Functional diversity and dynamics of bacterial communities in a membrane bioreactor for the treatment of metal-working fluid wastewater.

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.

Bacterial bioeffectors modify bioactive profile and increase isoflavone content in soybean sprouts (Glycine max var Osumi).

The effect of two bacterial strains to enhance bioactive contents (total phenolic compounds, total flavonoid compounds and isoflavones) and antioxidant activity on 3-day-old soybean sprouts were investigated. To identify bacterial determinants responsible for these effects, viable and UV-treated strains were delivered to wounded seeds at different concentration. Multivariate analysis performed with all the evaluated parameters indicated the different effectiveness of Stenotrophomonas maltophilia N5.18 and Pseudomonas fluorescens N21.4 based on different structural and metabolic determinants for each. N21.4 increased total phenolics and isoflavones from the genistein family, while N5.18 triggered biosynthesis of daidzein and genistein families coupled to a decrease in total phenolics, suggesting different molecular targets in the phenilpropanoid pathway. Only extracts from N5.18 treated seeds showed an improved antioxidant activity according to the ß-carotene bleaching prevention method. In summary, bioeffectors from both bacterial strains are effective tools to improve soybean sprouts quality; structural elicitors from N5.18 also enhanced antioxidant activity, being the best alternative for further development of a biotechnological procedure.

Bacillus G7 improves adaptation to salt stress in Olea europaea L. plantlets, enhancing water use efficiency and preventing oxidative stress.

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.

Modulation of Photosynthesis and ROS Scavenging Response by Beneficial Bacteria in Olea europaea Plantlets under Salt Stress Conditions.

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.

Bacillus H47 triggers Olea europaea metabolism activating DOXP and shikimate pathways simultaneously and modifying leaf extracts' antihypertensive activity.

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.

Elicitation with Bacillus QV15 reveals a pivotal role of F3H on flavonoid metabolism improving adaptation to biotic stress in blackberry.

The aim of this study is to determine the involvement of the flavonol-anthocyanin pathway on plant adaptation to biotic stress using the B.amyloliquefaciens QV15 to trigger blackberry metabolism and identify target genes to improve plant fitness and fruit quality. To achieve this goal, field-grown blackberries were root-inoculated with QV15 along its growth cycle. At fruiting, a transcriptomic analysis by RNA-Seq was performed on leaves and fruits of treated and non-treated field-grown blackberries after a sustained mildew outbreak; expression of the regulating and core genes of the Flavonol-Anthocyanin pathway were analysed by qPCR and metabolomic profiles by UHPLC/ESI-qTOF-MS; plant protection was found to be up to 88%. Overexpression of step-controlling genes in leaves and fruits, associated to lower concentration of flavonols and anthocyanins in QV15-treated plants, together with a higher protection suggest a phytoanticipin role for flavonols in blackberry; kempferol-3-O-rutinoside concentration was strikingly high. Overexpression of RuF3H (Flavonol-3-hidroxylase) suggests a pivotal role in the coordination of committing steps in this pathway, controlling carbon flux towards the different sinks. Furthermore, this C demand is supported by an activation of the photosynthetic machinery, and boosted by a coordinated control of ROS into a sub-lethal range, and associated to enhanced protection to biotic stress.

Management of Plant Physiology with Beneficial Bacteria to Improve Leaf Bioactive Profiles and Plant Adaptation under Saline Stress in Olea europea L.

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 effects to valorize pruning residues. To analyze bacterial effects 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 efficiency to be the most affected 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 efficiency. In conclusion, among the ten strains, three improved water use efficiency and one increased values of pruning residues.

Identifying the Compounds of the Metabolic Elicitors of Pseudomonas fluorescens N 21.4 Responsible for Their Ability to Induce Plant Resistance.

In this work, the metabolic elicitors extracted from the beneficial rhizobacterium Pseudomonas fluorescens N 21.4 were sequentially fragmented by vacuum liquid chromatography to isolate, purify and identify the compounds responsible for the extraordinary capacities of this strain to induce systemic resistance and to elicit secondary defensive metabolism in diverse plant species. To check if the fractions sequentially obtained were able to increase the synthesis of isoflavones and if, therefore, they still maintained the eliciting capacity of the live strain, rapid and controlled experiments were done with soybean seeds. The optimal action concentration of the fractions was established and all of them elicited isoflavone secondary metabolism-the fractions that had been extracted with n-hexane being more effective. The purest fraction was the one with the highest eliciting capacity and was also tested in Arabidopsis thaliana seedlings to induce systemic resistance against the pathogen Pseudomonas syringae pv. tomato DC 3000. This fraction was then analyzed by UHPLC/ESI-QTOF-MS, and an alkaloid, two amino lipids, three arylalkylamines and a terpenoid were tentatively identified. These identified compounds could be part of commercial plant inoculants of biological and sustainable origin to be applied in crops, due to their potential to enhance the plant immune response and since many of them have putative antibiotic and/or antifungal potential.

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria.

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.

Transcriptomics, Targeted Metabolomics and Gene Expression of Blackberry Leaves and Fruits Indicate Flavonoid Metabolic Flux from Leaf to Red Fruit.

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 role of isoflavone metabolism in plant protection depends on the rhizobacterial MAMP that triggers systemic resistance against Xanthomonas axonopodis pv. glycines in Glycine max (L.) Merr. cv. Osumi.

Glycine max (L.) Merr. plays a crucial role in both the field of food and the pharmaceutical industry due to their input as plant protein and to the benefits of isoflavones (IF) for health. In addition, IF play a key role in nodulation and plant defense and therefore, an increase in IF would be desirable for better field performance. IF are secondary metabolites and therefore, inducible, so finding effective agents to increase IF contents is interesting. Among these agents, plant growth promoting rhizobacteria (PGPR) have been used to trigger systemic induction of plant's secondary metabolism through their microbe associated molecular patterns (MAMPs) that fit in the plant's receptors to start a systemic response. The aim of this study was to evaluate the ability of 4 PGPR that had a contrasted effect on IF metabolism, to protect plants against biotic stress and to establish the relation between IF profile and the systemic response triggered by the bacteria. Apparently, the response involves a lower sensitivity to ethylene and despite the decrease in effective photosynthesis, growth is only compromised in the case of M84, the most effective in protection. All strains protected soybean against Xanthomonas axonopodis pv. glycines (M84 > N5.18 > Aur9>N21.4) and only M84 and N5.18 involved IF. N5.18 stimulated accumulation of IF before pathogen challenge. M84 caused a significant increase on IF only after pathogen challenge and N21.4 caused a significant increase on IF content irrespective of pathogen challenge. Aur9 did not affect IF. These results point out that all 4 strains have MAMPs that trigger defensive metabolism in soybean. Protection induced by N21.4 and Aur9 involves other metabolites different to IF and the role of IF in defence depends on the previous metabolic status of the plant and on the bacterial MAMP.

Biotic elicitation of isoflavone metabolism with plant growth promoting rhizobacteria in early stages of development in Glycine max var. Osumi.

Nine plant growth-promoting rhizobacteria from different backgrounds were assayed on Glycine max var. Osumi to evaluate their potential as biotic elicitors to increase isoflavone (IF) levels. Strains were inoculated on 2 day old pregerminated seeds. Six days after inoculation, the seedlings were harvested. Biometric parameters were registered, and IFs were determined. Although only one strain (N21.4) increased total IF contents and only one (M84) caused significant decreases in total IF, five different behaviors were detected when the daidzein and genistein families were analyzed separately. All strains triggered IF metabolism so further studies have to be developed since the different beneficial effects of IF through the diet may be due to the different IF profiles. These are encouraging results from two points of view: (1) N21.4 increases IF in seedlings, and (2) all other beneficial strains trigger IF metabolism differentially; hence, both facts could be used to prepare food supplements or as enriched standardized foods after full development of the biotechnological procedure.