1. Investigación

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.

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 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.