1. Investigación

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.

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.”

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.

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.

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.

Aims: to discover the interrelationship between growth, protection and photosynthesis induced by Pseudomonas fluorescens N21.4 in tomato (Lycopersicum sculentum) challenged with the leaf pathogen Xanthomonas campestris, and to define its priming fingerprint. Methods: Photosynthesis was determined by fluorescence; plant protection was evaluated by relative disease incidence, enzyme activities by specific colorimetric assays and gene expression by qPCR. Changes in Reactive Oxygen Species (ROS) scavenging cycle enzymes and pathogenesis related protein activity and expression were determined as metabolic and genetic markers of induction of systemic resistance. Results: N21.4 significantly protected plants and increased dry weight. Growth increase is supported by significant increases in photochemical quenching together with significant decreases in energy dissipation (Non-Photochemical Quenching, NPQ). Protection was associated with changes in ROS scavenging cycle enzymes, which were significantly increased on N21.4 + pathogen challenged plants, supporting the priming effect. Superoxide Dismutase (SOD) was a good indicator of biotic stress, showing similar levels in pathogen- and N21.4-treated plants. Similarly, the activity of defense-related enzymes, ß-1,3-glucanase and chitinase significantly increased in post-pathogen challenge state; changes in gene expression were not coupled to activity. Conclusions: protection does not compromise plant growth; N21.4 priming fingerprint is defined by enhanced photochemical quenching and decreased energy dissipation, enhanced chlorophylls, primed ROS scavenging cycle enzyme activity, and glucanase and chitinase activity.

A novel Pseudomonas, designated strain BBB001T , an aerobic, rod-shaped bacterium, was isolated from the rhizosphere of Nicotiana glauca in Las Palmas Gran Canaria, Spain. Genomic analysis revealed that it could not be assigned to any known species of Pseudomonas, so the name Pseudomonas palmensis sp. nov. was proposed. A 16S rRNA gene phylogenetic analysis suggested affiliation to the Pseudomonas brassicae group, being P. brassicae MAFF212427 T the closest related type strain. Upon genomic comparisons of both strains, all values were below thresholds established for differentiation: average nucleotide identity (ANI, 88.29%), average amino acid identity (AAI, 84.53%), digital DNA-DNA hybridization (dDDH, 35.4%), and TETRA values (0.98). When comparing complete genomes, a total of 96 genes present exclusively in BBB001T were identified, 80 of which appear associated with specific subsystems. Phenotypic analysis has shown its ability to assimilate glucose, potassium gluconate, capric acid malate, trisodium citrate, and phenylacetic acid; it was oxidase positive. It is able to produce auxins and siderophores in vitro; its metabolic profile based on BIOLOG Eco has shown a high catabolic capacity. The major fatty acids accounting for 81.17% of the total fatty acids were as follows: C16:0 (33.29%), summed feature 3 (22.80%) comprising C16:1 ω7c and C 16:1 ω6c, summed feature 8 (13.66%) comprising C18:1 ω7c, and C18:1ω6c and C17:0 cyclo (11.42%). The ability of this strain to improve plant fitness was tested on tomato and olive trees, demonstrating a great potential for agriculture as it is able to trigger herbaceous and woody species. First, it was able to improve iron nutrition and growth on iron-starved tomatoes, demonstrating its nutrient mobilization capacity; this effect is related to its unique genes related to iron metabolism. Second, it increased olive and oil yield up to 30% on intensive olive orchards under water-limiting conditions, demonstrating its capacity to improve adaptation to adverse conditions. Results from genomic analysis together with differences in phenotypic features and chemotaxonomic analysis support the proposal of strain BBB001T (=LMG 31775T = NCTC 14418T ) as the type strain of a novel species for which the name P. palmensis sp. nov is proposed.

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.

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.

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.