Improving Flavonoid Metabolism in Blackberry Leaves and Plant Fitness by Using the Bioeffector Pseudomonas fluorescens N 21.4 and Its Metabolic Elicitors: A Biotechnological Approach for a More Sustainable Crop.

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.

Extracts from cultures of Pseudomonas fluorescens induce defensive patterns of gene expression and enzyme activity while depressing visible injury and reactive oxygen species in Arabidopsis thaliana challenged with pathogenic Pseudomonas syringae.

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.

Microbe associated molecular patterns from rhizosphere bacteria trigger germination and Papaver somniferum metabolism under greenhouse conditions.

Ten PGPR from different backgrounds were assayed on Papaver somniferum var. Madrigal to evaluate their potential as biotic elicitors to increase alkaloid content under the rationale that some microbe associated molecular patterns (MAMPs) are able to trigger plant metabolism. First, the 10 strains and their culture media at two different concentrations were tested for their ability to trigger seed germination. Then, the best three strains were tested for their ability to increase seedling growth and alkaloid levels under greenhouse conditions. Only three strains and their culture media enhanced germination. Then, germination enhancing capacity of these best three strains, N5.18 Stenotrophomonas maltophilia, Aur9 Chryseobacterium balustinum and N21.4 Pseudomonas fluorescens was evaluated in soil. Finally, the three strains were applied on seedlings at two time points, by soil drench or by foliar spray. Photosynthesis was measured, plant height was recorded, capsules were weighted and alkaloids analyzed by HPLC. Only N5.18 delivered by foliar spray significantly increased plant height coupled to an increase in total alkaloids and a significant increase in opium poppy straw dry weight; these increases were supported by a better photosynthetic efficiency. The relative contents of morphine, thebaine, codeine and oripavine were affected by this treatment causing a significant increase in morphine coupled to a decrease in thebaine, demonstrating the effectivity of MAMPs from N5.18 in this plant species. Considering the increase in capsule biomass and alkaloids together with the acceleration of germination, strain N5.18 appears as a good candidate to elicit plant metabolism and consequently, to increase productivity of Papaver somniferum.

Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens.

The aim of this study was two-fold: first, to characterize blackberry fruits from Rubus sp. var. Lochness along the year, and secondly, to evaluate the ability of a Pseudomonas strain (N21.4) to improve fruit yield and quality under field conditions in production greenhouses throughout the year. The strain was root or leaf inoculated to blackberry plants and fruits were harvested in each season. Nutritional parameters, antioxidant potential and bioactive contents were determined; total fruit yield was recorded. Blackberries grown under short day conditions (autumn and winter) showed significantly lower °Brix values than fruits grown under long day conditions. Interestingly, an increase in fruit °Brix, relevant for quality, was detected after bacterial challenge, together with significant and sustained increases in total phenolics and flavonoids. Improvements in inoculated fruits were more evident from October through early March, when environmental conditions are worse. In summary, N21.4 is an effective agent to increase fruit quality and production along the year in blackberry; this is an environmentally friendly approach to increase fruit quality.

Bacterial siderophores efficiently provide iron to iron-starved tomato plants in hydroponics culture.

Iron is one of the essential elements for a proper plant development. Providing plants with an accessible form of iron is crucial when it is scant or unavailable in soils. Chemical chelates are the only current alternative and are highly stable in soils, therefore, posing a threat to drinking water. The aim of this investigation was to quantify siderophores produced by two bacterial strains and to determine if these bacterial siderophores would palliate chlorotic symptoms of iron-starved tomato plants. For this purpose, siderophore production in MM9 medium by two selected bacterial strains was quantified, and the best was used for biological assay. Bacterial culture media free of bacteria (S) and with bacterial cells (BS), both supplemented with Fe were delivered to 12-week-old plants grown under iron starvation in hydroponic conditions; controls with full Hoagland solution, iron-free Hoagland solution and water were also conducted. Treatments were applied twice along the experiment, with a week in between. At harvest, plant yield, chlorophyll content and nutritional status in leaves were measured. Both the bacterial siderophore treatments significantly increased plant yield, chlorophyll and iron content over the positive controls with full Hoagland solution, indicating that siderophores are effective in providing Fe to the plant, either with or without the presence of bacteria. In summary, siderophores from strain Chryseobacterium C138 are effective in supplying Fe to iron-starved tomato plants by the roots, either with or without the presence of bacteria. Based on the amount of siderophores produced, an effective and economically feasible organic Fe chelator could be developed.

Systemic disease protection elicited by plant growth promoting rhizobacteria strains: relationship between metabolic responses, systemic disease protection, and biotic elicitors.

A study of plant defensive systemic responses induced by three plant growth promoting rhizobacteria (PGPR) on Arabidopsis thaliana Col 0 against Pseudomonas syringae pv. tomato DC3000 at the biochemical and transcriptional levels is reported in this paper. All three strains decreased disease severity when applied to A. thaliana prior to pathogen inoculation. At the biochemical level, each of the three strains induced ethylene (ET) when incubated with 1-amino-cyclopropane-1-carboxylic acid, and salicylic acid (SA) production in the plant. Plants treated with each of the three strains were also reduced in salicylic acid production after pathogen challenge compared to untreated controls. This effect was more marked in plants treated with Chryseobacterium balustinum AUR9, the strain most effective in decreasing disease severity. The expression level of PR1, a transcriptional marker of the SA-dependent pathway in C. balustinum AUR9-treated plants, is fourfold that of controls while the expression of PDF1.2, a transcriptional marker for the SA-independent pathway, is not induced. C. balustinum cell wall lipopolysaccharides, being putative bacterial elicitor molecules, are able to reproduce this systemic induction effect at low doses. From these observations, we hypothesize that certain PGPR strains are capable of stimulating different systemic responses in host plants. With C. balustinum AUR9, the SA-dependent pathway is stimulated first, as indicated by increases in SA levels and PR1 expression, followed by induction of the SA-independent pathway, as indicated by the increases in ET concentrations. The effects of both pathways combined with respect to disease suppression appear to be additive.

Effect of inoculation with putative plant growth-promoting rhizobacteria isolated from Pinus spp. on Pinus pinea growth, mycorrhization and rhizosphere microbial communities.

AIMS: In this study, 10 putative plant growth-promoting rhizobacteria (PGPR) were assayed for their ability to improve Pinus pinea growth and mycorrhization. METHODS AND RESULTS: After an inoculation assay, except for two, all strains stimulated plant growth. All bacteria altered rhizosphere microbial communities as revealed by phospholipid fatty acid analysis; associating plant growth promotion with a decrease in biological diversity. Three strains were tested for their ability to enhance pine mycorrhization with wild fungi species. Only strain BB1 increased the total number of mycorrhizal root tips. Mycorrhizas present in the roots of each treatment were identified by ribosomal RNA sequencing and denaturing gradient gel electrophoresis analysis, detecting specificity between mycorrhizal species colonizing the roots and the inoculated PGPR. CONCLUSIONS: In conclusion, BB1 appears to be a good candidate to be developed into a biofertilizer directed to enhance pine growth and mycorrhization, which should result in a better establishment rate for plants used in reforestation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of PGPR to improve fitness of forest tree specie. Moreover, the specificity between the bacteria inoculated and the mycorrhiza that the plant selects involve a potential biotechnological use in production of value-added fungi.