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.”

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.

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.

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.

Blackberries (Rubus spp.) are among the high added value food products relevant for human health due to the increasing evidence of the beneficial effects of polyphenols, which are very abundant in these fruits. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress. Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a de novo transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs. Transcripts were aligned against Fragaria vesca genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20,463 genes common to leaves and fruits, while 6,604 genes were significantly overexpressed only in fruits, while another 6,599 genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present. Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidin-3-glucoside as the only common compound identified. Next, RT-qPCR of the core genes in the flavonol anthocyanin pathway and regulatory MYB genes were carried out. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels. On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis upon ripening. This suggests that, in addition to the biosynthesis taking place in the fruits during ripening, translocation of flavonols from leaves to fruits contributes to the high amounts of bioactives starting to accumulate in fruits.

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.

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.