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.”

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.

A phyto-rhizoremediation system using corn and esparto fiber as rooting support to remediate degraded metal working fluids (dMWFs) has been developed in the present study. In order to improve the process, plants were inoculated at the root level with bacteria either individually, and with a consortium of strains. All strains used were able to grow with MWFs. The results show that this system significantly lowers the Chemical Oxygen Demand below legal limits within 5 days. However, results were only improved with the bacterial consortium. Despite the effectiveness of the phyto-rhizoremediation process, plants are damaged at the photosynthetic level according to the photosynthetic parameters measured, as well as at the ultrastructure of the vascular cylinder and the Bundle Sheath Cells. Interestingly, the bacterial inoculation protects against this damage. Therefore, it seems that that the inoculation with bacteria can protect the plants against these harmful effects.

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.

An extensive microbiological study has been carried out in a membrane bioreactor fed with activated sludge and metal-working fluids. Functional diversity and dynamics of bacterial communities were studied with different approaches. Functional diversity of culturable bacterial communities was studied with different Biolog™ plates. Structure and dynamics of bacterial communities were studied in culturable and in non-culturable fractions using a 16S rRNA analysis. Among the culturable bacteria, Alphaproteobacteria and Gammaproteobacteria were the predominant classes. However, changes in microbial community structure were detected over time. Culture-independent analysis showed that Betaproteobacteria was the most frequently detected class in the membrane bioreactor (MBR) community with Zoogloea and Acidovorax as dominant genera. Also, among non-culturable bacteria, a process of succession was observed. Longitudinal structural shifts observed were more marked for non-culturable than for culturable bacteria, pointing towards an important role in the MBR performance. Microbial community metabolic abilities assessed with Biolog™ Gram negative, Gram positive and anaerobic plates also showed differences over time for Shannon's diversity index, kinetics of average well colour development, and the intensely used substrates by bacterial community in each plate.

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.

For the first time in the literature, duckweed (Lemna minor) tolerance (alone or in combination with a consortium of bacteria) to spent metal-working fluid (MWF) was assessed, together with its capacity to reduce the chemical oxygen demand (COD) of this residue. In a preliminary study, L. minor response to pre-treated MWF residue (ptMWF) and vacuum-distilled MWF water (MWFw) was tested. Plants were able to grow in both residues at different COD levels tested (up to 2300 mg·l−1), showing few toxicity symptoms (mainly growth inhibition). Plant response to MWFw was more regular and dose responsive than when exposed to ptMWF. Moreover, COD reduction was less significant in ptMWF. Thus, based on these preliminary results, a second study was conducted using MWFw to test the effectiveness of inoculation with a bacterial consortium isolated from a membrane bioreactor fed with the same residue. After 5 days of exposure, COD in solutions containing inoculated plants was significantly lower than in non-inoculated ones. Moreover, inoculation reduced β+γ-tocopherol levels in MWFw-exposed plants, suggesting pollutant imposed stress was reduced. We therefore conclude from that L. minor is highly tolerant to spent MWF residues and that this species can be very useful, together with the appropriate bacterial consortium, in reducing COD of this residue under local legislation limits and thus minimise its potential environmental impact. Interestingly, the lipophilic antioxidant tocopherol (especially the sum of β+γ isomers) proved to be an effective plant biomarker of pollution.

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.