4, IAA production was reduced from 12.03 to 2.93 mg L −1 (Supplementary Table S3). Similar results were observed for phosphate solubilization as the number of phosphate solubilizing bacteria increased in bacteria isolated from L. vulgare (from 29% to 46%), L. daveaui (from 52% to 76%), and L. multiflorum (from 23% to 38%) (Figure 2). However, the diameter of the halo (representative of solubilized calcium phosphate) decreased in 84% of the strains. The best solubilizers in the presence of NaCl were LVR13 (1.9 cm), LDR13 (1.9 cm), and LDR20 (1.8 cm) (Supplementary Table S3). The number of siderophore producing bacteria also increased with NaCl, and this trait was expressed by 100% of the endophytes isolated from L. daveaui and L. multiflorum, and by 79% of the bacteria isolated from L. vulgare (Figure 2). In the presence of NaCl the strains with the highest halo diameters were LDR17 and LDR14 with 5.6 and 5.2 cm, respectively (Supplementary Table S3). Even though the number of bacteria that produced siderophores increased in the presence of NaCl, the diameter of the halo decreased in 56% of the strains. Finally, NaCl also induced an increase in the number of nitrogen fixers in LVR and LDR groups but not in endophytes from L. multiflorum (Figure 2). Based on the results obtained, three PGPB strains (Pantoea anthophila LDR2, Pantoea agglomerans LDR25, and Pantoea sp. LVR13) were selected to form a consortium and be further inoculated in grapevines. These strains did not antagonize each other when grown together in TSA medium plates and were all halotolerant. In the presence of 0.2 M NaCl they had the following characteristics: LDR2 showed one of the highest amounts of IAA; LDR25, besides producing high IAA amounts, expressed all the studied PGP properties under saline conditions; and LVR13 expressed all PGP properties and was one of the best phosphate solubilizers in such conditions. 3.5. Study of grapevine response to the inoculation with halotolerant bacteria and to NaCl treatment 3.5.1. Plant performance before and during NaCl treatment Four months after the first inoculation with the selected PGPB consortium, before NaCl treatment application (Supplementary Figure S3), inoculated grapevines showed significantly higher A, g s , E, NDVI and PRI values than noninoculated plants (Figure 3). However, no differences were found between inoculated and non-inoculated plants in the number of leaves (10.6 ± 0.06 and 10.1 ± 0.06, respectively) and shoot length (13.2 ± 3.41 and 16.2 ± 5.41, respectively). When reflectance indexes were measured one day after NaCl treatment application, before any visual symptom was observed in grapevine plants, a decrease was found in NDVI both, in inoculated and non-inoculated plants exposed to salt stress (21.4% and 22.2% decrease, respectively), which had closed stomata and zero photosynthesis rate (Figure 3). Furthermore, canopy temperature assessed by thermography images (Supplementary Figure S4) showed that salt stress induced an increase of 3.2°C and 2°C in inoculated and non-inoculated plants, respectively (Figure 4). At this same time point, in plants not exposed to salt stress we observed that the inoculated ones presented 33% and 8% higher NDVI and PRI, and 0.4°C lower canopy temperature (Figure 3(D,E); Figure 4), although no differences were found in A, g s and E (Figure 3). Two days after saline solution application, the first salt stress symptoms were observed (Figure 5). Symptomatic Figure 2. Percentage of bacterial showing the different plant growth promoting (PGP) properties in absence of NaCl (0 M NaCl) and in presence of 0.2 M NaCl of (A) endophytes from roots of Limonium vulgare (LVR), (B) endophytes from roots of L. daveaui (LDR), and (C) endophytes from roots of L. multiflorum (LMR). JOURNAL OF PLANT INTERACTIONS 7 Figure 3. Grapevine leaf gas exchange parameters. (A) Net photosynthesis rate, A; (B) stomatal conductance, g s ; (C) transpiration rate, E; (D) normalized difference vegetation index, NDVI; and (E) photochemical reflectance index, PRI measured at different time points in Aragonez cv. grapevines grafted onto Richter 110 rootstocks inoculated or not with a bacterial consortium and treated or not with a saline (NaCl) solution. Bars indicate the mean value per treatment ± standard error. Different letters at each time point indicate significant differences according to the Duncan multiple comparison test, and ‘ns’indicates non-significant differences in a particular measurement time. The dashes on July 29 and August 18 indicate that there were not enough plants with mature leaves in the corresponding treatments for performing the analysis. 8S. NAVARRO-TORRE ET AL. leaves of inoculated and non-inoculated plants showed small marginal and inter-nerval leaf burnt. At the third day after the saline treatment, plants started defoliating and by the sixth day, all leaves were senescent, and a high degree of defoliation was observed. 3.5.2. Plant recovery Ten days after salinity stress elimination, on July 28th, 62.5% of the bacteria-inoculated plants started to show new buds and leaves, while in the group of non-inoculated grapevines only 40% showed such signals of recovery. In both cases, news leaves were too small for gas exchange measurements (Supplementary Figure S5), and therefore, these parameters were only measured in inoculated and non-inoculated plants not-previously treated with salt. In these cases, no significant differences were found between the two experimental groups for A, g s , E, NDVI or PRI parameters (Figure 3). On the other hand, it was possible to perform infrared image analysis on PGPB-inoculated plants that had been exposed to salt stress, because they had already a small canopy with small leaves (Supplementary Figure S4). In those plants, canopy temperature was significantly higher than in plants nonexposed to salt stress (Figure 4). One month after NaCl elimination, on August 18th, in grapevines that had not been exposed to salt stress, the number of leaves was significantly higher in inoculated plants than in the non-inoculated ones (Figure 6). On the other hand, in plants that had been exposed to salt stress, 75% of the PGPB-inoculated plants showed new leaves and buds, while only 60% of the non-inoculated grapevines did. Besides, inoculated plants produced a high number of new leaves, similar to the one found in inoculated plants not subjected to salt stress, although with much smaller dimensions and not all mature (Figure 6). At that time point, four of these plants had sufficiently developed leaves for PRI, NDVI, gas exchange measurements and canopy temperature analysis, while only one non-inoculated plant from the salt stress treatment had mature leaves. Therefore, this experimental treatment could not be included in the subsequent statistical analyses. When physiological parameters were analyzed, no significant differences were observed in A and g s among the experimental groups, inoculated or not, exposed to salt stress or not (Figure 3(A,B)) presenting mean values (± standard error) of 7.93 ± 0.55 µmol CO 2 /m 2 s and 171.96 ± 22.24 mmol/m 2 s, respectively. However, PGPB-inoculated plants that had not been subjected to salt stress had the highest transpiration rates compared to the other experiment treatments (Figure 3(C)). These plants were also the ones showing the lowest canopy temperature (Figure 4). Concerning PRI, the highest values were found in PGPB-inoculated plants that were recovering after salt stress (Figure 3(D,E)) but no differences were found in NDVI values among the experimental treatments. On August 31st, one month after salt removal, the new leaves in stress-recovered plants were larger in size and measurements could be performed in both treatments (inoculated and non-inoculated). The length of the new green shoots was highest in inoculated plants recovering from the salt stress (70 cm ± 13.4), and no significant differences were found in that parameter among the remaining experimental treatments (Figure 6). The number of leaves was also the highest in inoculated plants recovering from the salt stress, although many of these leaves were still small in size. Non-inoculated plants non-exposed to salt stress were the ones with the lowest number of leaves (Figure 6). At that same time point, the two-way ANOVA showed a significant negative effect of salt stress factor in NDVI, canopy temperature, shoot and root dry biomass, while PGPB-inoculation factor did not show a significant effect Figure 4. 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