Browsing by Author "Ramos Solano, Beatriz"

In addition to genetic adaptative mechanisms, plants retrieve additional help from the surrounding microbiome, especially beneficial bacterial strains (PGPB) that contribute to plant fitness by modulating plant physiology to fine-tune adaptation to environmental changes. The aim of this study was to determine the mechanisms by which the PGPB Bacillus G7 stimulates the adaptive mechanisms of Olea europaea plantlets to high-salinity conditions, exploring changes at the physiological, metabolic and gene expression levels. On the one hand, G7 prevented photosynthetic imbalance under saline stress, increasing the maximum photosynthetic efficiency of photosystem II (Fv/Fm) and energy dissipation (NPQ) and protecting against photooxidative stress. On the other hand, despite the decrease in effective PSII quantum yield (ΦPSII), net carbon fixation was significantly improved, resulting in significant increases in osmolytes and antioxidants, suggesting an improvement in the use of absorbed energy. Water use efficiency (WUE) was significantly improved. Strong genetic reprogramming was evidenced by the transcriptome that revealed involvement of the ABA-mediated pathway based on upregulation of ABA synthesis- and ABA-sensing-related genes together with a strong downregulation of the PLC2 phosphatase family, repressors of ABA-response elements and upregulation of ion homeostasis-related genes. The ion homeostasis response was activated faster in G7-treated plants, as suggested by qPCR data. All these results reveal the multitargeted improvement of plant metabolism under salt stress by Bacillus G7, which allows growth under water limitation conditions, an excellent trait to develop biofertilizers for agriculture under harsh conditions supporting the use of biofertilizers among the new farming practices to meet the increasing demand for food.

Improvement of plant adaptation by beneficial bacteria (PGPB) may be achieved by triggering multiple pathways to overcome the environmental stress on plant’s growth cycle, activating plant’s metabolism. The present work reports the differential ability of three Bacillus strains to trigger olive tree metabolism, among which, only H47 was outstanding increasing iridoid and flavonol concentration. One-year old olive seedlings grown open air, under harsh conditions of water shortage in saline soils, were root-inoculated with three Bacillus PGPB strains throughout a 12-month period after which, photosynthesis was determined; photosynthetic pigments and bioactive secondary metabolites (iridoids and flavonols) were analyzed, and a study of gene expression of both pathways involved was undertaken to unravel molecular targets involved in the activation. All three strains increased plant fitness based on photosynthetic values, increasing energy dissipation capacity to lower oxidative stress; only H47 increased CO2 fixation and transpiration. Bacillus H47 was found to trigger synthases in the DOXP pathway (up to 5-fold in DOXP-synthase, 3.5-fold in Iridoid synthase, and 2-fold in secologanin synthase) associated to a concomitant increase in iridoids (up to 5-fold in oleuropein and 2-fold in its precursor secologanin). However, despite the 2-fold increases detected in the two predominant flavonols, gene expression was not enhanced, suggesting involvement of a pulse activation model proposed for innate immunity. Furthermore, the activity of leaf extracts to inhibit Angiotensin Converting Enzyme was evaluated, to explore further uses of plant debris with higher added value. Despite the increases in iridoids, leaf extracts from H47 did not increase ACE inhibition, and still, increased antihypertensive potential in oil obtained with this strain is to be explored, as leaves are the source for these metabolites which further translocate to fruits. In summary, Bacillus H47 is an effective strain to increase plant adaptation to dry and saline environments, activates photosynthesis and secondary metabolism in olive tree.

Blackberries are naturally rich in functional components beneficial for human health. The postharvest period of these fruits is very short due to fungal development, therefore, it is of great economic interest. Flavonoids and anthocyanins are secondary metabolites, and thus, strongly inducible. The aim of this study was to evaluate the ability of 6 bacteria with biocontrol traits and demonstrated Induced Systemic Resistance capacity, to prevent fungal growth during the postharvest period; the secondary aim was to identify whether the bacterial determinant was structural or metabolic, and if the treatment would affect flavonoid and anthocyanin levels. To achieve this goal, bacterial strains were sprayed dead or alive; fungal growth and phytochemicals were recorded. Only one strain delayed fungal growth by 50%, being structural and metabolic elicitors independently as efficient as the strain itself (dead or alive). This protection was associated to a decrease in the evaluated metabolites (28% total phenolics, 33% total flavonoids, 24% anthocyanins), suggesting transformation of flavonoids and anthocyanins (phytoanticipins) onto other molecules (phytoalexins) involved in defense and confirming induction of natural immunity. This study shows the potential of beneficial bacteria to develop a biological product to extend fruitshelf life of blackberries, increasing benefits for health and economic profit.

The use of beneficial rhizobacteria (bioeffectors) and their derived metabolic elicitors are efficient biotechnological alternatives in plant immune system elicitation. This work aimed to check the ability of 25 bacterial strains isolated from the rhizosphere of Nicotiana glauca, and selected for their biochemical traits from a group of 175, to trigger the innate immune system of Arabidopsis thaliana seedlings against the pathogen Pseudomonas syringae pv. tomato DC3000. The five strains more effective in preventing pathogen infection were used to elucidate signal transduction pathways involved in the plant immune response by studying the differential expression of Salicylic acid and Jasmonic acid/Ethylene pathway marker genes. Some strains stimulated both pathways, while others stimulated either one or the other. The metabolic elicitors of two strains, chosen for the differential expression results of the genes studied, were extracted using n-hexane, ethyl acetate, and n-butanol, and their capacity to mimic bacterial effect to trigger the plant immune system was studied. N-hexane and ethyl acetate were the most effective fractions against the pathogen in both strains, achieving similar protection rates although gene expression responses were different from that obtained by the bacteria. These results open an amount of biotechnological possibilities to develop biological products for agriculture.

The Pseudomonas fluorescens strain used in this work (Aur 6) has demonstrated its ability to improve fitness of different plant species upon biotic and abiotic stress conditions. Random mutants of this strain were constructed with the Tn5 transposon technology, and biological tests to evaluate loss of salt protection were conducted with all the mutants (104 mutants) on rice seedlings. Mutant 33 showed an evident reduction in its ability to protect plants upon salt stress challenge, whereas mutant 19 was more effective than the wild type. Enzymes related with oxidative stress were studied in both mutants and wild type. Enzyme activities were decreased with mutant 33 with regard to wild type, whereas mutant 19 did not produce important changes suggesting involvement of redox balance associated to the observed modifications in these antioxidant enzymes as one of the probable mechanisms used by these strains. Data of malondialdehyde (MDA) were consistent with this fact. Mutants also affected accumulation of proline, the most common osmolyte in plants. A second experiment to evaluate the ability of both mutants and wild type to stimulate growth on tomato plants was conducted, as this feature was previously demonstrated by wild type. Similar results were obtained in growth of both species, suggesting that mutations of both mutants are related with the capacities of the wild type to stimulate growth. To reveal mutated genes, both mutants were mapped. Three mutated genes were found in mutant 33. A gene related with a general secretion pathway protein D, a gene related with a putative two-component system sensor kinase (ColS), and a gene related with flagellar motor switch protein (FliG). In mutant 19, two mutated genes were found. One gene related with heavy metal efflux pump Czca family, and other gene of 16s rRNA.

The use of plant growth-promoting rhizobacteria (PGPR) inoculated on plants has shown that it can increase the success of reforestation and accelerate soil recovery by improving soil microbial diversity. Three PGPR isolated from natural pine populations were selected for their metabolic capabilities and taxonomic affiliation (Z4.3; Bacillus sp., Z5.4; Arthobacter sp., and Z7.15; and Pseudomonas sp.) when inoculated alone or in combination (consortium) on stone pine seedlings before transplanting to the field. Before transplanting and after nine months, rhizospheric soil samples were collected for structural and functional metagenomic studies. First, the data were analyzed using EasyMAP. Neither alpha nor beta diversity showed significant differences between the samples, although unique taxa representative of each sample were detected. The predominant phylum in all cases was Proteobacteria, followed by Bacteroidetes and Acidobacteria. The linear discriminant analysis (LDA) effect size (LEfSe) found significantly over-represented taxa in some samples, highlighting different representatives of the order Sphingomonadales in several of them. Functional inference performed with PICRUSt also showed significantly over-represented functions in some samples. The study demonstrates that PGPR have a positive effect on plants and cause detectable changes in microbial communities in terms of both structure and function.

For the first time in the literature, a relationship between the root application of melatonin and the greater capacity for resistance against Psedomonas syringae DC3000 in tomato plants has been established. Root delivered melatonin (100 μM), induced systemic resistance against pathogen reducing disease incidence by 51%. Mechanisms of action used by melatonin were assessing through different physiological, metabolic, and genetic markers. As a physiological marker, photosynthetic efficiency was studied, with a TARGAS 1 portable photosynthesis system. Metabolic markers were analysed on leaf powder collected 1 week after the pathogen challenge. These markers analysed were grouped into those related to the scavenging of Reactive Oxygen Species (ROS) and oxidative stress (ascorbate peroxidase (APX) activity, hydrogen peroxide (H2O2) concentration, malondialdehyde (MDA) concentration, and proline concentration) and those related to defence mechanisms (ß-1,3-glucanase and chitinase). Genetic markers were studied on leaf powder collected 6 h and 10 h after pathogen challenge. For this, the differential expression of the genes PR1, PR2 and PR3 was studied. Upon pathogen challenge, melatonin reverted the negative effects of the pathogen in net photosynthesis rate achieving similar values to healthy plants. Melatonin reduced oxidative stress, according to lower MDA (29%) and H2O2 (46%), improving ROS scavenging potential by enhancing APX activity (83%) and proline concentration (44%). Melatonin simultaneously triggered the salicylic acid (SA)-mediated pathway and the jasmonic acid/ethylene (JA/ET)-mediated pathway as the enzymatic activities ß-1,3-glucanase (Pathogenesis-Related protein 2; PR2; 103%) and chitinase activitiy (Pathogenesis-Related protein 3; PR3; 44%), markers of the first and second pathways respectively, were enhanced. This enhanced activity was consistent with enhanced expression of genes encoding PR2 and PR3. Results obtained indicate that melatonin, a natural plant compound, could be used in tomato cultivation as an economical and ecofriendly chemical agent against biotic stress.

We evaluated the ability of metabolic elicitors extracted from Pseudomonas fluorescens N21.4 to induce systemic resistance (ISR) in Arabidopsis thaliana against the pathogen Pseudomonas syringae DC3000. Metabolic elicitors were obtained from bacteria free culture medium with n-hexane, ethyl acetate and n-butanol in three consecutive extractions. Each extract showed plant protection activity. The n-hexane fraction was the most effective and was used to study the signal transduction pathways involved by evaluating expression of marker genes of the salicylic acid (SA) signalling pathway (NPR1, PR1, ICS and PR2) and the jasmonic acid/ethylene (JA/ET) signalling pathway (PDF1, MYC2, LOX2 and PR3). In addition, the level of oxidative stress was tested by determining the activity of enzymes related to the ascorbate-glutathione cycle. N-hexane extracts stimulated both pathways based on overexpression of ICS, PR1, PR2, PDF1 and LOX2 genes. In addition, activity of the pathogenesis-related proteins glucanase (PR2) and chitinase (PR3), lipoxygenase and polyphenol oxidase was enhanced together with an increased capacity to remove reactive oxygen species (ROS). This was associated with less oxidative stress as indicated by a decrease in malondialdehyde (MDA), suggesting a causative link between defensive metabolism against P. syringae and ROS scavenging.

A 72,000 recombinant phages metagenomic library was constructed from rice rhizosphere. An esterase screening was performed and resulted in the identification of 6 positive esterase clones. Two of them, Ela1 and Ela2, were selected for a further characterization. Sequence analysis revealed that Ela1 exhibits a high homology with proteins annotated as acetyl xylan esterase (AXE) and Ela2 with SGNH hydrolases. Both enzymes are carboxylic ester hydrolases, with a high stability, an alkaline optimum pH 8-9 and active at high temperatures (75°C). Additionally, a 16S rRNA library was performed in order to characterize the biodiversity and biological diversity of the ecosystem source of this gene. It confirmed the predominance of thermophilic groups of bacteria matching with the esterases Ela1 and Ela2 annotation results and biochemical characterization. Thus, rice rhizosphere, which is a high-pressure selective ecosystem, arises as a very appropriate source of novel enzymes with a great potential for biotechnological and industrial applications.

Beneficial rhizobacterium Pseudomonas fluorescens N 21.4 and its metabolic elicitors inoculated to cultivars of blackberry (Rubus spp. Var. Loch Ness) reinforced the plants’ immune system and improved their fitness by increasing photosynthesis, decreasing oxidative stress, and activating pathogenesis-related proteins. They also triggered the leaves’ flavonoid metabolism, enhancing the accumulation of beneficial phenolic compounds such as kaempferols and quercetin derivatives. The elicitation of leaf secondary metabolism allows one to take advantage of the blackberry leaves (a current crop waste), following the premises of the circular economy, to isolate and obtain high added value compounds. The results of this work suggest the use of N 21.4 and/or its metabolic elicitors as plant inoculants as an effective and economically and environmentally friendly agronomic alternative practice in the exploitation of blackberry crops to obtain plants with a better immune system and to revalorize the leaf pruning as a potential source of polyphenols.

Plant bioactives are unique sources for pharmaceuticals, food additives, flavors, and other industrial materials. Since a great part of beneficial foods and food components are from plant origin, improving agricultural production of crops with a high bioactive content is of increasing interest. On the other hand, a great part of plant bioactives are secondary metabolites, and therefore synthesized by plants only to overcome environmental changes along the plant‟s biological cycle; hence, since secondary metabolism is inducible, bioactive levels change constantly on field produced foods. In view of the above, identification of biotic elicitors from microbial origin is a topic with increasing interest due to its potential application in cell and tissue culture to obtain functional ingredients, or even in fresh functional foods directly to consumers. In this sense the literature reports a number of studies in which elicitors from pathogenic microorganisms are used, but the use of beneficial microorganisms as plant growth promoting rhizobacteria (PGPR) or their metabolic elicitors are still to see an outstanding application on the field of functional foods. Two case studies are presented to illustrate the rationale of our working hypothesis, showing how the inoculants can improve contents of bioactives: one dealing with Hypericum perforatum hipericins, another one on Glycine max. with isoflavones.

Exposure to ultraviolet-B (UV-B) radiation can lead to oxidative damage in plants, increasing reactive oxygen species (ROS) production. To overcome ROS burst, plants have antioxidant mechanisms related to ROS scavenging which can be improved by elicitation with biological agents or derived molecules (elicitors), as they can trigger a physiological alert state called “priming”. This work describes the effects of lipo-chitooligosaccharides (LCOs) treatment applied to tomato plants under UV-B stress. The LCOs used in the study are produced by three species of the genus Ensifer (formerly Sinorhizobium) (SinCEU-1, SinCEU-2, and SinCEU-3) were assayed on tomato plants under UV-B stress. LCOs were able to significantly increase most of the enzymatic activities related to ROS scavenging while non-enzymatic antioxidants were not modified. This response was associated with a lower oxidative stress, according to malondialdehyde (MDA) levels and the higher antioxidant capacity of the plants. Furthermore, the photosynthetic efficiency of LCOs-treated plants indicated a better physiological state than the control plants. Therefore, although more studies and deepening of certain aspects are necessary, LCOs have shown great potential to protect plants from high UV-B radiation conditions.

Global climate change has increased warming with a concomitant decrease in water availability and increased soil salinity, factors that compromise agronomic production. On the other hand, new agronomic developments using irrigation systems demand increasing amounts of water to achieve an increase in yields. Therefore, new challenges appear to improve plant fitness and yield, while limiting water supply for specific crops, particularly, olive trees. Plants have developed several innate mechanisms to overcome water shortage and the use of beneficial microorganisms to ameliorate symptoms appears as a challenging alternative. Our aim is to improve plant fitness with beneficial bacterial strains capable of triggering plant metabolism that targets several mechanisms simultaneously. Our secondary aim is to improve the content of molecules with bioactive e ects to valorize pruning residues. To analyze bacterial e ects on olive plantlets that are grown in saline soil, photosynthesis, photosynthetic pigments, osmolytes (proline and soluble sugars), and reactive oxygen species (ROS)-scavenging enzymes (superoxide dismutase-SOD and ascorbate peroxidase-APX) and molecules (phenols, flavonols, and oleuropein) were determined. We found photosynthetic pigments, antioxidant molecules, net photosynthesis, and water use e ciency to be the most a ected parameters. Most strains decreased pigments and increased osmolytes and phenols, and only one strain increased the antihypertensive molecule oleuropein. All strains increased net photosynthesis, but only three increased water use e ciency. In conclusion, among the ten strains, three improved water use e ciency and one increased values of pruning residues.

La presente Tesis Doctoral se centra en el efecto de cepas PGPB sobre la mejora de la adaptación a situaciones de estrés biótico y abiótico, sobre planta modelo Arabidopsis thaliana y Olea europaea L. Los efectos de las bacterias sobre la variedad Arbequina son mucho más acusados que en la variedad Arbosana y en los estudios realizados se comprueba que los pigmentos fotosintéticos son una diana común de las PGPB ensayadas. Teniendo en cuenta estos resultados se seleccionaron las 3 cepas más efectivas en ambas especies que presentaban una mayor divergencia genética en base al gen 16s ARN, para profundizar en el estudio de los mecanismos de adaptación a estrés propios de cada cepa. En el estudio en A. thaliana, se demostró que las tres cepas seleccionadas activan simultáneamente las dos rutas de transducción de señal implicadas en defensa, Et/J y SA, demostrando la intenelación entre ambas rutas de señalización. En cuanto a los resultados obtenidos en la variedad Arbequina se ha puesto de manifiesto que la mejora en la adaptación a estrés salino inducida por la cepa G7 implica la activación de la ruta del Ácido Abcísico, y que la cepa H47 mejora la adaptación de las plantas de olivo a estrés salino estimulando el metabolismo secundario. Por último, la aplicación de las 3 cepas, tanto en planta de olivo como en restos de poda, inducen un aumento de flavonoles e iridoides, aumentando su potencial de uso con fines nutricionales o farmacológicos y contribuyendo así a la economía circular del olivo.

Background: The beneficial rhizobacterium, Pseudomonas fluorescens N 21.4, and its metabolic elicitors were inoculated in com-mercial cultivars of blackberry plants (Rubus cv. Loch Ness). Phenolic compounds present in red and black fruit and the expres-sion of structural marker genes of the phenylpropanoid pathway during fruit ripening were studied.Results: An inverse relationship between gene expression and accumulation of metabolites was seen, except for the RuDFRgene, which had a direct correlation with cyanidin 3-O-glucoside synthesis, increasing its content 1.3 times when RuDFR wasoverexpressed in the red fruit of plants inoculated with the metabolic elicitors of P. fluorescens N 21.4, compared with red fruitof plants inoculated with N 21.4. The RuCHS gene also had a fundamental role in the accumulation of metabolites. Both rhizo-bacterium and metabolic elicitors triggered the flavonoid metabolism, enhancing the catechin and epicatechin contentbetween 1.1 and 1.6 times in the case of red fruit and between 1.1 and 1.8 times in the case of black fruit. Both treatments alsoboosted the anthocyanin, quercetin, and kaempferol derivative content, highlighting the effects of metabolic elicitors in redfruit and the effects of live rhizobacterium in black fruit.Conclusion: The metabolic elicitors' capacity to modulate gene expression and to increase secondary metabolites content wasdemonstrated. This work therefore suggests that they are effective, affordable, easily manageable, and ecofriendly plant inoc-ulants that complement, or are alternatives to, beneficial rhizobacteria.

Climate change consequences for agriculture involve an increase of saline soils which results in lower crop yields due to increased oxidative stress in plants. The present study reports the use of Plant Growth Promoting Bacteria (PGPB) as a tool to modulate plant innate mechanisms of adaptation to water stress (salinity and drought) in one year-old olive plantlets var. Arbosana and Arbequina. Integration of external changes in plants involve changes in Reactive Oxygen Species (ROS) that behave as signals to trigger plant adaptative mechanisms; however, they become toxic in high concentrations. For this reason, plants are endowed with antioxidant systems to keep ROS under control. So, the working hypothesis is that specific beneficial strains will induce a systemic response able to modulate oxidative stress and improve plant adaptation to water stress. Ten strains were assayed, evaluating changes in photosynthesis, pigments, ROS scavenging enzymes and antioxidant molecules, osmolytes and malondialdehyde, as oxidative stress marker. Photosynthesis and photosynthetic pigments were the most affected variables. Despite the specific response of each variety, the favorite targets of PGPBs to improve plant fitness were photosynthetic pigments and the antioxidant pools of glutathione and ascorbate. Our results show the potential of PGPBs to improve plant fitness modulating oxidative stress.

A gram-positive, nonpathogenic, central endospore-forming, flagellated strain, was successfully isolated from the rhizosphere of Pinus pinaster in Aracena (Spain). Its optimal growth conditions are 28 ◦C, pH 6, and 0 % salinity. It is able to assimilate glucose, L-fucose, L-arabinose, b-metil-Dxylose and shows high catabolic capacity. The major fatty acids (>79.20 % of the total fatty acids) are anteiso C15:0 > iso C15:0 > C14:0. A phylogenetic analysis based on the 16S rRNA gene sequence revealed similarity to P. frigoritolerans DSM8801T (99.9 %), P. castrilensis CECT30509T (99.8 %), and P. simplex DSM1321T (99.6 %). Comparison of whole genomes revealed that strain BBB004T is more similar to P. simplex DSM1321T. According to ANI (93.54 %), AAI (94 %), dDDH (60.6), %G + C (0.12), TETRA (0.99822) and intergenomic distance (0.2835) values, therefore this species is different to the closest. A total of 133 genes unique to Peribacillus BBB004T were identified. Supported by these analyses, strain BBB004T (=LMG32742T = CCUG76477T) was proposed as the type strain for a new species, named “Peribacillus aracenensis sp. nov.”

Physiological, metabolic, and genetic changes produced by two plant growth promoting rhizobacteria (PGPR) Pseudomonas sp. (internal code of the laboratory: N 5.12 and N 21.24) inoculated in tomato plants subjected to moderate water stress (10% polyethylene glycol-6000; PEG) were studied. Photosynthesis efficiency, photosynthetic pigments, compatible osmolytes, reactive oxygen species (ROS) scavenging enzymes activities, oxidative stress level and expression of genes related to abscisic acid synthesis (ABA; 9-cis-epoxycarotenoid dioxygenase NCDE1 gene), proline synthesis (Pyrroline-5-carboxylate synthase P5CS gene), and plasma membrane ATPase (PM ATPase gene) were measured. Photosynthetic efficiency was compromised by PEG, but bacterial-inoculated plants reversed the effects: while N5.12 increased carbon fixation (37.5%) maintaining transpiration, N21.24 increased both (14.2% and 31%), negatively affecting stomatal closure, despite the enhanced expression of NCDE1 and plasma membrane ATPase genes, evidencing the activation of different adaptive mechanisms. Among all parameters evaluated, photosynthetic pigments and antioxidant enzymes guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) responded differently to both strains. N 5.12 increased photosynthetic pigments (70% chlorophyll a, 69% chlorophyll b, and 65% carotenoids), proline (33%), glycine betaine (4.3%), and phenolic compounds (21.5%) to a greater extent, thereby decreasing oxidative stress (12.5% in Malondialdehyde, MDA). Both bacteria have highly beneficial effects on tomato plants subjected to moderate water stress, improving their physiological state. The use of these bacteria in agricultural production systems could reduce the amount of water for agricultural irrigation without having a negative impact on food production.

The aim of this study is focused on determining the Bacillus amyloliquefaciens QV15 priming fingerprint in two different plant species, Arabidopsis and blackberry as a crop of agronomic interest, associated with protection upon pathogen challenge. To achieve this goal, Arabidopsis thaliana plants were challenged with Pseudomonas syringae DC3000 under controlled conditions, and field-grown blackberries were challenged by a powdery Mildew outbreak, finding plant protection in plants treated with QV15, in both conditions. Changes in ROS scavenging enzymes’ activity, defense-related enzymes’ activity and gene expression were evaluated in both plant species, before and after pathogen challenge, revealing the ability of this strain to prime both. As a result of this analysis, the priming fingerprint induced by QV15 was defined by a decrease in ROS scavenging enzymes’ activity in pre- and post-challenged plants, an increase in glucanase and chitinase activity after pathogen challenge, significantly increasing the expression of PR1, indicating a salicylic acid (SA)-mediated pathway activation. These results suggest an excellent potential of B. amyloliquefaciens QV15 to protect different plant species against different pathogens in field conditions.

Two plant growth promoting rhizobacteria (PGPR) were tested to evaluate their capacity to prime rice seedlings against stress challenge (salt and Xanthomonas campestris infection). As is accepted that plants respond to biotic and abiotic stresses by generation of reactive oxygen species (ROS), enzyme activities related to oxidative stress (ascorbate peroxidase (APX, EC 1.11.1.11), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione reductase (GR, EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1)) as well as the pathogenesis-related proteins (PRs) ß-1,3-glucanase (PR2, EC 3.2.1.6) and chitinase (PR3, EC 3.2.1.14) weremeasured at 3 timepoints after stress challenge.Inaddition,photosyntheticparameters related with fluorescence emission of photosystem II (F0, Fv/Fm, PSII and NPQ) were also measured although they were barely affected. Both strains were able to protect rice seedlings against salt stress. AMG272 reduced the salt symptoms over 47% with regard to control, and L81 over 90%. Upon pathogen challenge, 90% protection was achieved by both strains.All enzyme activities related to oxidative stress were modified by the two PGPR, especially APX and SOD upon salinity stress challenge, and APX and GR upon pathogen presence. Both bacteria induced chitinase activity 24 and 48 h after pathogen inoculation, and L81 induced ß-1,3-Glucanase activity 48 h after pathogen inoculation, evidencing the priming effect. These results indicate that these strains could be used as bio-fortifying agents in biotechnological inoculants in order to reduce the effects of different stresses, and indirectly reduce the use of agrochemicals.